Truncated platelet-activating factor acetylhydrolase

专利摘要:
The present invention provides purified and isolated polynucleotide sequences encoding plasma platelet-activating factor acetylhydrolase. Also provided are methods and materials for the recombinant production of platelet-activating factor acetylhydrolase products which are predicted to be useful for modulating pathological inflammation.
公开号:KR20000068780A
申请号:KR1019997003367
申请日:1997-08-13
公开日:2000-11-25
发明作者:커센스로렌스에스；에버하트크리스틴디；그레이패트릭；트롱하이르；조엘커래리더블류；윌더체릴엘
申请人:게리 엘. 윌콕스；이코스 코포레이션；
IPC主号:

专利说明:

Truncated platelet-activating factor acetylhydrolase {TRUNCATED PLATELET-ACTIVATING FACTOR ACETYLHYDROLASE}
[2] Platelet-activating factor (PAF) is a biologically active phospholipid synthesized by various cell types. At normal concentrations and in vivo concentrations of 10 ⁻¹⁰ to 10 ⁻⁹ M, PAF activates target cells such as platelets and neutrophils by binding to specific G protein-binding cell surface receptors [Venable et al. J. Lipid Res. , 34: 691-701 (1993). PAF has the structure 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine. For optimal biological activity, the sn-1 position of the PAF glycerol backbone should ether link with fatty alcohol and the sn-3 position should have a phosphocholine head group. PAF acts in normal physiological processes (eg inflammation, hemostasis and powder) and is an indicator of pathological inflammatory responses (eg, asthma, hypersensitivity, sepsis and arthritis) [Venable et al. And Lindsberg et al. Ann. Neurol., 30: 117-129 (1991). The possibility that PAF can improve pathological responses has been heightened by attempts to mutate PAF activity, and the main focus of this study has been to develop antagonists of PAF activity that interfere with PAF binding to cell surface receptors. For example, see Helin et al. Clin, Exp. Allergy, 22: 980-983 (1992).
[3] The synthesis and secretion of PAF as well as its degradation and purification rates appear to be well controlled. Photoreaction Reactions As long as the PAF regulatory mechanisms causing the lack of appropriate products or degradation result in a mutant inflammatory action of PAF, alternatives to altering PAF activity include mimicking or increasing the neutralization process that causes inflammatory degradation. Macrophages [J. Biol. Chem., 265 (17): 9682-9687 (1990)], hepatocyte and human hepatoma cell line HepG2 [Satoh et al. J. Clin. Invest., 87: 476-481 (1991) and Tarbet et al. J. Biol. Chem., 266 (25): 16667-16673 (1991), have been reported to secrete PAF acetylhydrolase (PAF-AH), which has an enzymatic activity that inactivates PAF. In addition to inactivating PAF, PAF-AH also inactivates oxidatively fragmented phospholipids, as well as byproducts of arachidonic acid cascades that regulate inflammation. J. Biol, Stremler et al. See Chem., 226 (17): 11095-11103 (1991). PAF inactivation by PAF-AH initially consists of hydrolysis of PAF sn-2 acetyl groups, which PAF-AH metabolizes oxidatively fragmented phospholipids by removing sn-2 acyl groups. Two forms of PAF-AH have been identified: cytoplasmic forms found in various cell types and tissues, such as endothelial cells and hemocytes, and extracellular forms found in plasma and serum. Plasma PAF-AH does not hydrolyze intact phospholipids except for PAF, and this substrate specificity allows enzymes to circulate in vivo while having currency exchange activity without side effects. Plasma PAF-AH can account for all PAF degradation of human blood in vitro [Stafforini et al. J. Biol. Chem., 262 (9): 4223-4230 (1987).
[4] While PAF-AH in the cytoplasmic form and PAF-AH in the plasma form have the same substrate specificity, plasma PAF-AH has the biochemical characteristics that distinguish it from the cytoplasmic PAF-AH and other characteristic rapase. In particular, plasma PAF-AH is associated with lipoprotein particles and inhibited by diisopropyl fluorophosphate and is relatively insensitive to proteolysis without being affected by calcium ions, with an apparent molecular weight of 43,000 Daltons. See Stafforini et al. (1987), supra. The paper by Stafforini et al. Describes a method for partial purification of PAF-AH from human plasma and the amino acid composition of the plasma material obtained by this method. Cytoplasmic PAF-AH is described by J. Biol. Purified from erythrocytes as reported in Chem., 268 (6): 3857-3865 (1993), 10 amino terminal residues of cytoplasmic PAF-AH are also described in this paper. J. Biol, Hattori et al. Chem., 268 (25): 18748-18753 (1993) describe plasma PAF-AH purified from bovine brains. Following the present application, the nucleotide sequence of cerebellar plasma PAF-AH is described in J. Biol. Chem., 269 (237): 23150-23155 (1994). On January 5, 1995, three months after the present application, the nucleophilic sequence of lipoproteins associated with phospholipase A2 (L _p -PLA ₂ ) was found in Smithklein Beckham PLC Patent Cooperation Treaty (PCT) International Publication No. WO95 / 00649 published. The nucleotide sequence of L _p -PLA ₂ differs in one position as compared to the PAF-AH nucleotide sequence of the present invention. Nucleotide differences (corresponding to position 1297 of SEQ ID NO: 7) result in amino acid differences between the enzymes encoded by the polynucleotides. The amino acid at position 379 of SEQ ID NO: 8 is valine while the corresponding amino acid of L _p -PLA ₂ is alanine. In addition, the PAF-AH nucleotide sequence of the present invention includes 124 bases at the 5 'end, and 20 bases are not present at the 3' end in the L _p -PLA ₂ sequence. Three months later, on April 10, 1995, the L _p -PLA ₂ sequence was deposited in Genebank under Accession No. U24577, which differed 11 positions when compared to the PAF-AH nucleotide sequence of the present invention. The nucleotide difference (corresponding to 79, 81, 84, 85, 86, 121, 122, 904, 905, 911, 983 and 1327 of SEQ ID NO: 7) results in four amino acid differences between the enzymes encoded by the polynucleotides. The 249, 250, 274 and 389 amino acids of SEQ ID NO: 8 are lysine, aspartic acid, phenylalanine and leucine, respectively, while the corresponding amino acids in the Genbank sequence are isoleucine, arginine, leucine and serine, respectively.
[5] PAF-AH recombinant products will make exogenous PAF-AH available to mimic or increase normal in vivo inflammatory degradation processes. Since PAF-AH is a product normally found in plasma, administration of PAF-AH has a physiological advantage over administration of PAF receptor antagonists. Moreover, PAF receptor antagonists structurally related to PAF inhibit the desired metabolism of PAF and oxidatively fragmented phospholipids because they inhibit native PAF-AH activity. Thus, inhibition of PAF-AH activity by PAF receptor antagonists interferes with competitive blockade of PAF receptors by antagonists. See Stremler et al., Supra. In addition, at the site of acute inflammation, for example, the release of oxidants inactivates natural PAF-AH enzymes that raise local levels of PAF and PAF-like compounds, which are all exogenous that bind to PAF receptors. Compete with the administered PAF receptor antagonist. In contrast, treatment with recombinant PAF-AH will increase endogenous PAF-AH activity and supplement all inactivated endogenous enzymes.
[6] Therefore, the production of reagents for detecting PAF-AH in plasma and the development of materials and methods useful for recombinant production of PAF-AH, necessitate the identification and isolation of polynucleotide sequences encoding human plasma PAF-AH.
[7] Summary of the Invention
[8] The present invention provides purified novel polynucleotides (ie, sense and antisense strands of DNA and RNA) or enzymatically active fragments encoding human plasma PAF-AH. Preferred DNA sequences of the invention include genomic sequences and cDNA sequences as well as totally or partially chemically synthesized DNA sequences. The PAF-AH coding DNA sequence set forth in SEQ ID NO: 7 and a DNA sequence which hybridizes if there is no excess of a DNA sequence or genetic code hybridized to its unencrypted strand under standard conditions are also included in the present invention. The invention also includes biological replicas of the DNA sequences of the invention (ie, DNA sequence copies produced and isolated in vivo or in vitro). Provided are self-replicating recombinant constructs, such as plasmid DNA vectors containing viral PAF-AH sequences and viral DNA vectors, wherein the DNA encoding PAF operably binds to endogenous or exogenous expression regulatory DNA sequences and transcription terminators. .
[9] According to another aspect of the present invention, eukaryotic or prokaryotic host cells are stably hemophilized with the DNA sequences of the present invention such that the desired PAF-AH is expressed to some extent. Host cells expressing PAF-AH products can serve several useful purposes. Such cells consist of PAF-AH and available materials of immunogens for developing antibody material that are particularly immunoreactive. The host cells of the present invention are particularly useful for large scale PAF-AH production methods, where the cells are grown in a suitable culture medium, and the preferred polypeptide product is isolated from the cell or the medium in which it is grown by methods such as immunoaffinity purification. .
[10] Non-immunological methods of the invention for purifying PAF-AH in plasma include the following steps:
[11] (a) separating the low density lipoprotein particles; (b) dissolving the low density lipoprotein particles in a buffer containing 10 mM CHAPS to prepare a first PAF-AH enzyme solution; (c) adding said first PAF-AH enzyme solution to a DEAE anion exchange column; (d) washing the DEAE anion exchange column with a buffer of about pH7.5 containing 1 mM CHAPS; (e) eluting the PAF-AH enzyme from the DEAE anion exchange column in a fraction using a buffer of about pH7.5 containing a 0 to 0.5 M NaCl gradient; (f) flinging the fraction eluted in a DEAE anion exchange column with PAF-AH enzymatic activity; (g) adjusting the active fraction polled from the DEAE anion exchange column for 10 mM CHAPS to produce a second PAF-AH enzyme solution; (h) adding the second PAF-AH enzyme solution to the blue dye ligand affinity column; (i) eluting PAF-AH from the blue dye ligand affinity column using a buffer containing 10 mM CHAPS and a chaotropic salt; (j) adding the blue dye ligand affinity column eluent to a Cu ligand affinity column (k) eluting PAF-AH from the Cu ligand affinity column using a buffer containing 10 mM CHAPS and imidazole: (l ) Adding the Cu ligand affinity column eluent to SDS-PAGE; And (m) separating about 44 kDa PAF-AH enzyme from the SDS-polyacrylamide gel. Preferably, the buffer of step (b) is 25 mM Tris-Hcl, 10 mM CHAPS, pH 7.5; (d) step buffer is 25 mM Tris-HCl, 1 mM CHAPS; the column of step (h) is a Blue Sepharose Fast Flow column; buffer in step (i) is 25 mM Tris-HCl, 10 mM CHAPS, 0.5 M KSCN, pH7.5; the column of step (j) is a Cu chelating Sepharose column; The buffer of step (k) is 50 nM imidazole with 25 mM Tris-HCl, 10 mM CHAPS, 0.5 M NaCl, pH about 7.5-8.0.
[12] The method of the present invention for purifying enzymatically active PAF-AH in E. coli producing PAF-AH comprises the following steps: (a) Centrifugation from lysed E. coli producing PAF-AH enzyme Preparing a separated supernatant; (b) adding the centrifuge supernatant to a blue dye ligand purification column; (c) eluting PAF-AH enzyme to the blue dye ligand affinity column using a buffer containing 10 nM CHAPS and a chaotropic salt; (d) adding the blue dye ligand affinity column eluent to a Cu ligand affinity column; And (e) eluting the PAF-AH enzyme from the Cu ligand affinity column using a buffer containing 10 mM CHAPS and imidazole. Preferably, the column of step (b) is a blue Sepharose fast flow column; buffer in step (c) is 25 mM Tris-HCl, 10 mM CHAPS, 0.5 M KSCN, pH 7.5; the column of step (d) is a Cu chelating Sepharose column; The buffer of step (e) is 25 mM Tris-HCl, 10 mM chomene, 0.5 M NaCl, 100 mM imidazole, pH 7.5.
[13] The method of the present invention for purifying enzymatically active PAF-AH in E. coli producing PAF-AH comprises the following steps: (a) Centrifugation in lysed E. coli producing PAF-AH enzyme Preparing a supernatant; (b) diluting the centrifuge supernatant with low pH buffer containing 10 mM CHAPS; (c) adding the centrifuge supernatant to a cation exchange column equilibrated to about pH 7.5; (d) eluting the PAF-AH enzyme in the cation exchange column using 1 M salt; (e) raising the pH of the eluent in the cation exchange column and adjusting the salt concentration of the eluent to about 0.5 M salt; (f) adding the cation exchange column eluent to a blue dye ligand affinity column; (g) eluting PAF-AH in the blue dye ligand affinity column using a buffer containing about 2 M to 3 M salts; And (h) dialysis of the blue dye ligand affinity column eluent with a buffer containing about 0.1% Tween. Preferably, the buffer of step (b) is 25 mM MES, 10 mM CHAPS, 1 mM EDTA, pH4.9; the column of step (c) is an S Sepharose column equilibrated with 25 mM MES, 10 mM CHAPS, 1 mM EDTA, 50 mM NaCl, pH 5.5; PAF-AH is eluted in step (d) with 1 mM NaCl; the pH of the eluent of step (e) is adjusted to pH 7.5 using 2 M Tris base; the column of step (f) is a Sepharose column; buffer in step (g) is 25 mM Tris, 10 mM CHAPS, 3 M NaCl, 1 mM EDTA, pH7.5; And buffer of step (h) is 25 mM Tris, 0.5 M NaCl, 0.1% Tween 80, pH7.5.
[14] The method of the present invention for purifying enzymatically active PAF-AH in E. coli comprises the following steps: (a) E to obtain dissolved PAF-AH supernatant after dissolution in buffer containing CHAPS. preparing a coli extract; (b) diluting the supernatant and adding it to an anion exchange column equilibrated at about pH 8.0; (c) eluting PAF-AH in the anion exchange column; (d) adding said anion exchange column eluent to a blue dye ligand affinity column; (e) eluting the blue dye ligand affinity column with a buffer containing 3.0 M salt; (f) diluting the blue dye eluent into a buffer suitable for the hydroxyapatite chromatography run; (g) performing hydroxyapatite chromatography when washed and eluted with buffer (with or without CHAPS); (h) diluting the hydroxyapatite eluate to a salt concentration suitable for cation exchange chromatography; (i) adding said diluted hydroxyapatite eluate to a cation exchange column within a pH range of about 6.0 to 7.0; (j) eluting PAF-AH in the cation exchange column with a suitable formulation buffer; (k) conducting cation exchange chromatography on ice; And (l) preparing a liquid or frozen PAF-AH formulation in the absence of CHAPS.
[15] Preference is given to the lysing 25 mM Tris, 100 mM NaCl, 1 mM EDTA, 20 mM CHAPS, pH8.0 in step (a); In step (b), the anion exchange chromatography supernatant is preferably diluted 3-4 times with 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH8.0 and the column is 25 mM Tris, 1 mM EDTA, 50 mM NaCl. , Q-Sepharose column equilibrated with 10 mM CHAPS, pH 8.0; the anion exchange column of step (c) is eluted with 25 mM Tris, 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH8.0; In step (d), the step (c) eluent is added directly to the blue dye affinity column; column of step (e) is eluted with 3 M NaCl, 10 mM CHAPS, 25 mM Tris, pH8.0 buffer; Dilution of the hydroxyapatite chromatography blue dye eluent of step (f) is obtained by dilution in 10 mM sodium phosphate, 100 mM NaCl, 10 mM CHAPS, pH 6.2; The hydroxyapatite chromatograph of step (g) was carried out with a hydroxyapatite column equilibrated with 10 mM sodium phosphate, 100 mM NaCl, 10 mM CHAPS and with or without 50 mM sodium phosphate, 100 mM NaCl (10 mM CHAPS). ), eluted to pH 7.5; The dilution of the hydroxyapatite eluate for cation exchange chromatography in step (h) is obtained by dilution with a buffer of pH DIR 6.0 to 7.0 containing sodium phosphate (with or without CHAPS); Sepharose column of step (i) is equilibrated with 50 mM sodium phosphate (with or without 10 mM CHA P5), pH 6.8 FH; in step (j), eluting with a suitable formulation buffer such as 125 mM NaCl, pH7.5 containing 50 mM potassium phosphate, 12.5 mM aspartic acid, 0.01% Tween-80; Cation exchange chromatography in step (k) is carried out at 2-8 ° C. Formulation buffers that stabilize PAF-AH, examples of suitable buffers for use in step (i) include 50 mM potassium phosphate, 12.5 mM aspartic acid, 125 mM NaCl pH7.4 (Twin-80 or Pluronic F68) Or 25 mM potassium phosphate buffer containing (at least 125 mM NaCl, 25 mM arginine and 0.01% Tween-80 (with or without about 0.1-0.05% Pluronic F68)).
[16] The method of the present invention for purifying enzymatically active rPAF-AH product from E. coli comprises the following steps: (a) lysate rPAF-AH product supernatant after lysis in buffer containing Triton X-100. Preparing an E. coli extract capable of obtaining the same; (b) diluting the supernatant and adding it to a fixed metal affinity exchange column equilibrated to a pH of about 8.0; (c) eluting rPAF-AH in the fixed metal affinity exchange chromatography with a buffer containing imidazole; (d) adjusting the salt concentration to add the fixed metal affinity column eluent to the hydrophobic interaction column (HIC # 1); (e) eluting the HIC # 1 by lowering the DUA concentration and / or increasing the detergent concentration; (f) titrating the HIC # 1 eluent with pH DIR 6.4 (g) adding the appropriate HIC # 1 eluent to the cation exchange column (CEX # 1) equilibrated to pH about 6.4; (h) Concentration Eluting the CEX # 1 with sodium chloride; (i) adjusting the concentration of CEX # 1 eluent to about 2.0 M with sodium chloride; (j) adding the adjusted CEX # 1 eluent to a hydrophobic interaction column (HIC # 2) equilibrated with sodium chloride at pH about 8.0 and pH about 2.0 M; (k) eluting the HIC # 2 by lowering salt concentration and / or increasing detergent concentration; (l) diluting the HIC # 2 eluent to adjust the pH to about 6.0; (m) adding the HIC # 2 eluent to a cation exchange column (CEX # 2) equilibrated to pH DIR 6.0; (h) eluting the rPAF-AH product of CEX # 2 with a suitable formulation buffer.
[17] Preferably, the lysis buffer of step (a) is 90 mM Tris, 0.125% Triton X-100, 0.6 M NaCl, pH8.0, and the lysis is carried out in a high pressure homogenizer; The supernatant of step (b) is diluted in equilibration buffer (20 mM Tris, 0.5 M NaCl, 0.1% Triton X-100, pH8.0) and purchased from a zinc chelate column (chelating Sepharose Fast Flow, Pharmacia, Uppsala, Sweden). ) Was charged, equilibrated with equilibration buffer and added to the diluted supernatant, washed with 20 mM Tris, 0.5 M NaCl, 4 M Urea, 0.1% Triton X-100, pH 8.0, followed by 20 mM Tris, 0,5 M NaCl. , 0.02% Triton X-100, pH 8.0; in step (c) eluted with 20 mM Tris, 50 mM imidazole, 0.02% Triton X-100, pH 8.0; Eluate in step (d) was adjusted with 1 mM EDTA and 2 M NaCl FH and phenyl Sepharose 6 fast flow (purchased from Pharmacia) was equilibrated buffer (2.0 M NaCl, 25 mM Tris, 0.2% Triton X-100, pH8.0). ), Equilibrated with (c) step eluent adjusted at room temperature, washed with equilibration buffer and washed with 25 mM NaPO ₄ , 0.02% Triton X-100, pH6.5 FH at a flow rate of 30 cm / hour; in step (e) elutes 25 mM NaPO ₄ , 3% Triton X-100, pH6.5 FH; Equilibrate the (g) macro-prep high speed S column (purchased from Bio-Rad Labs, Richmond, CA) with equilibration buffer (20 mM NaPO ₄ , 0.02% Triton X-100, pH 6.4), and (f) Was added dropwise with the adjusted eluent, followed by washing with equilibration buffer and washing with 25 mM Tris, 0.02% Triton X-100, pH 8.0; in step (h) eluted with 25 mM Tris, 0.02% Triton X-100, 1.3 M NaCl, pH 8.0; In step (j), Bakerbond Wide Pore High-Profile C ₃ (purchased from Baker, Philipsburg, NJ) is equilibrated with equilibration buffer (20 M NaCl, 25 mM Tris, 0.02% Triton X-100, pH 8.0), (l) was added dropwise at room temperature with the adjusted eluent and then washed with equilibration buffer, followed by washing at 25 cm / h with 25 mM Tris, 0.02% Triton X-100, pH 8.0; in step (k) eluting with 10 mM Tris, 3.0% Triton X-100, pH 8.0; in step (l), equilibrate with equilibration buffer (20 mM succinate, 0.1% Pluronic F68, pH 6.0); The SP Sepharose Fast Flow column (purchased in Pharmasa) of step (m) is equilibrated with the equilibration buffer of step (l), dropped into the eluent of step (l) and washed with equilibration buffer; In step (h) elute with 50 mM NaPO ₄ , 0.7 M NaC, 0.1% Pluronic F68, 0.02% Tween 80, pH7.5.
[18] The PAF-AH product can be obtained separately from natural cell sources or chemically synthesized, but is preferably produced by recombinant processes involving eukaryotic or prokaryotic host cells of the invention. Also included are PAF-AH products having some or all of the amino acid sequence set forth in SEQ ID NO: 8. In particular a fragment in which the first 12 N-terminal amino acids of the mature human PAF-AH amino acid sequence set forth in SEQ ID NO: 8 are deleted, more particularly Met ₄₆ , Ala ₄₇ or Ala ₄₈ as the starting N-terminal amino acids of SEQ ID NO: 8 It also includes those having. Also included are fragments thereof which lack 30 C-terminal amino acids of the amino acid sequence of SEQ ID NO: 8, especially those having Ile ₄₂₉ and Leu ₄₃₁ as C-terminal residues. The invention also provides amino acids within the sequence of SEQ ID NO: 8, selected from S108A, S273A, D286A, D286N, D296A, D304A, D338A, H351A, H395A, H399A, C67S, C229S, C291S, C334S, C407S, D286A, D286N and D304A. Also included are PAF-AH or PAF-AH variants with substituents. As noted above, the present invention provides polynucleotides (including DNA) encoding such fragments or variant fragments, and also provides methods for recombinantly producing such fragments or variants by growing host cells containing such DNA. . Currently preferred PAF-AH products include prokaryotic polypeptide DNA expression products encoding amino acid residues Met ₄₆ to Asn ₄₄₁ of SEQ ID NO: 8, designated rPH.2. Preferred PAF-AH products also include prokaryotic polypeptide DNAm expression products encoding amino acid residues Met ₄₆ to Iel ₄₂₉ of SEQ ID NO: 8 that are rPH. 9 was named. Both rPH.2 and rPH.9 products exhibit lower amino-terminal heterogeneity than the corresponding prokaryotic DNA expression products encoding, for example, the fully mature sequence of PAF-AH produced by the translation initiation codon. Moreover, the rPH.9 product shows higher carboxy terminal homogeneity (conformity). It is necessary to confer optimal biological activity on the recombinant expression products of the invention by using mammalian host cells, so post-translational variations (e.g., myristol, glycosylation, truncation, lipidation and tyrosine, serine or threonine phosphorylation). Is expected to be possible. The PAF-AH product of the present invention may be a full length polypeptide, fragment or variant. Variants include PAF-AH analogs, wherein one or more specific (ie, naturally encoded) amino acids are deleted or substituted, and one or more nonspecific amino acids are added: (I) to PAF-AH It lacks one or more of its specific immunological properties or enzyme activities, or (2) specifically lacks certain biological activities of PAF-AH. Proteins or other molecules that bind to PAF-AH will be used to alter its activity.
[19] The invention also includes antibody materials (eg, monoclonal antibodies, polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-transfected antibodies, etc.) and other proteins that specifically bind to PAF-AH. . In particular, the binding proteins of the present invention are monoclonal produced by hybridomas 90G11D and 90F2D, deposited September 30, 1994, at the American Type Culture Collection (ATCC), Rockville Parkrow Drive 12301, 202852, Maryland, USA. It is described in detail by the antibody and its accession number is HB11274 and HB11725, respectively. The binding proteins of the present invention are also described in detail by monoclonal antibodies produced by hybridoma 143A deposited with ATCC on June 1, 1995, the deposit number of which is HB11900. Proteins or other molecules (e.g., lipids or small molecules) that specifically bind to PAF-AH can be purified using plasma, recombinant PAF-AH, PAF-AH variants, or PAF-AH isolated from cells expressing these products. You can check it. Binding proteins are useful for PAF-AH purification as well as compositions for immunization and for detecting or quantifying PAF-AH in bodily fluid samples and tissue samples by known immunological methods. Included in the present invention are anti-genetic antibodies specific for PAF-AH-specific antibodies.
[20] The scientific value of the information given by describing the DNA and amino acid sequences of the present invention is clear. As a series of examples, it was possible to isolate by DNA / DNA hybridization of genomic DNA sequences encoding PAF-AH by subtracting the cDNA sequence for PAF-AH and controlling PAF-AH expression control regulatory sequences such as promoters, effectors, etc. It is now possible to specify. DNA / DNA hybridization processes with the DNA sequences of the present invention under standard conditions of the art, other alleles sharing one or more of the allelic variants of PAF-AH, the biochemical and / or immunological properties of PAF-AH It is expected to be able to isolate DNA encoding structurally related proteins and proteins of non-human species homologous to PAF-AH.
[21] Due to the DNA sequence information provided herein, it has failed to express functional PAF-AH enzymes by homologous recombination or "knockout" strategies (see, eg, Kapecchi, Science, 244: 1288-1292 (1989)). Rodents can be developed that express PAF-AH mutant enzymes. Suitably labeled polynucleotides of the present invention are useful in hybrid assays that detect the cellular ability to synthesize PAF-AH. The polynucleotides of the present invention will be the basis of a diagnostic method useful for identifying genetic variations in diseased PAF-AH sites. The present invention also provides antisense polynucleotides related to modulating the expression of PAF-AH by cells that normally express PAF-AH.
[22] The present invention includes administering a PAF-AH agent of the present invention to a mammalian species, particularly the human body, to ameliorate pathological inflammatory symptoms. Based on the fact that PAF is involved in pathological inflammatory symptoms, the following diseases can be treated by administering PAF-AH: Asthma [Miwa et al. J. Clin. Invest., 82: 1983-1991 (1988); J. Allergu Clin, Hsieh et al. Immunol., 91: 650-657 (1993); And Allergy, 49 from Yamashita, et al .; 60-63 (1994)], perfusion injury and central nervous system ischemia [Lindsberg et al. (1991), supra], antigen-induced arthritis [Zarco et al. Clin. Exp. Immunol., 88: 318-323 (1992)], atheromatous formation [Handley et al., Drug Dev. Res., 7: 361-375 (1986)], Crohn's disease [see Digestive Diseases and Sciences, Denizot et al., 37 (3): 432-437 (1992)], ischemic intestinal necrosis / necrotic enterocolitis [Denizot et al. , Supra, and Acta Paediatr., Suppl. 396: 11-17 (1994)], ulcerative colitis [see above by Denizot et al.], Ischemic seizures [see Stroke et al., 23: 1090-1092 (1992)], ischemic brain injury [Stroke by Lindsberg et al. , 21: 1452-1457 (1990) and Lindsberg et al. (1991), supra), systemic lupus erythematosus [see Clinica Chimica Acta, Matsuzaki et al., 210: 139-144 (1992)], acute pancreatitis [Kald et al. Pahcrea, 8 (4): 440-442 (1993)], sepsis (see above by Kald et al.), Acute streptococcal glomerulonephritis [mezzano et al. J. Am. Soc. Nephrol., 4: 243-242 (1993)], pulmonary edema resulting from IL-2 treatment [Rabinovici et al. J. Clin. Invest., 89: 1669-1673 (1992)], allergic inflammation [Watanabe et al. Br. J. Pharmacol., 111: 123-130 (1994)], ischemic renal failure [see Annals of Internal Medicine, Grino et al., 121 (5): 345-347 (1994)], early delivery [Hoffman et al. J. Obstet. Gynecol., 162 (2): 525-528 (199) and Maki et al. Proc. Natl. Acad. Sci. USA, 85: 728-732 (1988); Adult respiratory distress syndrome [Rabinovici et al. J. Appl. Physiol., 74 (4): 1791-1802 (1993); Clin, Matsumoto et al. Exp. Pharmacol. Physiol., 19 509-515 (1992); And Rodriguez-Roisin et al. J. Clin. Invest., 93: 188-194 (1994). The invention also encompasses the use of PAF-AH formulations to treat human immunodeficiency virus (HIV) infection of the central nervous system. The term "treatment" as used herein includes both prophylaxis and treatment.
[23] Many animal models with ongoing pathological serious injury have been described in the art. For example, mouse models for asthma and arthritis are described in Example 16 herein; Earthenware models for arthritis are described in Zarco et al., Supra; Rat models of ischemic intestinal necrosis / necrotic small intestinal colitis are described in Fed. Res., 34 (2): 237-241 (1993) and Caplan et al .; Rabbit models of seizures are described in Lindsberg et al. (1990), supra; Mouse models for lupus are described in Matsuzaki et al., Supra; Rat models of acute pancreatitis are described in Kald et al., Supra; Rat models for pulmonary edema resulting from IL-2 treatment are described in Rabinovici et al., Supra; Rat models of allergic inflammation are described in Watanabe et al., Supra;
[24] Dog models for renal allografts are described in Watson et al. Transplantation, 56 (4): 1047-1049 (1993); Described in the literature; The rat and guinea pig models of adult respiratory distress syndrome are described by Rabinovici et al. And Lellouch-Tubiana, Am. Rev. Respir. Dis., 137: 948-954 (1988).
[25] The present invention particularly suffers from PAF-mediated pathological symptoms comprising administering to the mammal an amount of PAF-AH sufficient to inactivate a pathological amount of PAF in the mammal and have endogenous PAF-AH activity. PAF-AH compositions useful for mammals or methods of treating mammals that are sensitive thereto.
[26] Therapeutic / pharmaceutical compositions provided by the present invention include PAF-AH products and physiologically acceptable diluents or carriers, and may also include drugs with anti-inflammatory effects. The dosages described will be sufficient to add cold PAF-AH activity and inactivate pathological amounts of PAF. See Reminington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, PA (1990) for general administration instructions. Dosages will vary from about 0.1 to about 1000 ug PAF-AH / kg body weight. Therapeutic compositions of the invention can be administered by various routes depending on the pathological condition to be treated. For example, the route of administration may be intravenous, subcutaneous, suppositories, and / or lungs, and the like.
[27] In the case of pathological symptoms of the lung, administration of PAF-AH by the pulmonary route is particularly preferred. For example, pulmonary administration can be made using a variety of delivery devices, such as nebulizers, inhalers capable of measuring the dose, and powder inhalers, which are known in the art. The transport of several proteins to the lungs and the circulatory system by inhalation of an aerosol formulation is described in the following literature: Adjei et al. Pharm. Res., 7 (6): 565-569 (199) (leuprolide acetate); Braquet et al. J. Cardio. Pharm., 13 * Supp. 5): s. 143-146 (1989) (endothelin-1); Annals of Internal Medicin, III (3), 206-212 (1989) (α1-antitrypsin) by Hubbard et al .; Smith et al. J. Clin. Inrest., 84: 1145-1146 (1989) (α-1-proteinase inhibitors); J. Immunol., 140: 3482-3488 (1933) (recombinant gamma interferon and tumor necrosis factor alpha); Patent Cooperation Treaty (PCT) International Publication No. WO 941 20069 published September 15, 1994 (which is a polyethylene glycolated recombinant granulocyte colony stimulus).
[1] The present invention relates generally to isolated novel polynucleotides encoding platelet-activating factor acetylhydrolase and in particular human plasma platelet-activating factor acetylhydrolase. The present invention also relates to recombinant production materials and methods of platelet-activating factor acetylhydrolase products and platelet-activating factor acetylhydrolase products encoded by this polynucleotide, and additionally to platelet-activating factor acetylhydrola It relates to an antibody substance which specifically acts on the agent.
[29] Numerous aspects and advantages of the invention will be apparent from the following detailed description and drawings.
[30] 1 is a photograph of a PVDF membrane containing PAF-AH purified from human plasma;
[31] 2 is a graph showing the enzymatic activity of recombinant human plasma PAF-AH;
[32] 3 is a schematic showing the recombinant PAF-AH fragment and its catalytic activity;
[33] 4 shows rPH as a recombinant PAF-AH product. 2 shows the results of mass spectrometry.
[34] 5 shows rPH as a recombinant PAF-AH product. 9 shows the results of mass spectrometry.
[35] FIG. 6 is a bar graph illustrating the localization of recombinant PAF-AH of the present invention to block PAF-induced foot edema in rats.
[36] 7 is a bar graph illustrating intravenous administration of PAF-AH to block PAF-induced foot edema in rats.
[37] 8 is a bar graph illustrating that PAF-AH also blocks PAF-induced edema, but not Zymosan A-DB.
[38] 9A and 9B show the dose response results of PAF-AH anti-inflammatory activity in rat paw edema.
[39] 10A and 10B show the results of in vivo efficacy of one administration of PAF-AH outside of defined time;
[40] Eleventh is a line graph showing PAF-AH pharmacokinetics in blood circulation of rats.
[41] 12 is a bar graph showing the anti-inflammatory effects of less effective PAF antagonists and PAF-AH in rat paw edema.
[42] FIG. 13 shows that PAF-AH neutralizes apoptosis effects of HIV-1-salts and activated monocyte conditioned media.
[28] The invention is illustrated by the following examples. Example 1 describes a new method of purifying PAF-AH in human plasma. Example 2 describes amino acid microsequencing of purified human plasma PAF-AH. Example 3 describes a full length cDNA cloning encoding human plasma PAF-AH. The identification of putative splice variants of the human plasma PAF-AH gene is described in Example 4. Example 6 describes the cloning of dogs, mice, cows, chickens, rodents and Merca cDNAs homologous to human plasma PAF-AH cDNA. Example 7 shows assay results demonstrating the enzymatic activity of recombinant PAF-AH transiently expressed in COS7 cells. Example 8 contains E. coli, S. a. Full length truncated chimeric human PAF-AH DNA expression in S. Cerevisiae and mammalian cells is described. Example 9 describes a protocol for purifying recombinant PAF-AH in E. coli and an assay to confirm its enzymatic activity. Example 10 describes a variety of recombinant PAF-AH products, including amino acid substitution analogs and amino- and truncated products, and describes experiments demonstrating glycolylation of native PAF-AH isolated from plasma. . Northern blot analysis of human plasma PAF-AH RNA expression in various tissues and cell lines is shown in Example 11, and Example 12 shows the in situ hybridization results. Example 13 describes the development of monoclonal and polyclonal antibodies specific for human plasma PAF-AH. Examples 14, 15, 16, 17, 18 and 19 are the biological effects on acute inflammation, pleurisy, tent, necrotic small intestinal colitis, adult respiratory distress syndrome and pancreatitis by administering the recombinant PAF-AH product of the present invention to animal models, respectively. My therapeutic effect is described. Example 20 describes the in vitro effects of recombinant PAF-AH products on neurotoxins associated with HIV infection. Example 21 shows the results of an immunoassay of the serum of a patient (human) lacking PAF-AH activity and describes the identification of genetic lesions in a patient sensitive to deficiency.
[43] Example 1
[44] PAF-AH was purified from plasma to provide material for amino acid sequencing.
[45] A. Optimization of Purification Conditions
[46] First, low-density lipoprotein (LDP) particles were precipitated from plasma with phosphotumsten and dissolved in 0.1% Tweem 20, followed by incomsistemt elutiom of PAF-AH activity on a DEAE column requiring dissolution and reassessment of subsequent purification conditions. ) Was chromatographed on a DEAE column (Pharmacia, Uppsala, Sweden) according to the method of Stafforini et al. (1987) described above.
[47] Tweem 20, CHPS (Pierce Chemical, Inc., Rockford, Milinomi) and octyl glucoside were evaluated by centrifugation and gel filtration chromatography for their ability to dissolve LOL particles. CHAPS showed 25% more solubility recovery than Tweem 20 and 300% more solubility recovery than octyl glucoside. 10 mM CHAPS dissolved LOL precipitates with DEAE Sepharuse Fast Flow column (anion exchange column) with a left buffer containing 1 mM CHAPS to provide a large pool of partially purified PAF-AH ("DEAE pool") for the evaluation of additional columns. ; Pharmacia).
[48] The DEAE pool was used as a starting material for the testing of several chromatographic columns for further purification of PAF-AH activity. Columns tested were as follows: Blue Sepharose Fast Flow (Pharmacia), dye ligand affinity column; S-Sepharose Fast Flow (Pharmacia), cation exchange column; Cu Chelatimg Sepharose (Pharmacia), metal ligand affinity column; Fractogel S (EM Seperracences, Gibbstown, NJ), cation exchange column; And Sephacryl-200 (Pharmacia), gel filtration column This chromatographic analysis showed a low level of purification which was unsatisfactory when working at 1 mM CHAPS. The enzyme activity fraction eluted over a larger size range than the 44 KDa approximate size predicted by gel filtration chromatography on Sephacryl S-200 in the subsequent 1 mM CHAPS. Taken together, these results indicate that LOL proteins are aggregated in solution.
[49] Therefore, different LOL samples were evaluated by analytical gel filtration chromatography for collection of PAF-AH activity. Samples from DEAE plaques and samples of freshly dissolved LOL precipitates were analyzed on Superuse 12 (Pharmacia) equilibrated in the rounds with 1 mM CHAPS. Both samples eluted over a very wide range of molecular weights with the best activity eluting above 150 KDa. When samples were analyzed on Superese 12 equilibrated with 10 mM CHAPS, most activity was eluted at 44 KDa near as expected for PAF-AH activity. However, the samples contained some DAF-AH activity in the high molecular weight region corresponding to aggregation.
[50] Other samples eluted by DAF-AH activity at approximately 44 KDa when tested by Wiyer gel filtration. These samples were LOL precipitates dissolved in 10 mM CHAPS after 1 day from DEAE column. The data indicate that at least 10 mM CHAPS is required to maintain the non-collection PAF-AH. The increase in CHAPS concentration from 1 mM to 10 mM after chromatographic analysis on DEAE showed a surprising difference in purification. For example, chromatographic analysis on Cu Chelatimg Sepharose after S-Separuse Fast Flow column enhanced PAF-AH activity by 15-fold. It was also determined that PAF-AH activity could be recovered from the reduced SDS-polyacrylamide gel as long as the sample was not broken. The activity of the material eluted from the Cu Chelatimg Sepharose column during SDS-polyacrylamide gel writing salt coincided with the main protein band when the gel was silver colored.
[51] B PAF-AH Purification Protocol
[52] The new protocol used for PAF-AH purification for amino acid sequencing consists of the next step performed at 4 ° C. Plasma was divided into 900 ml aliquots in 1 liter Nalgene bottles and adjusted to PH8.6. LDL particles were then precipitated by adding 90 mL of 3.85% sodium phosphostatastrol and 2M MgCl ₂ to 23. The plasma was then centrifuged at 3600 g for 15 minutes. The pellets were resuspended in 800 ml of 0.2% sodium citrate. LDL was precipitated again by adding 10 g NaCl and 24 ml 2M MgCl ₂ . LDL particles were centrifuged for 15 minutes to 3600 g of pellets. This process was repeated twice. The pellet was then frozen at -20 ° C. LDS particles obtained in 5 L of plasma were resuspended in 5 L of buffer A (25 mM Tris-HCl, 10 mM CHAPS, pH7.5) and stirred overnight. The dissolved LDL particles were centrifuged at 3600 g for 1.5 hours. The supernatants were combined and filtered with Whatman 113 filter paper to remove residual solid phase. The dissolved LDL supernatant was infiltrated on a DEAE Sepharose Fast Flow column (11Cm x 10Cm; 1 L resin rot; 80 mL / min) equilibrated in buffer BC 25 mM Tris-HCl, 1 mM CHAPS, pH7.5). The column was washed with buffer B until absorbance returned to baseline. The protein was eluted with 8 L, 0-0.5 M NaCl gradient to give 480 mL fraction. This step is necessary to obtain the binding for the Blue Sepharose Fast Flow column. Fractions were evaluated for acetylhydrolase activity as follows by the method described in Example 4.
[53] The fractions were made into pools and enough CHAPS was applied to freeze the pools of DIR 10 mM CHAPS. DEAE pool was loaded overnight at a rate of 4 ml / min on a Blue Sepharose Fast Flow column (5 cm × 10 cm; 20 ml bed volume) equilibrated with Buffer A containing 0.5 M Aacl. The column was washed with equilibration buffer at a rate of 16 ml / min until absorbance returned to baseline. PAF-AH activity was eluted with Buffer A containing 0.5M KSCN (Chaotropic Salt) at a rate of 16 ml / min and a 500 ml fraction was obtained. This step yielded more than 100-fold purification. The active fraction was pooled and adjusted to pH 8.0 with / M Tris-Hcl at pH 8.0. Cu Chelatimg Sepharose column (2.5cm × 2cm; 10ml) equilibrated in activated grasshopper solution from Blue Sephorose Fost Flow Chromatography ([25mM Tris-Hcl, 10mM CHAPS, 0.5M oclacl, pH 8.0 (applied with pH 7.5)] Bed volume; 4 ml / min) and the column was washed with 500 ml Buffer C. PAF-AH activity was eluted with 100 ml of 500 mM imidazole in Buffer C and a 100 ml fraction was obtained. The PAF-AH activity was pooled and soured against buffer A. The Cu Chelating Sepharose column allowed some purification.
[54] Cu Chelating Sepharose paste was reduced in 500 mM DTT at 37 ° C. for 15 minutes and loaded onto a 0.75 mm 7.5% polyacrylamide gel. The gel was cut into 0.5 cm slices and placed in a disposable microfuse duke containing 200 ul of 25 mM Tris-Hcl, 10 mM CHAPS, 15 cm M Aacl. Slices were yellowed at 4 ° C. overnight in Galanta. Supernatants from each cell were assessed in addition to PAF-AH activity to determine which protein bands on 575-PASE contained PAS-AH activity. PAS activity was found in about 44 KAa bands and was deleted by double Blu.
[55] As shown in Table 1, a 3 × 10 4 fold suspension of 5I45 plasma PAF-AH triactivation of about 200 ug PAF-AH is described in Stakorini et al. (1989) described above.
[56] samplevolumeActivityDynamic activityproteinInactivity% Recovery of activityTablet drainage(Ml)(copm x 10 ⁶ )(cpm x 10 ⁹ )Concentration (mg / ml)cpm x 10 ⁶ )srep Cum.srep Cum. plasma50002316620.37100100OneOne LDL450022971.761284843333 DEAE4200492071.08462121783.7124 biue161881140.025420070126119015 x 10 ⁵Cu12127001520.15822001041311.52.2 x 10 ⁵SDS-PAGE---------------------To 102.2 x 10 ⁶
[57] In summary, an important and specific step in the successful purification of plasma PAF-AH for microsequencing is as follows: ("Blue mM affinity column such as 10 mM (HAPS and chromatolysis in HAPS (2) Blue Sepharose Fast Flow). Chromatography on (3) Chromatographic analysis on Cu ligand affinity column, such as Cu Chelatimg Sepharose, and (4) Elution of PAF-AH from SDS-PAGE
[58] Example 2
[59] Amino acid sequencing was performed by Approxel Biosystems 473 A Proteim seguemcer with approximately 44 KDa protein bands obtained from the PAF-AH comprising the PUDF embrain described in Example 1 and sequenced. N-terminal sequencing of about 44 KDa protein bands corresponding to PAF-AH activity indicated that the bands contained two major and two subsequences. The ratio of the two sequences was 1:10, making it difficult to interpret the sequence data.
[60] To separate the sequences of the two major proteins degraded on the SDS gel, a double PVDF membrane containing about 44 KDa bands was cut in half to separate the three and bottom portions of the membrane.
[61] The N-terminal sequence obtained for the lower half of the membrane is as follows:
[62] SEQ ID NO: 1
[63] FKDLGEENFKALVLIAF
[64] A search of the protein database indicated that this sequence is part of human serum albumin. Correlations of the same PVDF membrane were also sequenced and the N-terminal amino acid sequence was as follows:
[65] SEQ ID NO: 2
[66] IQVLMAAASFGQTKIP
[67] This sequence did not match any protein in the searched database and differs from the N-terminal amino acid sequence:
[68] SEQ ID NO: 3
[69] MKPLVVFVSGG
[70] This has been reported as Staphyrini et al (1993) described above as erythrocyte cytoplasmic PAF-AH. The new sequence (SEQ ID NO: 2) was applied for plasma PAF-AH cDHA cloning as described in Example 3.
[71] Example 3
[72] Full-length clones encoding plasma PAF-AH were isolated from the macrophage cDHA library.
[73] A. Construction of Macrophage cDNA Library
[74] Poly A ⁺ RNA was collected from peripheral blood monocyte-induced macrophages. A blunted double-helix cDNA was generated using the Imvitrogem Copy kit (Sam Diego, Canada) and ligated into the cDNA prior to insertion with a mammalian expression vector, PRc / CNV (Imvitroegm). The obtained plasmid was electrolyzed by E. Col; Introduced with the Straim XL-1 Blue.
[75] The modified bacteria were plated at a total density of about 300 clones per agarose plate on a total of 978 plates. Plasmid DNA prepared separately from each plate was retained in each pool and bound to a large pool representing each 300,000 clones.
[76] B. Library Selection by PCR
[77] Macrophage libraries were selected by polymerase chain reaction using degenerate antisense oligonucleotide PCR based on the novel N-terminal amino acid sequence described in Example 2. The sequence of the primer is speculative in IUPAC nomenclature, where "I" is inosine.
[78] SEQ ID NO: 4
[79] 5 'ACATGAATTCGGIATCYTTIGTYTGICCRAA 3'
[80] Wearer [Nuc. Acid Ros., 195; 1981-1986 (1991)] was used to select nucleotides at the third position of each condon of the primer. Primers were used to bind specific primers for SP6 or T7 promoter sequences, both flanking the pRc / NH ₂ flanking region, to select 300,000 clones of macrophage library pools. All PCR reactions included template cDHA, 1 μg of 100 mg each primer, 0.125 mM each predominant, 10 mM Tris-Hcl of pHα4, 50 mM MgCl ₂ and 2.5 units of Tug polymerase. 30 cycles of amplification at 94 ° C. for 1 minute, 1 minute at 60 ° C. and his amplification at 72 ° C. were followed by an initial denaturation step of 94 ° C. for 4 minutes. The resulting PCR product was cloned into PBlUescript SK (stratazini, Lazola, Canada) and the nucleotide sequence was determined by dideoxy chain termination. The PCR product contained the sequence predicted by the new peptide sequence and corresponds to nucleotides 1-331 of SEQ ID NO: 7.
[81] PCR primers are as follows and were designed to identify full-length clones as specific for the cloned PCR fragments described above.
[82] Sense primer (SEQ ID NO: 5)
[83] 5 'TATTTCTAGAAGTGTGGTGGAACTCGCTGG3'
[84] Intersense Primer (SEQ ID NO: 6)
[85] 5 'CGATGAATTCAGCTTGCAGCAGCCATCAGTAC3'
[86] PCR reactions using primers were performed as described above to first select cDNA pools of 300,000 clones followed by a suitable subset of smaller pools of 3000 clones. The pool of 3000 clones that produced the PCR product of expected size was then used to modify the bacteria.
[87] C. Library Screening by Hybridization
[88] DNA obtained from the modified bacteria was subsequently cloned with the probe and selected by hybridization using PCR fragments. Colonies were blunted with nitrocellulose, 50% formamide, 0.75 M sodium chloride, 0.075 M sodium citrate, 0.05 M sodium phosphate pH6.5, 1% polyvinyl pyridine, 1% picol, 1% bobbin serum Pre- and hybridized in albumin and 50 mg / ml sonicated salmon sperm DNA. The collagen probe was labeled by random hexamer priming. After hybridization overnight at 42 ° C., the broths were washed extensively at 42 ° C. with 0.03 M sodium chloride, 3 mM theldna citrate, 0.1% SDS. Nucleotide sequences of 10 glia clones were determined. Clone SAH 406-3, one of the clones, contained a sequence predicted by the original peptide sequence of PAF-AH activity purified from plasma. The DNA and derived amino acid sequences of plasma PAF-AH are shown in SEQ ID NOs: 7, 8, respectively.
[89] Clone SAH 406-3 contains a 1.52 kb insert with an opem reading fram that encodes a predicted protein of 441 amino acids. In amino terminus, the relatively hydrophobic segment of 41 recipes precedes the N-terminal amino acid (isosolin at position 42 of SEQ ID NO: 8) identified by microsequencing of the protein. The encoded protein may have an additional peptide that cleaves to produce such a long signal sequence or signal sequence + mature functional enzyme. The presence of signaling concerns is a property of secreted proteins. In addition, the protein encoded by the clone SAH 406-3 includes a consensus GxSxG motif (amino acids 271-275 of SEQ ID NO: 8) believed to include the active site serine of all mammalian lipases, microspray lipases and serine proteases. do. Chapus et al., Biochimie, 70; See 1223-1334 (1988) and Brenner, Hature, 334: 528-530 (1988).
[90] Table 2 is a comparison of the amino acid composition of the plasma PAF-AH of the present invention predicted from SEQ ID NO: 8 with the amino acid composition of the intentionally purified material described by Stafforini et al. (1987) described above.
[91] Clone sAH406-3Stiffini Ala2624 Asp & Asn4837 Cys514 Glu & Gln2642 Phe2212 Gly2958 His1324 Ile3117 Lys2650 Leu4026 Met107 Pro1511 Arg1816 Ser2736 Thr2015 Val1314 Trp7Undefined Tyr1413
[92] Differences in the amino acid composition of the substance are evident even in the mature amino acid composition of the plasma PAF-AH and the plasma purified beforehand.
[93] The alignment of the amino acid sequence of the plasma PAF-AH of the present invention and the derived amino acid sequence of the bobbin brain cytoplasmic PAF-AH having nucleotides and nucleotides, such as Hartji, was attempted, but no significant structural similarity was observed in the sequence. .
[94] 4 to implementation
[95] The putative splice variant of the human PAF-AH gene was taken from the untranscribed 5 ′ region (nucleotides 31-52 of SEQ ID NO: 7) and the 3 ′ end of the PAF-AH cDNA (nucleotides 1465-1487 of SEQ ID NO: 7) The detection was performed when PCR was performed on macrophages and stimulated PBMC cDNA using primers hybridized to the region terminating the terminating condon. The PCR reaction produced two bands on the gel, one corresponding to the expected size of the PAF-AH cDNA of Example 3 and the other about 108 bp short. The sequencing of both bands showed that the large band was the PAF-AH cDNA of Example 3 and the exon 2 of the PAF-AH sequence encoding a probanded sequence of the short band plasma PAF-AH and the putative signal (Example described below) 5) showed a deficiency. The predicted catalytic trabird and all cysteines were present in short clones, so the biochemical activity of the protein encoded by the clones seemed to match that of the plasma enzyme.
[96] To begin evaluating the biological tritubules of the PAF-AH splice variant predicted to encode cytoplasmic active enzymes, relatively many two forms in blood mononuclear cell-induced macrophages were evaluated by RNase protection. Any message was obtained for the newly isolated mononuclear cells, but both messages were found to be mononuclear macrophages on day 2 of the in vitro session and were present throughout the 6 days of culture. Both messages were nearly identical throughout the positive differentiation period. In contrast, a similar analysis of neural tissue showed that only full-length messages predicted to encode the full-length cell morphology of PAF-AH were represented.
[97] Example 5
[98] Plasma PAF-AH genomic sequence was also isolated. The structure of the PAF-AH gene was determined by separating lambda and PI phage clones containing human genomic DNA by DNA capture under highly controlled conditions. Fragments of phage clones were subcloned and sequenced using DesignDOS primers to anneal at intervals throughout the cDNA clone SAH 406-3. In addition, new sequencing primers designed to anneal to intron regions flanking the exons were used to backcross sequencing the exon-intron boundaries to identify sequences. Exon / intron boundaries were defined as points at which the genome and cDNA sequences diverge. Diddle analysis showed that the distant PAF-AH gene consists of 12 exons.
[99] Some of the exons 1, 2, 3, 4, 5, 6 and 7 were isolated from the male large placental library constructed in lambda FIX (Strataqeme). Phage plaque is bloated on nitrocellulose and 50% formamide, 0.75M sodium chromide, 75mM sodium citrate, 50mM sodium forstate (PH6.5), 1% polyvinyl pyridine, 1% picol, 1 Pre-hybridized and hybridized in% bovine serum albumin and 50 mg / ml superdermalized salmon sperm DNA. Hybridization probes used to identify phage clones comprising parts of exons 2.6 and 7 consisted of the entire cDNA clone sAH 406-3. Clones comprising exon 1 were identified using fragments derived from the 5 ′ end of the cDNA clone (nucleotides 1-312 of SEQ ID NO: 7). Both probes were labeled 32p by hexamer random priming. Incubated overnight at 42 ° C., the blots were washed extensively in 42 ° C., 30 mM sodium chromide, 3 mM sodium citrate, 0.1% SDS. The DNA sequences of exons 1, 2, 3, 4, 5. and 6 along the partially enclosed indoron sequences are shown in SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively.
[100] Example 6
[101] Exons 8, 9, 10, 11 and 12 and the rest of exon 7 were subcloned from PI clones isolated from human PI genomic library. PI phage plaques were blotted on nitrocellulose and were 0.75M sodium chloride, 50mM sodium fourstate (PH 7.1), 5mM EDTA, 1% polyvinyl pyridine, 1% picol, 1% bovine serum albumin, 0.5% SDS and total Pre- and hybridized in 0.1 mg / ml human DNA. Hybridization probes labeled 32 p by hexqmer rondom primimg consisted of 2.6 Kb EcoRl fragments of genomic DNA derived from the 3 ′ end of the isolated lambda clone. This fragment contained exon 6 and part of exon 7 was present in phageclone. After overnight hybridization at 65 ° C., the blots were washed as described above. The DNA sequences of exons 7, 8, 9, 10, 11 and 12 along with partially enclosed indron sequences are given in SEQ ID NOs: 15, 16, 17, 18, 19 and 20, respectively.
[102] Example 6
[103] Full length Plasma PAF-AH cDNA clones were isolated from spleen cDNA libraries of mice, dogs, cows and chickens, and semi-rodent clones were isolated from rat thymic cDNA libraries. Clones were identified by low stringency hybridization up to human cDNA (high condensation conditions were the same as described for exons 1-6 of Example 5 above, except that 20% formamide was used instead of 50% formamide). A 1 Kb Himd III fragment (nucleotides 309-1322 of SEQ ID NO: 7) of human PAF-AH sAH 406-3 cDNA clone was used as the DL probe. Semi-monkey clones were also isolated from Mercak brain cDNA by PCR using primers based on nucleotides 285-303 and 851-867 of SEQ ID NO: 7. The nucleotide and putative amino acid sequences of the mouse, dog, bovine, chicken, rat and Mercak cDNA clones are shown in SEQ ID NOs: 21, 22, 23, 24, 25 and 26, respectively.
[104] A comparison of the putative amino acid sequences of human cDNA and cDNA clones shows the amino acid percentage identification values shown in Table 3.
[105] humandogmousesmallchicken dog80106648250 rat66641006447 monkey9282698052 Rat7469826955 small828264100850 chicken50504750100
[106] About 38% of the recipe is fully conserved in all sequences. The maximum divergence region is at the carboxyl terminal end and amino terminal end (including signal sequence) shown in Example 10 as not critical for surface activity. The Gly-Xaa-Ser-Xaa-Gly motif (SEQ ID NO: 27) found in neutral lipase and other esterases was conserved in cow, dog, mouse, rat and chicken PAF-AH. The central serine of this motif serves as an active site nucleophile for these enzymes. The predicted aspartate and histidine components of the active side (Example 10 A) were also preserved. Therefore, the plasma PAF-AH of the present invention appears to use a catalytic trimad and does not show another sequence homologous to lipase, but seems to have α / β hydrolase guilt of neutral lipase.
[107] Furthermore, plasma PAF-AH is expected to have regions that coordinate measurement interaction with the low and high density lipoprotein particles of plasma. Interactions with these particles can be established by the N-terminal half of the molecule having a large stretch of highly conserved amino acids in the middle but not including the catalytic tribad of the enzyme.
[108] Example 7
[109] Plasma PAF-AH cDNA Clone sAN 406-3 (Example 3) To determine whether this encodes a protein with PAF-AH activity, the PRC / CMV expression construct was tentatively expressed in COS 7. After 3 days of transfection by the DEAE dEXTRAM method, COS cell media was assessed by PAF-AH activity.
[110] The cells were seeded at 300,000 cell density per 60 mm tissue culture dish. The next day, the cells were incubated in DMEM containing 0.5 mg / ml DEAD dextran, 0.1 mM chloroquine and 5-10 mg of plasmid DNA for 2 hours. Cells were then incubated in DNEM containing 10% fetal calf serum treated with 10% DMSO in four-state-buffered saline, washed with media and pretreated with diisopropyl fluorophosphate (DFP) for 1 minute. Endogenous serum PAF-AH was inactivated. After 3 days of incubation, the media obtained from the transfected sieves was assessed for PAF-AH activity. The synthetase is inhibited by a serine esterase inhibitor DFP such as that described above for Plasma PAF-AH by Steforini et al. (1987) described above and has a SAH 406-3 insert in the reverse direction whether calcium independent. Or cells transfected with PRc / CMV without or with an insert.
[111] The PAF-AH activity of the transfected trisulfate solution was determined by the method of Staffonini et al. (1990) described above by modifying as follows. Briefly, PAF-AH activity was determined by measuring 3H-exacetic acid hydrolysis from [acetyl-3H] PAF (New England Nuclear, Moston Miami). Many 3H-acetates were separated from the substrates labeled by reverse osmosis column chromatographic analysis over an octadecylacakel cartridge (Benker Research Products, Phillipsburg, Pennsylvania). 50 mL Pmoles were labeled (cold PAF) in a 1: 5 ratio and used per reaction. The reaction was incubated at 37 ° C. for 30 minutes and stopped by the addition of 40 ml of 10 M acetic acid. The solution was then washed through an octadecyl silica gel cartridge rinsed with 0.1 M sondium acetate. The aqueous solution eluted from each sample was collected and counted in the liquid scintillation count over one minute. Enzyme activity was expressed in counts per minute.
[112] As shown in FIG. 2, the media obtained from cells transfected with SAN 406-3 contained PAF-AN activity at levels four times higher than background. This activity is not affected by the presence of EETA but shows a matching activity with the code.
[113] Example 8
[114] Full-length and truncated plasma PAF-AH DNA and chimeric mouse-human PAF-AH DNA were expressed in E.W and yeast and stably expressed in mammalian cells by recombinant methods.
[115] Expression in A. E. Coli
[116] PCR was used to generate protein codem fragments of plasma PAF-AH cDNA from clone sAH 406-3, which could be subcloned into an E.Wli expression vector. The subcloned fragment begins with a codon encoding Ile ₄₂ (SEQ ID NO: 8), an N-terminal recipe of purified enzyme from plasma at 5 ′ of the human gene. The rest of the gene through the original stop codon was included in the construct. The 5'sense PCR primers used were as follows:
[117] SEQ ID NO: 28
[118] 5TATTCTAGAATTATGATACAAGTATTAATGGCTGCTGCAAG3 '
[119] And transcription initiation codons (underlined) as well as Xba cloning sites. The 3 'antisense primers used were as follows:
[120] SEQ ID NO: 29
[121] 5 'ATTGATATCCTAATTGTATTTCTCTATTCCTG3'
[122] And an EwRV cloning site surrounding the end codon of sAH 406-3. PCR reaction was carried out as in Example 3. The resulting PCR product was digested with Xba and EcoRV and subcloned into a pBR ₃₂₂ vector containing a Trp promoter (deBoer et al., PNAS, 80: 21-25 (1983)) located directly upstream of the cloning site. Incubated in L broth containing 100 μl / ml. Transformants from overnight cultures were pelleted and resuspended in Lysis buffer containing 0.05 trypsin-inhibiting unit (TIU) / ml aprotinin. After 1 hour incubation on ice and 2 min sonication, the acetate was assessed for PAF-AH activity by the method described in Example 4. E. coli modified with the expression construct (named trp AH) gave rise to products with PAF-AH activity. See Table 6 of Example 9.
[123] Three additional promoters, the tacII promoter (de Boer, tactical), the arabinose B promoter from Salmonella typhimurium (Horwitz et al., Gene, 14: 309-319 (1981)) and the alc bacteriophage T7 promoter. The construct was used to induce the expression of the PAF-AH sequence of E. Coli. A configuration comprising the Trp promoter (PUC trp AH), the tacII promoter (PUC tac AH) and the araB promoter (PUC ara AH) was combined in plasmid PUC19 (New England Biolabs, Miami) and included the T7 promoter (PPET AH). The configuration was combined with plasmid PET15B (Novagen, Madison, Wisconsin). A construct comprising the hybrid promoter PHAB / pH consisting of the araB promoter dissolved at the ribosomal binding site of the T7 promoter region was also combined in PET15B. All E. cOLI constructs produced PAF-AH activity in the range of 20-50 U / ml / OD ₆₀₀ . DL activity corresponds to a total recombinant protein mass equal to or greater than 1% of total cell protein.
[124] It was evaluated whether some E. coli expression constructs produced PAF-AH with extended amino termini. The N-terminus of native plasma PAF-AH was identified as Ile ₄₂ by amino acid sequencing (see Example 2). However, the sequence immediately upstream of Ile ₄₂ is not synonymous with the amino acid found at the signal sequence cleavage site (ie, the -1 position does not follow "-3-1-rule" by lysine; Von Heijne, Nuc. See Acids Res., 14: 4683-4690 (1986). More typical signal sequences (M ₁ -A ₁₇ or M ₁ -P ₂₁ ) are recognized by the cell secretory followed by endoproteolytic cleavage. The entire coding sequence for PAF-AH starting at starting methionine (nucleotides 162-1487 of SEQ ID NO: 7) was processed to be expressed in E. coli using the trp promoter. As shown in Table 4, this construct produced active PAF-AH but expressed only about 1/50 of the original construct starting at Ile ₄₂ . Another expression construct starting at Val ₁₈ (nucleotides 213 to 1487 of SEQ ID NO: 7) produced active PAF-AH at about one third the level of the original construct. These results suggest that the amino terminus and its extensions are not critical or essential for the recombinant PAF-AH activity produced in E. coli.
[125] edificePAF-AH Activity (U / ml / OD ₆₀₀ ) Lysatebadge pUC trp AH (He ₄₂ N-terminus)177.70.030 pUC trp AH He ₁ 3.10.003 pUC trp AH He ₁₈ 54.60.033
[126] The truncated recombinant human PAF-AH product was produced in E. coli using a low copy number plasmid and a promoter inducible by adding marabinose to the culture. This N-terminal truncated PAF-AH product is a DNA recombinant expression product encoding the Met ₄₆ to Asn ₄₄₁ amino acid residues of the polypeptide encoded by the full length PAF-AH cDAN (SEQ ID NO: 8), rPH. Name it 2 RPH in bacterial cells. 2 The plasmid used for production is pB322-based plasmid pBAR2 / PH. As 2 this plasmid was (1) from nucleotides 297 to 1487 of SEQ ID NO: 7 encoding human PAF-AH starting with methionine codon at position 46, (2) from arabinose operon of Salmonella typimurium araB-C promoter and araC gene, (3) transcription termination sequence from bacteriophage T7 and (4) replication origin of bacteriophage f1.
[127] In particular, pBAR2 / PH. 2 contained the following DNA fragments: (1) the vector sequence containing the ampicillin resistance or tetracycline resistance gene derived from bacterial plasmid pBR322 and the origin of replication, from the broken AatII site at position 1994 to the EcoRI site at nucleotide 6274; (2) the EcoRI site at position 6274 to the Xbal site at position 131, wherein the Salmonella typhimurium arabinose operon DNA (Genbank Accession Nos. M11045, M11046, M11047, J01797); (3) a DNA containing a ribosome binding site of pET21b (purchased from Novagen, Madison, Wisconsin, USA), from the Xbal site at position 131 to the Ncol site at position 170; (4) the Ncol region at position 170 to the Xhol region at position 1363, wherein the human PAF-AH cDNA sequence; And (5) a DNA fragment of pET-21 b (obtained from Novagen), comprising an Xter site at position 1363 to a broken AatII site at position 1993, containing the electron termination sequence of bacteriophage T7 and the origin of replication of bacteriophage f1.
[128] RPH as another PAF-AH product. The product named 9 is a recombinant DNA expression product encoding amino acid residues Met ₄₆ to Ile ⁴²⁹ in a polypeptide encoded by full length PAF-AH cDNA (SEQ ID NO: 8). rPH. DNA encoding 9 was expressed as rPH in bacterial cells. 2 were inserted into the same vector used for production. This plasmid was transferred to pBAR2 / PH. Named 9, this plasmid contained the following DNA fragment: (1) EcoRI site of broken AatII to nucleotide 6239 at position 1958), ampicillin resistance gene or tetracycline resistance gene derived from bacterial plasmid pBR322 and origin of replication Vector sequence containing the; (2) the EcoRI site at position 6239 to the Xbal site at position 131, comprising: Salmonella typhimurium arabinose operon (Genbank Accession Nos. M11045, M11046, M11047, JO1797) DNA; (3) a DNA containing a ribosome binding site of pET-216 (obtained from Novagen, Edison, WI), from the Xbal site at position 131 to the Ncol site at position 170; (4) the Ncol region at position 170 to the Xhol region at position 1328, wherein the human PAF-AH DNA sequence; (5) a pET-21b DNA fragment containing the transcription termination sequence of bacteriophage T7 and the origin of replication of bacteriophage f1, from the Xhol site at position 1328 to the broken AatII site at position 1958.
[129] pBAR2 / PH. 2 and pBAR2 / PH. Expression of the PAF-AH product at 9 is under the control of the araB promoter, which is highly inhibited in the presence of glucose and in the absence of arabinose, except for its role as a potent promoter when L-arabinose is added to a culture without glucose. . A method of selecting cells containing plasmids is to add ampicillin (associated with antibiotics) or tetracycline to the culture medium. Various E. coli species can be used as recombinant expression hosts for PAF-AH objects, which include basic organic nutrients and derivatives thereof for arabinose metabolism such as W3110, DH5α, BL21, C600, J101, CAG629, Mutant-containing strains that reduce proteolysis, such as KY1429, include strains that lack the ability to degrade arabinose, such as SB7219 and MC1061. By using a strain that cannot destroy arabinose, there is an advantage in that the inducer for producing PAF-AH (Arabinose) is not removed from the medium during the induction period, which results in the yield of a strain capable of metabolizing arabinose. Higher levels of PAF-AH can be obtained. All suitable media and culture conditions can be used to express the active PAF-AH product in several E. coli strains. For example, a rich media formulation such as LB, EDM295 (M9 basal minimal medium supplemented with yeast extract and acid hydrolyzed casein) or a pH6.75 basic minimal medium supplemented with trace elements and vitamins and using glycerol as a carbon source. "Limited" media, such as A675, can produce sufficient rPAF-AH products. Tetracycline is also added to the medium to select plasmids.
[130] Plasmid pBAR2 / PH. 2 was transformed into E. coli strain MC1061 (ATCC 53338), which was unable to metabolize arabinose due to the determination of arabinose operon. MC1016 was lysine nutritionally modified strain and cultured in a batch operation using a limited medium containing casamino acid complementing the leucine mutation.
[131] pBAR2 / PH. E. coli M1061 cells transformed with 2 were grown in batch medium containing 2 gm / L glucose at 30 ° C. Glucose plays a dual role as an inhibitor of the carbon source and arabinose promoter necessary for cell growth. Glucose ash feed was stopped (<50 mg / L) and nourishment (containing 300 gm / L glucose) was started. The nutrient supply was increased primarily for 16 hours at which rate acid byproduct formation was limited. At this point, the medium was replaced with a medium containing glycerol instead of glucose. At the same time, 500 gm / L of L-arabinose was added so that the final concentration was 5 gm / L. Glycerol was fed at a constant feed rate for 22 hours. Cells were collected using a hollow fiber filter and concentrated to approximately 10-fold suspension. The cell paste was stored at -70 ° C. Cells with a PAF-AH activity of 65-70 U / OD / ml, about 10% of total cellular protein, and a final mass of about 80 gm / L were obtained (OD ₆₀₀ = 50-60). The final media volume of about 75 liters contained 50-60 gm PAF-AH.
[132] pBAR2 / PH. 2 or PH. High levels of rPAF-AH products were produced when 9 was expressed in SB 7219 or MC1061 strains. Other strains that do not degrade arabinose are also suitable for high density cell production. Cells are preferably cultured under the following conditions. Exponentially produced SB7219; pBAR2 / PH. 2 and SB7219; The pBAR2 / PH.9 strain was inoculated into a batch medium containing fermentor containing 2 g / L glucose. When glucose is consumed, the solution is fed to the tank with a solution of glycerol containing trace elements, vitamins, magnesium salts and ammonium salts for healthy growth. The tank was maintained at 30 ° C. and air was supplied to supply oxygen, and stirred to maintain an oxygen dissolution level of at least about 15% saturation. When the cell density is at least 110 g / L (wet cell mass), feed at a constant feed rate and add L-arabinose mass adduct to the culture (about 0.5% with final jug). Product formation was observed for 16-22 hours. Cultures generally reach 40-50 g / L (dry cell mass). Cells are collected by centrifugation, stored at -70 ° C, and the rPAF-AH product is purified before analysis. Usually a specific product of at least 150 units / ml / OD ₆₀₀ is obtained.
[133] B. Expression in Yeast Cells
[134] Recombinant human PAF-AH was also expressed in Saccharomyces cerevisiae. The yeast ADH2 promoter was used to regulate rPAF-AH expression and produce 7U / ml / OD ₆₀₀ (see Table 5 below).
[135] edificePromoterStrainEnzyme Activity (U / ml / OD) pUC tac AHpUC tac AHpUC tac AHpET AHpHAB / PHpBAR2 / PH.2pYep ADH2 AHtactrparaBT7araB / T7araBADH2E. coli W3110E. coli W3110E. coli W3110E. coli BL21 (DE3) (Novagen) E. coli XL-1MC1061 yeast BJ2.283040205034907
[136] C. PAF-AH expression in stray animal cells
[137] 1. Expression of human PAF-AH cDNA constructs
[138] Plasmids constructed to express PAF-AH, except pSFN / PAFAH.1, replicated multiple plasmids in COS cells using the potent viral promoter of cytomegalovirus, the polyadenylation site of bovine growth hormone gene, and the SV40 replication origin. Is done. Plasmids were electroporated into cells.
[139] Substitution of the 5 'flanking sequence (pDC1 / PAFAH.1) or 5' or 3 'flanking sequence (PDC1 / PAFah.2) of human PAF-AH cDNA with flanking sequences of other genes known to be expressed at high levels in mammalian cells The first set of plasmids was thereby constructed. Transfection into COS cells by transient transformation of this plasmid into COS, CHO or 293 cells followed by nearly the same levels as mentioned in clone sAH 406-3 of Example 7 (0.01 units / ml or 2-4 in the background above) To produce PAF-AH. Instead of the cytomegalovirus promoter, other plasmids were constructed that included a friend splenic focus-forming virus captive motor. The plasmid pmH-neo [Hahn et al., Gene, 127; 267 (1993)] human PAF-AH cDNA was inserted. Two transfectants (4B11 and 1C11) with PAF-AH activity of 0.15-0.5 units / ml were isolated by transfecting myeloma cell line NSO with a plasmid designated pSFN / PAFAH.1 and screening hundreds of clones. It has an estimated specific activity of 5000 units / mg and the productivity of these two NSO transfectants corresponds to about 0.1 mg / liter.
[140] 2. Expression of mouse-human chimeric PAF-AH gene construct
[141] Mammalian expression vector pRc / CMV versus secretion at 5-10 units / ml (more than 100-fold background) after transfection into COS cells due to a construct containing pDc encoding mouse PAF-AH (pRc / MS9) Produced PAF-AH. Since the estimated inactivation of mouse PAF-AH is almost the same as human enzymes, mouse cDNA is expressed at levels 500-1000 times higher than human PAF-AH cDNA.
[142] To test the difference in PAF-AH expression levels in humans and mice in COS cells, two mouse-human chimeric genes were constructed and expressed in COS cells. One of these constructs is pRc / PH.MHC1, an AUS PAF-AF polypeptide fused with the C-terminal 343 amino acids of human PAF-AH in the expression vector pRc / CMV (available from Invitrogen, San Diego, Calif.) (SEQ ID NO: : 21) contains the coding sequence of the N-terminal 97 amino acids. The second chimeric gene is the pRc / PH.MHC2 plasmid, wherein the mouse PAF-AH polypeptide fused with 400 C-terminal residues of human PAF-AH in pRc / CMV contains a N-terminal 40 amino acid coding sequence. Transfection of COS cells with pRc / PH.MHC1 accumulates 1-2 units / ml of PAF-AH activity in the medium. Modulating medium derived from cells transfected with pRc / PH.MHC2 was found to have RAF-AH activity of only 0.01 units / ml. In these experiments, it was evident that the difference in expression levels between the mouse and human PAF-AH genes was due at least in part to the polypeptide fragments between residues 40 and 97 or the corresponding RNA or DNA fragments encoding this region of the PAF-AH protein.
[143] 3. Recoding the First 290 bP of PAF-AH Coding Sequence
[144] One hypothesis about human PAF-AH synthesized at low levels in transfected mammalian cells is that codons used by natural genes are not suitable for efficient expression. However, since optimal codons generally have a 10-fold effect on expression at most, it is not thought that there may be a 500-1000 fold difference due to the use of codons in expression levels between mouse and human genes. Second Hypothesis Explaining the Difference Between Mouse and Human PAF-AH Expression Levels Human PAF-AH mRNA at the 5 ′ coding site results in an ineffective translation initiation or progression, or a secondary structure that induces relatively rapid mRNA degradation. To form.
[145] To examine this hypothesis, synthetic fragments encoding human PAF-AH proteins from amino-terminus to residue 96 that substituted ("recoded") most of the codons with codons of different sequences, except for encoding the same amino acids. It was configured. Substitution of the second codon from GTG to GTA generated the Asp718 site, which is present at one end of the synthetic fragment and in the mouse cDNA. The other end of the fragment contained the BamHI site normally found in codon 97 of the human gene. An Asp718 / BamHI fragment of approximately 290 bp was derived from a PCR fragment prepared by a double asymmetric PCR method for synthetic gene construction as described in Biotechinques, Sandhu et al., 12: 14-16 (1992). The DNA fragment encoding the residue of the human PAF-AH molecule starting at nucleotide 453 of SEQ ID NO: 7 and the synthetic Asp718 / BamHI fragment were combined, and the sequence encoding the original human PAF-AH enzyme was converted into the mammalian expression vector pRc. Plasmid pRc / HPH.4 was prepared by inserting into / CMV (purchased from Invitrogen). The 5 'flanking sequence adjacent to the human PAF-AH coding sequence in pRc / HPH.4 is derived from that of mouse cDNA (nucleotides 1 to 116 of SEQ ID NO: 21) encoding PAF-AH in pRc / MS9.
[146] pRc / HPH. To test human PAF-AH expression in 4, pRc / HPH. 4 (recoded human gene), pRc / HPH. 4 (recoded human gene), pRc / HPH. COS cells were transiently transfected with 4 (mouse PAF-AH) or pRc / HPH.MHC1 (mouse-human hybrid 1). PAF-AH activity of the transfected cell conditioned media was tested and it contained 5, 7 units / ml (mouse gene), 0.9 units / ml (mouse-human hybrid 1) or 2.6 units / ml (recoded human gene) Turned out to be. Thus, the recoding strategy of the first 290 bp coding sequence of human PAF-AH has successfully raised human PAF-AH expression levels from 2-3 ng / ml to about 0.5 ug / ml upon transient COS cell transfection. pRc / HPH. The PAF-AH gene, recoded at 4, will be inserted into a mammalian expression vector carrying the dehydrofolate reductase (DHFR) gene and the VIII-negative Chinese hamster ovary cells will be transfected with the vector. Methotrexate selection will be performed on the transfected cells to obtain clones that produce high levels of human PAF-AH which are responsible for gene amplification.
[147] Example 9
[148] Recombinant human plasma PAF-AH (initiated in Ile42) expressed in E. coli was purified once by Coomassie-stained SDS-PAGE by various methods and then analyzed for activity exhibited by the native PAF-AH enzyme.
[149] A. Recombinant PAF-AH Purification
[150] The purification method used is similar to the method described in Example 1 for natural PAF-AH. The following steps were carried out at 4 ° C. 50 ml of PAF-AH producing E. coli (transformed with trp AH expression construct) pellets were lysed as described in Example 8. The supernatant was added dropwise at 0.8 ml / min on a Blue Sepharose Fast Flow column (2.5 cm X 4 cm; 20 ml bed volume) equilibrated with Buffer D (25 mM Tris-HCl, 10 mM CHAPS, 0.5 M NaCl, pH7.5). It was. The column was washed with 100 ml of Buffer D and then eluted with 100 ml of Buffer A containing 0.5 M KScN at 3.2 ml / min. 15 ml of active fraction was added dropwise onto 1 ml of Cu chelating Sepharose column equilibrated with Buffer D. The column was washed with 5 ml of buffer D and then eluted naturally with 5 ml of buffer D containing 100 mM imidazole. Fractions with PAF-AH activity were analyzed by SDS-PAGE.
[151] The purification results are shown in Table 6, where the unit is umol PAF hydrolysis / hour. The purification product obtained at 4 ° C. appears on the SDS-PAGE as one strong band under the 43 kDa marker which stains it up and down. Recombinant material is significantly pure and exhibits higher specific activity compared to the PAF-AH preparation of plasma described in Example 1.
[152] sampleVolume (ml)Activity (U / ml)Total activity (Ux10 ³ )Prot Conc (mg / mL)Inactivity (U / mg)Activity recovery rate (%)Folding tablets StepCumStepCum. Lysate4.5989445115.663100100OneOne blue15649600.07914222214.414.4 CuOne212821280.553869220484.261
[153] When the same purification protocol was performed at ambient temperature, the band group below the 29 kDa label in addition to the band below the 43 kDa label correlated with the PAF-AH activity of the assay gel slice. These low molecular weight bands will be proteolytic fragments of PAF-AH with enzymatic activity.
[154] Different purification methods were also performed at ambient temperature. PAF-AH-producing E. coli (transformed with expression construct PUC trp AH) pellets (100 g) were added to 200 ml of lysis buffer (25 mM Tris, 20 mM CHAPS, 50 mM NaCl, 1 mM deta, 50 ug / resuspended in ml benzamidine, pH 7.5) and dissolved through three passes of the microfluidizer at 150,000 psi. The solid was removed by centrifugation at 14,300 g for 1 hour. S Sepharose Fast diluted 10-fold in supernatant in dilution buffer (25 mM CHAPS, 1 mM EDTA, pH 4.90) and equilibrated with Buffer E (25 mM MES, 10 mM CHAPS, 1 mM EDTA, 50 mM NaCl, pH 5.5) 25 ml / min dropwise on a flow column (200 ml) (cation exchange column) Wash the column with 1 I of buffer E, elute with 1 M NaCl and adjust pH to 7.5 FH with 0.5 ml 2 M Tris base. Eluates were collected in 50 ml fractions fractions with PAF-AH activity were pooled to 0.5 M NaCl Equilibrated with Buffer F (25 mM Tris, 10 mM cHAPS, 0.5 M NaCl, 1 mM EDTA, pH7.5) S pool was added dropwise at 1 ml / min on a Blue Sepharose Fast Flow column (2.5 cm × 4 cm; 20 ml) Wash the column with 100 ml Buffer F and then with 100 ml Buffer F containing 3 M NaCl. Eluted at 4 ml / min Blue Sepa The fast flow chromatography step was repeated to reduce endotoxin levels in the sample Buffer G (25 mM Tris pH 7.5, 0.5 M NaCl, 0.1% Tween 80, 1 mM EDTA) was pooled by fractions with PAF-AH activity. Dialysis against.
[155] The purification results are shown in Table 7, and the unit is umol PAF hydrolysis / hour.
[156] sampleVolume (ml)Activity (U / ml)Total activity (Ux10 ³ )Prot Conc (mg / mL)Inactivity (U / mg)Activity recovery rate (%)Folding tablets StepCumStepCum. Lysate2005640112857.4698100100OneOne S11157426373.69155757561616 blue10039443940.8446763562348
[157] The resulting purification product appeared on SDS-PAGE as one dark band under the 43 kDa marker, which directly stained it up and down. Recombinant material is significantly pure and exhibits higher specific activity compared to the PAF-AH preparation of plasma described in Example 1.
[158] Other purification methods belonging to the present invention include the following cell lysis, clarification and column steps. Dilute the cells 1: 1 in lysis buffer (25 mM Tris pH7.5, 150 mM NaCl, 1% Tween 80, 2 mM EDTA). By lysing the material three times through a 15,000-20,000 psi cold microfluidizer,> 99% crushed cells were obtained. The lysate was diluted 1:20 in dilution buffer (25 mM Tris pH8.5, 1 mM DETA), then filled with Q-Sepharose Big Bead Chromatography Medium (available from Pharmacia) and 25 mM Tris pH8.5, 1 It was added to a column equilibrated with mM EDTA, 0.015% Tween 80. The eluate was diluted 1: 10 with 25 mM MES pH 5.5, 1.2 M ammonium sulphate, 1 mM EDTA and added to butyl sepharose chromatography medium (Pharmacia) equilibrated with the same buffer. PAF-AH activity is eluted in 25 mM MES pH 5.5, 0.1% Tween 80, 1 mM DETA.
[159] Another method of the present invention for purifying enzymatically active PAT-AH in E. coli comprises the following steps: (a) E. to obtain a dissolved PAFT-AH supernatant after lysis in a buffer containing CHAPS. Preparing a COLI extract; (b) diluting the supernatant and adding it to an anion exchange column equilibrated to pH about 8.0; (c) eluting the PAF-AH enzyme in the anion exchange column; (d) adding said anion exchange column adjustment eluent to a blue dye ligand affinity column; (e) eluting the blue dye ligand affinity column with a buffer containing 3.0 M salt; (f) diluting the blue dye eluent into a buffer suitable for the hydroxyapatite chromatography run; (g) performing hydroxyapatite chromatography when washed and eluted with a buffer (with or without CHAPS); (h) diluting the hydroxyapatite eluate to a salt concentration suitable for cation exchange chromatography; (i) adding the diluted hydroxyapatite eluate to a cation exchange column at a pH in the range of about 6.0 to 7.0; (j) eluting PAF-AH in the cation exchange chromatography with a suitable formulation buffer; And (l) formulating liquid or frozen PAF-AH in the absence of CHAPS.
[160] Preferably, the lysis buffer of step (a) is 25 mM Tris, 100 mM NaCL, 1 mM DETA, 20 mM CHAPS, pH 8.0; In step (b) the anion exchange chromatography supernatant is diluted 3-4 times with 25 mM Tris 1 mM EDTA, 10 mM CHAPS, pH8.0 and the column is 25 mM Tris, 1 mM DETA, 50 mM NaCl, 10 mM CHAPS , Q-Sepharose column equilibrated to pH8.0; the anion exchange column of step (c) is eluted with 25 mM Tris, 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH8.0; in step (d) the eluent of step (C) is added directly to the blue dye-friendly column; column of step (e) is eluted with 3 M NaCl, 10 mM CHAPS, 25 mM Tris, pH8.0 buffer; In step (f), the blue dye solution of hydroxyapatite chromatography is diluted with 10 mM sodium phosphate, 100 mM NaCl, 10 mM CHAPS, pH6.2; The hydroxyapatite chromatography in step (g) was performed with a hydroxyapatite column equilibrated with 10 mM sodium phosphate, 100 mM NaCl, 10 mM CHAPS, with or without 50 mM sodium phosphate, 100 mM NaCl (10 mM CHAPS). ); elute pH 7.5; in step (h) the hydroxyapatite eluate of cation exchange chromatography is diluted with a buffer in the range of about 6.0 to 7.0 pH containing sodium phosphate (with or without CHAPS); The S Sepharose column of step (i) is equilibrated with 50 mM sodium phosphate (with or without 10 mM chomenes), pH6.8. in step (j) elute with a suitable formulation buffer such as pH 7.5, 125 mM NaCl, 50 mM potassium phosphate, 12.5 mM aspartic acid, containing 0.01% Tween-80; In step (k) cation exchange chromatography is carried out at 2-8 ° C. Examples of formulation buffers suitable for use in step (l) that stabilize PAF-AH include 50 mM potassium phosphate, 12.5 mM aspartic acid, 125 mM NaCl pH7.4 (EOFIR Tween-80 and Pluronic F68 adducts). 25 mM potassium phosphate buffer containing or not) or (at least) 125 mM NaCl, 25 mM arginine and 0.01% Tween-80 (with or without about 0.1 and 0.5% of Pluronic F68). .
[161] B. Recombinant PAF-AH Activity
[162] The most prominent characteristic of PAF acetylhydrolase is that it has significant specificity for fruitiness involving short residues at the sn-2 position of the substance. This specificity distinguishes PAF acetylhydrolase from other forms of PLA2. Thus, to determine if recombinant PAF-AH degrades phospholipids with long chain fatty acids at the sn-2 position, 1-palmitoyl-2-arachidonoyl-sn as this is a preferred substance for the well-characterized PLA ₂ form. Hydrolysis of glycerpo-3-phosphocholine (arachidonoyl PC) was analyzed. As in previous studies with native PAF-AH, these phospholipids were not hydrolyzed when incubated with recombinant PAF-AH. In an additional experiment, ArikidonoylPC was included in a standard PAF hydrolysis assay at concentrations in the range of 0-125 uM to determine if it inhibited PAF hydrolysis by recombinant PAF-AH. There was no inhibition of PAF hydrolysis even at the highest concentration of PAF-AH, which was five times greater than the concentration of PAF. Thus, recombinant PAF-AH exhibits the same substrate selectivity as natural enzymes; Long chain substrates are not recognized. Moreover, recombinant PAF-AH enzymes rapidly degraded the oxidized phospholipids (glutaroyl PC) that oxidatively cleave sn-2 fatty acids. Natural plasma PAF-AH has several properties that make it distinguishable from other phospholipases, and is resistant to compounds that break disulfides or mutate sulfhydryl groups.
[163] Both recombinant and native plasma PAF-AH enzymes are sensitive to DFP, suggesting that serine is part of its active site. A unique feature of native plasma PAF is that it is closely related to lipoproteins in the bloodstream and its catalytic activity is influenced by the lipoprotein environment. When incubated with recombinant PAF-AH of the present invention in human plasma (pretreated with DFP to remove endogenous enzyme activity), it relates to high and low density lipoproteins in the same manner as natural activity. This is important because there is substantial evidence that protein variation is essential for the cholesterol accumulation observed in bamboo shoots and that the oxidation of lipids is an initiation factor in this process PAFH-AH is a low-density lipodanac variant under in vitro oxidative conditions. It plays a role in vivo and prevents inflammation by administering PAF-AH, which not only resolves inflammation but also inhibits the oxidation of lipoproteins in atherosclerotic plaques.
[164] All these results confirmed the coding of a protein having cDNA clone sA 406-3 DL human plasma PAF acetylhydrolase activity.
[165] Example 10
[166] Several different recombinant PAF-AH products were expressed in E. coli. These products included PAF-AH analogs with single amino acid mutants and PAF-AH fragments.
[167] A. PAH-AH Amino Acid Substitution Products
[168] PAH-AH is a lipase because it hydrolyzes phospholipid PAF. While it is not clear that there is an overall similarity between PAH-AH and other specified lipases, there are conserved residues compared to structurally specified lipases. Serine was identified as the number of active sites. Serine, together with aspartic acid residues and histidine residues, forms three sets of catalysts representing active sites of lipase. Although three residues are not in close proximity to the primary protein sequence, structural studies have demonstrated that the three residues are in close proximity to three-dimensional space. Structural comparisons of mammalian lipases suggest that aspartic acid residues are generally 24-amino acid C-terminus for the active site serine. In addition, histidine is generally 109-111 amino acids C-terminal to the active site serine.
[169] By site-directed mutagenesis and PCR, individual codons in the human PAH-AH coding sequence were modified to encode alanine residues and expressed in E. coli. As shown in Table 8 below, for example, where the abbreviation "S108A" means that the serine residue at position 108 has been replaced with alanine, a point mutation of Ser ₂₇₃ , Asp ₂₉₆ or His ₃₅₁ completely reduces the PAH-AH activity. Destroy. The distance between active site residues is similar to PAH-AH (Ser to Asp, 23 amino acids; Ser to His, 78 amino acids) and other rapases. These experiments demonstrate that Ser ₂₇₃ , Asp ₂₉₆ and His ₃₅₁ are critical residues for activity and are therefore likely candidates for triple catalyst residues. Cysteine is often critical for the protein's functional integrity because of its ability to form disulfide bonds. The plasma PAF-AH enzyme contains five cysteines. Each cysteine was individually mutated for serine and the resulting mutants were expressed in E. coli to determine whether any of the five were critical for enzyme activity. Primary activity results using partially purified preparations of these recombinantly produced mutants are shown in column 2 of Table 8, while results using more purified agents are shown in column 3 of Table 8. . This data indicates that all of the cysteine mutants have high equivalent activity, and none of these cysteines are required for RAF-AH activity. Other point mutations also had little or no effect on RAF-AH activity. In Table 8, "++++" indicates wild type PAF-AH activity of about 40-60 μ / mL / OD ₆₀₀ , and "+++" indicates about 20-40 μ / mL / OD ₆₀₀ activity, "++" is about 10 - shows a 20 μ / ㎖ / OD ₆₀₀ activity, "+" is 1- 10 μ / ㎖ / OD ₆₀₀ represents the activity, "-" in the <1 μ / ㎖ / OD ₆₀₀ activity Indicates.
[170] MutationPAF-AH activityIntrinsic PAH-AH Activity of Purified Formulations Wild type++++6.9 mmol / mg / hr S108A++++S273A-D286A-D286N++D296A-D304A++++D338A++++H351A-H395A, H399A++++C67S+++5.7 mmol / mg / hr C229S+6.5 mmol / mg / hr C291S+5.9 mmol / mg / hr C334S++++6.8 mmol / mg / hr C407S+++6.4 mmol / mg / hr C67S, C334S, C407S 6.8 mmol / mg / hr
[171] B. PAF-AH Fragment Product
[172] C-terminal deletions were prepared by cleaving the 3 'end of the PAF-AH coding sequence with endonuclease III at various time frames and then binding the shortened coding sequence to the plasmid DNA encoding stop codons in all three reading frames. . Ten different deletion constructs were characterized by DNA sequencing, protein expression and PAF-AH activity. The removal of 21 from the 30 C-terminal amino acids greatly reduced the catalytic activity and the 52 residues removed to completely destroy the activity. See FIG. 3.
[173] A pseudo deletion was made at the amino acid terminus of PAF-AH. PAF-AH and E. coli thioredoxin were fused at the N-terminus to promote constant high expression PAF-AH activating stones (see LaVallie et al., Bio / technology, 11: 187-193 (1993)). . 19 amino acids were removed from the naturally-processed N-terminus (Ile ₄₂ ) to reduce the activity by 99%, while 26 amino acids were removed to completely destroy the enzymatic activity of the fusion protein. See FIG. 3. Deletion of twelve amino acids resulted in a fourfold increase in enzyme activity.
[174] By a similar method as described in Example 1 (microcon 30 filters purchased from Amicon instead of Cu columns were used to concentrate the blue Sepharose eluate), PAF-AH was continuously purified from fresh human plasma. In addition to Ile ₄₂ , two N-terminus were identified, Ser ₃₅ and Lys ₅₅ . Heterogeneity may be natural to the enzymes in the plasma or may occur during purification.
[175] The purified material described above was also analyzed for glycosylation. Purified natural PAF-AH was incubated in the presence or absence of N-glycanase, an enzyme that removes N-linked carbohydrates from glycoproteins. Treated PAF-AH samples were visualized by electrophoresis through a 12% SDS polyacrylamide gel and then western blotting using rabbit polyclonal antiserum. Proteins not treated with N-glycanase migrated as diffusion bands of 40-50 kDa, while proteins treated with glycanases moved as tight bands of about 44 kDa, demonstrating that natural PAF-AH is glycosylated. It is.
[176] N-terminal heterogeneity was also eliminated in the purified formulation of recombinant PAF-AH (Ile ₄₂ N-terminal). These formulations were mixtures of polypeptides with N-terminus starting at Ala ₄₇ , Ile ₄₂ or at the Met- ₁ initiation close to Ile ₄₂ .
[177] 1. Primary comparison of PAF-AH fragments and PAF-AH
[178] In view of the observed heterogeneity of recombinantly produced PAF-AH, other recombinant products were prepared and tested for homogeneity after recombinant expression and purification. Compositions of the recombinant expression products of pBAR2 / PH.2 and pBAR2 / PH.9 in E. coli strain MC1061 were analyzed at different time points during the production phase of cell fermentation. Partially purified samples of recombinant PH.2 and PH.9 from cells collected at time points ranging from 5 to 22 hours after induction of protein expression were subjected to matrix assisted laser desorption ionization mass spectrometry (MALDI-). MS).
[179] With the PH.2 expression vector, two peaks were observed in the spectra of the partially purified protein at the expected mass values for the rPAF-AH protein. Two peaks were observed at all time points, with more heterogeneity observed when fermentation was stressed, as indicated by accumulation of acetate in the medium and / or oxygen depletion. In this mass range, the accuracy of the MALDI-Ms technique was about ± 0.3% for the mass of one amino acid. The higher mass peaks observed were consistent with the presence of expected full length translation products for the PH.2 vector, except for translation initiation methionine, which is expected to be removed after translation. Lower mass peaks were less than approximately 1200 atomic weight units.
[180] When the PH.9 expression vector was used, a single peak appeared mainly in the spectrum of the partially purified protein at the expected mass values for the rPAF-AH protein. This single peak was observed at all time points, with no increase in heterogeneity at other time points. The observed mass of this protein was consistent with the presence of the expected full length translation product for the PH.9 vector, except for the starting methionine.
[181] 2. Purification of PAF-AH Fragments
[182] Recombinantly expressed rPH.2 (expression product of DNA encoding Met ₄₆ -Asn ₄₄₁ ) and rPH.9 (expression product of DNA encoding Met ₄₆ -Ile ₄₂₉ ) preparations were purified by rPAF-AH (Ile _42). -Asn ₄₄₁ expression product of the DNA encoding) for further comparison. rPH.9 was produced by E. coli strain SB7219 and generally purified according to the zinc chelate compound purification procedure as described above, while rPH.2 was produced by E. coli strain MC1061 and purified as described below. Transformed cells were lysed by diluting the cell paste with lysis buffer (100 mM succinate, 100 mM NaCl, 20 mM CHAPS, pH 6.0). The slurry was mixed and lysed by high pressure collapse. The lysed cells were centrifuged and the supernatant containing rPH.2 was maintained. The cleared supernatant was diluted in 25 mM sodium phosphate buffer containing 1 mM EDTA, 10 mM CHAPS, pH 7.0. Diluted supernatant was added to a Q Sepharose column. Wash the column once with 3 column volumes of 25 mM sodium phosphate buffer containing 1 mM EDTA, 50 mM NaCl, 10 mM CHAPS, pH 7.0 (Wash 1), then contain 1 mM EDTA, 10 mM CHAPS, pH 8.0 Was washed with 10 column volumes of 25 mM Tris buffer (Wash 2) and 10 column volumes of 25 mM Tris buffer containing 1 mM EDTA, 100 mM NaCl, 10 mM CHAPS, pH 8.0 (Wash 3). Elution was performed with 25 mM Tris buffer containing 1 mM EDTA, 350 mM NaCl, 10 mM CHAPS, pH 8.0. The Q Sepharose Eluate was diluted 3-fold in 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0 and then added to a Blue Sepharose column. The column was first washed with 10 column volumes of 25 mM Tris, 1 mM EDTA, 10 mM CHAPS, pH 8.0. The column was then washed with 3 column volumes of 25 mM Tris, 0.5 M NaCl, 10 mM CHAPS, pH 8.0. Elution was performed with 25 mM Tris, 3.0 M NaCl, 10 mM CHAPS, pH 8.0. Blue Sepharose eluate was diluted 5-fold in 10 mM sodium phosphate, 10 mM CHAPS, pH 6.2 and then added to the chromatography column. This column was washed with 10 column volumes of 10 mM sodium phosphate, 100 mM NaCl, 0.1% Pluronic F68, pH 6.2. RPH.2 was eluted with 120 mM sodium phosphate, 100 mM NaCl, 0.1% Pluronic F-68, pH 7.5. Hydroxyapatite eluate was diluted 6-fold with 10 mM sodium phosphate, 0.1% Pluronic F68, pH 6.8. The diluted hydroxyapatite eluate was adjusted to pH 6.8 with 0.5 N succinic acid and then added to the SP Sepharose column. The SP Sepharose column was washed with 10 column volumes of 50 mM sodium phosphate, 0.1% Pluronic F68, pH 6.8 and eluted with 50 mM Sodium Phosphate, 125 mM NaCl, 0.1% Pluronic F68, pH 7.5. Eluted rPH.2 is formulated by diluting to a final concentration of 4 mg / ml in 50 mM sodium phosphate, 125 mM NaCl, 0.15% Pluronic F68, pH 7.5, and Tween 80 to a final concentration of 0.02% Tween 80. Was added. The formulated product was then filtered through a 0.2 μ membrane and stored before use.
[183] 3. Comparison of RAF-AH fragment and RAF-AH by sequencing
[184] Purification by N-terminal sequencing using Applied Biosystems Model 473A protein sequencer (purchased from Applied Biosystems, Foster City, CA) and C-terminal sequencing using Hewlett-Packard model G1009A C-terminal protein sequencer RPH.2 and rPH.9 preparations were compared with purified rRAF-AH preparations. The rPH.2 formulation had less N-terminal heterogeneity than rRAF-AH. The N-terminal analysis of the rPH.9 preparation was similar to the N-terminal analysis of rPH.2, but less C-terminal branching was observed in the rPH.9 preparation than in rPH.2.
[185] The purified rPH.2 preparation includes a plurality of sequences having the N- terminus of Ala ₄₇ (about 86 - 89%) to the sequence of a bit having the N- terminus of Ala ₄₈ (about 11 - 14%) was contained, both N The terminal ratios were quite consistent under different fermentation conditions. The purified rPH.9 preparation also a number of the sequence having the N- terminus of Ala ₄₇ and contained (approximately 83 - 90%) to the sequence of a bit having the N- terminus of Ala ₄₈ (17% 10). In contrast, attempts to produce polypeptides starting at Ile ₄₂ (rRAF-AH) in bacteria resulted in artificially initiated Met- ₁ methionine (37-A) close to Ala ₄₇ (20-53%), Ile ₄₂ (8-10) or Ile ₄₂ (37). Various mixtures of polypeptides having N-terminus, starting at 72%). In the case of rPH.2 and rPH.9, the starting methionine is effectively removed by amino-terminal peptidase after bacterial synthesis of the polypeptide, while maintaining alanine at position 47 (or alanine at position 48) as the N-terminal residue. .
[186] C-terminal sequencing of rPH.2 showed that it had a C-terminus of HOOC-Asn-Tyr as a majority sequence (about 80%), which is expected HOOC-Asn ₄₄₁ -Tyr of the translation product. While coinciding with the ₄₀₀ C-terminus, about 20% was HOOC-Leu. Fractionation of the rPH.2 preparation by SDS-PAGE followed by additional sequencing of the primary and secondary bands, resulting in lower secondary bands consistent with products with not only 10 amino acids shorter than full length translations but also with lower levels of HOOC-His. The C-terminal sequence of HOOC-Leu-Met was obtained from (AH _L , described in Section B.5. Below). The mapping of the peptides also revealed that additional C-terminus is present in the PH.2 protein. The C-terminus of rPH.9 was primarily HOOC-Ile-His by direct sequencing (approximately 78-91%, dependent on total), with the expected HOOC-Ile ₄₂₉ -His ₄₂₈ C-terminus of the translation product. Matches. Since some background (“noise”) appears in this technique, low levels of other sequences cannot be excluded.
[187] 4. Comparison of RAF-AH fragment and RAF-AH by MALDI-MS
[188] MALDI-MS was performed on purified rPH.2 and rPH.9 formulations. The rPH.2 spectrum showed two peaks in the spectrum at the mass values expected for the rRAF-AH product (see FIG. 4), which is described in section B.1. Similar to the type observed with partially purified protein in. Secondly, lower molecular weight peaks were typically present at about 20% to 30% of the total. The rPH.9 spectrum showed a dominant peak at masses consistent with the expected mass for the full length translation product for the PH.9 vector, excluding translation initiation methionine (see FIG. 5). Small shoulder peaks with slightly lower molecular weights were also observed for rPH.9, which is about 5% of the total.
[189] 5. Comparison of RAF-AH fragment and RAF-AH by SDS-PAGE
[190] Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) was performed on the purified rRAF-AH, rPH.2 and rPH.9 formulations. Based on protein molecular weight standards, overlapping band types were observed for rPH.2 near the expected electrophoretic shift range for the rRAF-AH product. One or some gels showed two predominant bands, which were easily observed in the secondary band and the following primary bands. Each of these upper, middle primary and lower secondary bands were named AH _U , AH _M and AH _L. All of these bands reacted with anti-rRAF-AH monoclonal antibodies against western blot and thus were identified as rRAF-AH related products. The upper secondary band AH _U increased in strength during the storage time of the protein, possibly indicating a modified form of the rRAF-AH product. SDS-PAGE of the rRAF-AH preparation is similar to SDS-PAGE of rPH.2. There are two major bands moving near the expected molecular weight for rRAF-AH as well as the minor band and traces below the major band. In contrast, rPH.9 showed a single dominant band that did not diverge for SDS-PAGE. At slightly lower molecular weights and expected dimer positions, faint bands were seen. AH _U -like bands were not observed.
[191] Compositions of the purified rPH.2 and rPH.9 preparations were analyzed by 2D gel [Isoelectric Point Electrophoresis (IEF) in Urea and SDS-PAGE in two dimensions]. In the case of rPH.9, the 2D gel showed five main points separated from the IEF. The charge heterogeneity shown is consistent between the lots of rPH.9. In contrast, the 2D gel type of rPH.2 was more complicated as it contained about 15 points separated at the IEF and SDS-PAGE dimensions.
[192] 6. Comparison of activity of RAF-AH fragment and RAF-AH
[193] Purified rPH.2 and rPH.9 have an indistinguishable activity from the enzymatic activity of purified endogenous RAF-AH in serum, and rPH.2 and rPH.9 are lipoproteins in a manner similar to purified endogenous RAF-AH. Combine with
[194] Example 11
[195] Expression types of human plasma RAF-AH mRNA in human tissues were primarily analyzed by Northern blot hybridization.
[196] RNA was prepared from the human cerebral cortex, heart, kidney, placenta, thymus and tonsils using RNA Stat 60 (Tel-Test "B", purchased from Friendswood, Texas, USA). In addition, RNA was prepared from the human hematopoietic progenitor-like cell line, THP-1 (ATCC TIB 202), which distinguishes it from the macrophage-like phenotype using phorbol ester phorbolmyristylacetate (PMA). Induced to. Tissue RNA and RNA prepared from promyeloid THP-I cell line 1 to 3 days after and before induction were electrophoresed through 1.2% agarose formaldehyde gel and then transferred to nitrocellulose membrane. Full length human plasma RAF-AH cDNA, SAH 406-3 was labeled by random detection and hybridized to the membrane under the same conditions as described in Example 3 for library screening. Initial results indicate that the RAF-AH probe hybridizes with thymus, 1.8 kb band of tonsils, and lesser placental RNA.
[197] RAF is synthesized in the brain under normal physiological as well as pathological conditions. It is expected that the known pro-inflammatory and potential neurotoxin properties of this molecule, the ubiquity of PAF synthesis or the mechanism for its rapid metabolism are critical to the health of neural tissues. The presence of PAF acetylhydrolase in nervous tissue is consistent with its protective role. Interestingly, RAF-AH in bovine heterotrimeric cells [Hattori et al., J. Biol. Chem., 269 (37): 23150-23155 (1994), both the cloning described herein and the RAF-AH of the present invention have been identified in the brain.
[198] To determine whether the two enzymes are expressed in similar or different compartments of the brain, we cloned the human epithelium of PAF-AH cDNA in bovine brain cells and essentially described the type of mRNA expression in the brain as described in the previous paragraph. By comparing the mRNA expression type of PAF-AH of the present invention by the same method by Northern blotting. The areas of the brain examined by Northern blotting were cerebellum, medulla, spinal cord, cortex, amygdala, caudate nucleus, thalamus and cerebral cortex, frontal lobe and temporal lobe. Although the heterotrimeric intracellular form appeared to be more abundant than the secreted form, messages from both enzymes were detected in each of these tissues. Northern blot analysis of additional tissues shows that heterotrimeric intracellular morphology includes thymus, prostate, testis, ovary, small intestine, colon, peripheral leukocytes, macrophages, brain, liver, skeletal muscle, kidney, pancreas and adrenal gland. Expressed in cells. Such ubiquitous expression suggests that PAF-AH in heterotrimeric cells has a common housekeeping function in cells.
[199] Expression of PAF-AH RNA was investigated in monocytes isolated from human blood and during spontaneous differentiation into macrophages in culture. Little or no RNA was detected in fresh monocytes, but expression was induced and maintained during differentiation into macrophages. Incidental accumulation of PAF-AH activity was present in the medium of the differentiated cells. Expression of human plasma PAF-AH transcripts was also observed in THP-1 cell RNA at day 1 rather than 3 days after induction. THP-1 cells did not express mRNA for PAF-AH in the basal state.
[200] Example 12
[201] PAF-AH expression in human and mouse tissues was examined by in situ hybridization
[202] Human tissue was obtained from the National Desire Research Interchange and the Cooperative Human Tissue Network. Normal mouse brain and spinal cord, and EAE stage 3 mouse spinal cord were harvested from S / JLJ mice. Normal S / JLJ mouse embryos were harvested 11 to 18 days after fertilization.
[203] The tissue fragments were placed in Tissue Tek II cryomold_ with a small amount of OCT compounds (Miles Laboratories, Inc., Naperville, Indiana). The mold is filled with OCT compound and placed in a container with 2-methylbutane [C ₂ H ₅ CH (CH ₃ ) ₂ , Aldrich Chemical Company, Milwaukee, Wisconsin, USA] and placed in a container. Once the tissue and OCT compounds in the creole mold were frozen, the blocks were stored at −80 ° C. until the sections were sectioned, and the tissue blocks were sectioned to a thickness of 6 μm and stored at −70 ° C. to allow them to warm up. After 5 minutes at 50 ° C, the condensate was removed and fixed at 4 ° C for 20 minutes in 4% paraformaldehyde, dehydrated at 4 ° C for 1 minute in each grade (70%, 95%, 100% ethanol) at room temperature. Air dry for 30 minutes Turned on. Intercept of 70% formamide / 2 × 2 minutes and denaturation at 70 ℃ within SSC, 2 × SSC, rinsed twice with dehydrated for 30 minutes and air dried. A tissue, in vitro RNA transcription-containing water ³⁵ S- Heterotrimeric intracellular PAF-AH identified by Hattori et al or DNA derived from the internal 1 kb Hind III fragment (SEQ ID NOs: nucleotides 308-1323) of the PAF-AH gene by UTP (Amersham) In situ hybridization with radiolabeled single-stranded mRAN generated from DAN derived from cDNA, probes of various lengths from 250-500 bp were used overnight hybridization (12-16 hours) at 50 ° C. ³⁵ S-labeled to a final concentration of 50% formamide, 0.3 M NaCl, 20 mM Tris pH 7.5, 10% dextran sulfate, 1 × Denhardt solution, 100 mM dithiotretol (DTT) and 5 mM EDTA. Riboprobes (6 × 10 ⁵ cpm / fragment), tRNA (0.5 μg / fragment) and Diethylpyrocarbonate (depc) -treatment was added to hybridization buffer. After hybridization, sections were washed with 4 × SSC / 10 mM DTT for 1 hour at room temperature, then 50% formamide / 1 × SSC / 10 mM DTT for 40 minutes at 60 ° C., 2 × SSC for 30 minutes at room temperature, and room temperature. Washed with 0.1 × SSC for 30 min. Sections were dehydrated, air dried for 2 hours, coated with Kodak NTB2 photoemulsion, air dried for 2 hours, developed (after storage in complete darkness at 4 ° C.) and counterstained with hematoxylin / eosin. .
[204] A. Brain
[205] cerebellum. In both the mouse and human brain, strong signals were seen in the Purkinie cell layer of the cerebrum, basket cells and individual neuronal cell bodies (one of the four cardiac nuclei of the cerebrum) in the dentate nucleus. Messages for PAF-AH in heterotrimeric cells were also observed in these cell types. In addition, plasma PAF-AH signals were seen in individual cells in the granular and molecular layers of the gray matter.
[206] hippocampus. In human hippocampal sections, individual cells throughout the section, considered neuronal cell bodies, showed strong signals. These have been identified as polymorphic cell bodies and granule cells. Messages for PAF-AH in heterotrimeric cells were also observed in the hippocampus.
[207] Brainstem. In both human and mouse brain stem sections, strong signals existed for individual cells in the gray matter.
[208] cortex. Individual cells throughout the cortex showed strong signals for human cortical sections taken from the cerebral, laryngeal and temporal cortex and whole brain sections of mice. These cells have been identified as vertebral, stellate and polymorphic cell bodies. It is believed that there is no difference in expression type in other layers of the cortex. These in situ hybridization results are different from those for the cerebral cortex obtained by northern blotting. This difference is likely due to the greater sensitivity in in situ hybridization compared to the sensitivity of northern blotting. As in the cerebellum and hippocampus, a similar type of expression of PAF-AH in heterotrimeric cells was observed.
[209] Sewerage. Some weak signals appeared in individual cells interspersed within the distal ends of human tissue sections.
[210] B. Human Colon
[211] Both normal and Crohn's colon showed signals in lymphoid aggregates present in the mucous membrane of the sections, which were slightly higher than sections from Crohn's disease patients. Crohn's disease colon also had a strong signal to the lamina propria. Similarly, high levels of signal were observed in the diseased appendage fragments while normal appendages showed low but detectable signals. Sections of ulcerative colitis patients showed no obvious signal in lymphoid aggregates or lamina propria.
[212] C. Human tonsils and chest
[213] Strong signals were found in the ubiquitous group of individual cells inside the embryonic center of the amygdala and inside the thymus.
[214] D. Human Lymph Nodes
[215] Although slightly weaker signals were observed in the lymph nodes of sections from donors with sepsis, strong signals were observed in lymph node sections obtained from normal donors.
[216] E. Human Small Intestine
[217] Both normal and Crohn's disease small intestines had weak signals in the lamina propria of firepans and sections, and slightly higher in diseased tissues.
[218] F. Human Spleen and Lungs
[219] No signal was observed in either the spleen (normal and spleen sections) or lung (normal and emphysema sections) tissue.
[220] G. Mouse Spinal Cord
[221] In both normal and EAE stage 3 spinal cords, there was a strong signal on the gray matter of the spinal cord and its expression was slightly higher in the EAE stage 3 spinal cord. In the EAE stage 3 spinal cord, white matter and perivascular salivary cells, invasive macrophages and / or other white blood cells showed signals that were not present in the normal spinal cord.
[222] F. Mouse Fetus
[223] Signals in eleven-day-old embryos were evident in the central nervous system of the fourth ventricle, which remained constant throughout the fetus as it developed into the cerebellum and brainstem. Like mature fetuses, the signal was evident in the central nervous system of the spinal cord (day 12), primary cortex and pedicle ganglia (day 14) and the pituitary gland (day 16). Signals were observed on the peripheral nervous system (starting on day 14 or 15) and on day 17 for nerves in which the spinal cord was maintained, and strong signals appeared around the beard of the fetus. On day 14, expression occurred in the liver and lungs, in the intestine (starting on day 15), and posterior of the mouth / esophagus (starting on day 16). By day 18, expression types were differentiated into cortex, posterior brain (cerebellum and brainstem), nerves with the lumbar spine, signals from the posterior, mouth and esophagus of the mouth / esophagus, and possibly weak signals from the lungs and intestines.
[224] G. Summary
[225] PAF-AH mRAN expression in tonsils, thoracic, lymph nodes, pyriban, adjunct and colonic lymphoid aggregates is consistent with the result that macrophages predominate in vivo of PAF-AH, both of which are phagocytic and antigen-processing longitudinal. This is due to the aggregation of tissue macrophages that act as a forcer.
[226] Expression of PAF-AH in inflammatory tissue is compatible with the hypothesis that the role of monocyte-derived macrophages addresses inflammation. It is expected that PAF-AH will inactivate PAF and pro-inflammatory phospholipids, thus down-regulating the inflammatory cascade of events initiated by these mediators.
[227] PAF is detected in whole brain tissue and secreted by rat cerebellar granule cells in culture. In vitro and in vivo experiments demonstrate that PAF binds to specific receptors in neural tissues and induces functional and phenotypic changes such as calcium migration, upregulation of transcriptional activation genes and differentiation of neural progenitor cell lines, PC12. These observations suggest a physiological role of PAF in the brain, which is compatible with hippocampal tissue sections and recent experiments using PAF analogs and antagonists with PAF as an important retrograde messenger for the long term potential of the hippocampus. Thus, in addition to the pathological effects in its inflammation, PAF is involved in a consistent neuronal signaling process. Expression of PAF-AH in cells in the brain regulates the duration and scale of PAF-mediated signaling.
[228] Example 13
[229] Monoclonal antibodies specific for recombinant human plasma PAF-AH were generated using E. coli producing PAF-AH as an immunogen
[230] Recombinant PAF-AH was injected into mouse # 1342 on days 0, 19 and 40. For prefusion additional stimulation, mice were given an immunogen in PBS, and after 4 days the mice were sacrificed and their spleens removed aseptically and placed in 10 ml serum-free RPMI 1640. Matte of two glass microscope slides immersed in serum-free RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units / ml penicillin and 100 μg / ml streptomycin (RPMI) (Gibco, Canada) The spleen between the ends was ground to make a single-cell suspension. The cell suspension is filtered through a sterile 70-mesh Nitex cell filter (Becton Dickinson, Parsippany, NJ), centrifuged at 200 g for 5 minutes and washed twice to resuspend the pellet in 20 ml serum-free RPMI. It was. Thymic cells obtained from three young Balb / c mice were prepared in a similar manner. NS-1 myeloma cells maintained in the log phase in RPMI with 200% Fetal Bovine Serum (FBS) (Hyclone Lavoratoris, Inc., Logan, Utah) were added 200 days prior to fusion. Centrifuge at g for 5 minutes and wash the pellet twice as described in the previous paragraph.
[231] 1 × 10 ⁸ splenocytes were combined with 2.0 × 10 ⁷ NS-1 cells and centrifuged to extract the supernatant. The cell pellet was removed by tapping the test tube and 1 ml of 37 ° C. PEG 1500 (50% pH 8.0 in 75 mM Hepes) (Möllinger Mannheim) was added with stirring for 1 minute followed by 7 ml of serum-free RPMI for 7 minutes. An additional 8 ml RPMI was added and cells were centrifuged at 200 g for 10 minutes. After the supernatant was decanted, the pellet was quenched with 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Kapco), 25 units / ml IL-6 (Möllinger Mannheim) and Resuspend in 200 ml RPMI containing 1.5 × 10 ⁶ thymosite / ml and plate in flat bottom 96 well tissue culture plates (Corning, NY, USA).
[232] At 2, 4 and 6 days after fusion, 100 μl of medium was removed from the wells of the fusion plate and replaced with fresh medium. On day 8, fusions were screened by ELISA and tested for the presence of mouse IgG binding to recombinant PAF-AH. 100 μg / well of recombinant PAF-AH diluted in 25 mM Tris, pH 7.5 was coated on Emulon 4 plate (Dynatech, Cambridge, Mass.) For 2 hours at 37 ° C. The coating solution was removed and 200 μl / well of blocking solution [0.5% fish skin gelatin (Sigma diluted in CMF-PBS)] was added and incubated at 37 ° C. for 30 minutes. Plates were washed three times with PBS with 0.05% Tween 20 (PBST) and 50 μl culture supernatant was added. Incubate for 30 minutes at 37 ° C. and wash as above, and anti-mouse IgG (fc) of horseradish peroxidase-bound goat diluted 1: 3500 in PBST (Jackson Immunore Research, West Grove, Pennsylvania) 50 μl was added. Incubate the plate as above, wash 4 times with PBST and then 100 μL consisting of 1 mg / ml o-phenylene diamine (Sigma) and 100 mM citrate, 0.1 μl / ml 30% H ₂ O ₂ in pH 4.5. Substrate was added. 50 μl of 15% H ₂ SO ₄ was added to stop the color reaction within 5 minutes. I read A ₄₉₀ as a reputation reader (Dynatech).
[233] The selected fusion wells were cloned twice by dilution into 96 well plates and after 5 days the number of colonies / wells was visually calculated. Cloned hybridomas were 90 D1E, 10E3A, 90E6C, 90G11D (ATCC HB 11724) and 90F2D (ATCC HB 11725).
[234] Monoclonal antibodies produced by hybridomas were isotyped using the isotrip system (Möllinger Mannheim, Indianapolis, Indiana). As a result, all of the monoclonal antibodies produced by hybridoma from fusion 90 were IgG ₁ .
[235] All of the monoclonal antibodies generated by hybridomas from fusion 90 were functionalized in the ELISA assay but were unable to bind PAF-AH for Western blot. To generate, mouse # 1958 was immunized with recombinant enzyme. Hybridomas were generated as described for fusion 90, but were screened by Western blotting rather than ELISA to identify Western-sensitive clones.
[236] For Western analysis, recombinant PAF-AH was mixed with an equal volume of sample buffer containing 125 mM Tris, pH 6.8, 4% SDS, 100 mM dithiothreitol and 0.05% bromphenol blue and 12% SDS polyacrylic Boil for 5 minutes before dropping onto the amide gel (Novex). After electrophoresis at 40 mAmps, proteins were electrophoresed on a polyvinylidene fluoride membrane (Pierce) for 1 h at 125 V in 192 mM glycine, 25 mM Tris base, 20% methanol and 0.01% SDS. Membranes were incubated overnight at 4 ° C. in 20 mM Tris, 100 mM NaCl (TBS) containing 5% bovine serum installment (BSA, Sigma). Incubate the blot for 1 hour at room temperature with polyclonal antiserum of rabbit diluted 1/8000 in TBS containing 5% BSA, then wash with TBS and alkaline phosphatase-binding in TBS containing 5% $ BAS Incubated for 1 hour at room temperature with anti-mouse IgG of goat. The blot was washed again with TBS and then 0.03% nitroblue tetrazolium in 0.02% 5-bromo-4-chloro-3-indolyl phosphate with 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl ₂ . It was incubated with. The reaction was stopped by repeated rinsing with water.
[237] Selected fusion wells were prepared as described above where the supernatant was positive in Western analysis. Hybridoma 143A was reacted with PAF-AH in a western blot and cloned (ATCC HB 11900).
[238] Polyclonal antiserum specific for human plasma PAF-AH was increased in rabbits by immunization every 100 months with 100 μg of purified recombinant enzyme in Freund's adjuvant.
[239] Example 14
[240] Edema model of rats [Henriques et al., Br. J. Pharmacol., 106: 579-582 (1992)] was used to evaluate the in vivo therapeutic effect of the recombinant PAF-AH of the present invention on acute inflammation. The study revealed that rPAF-AH blocks PAF-induced edema. A parallel study was conducted to compare the effect of PAF-AH with two commercially acceptable PAF antagonists.
[241] A. Preparation of PAF-AH
[242] E. coli transformed with the PAF-AH expression vector puc trp AH were lysed in a microfluidizer, the solids were centrifuged and the cell supernatant was loaded on an S-Sepharose column (Pharmacia). The column was washed extensively with a buffer consisting of 50 mM NaCl, 10 mM CHAPS, 25 mM MES and 1 mM EDTA, pH 5.5. PAF-AH was eluted by increasing the NaCl concentration of the buffer to 1 M. Affinity chromatography using a Blue Sepharose column (Pharmacia) was then used as an additional purification step. Prior to loading the PAF-AH formulation on the Blue Sepharose column, the sealant was diluted 1: 2 to reduce the NaCl concentration to 0.5 M and adjust the pH to 7.5. PAF-AH was eluted by extensive washing of the Blue Sepharose column with buffer consisting of 0.5 M NaCl, 25 mM Tris, 10 mM CHAPS and 1 mM EDTA, pH 7.5, followed by increasing NaCl concentration to 3.0 M.
[243] The purity of PAF-AH isolated by this method is usually 95% as assayed by SDS-PAGE and has an activity in the range of 5000-10,000 U / mL. Additional qualitative controls were performed for each PAF-AH preparation by measuring endotoxin levels and hemolytic action of freshly obtained rat erythrocytes. Buffer containing 25 mM Tris, 10 mM CHAPS, 0.5 M NaCl, pH 7.5 serves as a carrier for administration as well as a storage medium of the enzyme. The dose used in the experiment was based on the enzyme activity assay performed immediately before the experiment.
[244] B. Induction of Edema
[245] 6-8 week old female Long Evans rats (Charles River, Wilmington, Mass.) Were used for all experiments. Prior to experimental manipulations, animals were mixed with a mixture of anesthetic brittlets (Port Dodge Laboratories, Port Dodge, Iowa, USA), Rompen (Miles, Shoun Mission, Kansas, USA), and Ace Promazine (Aveco, Iowa, Dodge, USA). Was anesthetized followed by administration of about 2.5 mg ketaset, 1.6 mg romfun, 0.2 mg ace promazine per animal / dose. Edema was induced in the feet by administering PAF or Jimosan as follows. PAF (Sigma # P-1402) was freshly prepared for each experiment from a 19.1 mM stock solution stored in chloroform / methanol (9: 1) at 20 ° C. The required volume was dried under N ₂ and sonicated for 5 min by diluting 1: 1000 in a buffer containing 150 mM NaCl, 10 mM Tris pH 7.5, and 0.25% BAS. Animal hind limbs were then injected with 50 μl PAF (final dose of 0.96 nmole), edema was assayed after 1 hour, edema after 2 hours in some serum, and again after 2 hours in some experiments. Jimosan A (Sigma # A-8800) was prepared fresh for each experiment as a suspension of 10 mg / ml in PBS. Edema was then assayed 2 hours after the injection of 50 μl of jimosane (500 μg final dose) between the hind legs of the animals. Edema was quantified by measuring the volume of the paw at the time of post-additional antigen administration with PAF or Jimosic, immediately prior to administration of PAF or Jimosan. Edema appeared to have increased foot volume in millimeters. Volume replacement measurements were made on anesthetized animals using a volume recorder (U sea Basil, Model # 7150) that measures the volume of replaced water in the immersed foot. To ensure that the immersion of the foot from one point to the next is comparable, it was marked with indelible ink on the hairless heel of the hind legs. Repeated measurements of the same foot using this technique resulted in accuracy of less than 5%.
[246] C. Route and Dosage of PAF-AH
[247] PAF-AH was topically injected between the feet or systemically IV injection into the tail vein. 100 μl PAF-AH (4000-6000 U / mL) was injected subcutaneously between the right hind limbs in the topically administered rats. The left foot received a 100 μl carrier (buffered saline solution) as a control. For systemic administration of PAF-AH, rats were injected with the indicated units of PAF-AH in a carrier administered 300 μl IV in the tail vein. The control was injected IV with a suitable volume of carrier in the tail vein.
[248] D. Administration of PAF-AH
[249] 100 μl of PAF-AH (4000-6000 U / ml) was injected subcutaneously between the right foot of the rat (N = 4). The left foot was injected with 100 μl of carrier (buffered salt solution). The other four rats were only injected with a carrier. Subcutaneous foot injections in all rats were immediately administered additional antigens with PAF and the paw volume was measured 1 hour after administration of the additional antigen. In FIG. 6, edema appeared as an average increase in foot volume (ml) ± SEM for each treatment group, indicating that PAF-induced foot edema is blocked by topical administration of PAF-AH. Groups treated with topical PAF-AH prior to administration of PAF booster antigens had reduced inflammation compared to the control injection group. An increase in paw volume of 0.08 ml ± 0.08 (sem) was seen in the PAF-AH group compared to 0.63 ± 0.14 (SEM) for the carrier treated control. Animals injected into the paw only with a carrier do not show an increase in paw volume and an increase in paw volume is a direct result of PAF injection.
[250] E. Intravenous Administration of PAF-AH
[251] Rats (N = 4 / group) were pretreated IV with PAF-AH (200 U in 300 μl carrier) or carrier alone 15 minutes prior to PAF challenge. Edema was assessed 1 and 2 hours after PAF challenge. In FIG. 7, edema for each treatment group appears as an average increase in volume (ml) ± SEM, demonstrating that IV administration of PAF-AH blocked PAF-induced paw edema 1 and 2 hours after challenge. will be. The group injected with 2000 U PAF-AH by the IV route reduced inflammation over the course of 2 hours. The mean volume increase at 2 hours for the PAF-AH treated group was 0.10 ml ± 0.08 (SEM) compared to 0.56 ml ± 0.11 for the carrier treated control.
[252] F. Comparison of PAF-AH Protection in Edema Induced by PAF or Jimosan
[253] Rats (N = 4 / group) were IV pretreated with PAF-AH (2000 U in 300 μL carrier) or carrier alone. Fifteen minutes after pretreatment, groups were injected with PAF or Jimosan A and foot volume was assessed after 1 and 2 hours, respectively. As shown in FIG. 8, edema is shown as an average increase in volume (mL) ± SEM for each treatment group, although systemic administration of PAF-AH (2000 U) is effective in reducing PAF-induced foot edema. In addition, they failed to block zymosan-induced edema. The average increase in volume of 0.08 ± 0.02 was seen in the PAF-AH treatment group, while 0.49 ± 0.03 in the control group.
[254] G. Effective amount titer of PAF-AH protection
[255] For two separate experiments, a series dilution or carrier control of PAF-AH was IV pretreated to a volume of 300 μl 15 minutes prior to PAF challenge. Both feet were further challenged with PAF (as described above) and edema was assessed after 1 hour. In FIG. 9 edema is shown as an average increase in volume (±) SEM for each treatment group, demonstrating increased protection from PAF-induced edema in injected rats with increasing dose of PAF-AH. do. In this experiment, the PAF-AH of ID ₅₀ provided by the IV route was found to be 40 to 80 U per FOT.
[256] H. In vivo efficacy of PAF-AH as a function of time after administration
[257] In two separate experiments, two groups of rats (N = 3 to 4 / group) were IV pretreated with PAF-AH (2000 U in 300 μl carrier) or carrier alone. After administration, groups were subcutaneous in the PAF at the time points 15 minutes to 47 hours after PAF-AH administration. Edema was then assessed 1 hour after PAF antigen administration. As shown in FIG. 10, edema is shown as an average increase in volume (mL) SEM for each treatment group, with administration of 2000 U PAF-AH protected rats from PAF-induced edema for at least 24 hours. .
[258] I. Pharmacodynamics of PAF-AH
[259] Four rats were given a volume of 300 μl of 2000 U PAF-AH. Plasma was collected at various time points and stored at 4 ° C., and then plasma concentrations of PAF-AH were measured by ELISA using a dual mAb capture assay. Briefly, monoclonal antibody 90G11D (Example 13) was diluted to 100 ng / ml in 50 mM carbonate buffer pH 9.6 and transferred overnight at 4 ° C. on an Emulon 4 ELISA plate. After extensive washing with PBS containing 0.05% Tween 20, the plates were blocked with 0.5% fish skin gelatin (Sigma) diluted in PBS for 1 hour at room temperature. PBS SO-diluted serum samples with 15 mM CHAPS were added in duplicate to the washed ELISA plates and incubated for 1 hour at room temperature. After washing, biotin conjugate of monoclonal antibody 90F2D (seal) 13) was added to the wells at a concentration of 5 μg / ml diluted PBS SO and incubated for 1 hour at room temperature. After washing, 50 μl of a 1: 1000 dilution of extraavidin (Sigma) was added to the wells and incubated for 1 hour at room temperature. After washing, wells were developed and quantified using OPD as substrate. The enzyme activity was then calculated from the standard curve. In FIG. 11, data points mean mean SEM, indicating that the expected concentration based on 5-6 ml plasma volume for 180-200 gram rats at 1 hour plasma enzyme level was approached. Mean = 374 U / ml ± 58.2. It consistently decreased past 1 hour plasma levels and reached a mean plasma concentration of 19.3 U / ml ± 3.4 at 25 hours, which is significantly higher than the endogenous rat PAF-AH levels found at about 4 U / ml by enzyme assay. .
[260] J. Effect of PAF-AH vs. PAF Antagonist
[261] Rat groups (N = 4 / group) were pretreated with one of three potential anti-inflammatory agents: PAF antagonist CV3988 (Biomol # L-103) IP administration (2 mg in 200 μl EtOH), PAF antagonist eggs Prazolam (Sigma # A-8800) IP administration (2 mg in 200 μl EtOH) or PAF-AH (2000 U) IV administration. Control FOTs were injected IV with a 300 μl volume of carrier. PAF antagonists were administered IP because they are soluble in ethanol. Rats injected with CV3988 or alprazolam were further challenged with PAF 30 min after PAF antagonist to circulate the PAF antagonist, whereas PAF-AH and carrier-treated rats were additional antigen 15 min after enzyme administration. Administered. FOT injected with PAF-AH showed a decrease in PAF-induced edema past that given by the established PAF antagonist CV3988 and alprazolam. Edema is shown in FIG. 12 as the average increase in volume (±) SEM for each treatment group.
[262] In summary, rPAF-AH is effective in blocking edema mediated by PAF in vivo. Administration of the PAF-AH product may be administered locally or systemically by IV injection. In dose studies, IV injections in the range of 160-2000 U / rat have been found to dramatically reduce PAF mediated inflammation, while ID ₅₀ Tour doses are considered to be in the range of 40-8-U / rat. Calculations based on plasma volume for 180-200 gram rats predict that plasma concentrations in the range of 25-40 U / mL should block PAF-induced edema. This prediction is supported by primary pharmacodynamic studies. A dose of 2000 U of PAF-AH was found to be effective in blocking PAF mediated edema for at least 24 hours. At 24 hours after administration of PAF-AH, the plasma concentration of the enzyme was found to be about 25 U / mL. It has been found that PAF-AH blocks PAF-induced edema more effectively than two known tested PAF antagonists.
[263] Collectively, these results demonstrate that PAF-AH effectively blocks PAF-induced inflammation and will be of therapeutic value for diseases in which PAF is the primary mediator.
[264] Example 15
[265] The recombinant PAF-AH of the present invention was treated in a second in vivo model, PAF-induced pleurisy. PAF is already known to induce catheter leakage when it is introduced into the pleural horn [Henriques et al., Supra]. 300 μl recombinant PAF-AH (1500 μmol / ml / hour, prepared as described in Example 14) or an equal volume of control buffer with 200 μl of 1% Evans Blue dye in 0.9% of female fot (Charles River, 180- 200 g) into the tail vein. Fifteen minutes later, 100 μl of PAF (2.0 nmol) was injected into the rat pleura. One hour after paf booster administration, the pleural fluid was collected by sacrificing rats and rinsing the cavity with 3 ml heparinized phosphate buffered saline. The amount of conduit leakage was determined by measuring the amount of Evans Blue dye in the pleural cavity, quantified by measuring absorbance at 620 nm. Rats treated with PAF-AH were found to have much less catheter leak than control animals (more than 80% reduction in inflammation).
[266] The foregoing results support the treatment of subjects suffering from pleurisy with the recombinant PAF-AH enzyme of the present invention.
[267] Example 16
[268] The recombinant PAF-AH of the invention was also tested for efficacy in an antigen-induced eosinophilic enhancement model. Eosinophilic accumulation in the airways is a hallmark of a late immune response that causes asthma, rhinitis and eczema. BALB / c mice were injected intraperitoneally with injections consisting of 4 μg of 1 μg of ovalbumin (OVA) in aluminum hydroxide (Pierce Lavoratoris, Imek Alum, Rockford, Ill.) Twice at two week intervals. Sensitized (Charles River). At 14 days after the second immunization, sensitized mice were dosed with additional antigen with aerosolized OVA or saline as a control.
[269] The previous challenging rats were randomly placed in four groups with four mice / group. Mice in groups 1 and 3 consisted of 0.1% Tween80 and 25 mM Tris, 0.5 M NaCl, 1 mM EDTA given by intravenous injection. Pretreatment with 140 μl of control buffer. Mice in groups 2 and 4 were pretreated with 750 units of PAF-AH (5,500 units / ml activity in 140 μg of PAF-AH buffer). After 30 min administration of PAF-AH or buffer, mice in groups 1 and 2 are exposed to aerosolized PBS as described below, while mice in groups 3 and 4 are exposed to aerosolized OVA. After 24 hours, mice were treated twice with either 140 μl of buffer (groups 1 and 3) or 750 units of PAF-AH in 140 μl of buffer given by intravenous injection.
[270] Exposure of animals to aerosolized OVA is induced in rats whose organ coral infiltration is susceptible. Sensitive rats were placed in 50 ml conical centrifuge tubes (Corning) and aerosolized OVA (50 mg / ml) dissolved in 0.9% saline for 20 minutes using a nebulizer (Model 646, Devilvis, Somerset, PA). Ml) is forced to breathe. Controlled mice are treated in a similar manner except that 0.9% saline was used in the nebulizer. 48 minutes after exposure to aerosolized OVA or saline, mice are sacrificed and organs are excited. Organs from each group were stored at -70 ° until they entered the OCT and the parts were cut off.
[271] To assess the eosinophilic infiltration of organs, cell parts from four groups of mice were stained with either luna solution, hematoxylin-eosin solution or peroxidase. Twelve 6 μm thick sections were cut from each rat group and thus numbered. Even-numbered portions were colored with Luna colorant as follows. The portions were fixed in foam-alcohol for 5 minutes at room temperature and rinsed through three changes of tap water for 2 minutes at room temperature and then rinsed in two changes of dM ₂ O for 1 minute at room temperature. Cell sections were stained with luna colourant for 5 minutes at room temperature (luna colourant, predominantly composed of 90 ml Weigal's iron hematoxylin and 10 ml 1% bibrich scarlet). The colored slides were immersed six times in 1% acid alcohol, rinsed in tap water for 1 minute at room temperature, immersed five times in 0.5% lithium carbonate solution and rinsed in running tap water for 2 minutes at room temperature. Slides were dehydrogenated each minute through 70% -95% -100% ethanol at room temperature, then cleared in two changes of xylene and fixed in cytosyl 60 for 1 minute at room temperature.
[272] During peroxidant staining, even-numbered portions were fixed in 4 ° C. acetone for 10 minutes and allowed to dry air. 200 μl of DAB solution was added to each portion and allowed to sit for 5 minutes at room temperature. The slides were rinsed in tap water for 5 minutes at room temperature and two drops of 1% osmium acid were used in each section for 3-5 seconds. The slides were rinsed in tap water for 5 minutes at room temperature and counterstained with Myers hematoxylin at room temperature 25 ° C. Slides were rinsed in running tap water for 5 minutes and dehydrogenated through 70% -95% -100% ethanol for 1 minute each at room temperature. The slides were cleared for 1 minute each and fixed in cytosyl 60 through two changes of xylene at room temperature.
[273] Eosinophilic water was assessed in the tracheal mucosal cells. Rat organs from groups 1 and 2 were found to have very little eosinophils scattered through the mucosal cells. As expected, the organs of group 3 mice pretreated with buffer and exposed to nebulized OVA were found to have a lot of eosinophilic water through the mucosal cells. In contrast, the organs of rats in group 4 exposed to OVA pretreated and sprayed with PAF-AH were found to have very little eosinophility compared to those seen in the two controlled groups, groups 1 and 2.
[274] Thus, treatment of PAF-AH treatment was directed to tasks that reveal the final phase immune response associated with eosinophilic accumulation in gout, such as causing asthma and rhinitis.
[275] Example 17
[276] The PAF-AH product of the present invention has also been tested in two different rat models for treatment of enterocolitis (NEC), the intestinal sharp hemorrhagic necrosis that occurs in low birth weight children and causes severe morbidity and mortality. Previous experiments have demonstrated that glucocorticoid treatment reduces the extent of NEC in animals and immature children, and suggests that glucocorticoid activity is caused by an increase in plasma PAF-AH activity.
[277] A. With NEC induced by PAF challenge
[278] Activity in rats
[279] 1. The invention of NEC
[280] Recombinant PAF-AH product, rPH.2 (25,500 units in 0.3 ml, groups 2 and 4), or vehicle / buffer (25 mM Tris, 0.5 M NaCl, 1 mM EDTA and 0.1% Tween 80) weighed 180-220 grams Was administered intravenously to the Wistar female rats (n = 3). Either one of BSA (0.25%)-saline (groups 1 and 2) or PAF (0.2 μg / 100 gm) suspended in BSA saline (groups 3 and 4) is rPH.2 or previously Furukawa [J. Pediatr. Res. 34: 237-241 (1993)] were injected into the abdominal aorta for 15 minutes at the level of the superior mesenteric artery following the mediator injection. The small intestine was examined in its entirety after two hours of removal of the caecum from the Trietz ligament and washing with cooled saline. Samples were obtained by microscopic examination from high, medium and sharp proportions of the small intestine. Cells were fixed in buffered formalin and samples were subjected to microscopic examination by staining with hematoxylin and eosin. The experiment was repeated three times.
[281] The global findings indicated the intestines that usually appeared in groups treated with BSA saline mediators. Similarly, rPH.2 injected without PAF did not affect the overall findings. In contrast, the injection of PAF into the descending aorta rapidly caused severe discoloration and bleeding on the intestinal membrane surface. Similar bleedings were recorded when the small intestine was examined on the mucosal surface and the intestine revealed severe necrosis. rPH.2 was injected 15 min intravenously prior to administration of PAF into the aorta and the intestine appeared standard.
[282] Microscopic examination of the intestines from groups 1, 2 and 4 showed the standard chorionic structure and standard cell population in the diaphragm propria. In contrast, the group treated with PAF alone showed sufficient thickness of necrosis and bleeding through the entire mucosa. Plasma PAF-AH activity was also determined in the mice used in the experiments described above. PAF-AH activity was determined as follows. Blood samples were obtained after the previous intravenous terminal injection. Subsequently blood samples were obtained from the vena cava immediately before and at the time of sacrifice of the PAF. Approximately 50 μl of blood was collected in heparinized capillaries. Plasma was obtained by next centrifugation (980 × g 5 min). The enzyme was analyzed as previously described by Yasuda and Johnston (Endocrinology, 130: 708-716 (1992)).
[283] Plasma PAF-AH activity measurements of all mice injected previously were found to be 75.5 ± 2.5 units (1 unit equal to 1 n moles × min ⁻¹ × ml ⁻¹ plasma). Plasma PAF-AH activity measurements at 15 minutes following the mediation were 75.2 ± 2.6 units for Group 1 and 76.7 ± 3.5 units in Group 3. Plasma PAF-AH activity of animals injected with 25,500 units rPH.2 after 15 minutes was 2249 ± 341 units for Group 2 and 2494 ± 623 units for Group 4. The activities of Groups 2 and 4 remaining until the time of sacrifice (2 1/4 hours after rPH.2 injection) increased (Group 2 = 1771 ± 308; Group 4 = 1939 ± 478). These results indicate that plasma PAF-AH activity in rats injected with media only (groups 1 and 3) did not change during the course of the experiment. All animals that received only PAF injection developed NEC, whereas all mice injected with rPH.2 by PAF injection were fully protected.
[284] 2. Dose dependence of NEC invention
[285] To determine whether protection against NEC in mice was dose dependent, animals were treated with increasing doses of rPH.2 15 minutes before PAF projection. Initially, rPH.2 in the range of 25.5 to 25,500 units was projected to the end of the rat vein. PAF (0.4 μg in 0.2 mL of BSA-salin) was injected into the abdominal aorta for 15 minutes after rPH.2 projection in succession. The small intestine was removed and examined for NEC development 2 hours after PAF projection. Plasma PAF-AH activity was determined before exogenous projection of the enzyme after rPH.2 projection and at 15 minutes, 2 1/4 hours. The results are measurements of 2-5 animals in each group.
[286] Large findings indicate that all mice receive less than 2,000 units of the enzyme-development NEC. Plasma PAF-AH activity in animals receiving the least amount of protection of the enzyme (2040 units) was 363 units per ml of plasma after 15 minutes, indicating a fivefold increase over baseline. When rPH.2 was added less than 1,020 total units, the resulting plasma enzyme activity was approximately averaged 160 or less and all animals developed NEC.
[287] 3. Continued protection against NEC
[288] To determine the length of exogenous PAF-AH product time that provides protection against NEC development, mice were injected once with a fixed amount of enzyme through the vein end and challenged with PAF at various time points in succession. Only rPH.2 (8,500 units in 0.3 ml) or mediators were projected into the intravenous ends of rats and PAF (0.36 μg in 0.2 ml of BSA-saline) was injected into the abdominal aorta at various times after enzyme administration. The small intestine was removed 2 hours after PAF injection and review of histology for the whole to assess NEC development. Plasma PAF-AH activity was determined at various time points two hours after enzyme administration and PAF administration. Measurements ± standard error for enzyme activity were determined for each group.
[289] The results showed that no mice developed NEC within the first 8 hours after rPH.2 injection, but 100% of animals challenged with PAF developed NEC at the next 24 to 48 hours of enzyme injection.
[290] 4. Development of NEC
[291] To determine whether administration of the PAF-AH product could reverse the development of NEC induced by PAF injection, 25,500 units of enzyme were administered via a PAF dose (0.4 μg) injection for 2 minutes into the vena cava. None of the animals developed NEC. However, when rPH.2 was administered through this route for 15 minutes after PAF injection, all animals developed NEC as early as Furukawa [Supra] reported by the administration of PAF, speeding up the process of NEC development. I was.
[292] The sum of these observations indicates that a relatively small (five-fold) increase in plasma PAF-AH activity can inhibit NEC. These observations are based on fetal rabbits [Maki, et al., Proc. Natl. Acad. Sci. (US) 85: 728-732 (1988)] and immature children [Caplan, et al., J. Pediatr. 116: 908-964 (1990), which showed that plasma PAF-AH activity gave relatively low implications to low birthrate infants that prophylactic administration of human recombinant PAF-AH products might be useful in the treatment of NEC. Linked to report.
[293] B. Activity in the Neonatal Model of NEC
[294] The efficacy of the PAF-AH product, rPH.2, was evaluated in the NEC model of neonatal mice stressed by fluid food and choking, two routine risk factors for human disease. In this model, approximately 70-80% of animals developed microscopic intestinal wounds similar to the NECs of newborns and newborns three days old. Newly born mice were obtained from pregnant Sprague-Toray rats (Harlan Sprague-Toray, Indianopolis, IN), anesthetized with CO ₂ and delivered via incisions. New born animals were collected, dried and maintained in a neonatal incubator during the entire experiment.
[295] First, separate groups of animals were used to assess dose and absorption of rPH.2. Standard newborn pups were given either 0 enters rPH.2 or three different doses intraperitoneally (3, 15, or 75) at 0 hours and blood was given 1 hour for plasma PAF-AH activity assessment. Collected at 6 hours or 24 hours. PAF-AH activity was measured using an ELISA using substrate incubation studies (Gray et al., Nature, 374: 549 (1995)) and anti-human rPAF-AH monoclonal antibodies against each sample (90F2D and 90G11D). Immunohistochemical analysis was performed on selected samples using two different monoclonal antibodies that developed against human rPAF-AH (90F2D and 90G11D, described in Example 13). Immunohistochemistry is performed with standard techniques using 1: 100 elution of the antibody and overnight incubation.
[296] Plasma PAF-AH activity could not be measured at any time using either substrate incubation or ELISA techniques following the enteral dose of rPH.2 in standard newborn mice. With intraperitoneal administration of rPH.2, significant circulating PAF-AH activity could be measured using both methods for 1 hour after dosing, the highest at 6 hours. High doses of rPH.2 (3 to 75 humans, 10 to 250 U) resulted in high plasma PAF-AH activity. Immunohistochemical analysis revealed the presence of rPAF-AH product in epithelial cells of the intestinal mucosa following enteral administration. Reactivity was usually concentrated in intestinal villi with minimal pigmentation present in the crypts. There was more pigmentation in the ileum than in the plant, and some rPAF-AH products were immunochemically identified in the proportion of clones. There was no evidence of evidence in any controlled or recovered samples from animals taken through the abdominal passage. Thus, enteral administration of rPAF-AH products resulted in enzyme accumulation in local mucosal epithelium without absorption of any measurable system, whereas intraperitoneal administration of rPAF-AH products resulted in high circulating enzyme levels but accumulation of local mucosa. Was not.
[297] In the NEC model, NEC is capran et al. Pattol., 14: 1017-1028 (1994), induced in neonatal mice. Briefly, animals were fed a newborn liquid food reconstituted from powder (Esville, Volden) via a tube every three hours. The food volume initially started at 0.1 ml / feed and progressed to 0.4 ml / feed until the fourth day of the protocol. All animals were allowed to choke twice a day by breathing 100% nitrogen for 50 seconds in a closed plastic chamber exposed to cooling (4 ° C.) for 10 minutes. Intestinal and bladder function were stimulated with gentle manipulations after each feeding. Animals were maintained for 96 hours or until they showed signs of suffering. Pathological animals had bloating, bloody stool, difficulty breathing, cyanosis and lethargy and were euthanized through the head. After sacrifice, the intestines of each rat were examined thoroughly for signs of necrosis and then formalin fixed for later histological analysis. Specimens were examined in a fashion containing paraffin, cut into microtomes, stained with hematoxylin and eosin and obscured by both observers. Intestinal wounds were rated as 1+ for epithelial cell lift or separation, 2+ for bleeding from epithelial cells to intermediate villi levels, 3+ for complete villi and 4+ for full layer necrosis.
[298] To assess the efficacy of rPH.2, three different groups of mice were treated with the compound via enteral delivery, intraperitoneal delivery, or both. Preparation of rPH.2 has a 4000 unit / mg PAF-AH activity approximating 0.8 mg / ml protein, with a <0.5 EU / mg endotoxin / protein ratio. Intradermal animals were given 25 humans (80 U) of rPH.2 through the ortho gastric tubes eluted with each meal (every 3 hours). Animals taken intraperitoneally were given 75 individuals by intraperitoneal injection twice a day. Controlled groups received an adequate volume of buffer (20 mM NaPO ₄ , pH 7.4) without rPH.2 and studied with each experimental group simultaneously. Death and NEC indications were assessed for each treatment group and differences were statistically analyzed using Fischer's exact test. P values less than 0.05 were considered important. The results are shown in Table 9 below.
[299] NECDead Control (intraperitoneal administration)7/108/10 rPH.2 (240 U abdominal twice a day)6/118/11 Control (intestinal administration)19/2621/26 rPH.2 (80 U enter every 3 hours)6/267/26 Control (abdominal + enteral administration)10/1712/17 rPH.2 (240 U abdominal twice a day and every 3 hours with 80 U enteral)3/147/14
[300] The data show the cumulative results from four different experiments for abdominal cavity, four experiments for cultivation, and three experiments for abdominal + enteral administration.
[301] Entering rPH.2 significantly induced the effects of both NEC and death compared to control animals. Results from four different enteral trials showed that pretreatment of rPH.2 reduced NEC from 19/26 (control) to 6/26 (P <0.001). Intestinal wounds varied between treated and controlled animals, but in most cases they were characterized by mesophilic necrosis in some fragments and total chorionic necrosis in other regions, areas where full necrosis occurs, and remaining portions of the standard intestinal structure. It was made. The worst degree of NEC was similar in treated and control animals with intestinal wounds (median score 2.8 in control vs. 2.4, P> 0.05 in rPH.2 treated rats).
[302] Intraperitoneal administration of rPH.2 had no significant effect on NEC or death in this model. The onset of symptoms was similar between this group and the control group (40 ± 5 hours in control vs. 36 ± 7 hours in rPH.2-treated rats) and the degree of NEC in the two groups was similar (2.6 vs midpoint in control). 2.5) in rPH.2-treated mice.
[303] Additional experiments were performed in rats taken in the intestinal and intraperitoneal groups with the same dose of rPH.2 as a group of treatment conditions (25 persons rPH.2 each feeding 3 hours each, 75 persons by intraperitoneal injection twice daily). Was done. The results are shown in Table 9 above. Although there was no significant difference in death coverage between the treated and control groups, rPH.2 treatment significantly reduced the NEC's coverage (10/17 in the control versus 3/14 in the rPH.2 treated mice). P = 0.04). In the records, 6 of 7 animals who died in the rPH.2-treated group had positive blood cultures for E. Coli obtained immediately before death.
[304] These results further support the protective role of PAF-AH products in the neoplastic model of non-PAF-induced NEC. Enteral treatment of the rPAF-AH product prevented NEC, whereas intraperitoneal treatment at these doses had no proven effect. These findings suggest that supplementation of PAF-AH products to immature newborns in liquid food may reduce the range of impact of the disease at risk for NEC.
[305] Example 18
[306] The efficacy of PAF-AH products was investigated in Guinea pigs with acute respiratory distress (ARDS).
[307] Intravenous platelet-activated factor (PAF) injection into Guinea pigs causes deep lung inflammation recall of early ARDS in humans. Within minutes of intravenous administration of PAF, pulmonary parenchyma congestes into a compressed bronchus and bronchiole [Reloch-Tubbana et al, supra]. Platelets and polymorphonuclear neutrophils assemble at the margins and cell aggregation is easily identified along the small arteries of the lungs [Reloch-Tubbana, Br. J. Exp Path., 66: 345-355 (1985). PAF injection also destroys bronchial epithelial cells isolated from the airways and accumulates in the airway lumen. This damage to airway epithelial cells is consistent with the formation of hyaline membranes in humans during the development of ARDS. Gathering to the edges of neutrophils and platelets occurs quickly by the leakage of these cells into the alveolar septum and the alveolar spaces of the lungs. Cellular infiltration induced by PAF was associated with organ edema [Kirch, Exp. Lung Res., 18: 447-459 (1992). In guinea pig lungs with disturbed PAF [Basran, Br. J. Pharmacol., 77: 437 (1982)] Evidence of edema when inducing dose-dependent (10-1000 ng / ml) extravasation of ¹²⁵ I labeled fibrinogen is further supported by in vitro studies. Lose.
[308] Based on this observation, an ARDS model was developed in Guinea pigs. The tubing was placed into the jugular vein of anesthetized male Hartley Guinea pig (approximately 350-400 grams) and eluted with a 500 μl volume of phosphate buffered saline with 0.25% bobbin serum albumin as carrier (PBS-BSA). Infusion over 15 minutes at a total dose in the range of -400 ng / kg. At various intervals in the next PAF injection animals are sacrificed and lung cells are collected. In PAF-injected Guinea pigs, doses dependent on lung injury and inflammation are clarified by 15 minutes and continue to be present at 60 minutes. Neutrophils and red blood cells are present in alveolar spaces of PAF-treated Guinea pigs, but not in animals injected with control or falsehood. Evidence of epithelial cell damage is also evident and suggests hyaline membrane formation in human ARDS patients. Protein crystals made on bronchoalveolar lavage (BAL) samples from PAF-injected Guinea pigs show dramatic accumulation of protein in the inflamed lung and clear evidence of catheter leakage.
[309] RPH.2 was found to be fully protected against PAF mediated lung injury in the Guinea pig model of ARDS. Guinea pig groups are pretreated with either rPH.2 (2000 units in 500 μl) or 500 μl of PAF-AH buffer alone. After 15 minutes these Guinea pigs were injected over 15 minutes with 400 ng / kg PAF in a 500 μl volume. In addition, the false group of Guinea pigs was injected with 500 μl of PBS-BSA. At full PAF infusion, BAL fluids were collected by washing the lung 2X with 10 ml saline containing 2 μ / ml heparin, which sacrificed and prevented coagulation. To determine protein concentration in BAL, samples were eluted at 1:10 in saline and OD 280 was determined. BAL fluid from false Guinea pigs was found to have a protein concentration of 2.10 ± 1.3 mg / ml. Sharply comparing the BAL body fluids of the PAF injected animals was found to have a protein concentration of 12.55 ± 1.65 mg / ml. In guinea pigs pretreated with rPH.2, BAL fluid had a protein concentration of 1.13 ± 0.25 mg / ml, demonstrating that PAF-AH products completely prevent pulmonary edema in response to PAF, comparable to false control. It turned out.
[310] Example 19
[311] The efficacy of rPH.2, a PAF-AH product, was evaluated in two different models of acute pancreatitis.
[312] A. Activity in the Rat Pancreatitis Model
[313] Male wistar rats (200-250 g) were followed from Charles River Laboratories (Wilmington, Mass.). They were allowed to live in a 12 hour day / night cycle in a 23 ± 2 ° C. controlled room climate and were given standard laboratory food with any water. Animals were randomly assigned to control or experimental groups. Mice were anesthetized with pentobarbital sodium 50 mg / kg intraperitoneally and polyvinyl catheter (size V3, Biolab product, Lake Havath, AZ) was placed by division into the jugular vein. The catheter was drilled subcutaneously to get out of the cervical region and allowed the animals to recover from anesthesia. The mice let the mole eat, but fasted all night. The next day the experiments were conducted on conscious animals. Catheter opening was maintained by constant saline (0.2 mL / h) injection for a while. On the day of the experiment animals were injected intravenously with rPH.2 or mediator control and either of the following injections were followed. (1) 5 μg / kg per cell of caerulein for 3.5 hours (2) 10 μg / kg per hour of cellulite for 5 hours (Research Plus, Bayonne, NJ) Immediately after complete infusion, the animals were pentobarbital Anesthetize with sodium and open the stomach to draw 5 ml blood from the lower vena cava for serial analysis. Then they were sacrificed by blood death. Serum amylase, serum lipase and serum bilirubin were measured and pancreas obtained. Pancreatic fragments are fixed with 4% phosphate buffered with formaldehyde solution for structural examination or immediately frozen deep at -80 ° C for myeloperoxidase activity measurements. Additional fragments of the pancreas were assessed for pancreatic water content and pancreatic amylase and trypsin as follows. Myeloperoxidase activity, a measure of neutral leukocyte clearance, was evaluated in the pancreas and lung as follows. Lung catheter permeability was also evaluated as follows. Statistical analysis of the data was performed using the untested t test. Reported data represent + S.E.M means of at least three different experiments. The difference in results was considered significant when P <0.05.
[314] 1. Water content of the pancreas
[315] The pancreatic pieces were sucked, dried and weighed (wet weight), then dried at 120 ° C. for 34 hours and then weighed again. The water content of the pancreas was calculated as the difference between wet and dry weight and expressed as a percentage of the wet weight of the pancreas. Rising water content of the pancreas was considered to indicate the development of edema.
[316] 2. Amylase of Serum and Pancreas
[317] Amylase activity in serum was determined by Pierre et al., Clin. Chem., 22: 4, 6-ethylidene (G ₇ ) -P-nitrophenyl (G ₁ ) -α ₁ D-maltoplasmid (ET-G ₇ PNP) (Sigma) as substrate by 1219 (1976) Chemical company, St. Louis, MO). Amylase activity in homogenized pancreatic cells in 10 mM phosphate buffer, pH 7.4, was also measured using the same method.
[318] 3. Trypsin of the pancreas
[319] Pancreatic activity was measured fluorescently using Boc-Gin-Ala-Arg-MCA as substrate. Briefly 2.7 ml of 50 mM Tris-buffer (pH 8.0) containing 200 μl sample and 150 mM NaCl, 1 mM CaCl ₂ and 0.1% bobbin serum albumin were mixed in a cuvette. After 20 seconds of preincubation to start the reaction, 100 μl of substrate was added to the sample. Reading out fluorescence (emission 380 nm, emission 440 nm) was performed and represented by the slope. Some trypsin activity was expressed as a percentage of total trypsin activity to allow for a lack of data from other experiments.
[320] 4. Organizational Structure and Morphology Measurement
[321] Under light microscopy, complete random cross-sections of the head, body and extremities of the pancreas were stained with 10% neutral phosphate-buffered formalin. Paraffin-filled 5 μm sections were stained with hematoxylin-eosin (H & E) and blinded by experienced morphologists. Acinar cell injury / necrosis is defined as either (a) the presence of acinar welcome cells, (b) the fear and bloating of the acinar cells and the destruction of the tissue structure of all or part of the acinar gland, both of which must be separated by an inflammatory response. do. The amount of acinar wounds / necrosis and the total area occupied by the acinar tissue are each computed image analysis video units (models CCD-72, Dage-MT1, Michigan, IN) installed with NIH-1200 image analysis software. It was quantified by morphology using. Ten randomly selected microscopic fields (125X) were examined for each tissue sample. The degree of acinar cell injury / necrosis was expressed as the percentage of total acinar tissue filled by the area meeting the criteria for injury / necrosis.
[322] 5. Determination of Pancreatic and Pulmonary Myeloperoxidase (MPO) Activity
[323] Neutrophil leukocyte replenishment in the pancreas and lung was assessed by measurement of tissue myeloperoxidase activity. Tissue samples were obtained at the time the sacrifice was stored at −70 ° C. by the time of the assay. Samples (50 mg) were fused and homogenized and centrifuged in 1 mL of 20 mM phosphate buffer (pH 7.4) (10,000 xg, 10 min 4 ° C.). The resulting pellet was 0.5% hexadecyltrimethylammonium bromide (Sigma, St. Lewis). , MO) was resuspended in 50 mM phosphate buffer (pH 6.0) and allowed to cycle four times of cooling-thawing. The suspension was triturated by sonication for 40 seconds and centrifuged (10,000 × g, 5 min, 4 ° C.). The reaction mixture, consisting of 1.6 mM tetramethylbenzidine (Sigma Chemical, St. Lewis, MO), 80 mM sodium phosphate buffer (pH5.4) and extracted enzyme of 0.3 mM hydrogen peroxide, was incubated at 37 ° C. for 110 seconds and the absorption was 655 nm. Was measured with a CobasBio automated analyzer at. This absorption rate was corrected for the dry weight of the debris of the tissue sample.
[324] 6. Measurement of catheter permeability of the lung
[325] Obstruction of the general biliary pancreatic duct also causes severe pancreatitis, which is typically associated with pulmonary canal permeability and wounds in the lung that can be measured by histological examination.
[326] An intravenous bolus injection of 5 mg / kg fluorescent isocyanate albumin (FITC-albumin, Sigma Chemical, St. Lewis, Mo.) was performed 2 hours before the animal died. Lung microtubule permeability was assessed by measuring the leakage of FITC-albumin from the catheter appendage to the bronchioles. Simply after the sacrifice, the right bronchus is blocked using a clamp and the airway is exposed. The right lung was subsequently washed using a cannula injected into the airways. Three washes of saline (60 ml wash) were lacking and FITC fluorescence in serum and washes was measured at luminescence 494 nm and emission 520 nm. The fluorescence ratio of the wash solution into the blood was calculated and the microtubule permeability measured in the lung. Lungs were also stained with H & E and examined histologically.
[327] 7. Effects of Serulein and rPH.2 Administration
[328] Injection of only 5 μg / kg / h of cerulein for 3.5 h was characterized by hyperamylaseemia, pancreatic edema measured by pancreatic content, and histostructural changes including marked acinar cell phobia and pancreatic edema. Typical mild secretariat-induced pancreatitis in. Saline infusions from control animals did not cause these biochemical or histological changes. Intravenous administration of rPH.2 at a 5, 10, or 20 mg / kg dose for 30 minutes prior to commencement of cerulein infusion of pancreatic edema (water content) and the magnitude of changes in tissue structure induced by infusion of cerulein alone Did not change significantly. Administration of rPH.2 also did not affect the cerulein-induced activity of the trypsinogen or amylase content of the pancreas.
[329] Infusion of high doses of 10 μg / kg / h of cerulein to rats for 5 hours resulted in a notable increase in trypsinogen and amylase activity in the pancreas, more significant increase in serum amylase activity and pancreatic edema and pancreatic MPO. More severe pancreatitis occurred, characterized by proportionality to the controls by a significant increase in activity. Pancreatic tissue structure indicated several combustible necrotic and some true swelling cells as well as pancreatic edema and acinar cell fear.
[330] Administration of rPH.2 (5 or 10 mg / kg, intravenously) 30 minutes prior to initiation of cerulein (10 μg / kg / h) improved the magnitude of many pancreatic changes induced by infusion of cerulein alone. The results are shown in Table 10. RPH.2 treatment at 5 mg / kg dose resulted in a decrease in serum amylase activity (from 10984 ± 1412 to 6763 ± 1256). High doses of rPH.2 of 10 mg / kg did not result in improvement of hyperamylaseemia. Treatment of 5 or 10 mg / kg rPH.2 also resulted in a slight decrease in cerulein-induced development of pancreatic edema measured by water content (90.61 ± 0.27 versus cerulein + 5 mg / for cerulein only). 88.21 for kg rPH.2). A 5 mg / kg dose of rPH.2 provided a notable improvement in pancreatic MPO activity (2.92 ± 0.32 fold increase over control of cerulein only versus 1.19 for cerulein with rPH.2, P <0.05 + 0.21). High doses of rPH.2 did not result in improvement of MPO activity. The use of rPH.2 did not significantly change both the trypsinogen activity or the pancreatic amylase content. Pancreatic tissue structure showed some improvement in microscopic necrosis and infiltration after rPH.2 pretreatment.
[331] Pancreatitis associated with lung injury has been observed both clinically and in severe models of pancreatitis. It did not cause mesoplasia of pancreatitis for 3.5 hours. However, 10 μg / kg / h of cerulein infusion, which causes more severe pancreatitis for 5 hours, also increased pulmonary catheter permeability (0.31 ± 0.04 to 0.79 ± 0.09), pulmonary MPO activity (pointing to neutral leukocyte satisfaction) and tissue Lung wounds measured by neutrophil infiltration in structural investigations were caused.
[332] Administration of rPH.2 at a dose of 5 mg / kg for 30 minutes prior to infusion of cerulein improved the elevation of MPO activity in lungs induced by infusion of cerulein alone (3.55 ± 0.93 versus rPH.2 for cerulein alone). 1.51 ± 0.26 for cerulein with). rPH.2 treatment significantly reduced the severity of microscopic changes observed in the lungs after infusion of cerulein. The cerulein-induced increase in pulmonary catheter permeability was not statistically significant but was reduced by rPH.2 treatment. Higher doses of 10 mg / kg of rPH.2 were not more effective than lower doses in reducing the severity of cerulein-induced lung injury.
[333] Control (no CER)Cerulean (CER) 10ug / kg / hCER + 5 mg / kgrPH.2CER + 10 mg / kgrPH.2 Serum Amylase (U / I)961 ± 17410984 ± 14126763 ± 12568576 ± 1024 Pancreatic water content (% wet weight)72.71 ± 06490.61 ± 0.2788.21 ± 0.6189.00 ± 0.94 Pancreatic MPO (fold increase over control)1.02.92 ± 0.321.19 ± 0.211.42 ± 0.19 Pancreatic trypsin activity (1000 x gradient ug DNA)0.12 ± 0.069.70 ± 2.508.33 ± 1.759.15 ± 1.28 Pancreatic amylase content (U / ug DNA)0.28 ± 0.060.42 ± 0.070.45 ± 0.040.46 ± 0.044 Lung catheter wash serum (%)0.31 ± 0.040.79 ± 0.090.70 ± 0.090.70 ± 0.07 Pulmonary MPO (increase in fold over control)1.03.55 ± 0.931.51 ± 0.261.64 ± 0.22
[334] B. Activity of the Opossum Pancreatitis Model
[335] Randomly caught, healthy American opossums (Didepis bulginia) were obtained from Scott-Maas and lived at 23 ± 2 ° C controlled atmosphere with a 12-hour day / night cycle, regardless of gender (2.0 kg to 4.0 kg). And was given standard laboratory food with water arbitrarily. After fasting overnight the animals were anesthetized with 50 mg / kg sodium-pentobabital i.p (Beterinary Laboratories, Renex, KS). Celiotomy was performed via midline cleavage under sterile conditions and usually the sugar pancreatic duct was trapped in all animals to induce severe necrotic pancreatitis. Animals were randomly assigned to control or experimental groups. On the second day after ligation of the pancreatic duct, the experimental group received 5 mg / kg body weight rPH.2 (supplied in 4 mg / ml eluent) per day via the intravenous end, whereas the control group received only the same amount of placebo mediation. Received an intravenous injection. One and two days after treatment (3 and 4 days after ligation of the pancreatic duct) animals were euthanized by sodium-pentobabital overdose. Blood samples were drawn from the chest for measurement of serum amylase, serum lipase and serum bilirubin, and pancreas was obtained. Pancreatic fragments are fixed in 4% phosphate buffered formaldehyde eluate for histological examination or frozen deep at -80 ° C just for the determination of myeloperoxidase activity. Additional fragments of the pancreas were evaluated for pancreatic water content and pancreatic amylase, as described in section A of this sample. Mailo peroxidase, a measure of neutrophil prosthesis, was evaluated in the pancreas as described above. Lung catheter permeability was also evaluated as above.
[336] Reported results are presented as standard error (SEM) measurements of measurements obtained from three or more separate experiments. The significance of the change was assessed by using the t-test of students when the data consisted of only two groups or by analysis of diversity (ANOVA) when comparing three or more groups. If ANOVA represents a significant difference, the data was analyzed using Tukey's method as a post hoc test for differences between groups. P values less than 0.05 are considered to indicate significant differences.
[337] The results are shown in Table 11. Normal obstruction of the Billy pancreatic duct caused severe necrotic pancreatitis characterized by hyperamylaseemia, hyperlipemia and widespread necrosis of the pancreas. Moreover, normal biliary pancreatic obstruction was associated with a marked increase in serum bilirubin levels. Intravenous administration of rPH.2 two days after pancreatic duct ligation improved the magnitude of many changes in the pancreas induced by vascular obstruction and placebo treatment alone. Although the difference is not numerically significant, the first day of rPH.2 treatment decreased serum amylase levels compared to placebo-treated animals, and the second day of rPH.2 treatment (day 4 post ligation of the pancreatic duct) compared to placebo. Decreased serum amylase levels. Although the difference is not numerically significant, one or second days of rPH.2 treatment decreased serum lipase levels in proportion to control. The first day of treatment resulted in an increase in pancreatic amylase, but the second day of rPH.2 treatment decreased pancreatic amylase content compared to control. Treatment of rPH.2 was not observed to affect serum bilirubin levels, pancreatic myeloperoxidase activity or pancreatic water content.
[338] Major characteristic histological changes induced by the occlusion of the Billy Pancreatic duct include significant necrosis, infiltration of inflammatory cells, phagocytosis of cells, and marked expansion of acinar alumina.
[339] Morphological examination of the pancreas for acinar cell wounds showed a major protective effect of rPH.2 in the pancreas one and two days after rPH.2 treatment. After one day of rPH.2 treatment, acupuncture wounds reduced about 23% of the total abundance compared to 48% wounds in placebo treated animals. This reduction in acinar cell wounds was more pronounced after the second day of treatment. This is true when rPH.2 treatment causes about 35% wounds of total acinar tissue compared to about 60% wounds in placebo treated animals.
[340] Pulmonary catheter permeability measured by FITC infusion showed a very high difference one day and two days after rPH.2 treatment compared to the placebo group. Histologic examination of the lung showed severe lung injury in all placebo treated animals. Pulmonary wounds are characterized by extensive inflammation with tissue gaps and alveolar infiltration of major macrophages, lymphocytes and neutrophils, and are characterized by thickening of the flammable alveolar membranes, with the exception of significant gap edema. Administration of rPH.2 resulted in a significant decrease in inflammatory cell infiltration and a decrease in gap edema at all times. In summary, these results indicate that administration of rPH.2 at a dose of 5 mg / kg / day at the beginning of 48 hours after intravenous pancreatic ligation results in an increase in amylase blood levels and a significant decrease in the severity of pancreatitis-induced lung injury. It caused a high improvement. Administration of rPAF-AH product in clinically revealed pancreatic models has shown a beneficial effect in reducing pancreatic severity.
[341] A direct correlation between pAF-AH activity and enzyme levels was observed. The lack of activity in the patient's serum reflects the absence of detectable enzymes. Similarly, plasma samples with half of normal activity contained half of normal levels of PAF-AH. These results suggest that pAF-AH activity deficiency is due to inactive enzymes that the monoclonal antibodies do not recognize or due to the inability to synthesize enzymes.
[342] Subsequent experiments revealed that the deficiency was due to genetic lesions in human plasma PAF-AH gene. Genomic DNA lacking individual PAF-AH was isolated and used as a template for PCR reaction with PAF-AH gene specific primers. Each coating sequence exon was initially amplified and sequenced from each. A single nucleotide substitution to exon 9 was observed (G → T at position 996 of SEQ ID NO: 7). The nucleotide substitutions resulted in the amino acid substitution of valine with phenylalanine at position 279 of the PAF-AH sequence (V279F). Exon 9 was amplified in genomic DNA from additional 11 PAF-AH deficient patients, which appeared to have the same point mutation.
[343] To test whether the mutations render the enzyme incapable of producing an E. coli expression construct carrying the mutation, the production method is similar to the technique and method in Example 10. When introduced into E. coli, the expression constructs lacked PAF-AH activity, while the control constructs without mutations were completely active. This amino acid substitution will result in structural variations that lack activity and lack immunoreactivity with the PAF-AH antibody of the invention.
[344] PAF-AH specific antibodies of the present invention can be used in diagnostic methods to detect abnormal levels of PAF-AH (the qualitative level is about 1 to 5 U / ml) in serum and to treat pathological symptoms with PAF-AH. Moreover, genetic screening for PAF-AH deficiency described in Japanese patents has been possible by identifying genetic lesions in the PAF-AH gene. Mutations resulted in restriction endonuclease sites (Mae II) and provide a simple method for analyzing restriction fragment length polymorphism (RELP) that distinguishes active alleles from mutant alleles.
[345] One day after treatment Two days after treatment (sacrifice on day 3) (sacrifice on day 4) rPH. 2 5 mg / kg rPH. 2 Placebo Placebo 5 mg / kg Serum Bilirubin 5.46 ± 0.96 7.10 ± 0.60 6.54 ± 0.55 7.10 ± 0.60 (ml / dl) Serum Amylase 5618 ± 899 4288 ± 675 6538 ± 1366 3106 ± 467 * (U / I) Serum Lipase 2226 ± 554 1241 ± 263 1424 ± 257 1023 ± 295 (U / I) Pancreatic water content (%) 81.10 ± 0.56 81.52 ± 0.79 80.05 ± 1.07 79.32 ± 0.49 Inserted MPO (OD / Fragment 1345 ± 286 1142 ± 83 1149 ± 232 1033 ± 130 dry weight) pancreatic amylase 706 ± 92 1101 ± 105 950 ± 85 712 ± 131 (U / ug DNA) lung conduit permeability 0.76 ± 0.09 0.21 ± 0.04 ** 0.57 ± 0.13 0.23 ± 0.04 * FITC wash serum) Wounds (total 48% 23% 60% 35%% of acinar cells) * P = 0.02 vs. Placebo ** P 〈0.001 vs. Placebo
[346] Example 20
[347] A study was conducted to evaluate the effect of the PAF-AH product, rPH.2, on neurotoxicity associated with HIV infection. Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system results in neuronal loss due to cell death. HIV-1-infected monocytes activated by various antigenic stimuli, including contact with neurons, secrete high levels of neurotoxic pro-inflammatory cytokines including PAF. The effect of rPH.2 on the neurotoxicity of conditioned media from HIV-infected and activated monocytes was assayed.
[348] Monocytes were infected with HIV and activated as follows. After leukopheresis, monocytes were recovered from peripheral bone marrow cells (PBMCs) of HIV- and hepatitis B-serum-reactive negative donors and were treated with Genis et al. Med. 176: 1703-1718 (1992) purified by reverse centrifugation elution (798%). Cells were cultured as a cohesive monolayer (T-75 culture flask 1 × 10 ⁴ cells / ml) in DMEM with recombinant human macrophage colony stimulator (MSCF) (Genetics Institute, Cambridge, Mass.). Under these conditions, monocytes were cultured with macrophages. Under these conditions, monocytes cultured macrophages. Under this zone, monocytes differentiate into macrophages. 7-10 days after culture, macrophages were exposed to HIV-1 _ADA (Accession No. M60472) at 0.01 infectious virion / target cell multiplicity of infection (MOI). Under these conditions, 20-50% of monocytes were infected 7 days after HIV-1 inoculation as determined by immunofluorescence and in situ hybridization techniques [kalter et al., J. Immunol., 146: 298-306 (1991). )]. Every 2 to 3 days fresh medium was fed to all mediums. Five to seven days after HIV-1 infection and kalter et al., In the peak of reverse transcriptase activity measured in the literature (107 cpm / ml), parallel cultures of HIV-1-infected medium and uninfected monocytes were LPS (10 ng). / Ml) or vehicle for 30 min at 37 ° C and then frozen at -80 ° C until use in neurotoxicity assays.
[349] Human cerebral cortical neuronal cell cultures were established as follows. According to a modified procedure of Banker and Cowan, Brain Res., 126: 397-425 C1977), human EH brain tissue was obtained from the cerebral brain of human fetal brain tissue aged 3 months (13-16 weeks gestation). Briefly, brain tissues were collected, washed with 30 ml of cold Hanks BSS (containing Ca + 2 and Mg + 2 + 25 mM HEPES and 5 X gentamycin) and then cut from the sticky meninges and blood and cut into 2 mm pieces. The tissue was forced through a 230 μm Nitex bag and titrated 10-15 times slowly with a fire polished Pasteur pipette. The tissue was centrifuged at 550 rpm, 5 min, 4 ° C. and the pellets were treated with N1 components (insulin, 5 mg / l; transferrin, 5 mg / l; selenite, 5 μg / l, progesterone 20 nM; putrescine, 100 μm) as well as 10% fetal calf serum (FCS), PSN antibiotic mixture (Tenicillin, 50 mg / l; Streptomycin, 50 mg / 1; Neomycin, 100 mg / l) and Punjone (2.5 mg / l) ) Was resuspended in 5-10 ml of MEM-Hip (D-glucose, 5 grams / liter; L-glutamine, 2 mM; HEPES, 10 mM; Na pyruvate, 1 mM; KCI, 20 mM). . Cell counts and visibility were determined by diluting Hanks BSS with 0.4% xmflqvks blue (1: 1 V / V) and counting with a hemocytometer. 10 ⁵ cells per 12 mm of glass coverslip pre-coated with poly-L-lysine (Sigma, St. Louis, Missouri, 70K-150K MW) placed in a 24-well culture dish 45-fold cells with a 10 ml pipette The density of 1 ml of medium was pipetted into each culture well. Cells were incubated at 37 ° C. for 10-28 days with medium replacement every 3 days in a humid atmosphere of 5% CO ₂ /95% air. Under these conditions, the cultures were> 60-70% homologous to neurons, had 20-30% astrocytes, and <1% microglia and ˜10% macrophages and microglia staining. After 14-28 days of culture, neuronal cultures express sufficient levels of N-methyl-D-aspartate (NMDA) or non-NMDA receptors and are NMDA or alpha-amino-3-hydroxy-5-methyl-4 bivalent It dies after excitatory administration of Zazole Proprionic Acid (AMPA).
[350] Neurotoxicity assays were performed as follows. Test samples were (a) conditioned media from LPS-stimulated HIV-1 infected monocytes, (b) control media, (c) conditioned media in which rPH.2 was added at 51 μg / ml or (d) rPH.2 Conditioned media with additional mediators were used for neurons at 1: 10 V / V concentration for 24 hours. Terminal deoxynucleotidyl transferase (TdT) used to bind digooxygenin-dUPT to the free 3'-OH terminus of 4% paraformaldehyde fixed neuron coverslip and freshly cleaved DNA (TUNEL staining) Neurotoxicity was measured by identifying apoptotic nuclei using a commercial kit (Apop Tag; ONCOR, Gaitherburg, MD). TUNEL-Stained Neurons Digitized Images in Randomly Selected ≥ 15 in Microscopy Using Computer Morphometrics (MCID, Image Survey, St. Catherine, Ontario, Canada) The total number of turbines was analyzed. Data is expressed in% neuronal nuclei positive for TUNEL staining ± SEM and is shown in Figure 13. Tests of numerical significance between control and experimental treatment were determined by paired t-tests of significance at ANOVA or P ≦ 0.05. Measurements of these cultures identifying conditioned media from HIV-infected and activated monocytes induced neuronal death in nearly 25% of the total population of cortical neurons and rPH.2 showed less than 5% toxicity of total neurons. Could be reduced. RPH.2 by itself was not neurotoxic when 50 μg / ml rPH.2 did not affect neuronal cell death in proportion to cultures treated with control medium. These results clearly indicate that the major neurotoxic component induced by the application of regulatory media from activated HIV-1 infected monocytes must be due to PAF. Since neurotoxicity can be almost completely eliminated by co-culture with PAF-AH products, this enzyme causes metabolism of PAF in the central nervous system. These findings suggest potential therapeutic interventions in the treatment of CNC neuropathy associated with HIV-1 infection.
[351] Example 21
[352] Nearly 4% of the Japanese population has low or nearly undetectable levels of PAF-AH activity in their plasma. This deficiency correlates with severe respiratory symptoms in asthmatic children who are believed to have a genetic deficiency in the autosomal recessive method [Miwa et al., J. Clin. Invest., 82: 1983-1991 (1988).
[353] To determine if the deficiency is due to the presence of enzymes but due to inactivity or the inability to synthesize PAF-AH, plasma from a number of patients deficient in PAF-AH activity was monoclonal by sandwich ELISA as follows: Antibodies 90G11D and 90F2D (Example 13) were used to assay for PAF-AH activity (as in the method described in Example 10 for transfectants) and the presence of PAF-AH. Four flat bottomed emulon plates (Dynatec, Chantilly, Va.) Were coated with 100 ng / well monoclonal antibody 90G11D and stored overnight. The plate was blocked for 1 hour at room temperature with 0.5% fish skin gelatin (Sigma) diluted in CMF-PBS and washed three times. 50 μl of 5 μg / ml monoclonal antibody 90F2D diluted in PBST biotinylated by standard method was added to each well, the plate was incubated for 1 hour at room temperature and washed three times. 50 μl of 1/1000 diluted extraavidin (Sigma) in CMF-PBST was then added to each well and the plate was incubated for 1 hour at room temperature before development.
[354] A direct relationship between PAF-AH activity and enzyme levels was observed. The lack of activity in the serum of the patient was reflected by the absence of detectable enzymes. Similarly, plasma samples with half of normal activity contained half of normal levels of PAF-AH. This observation suggests that the lack of PAF-AH activity is due to the inability to synthesize enzymes or to inactivating enzymes that do not recognize monoclonal antibodies.
[355] Other experiments have shown that the deficiency is due to genetic lesions of the human plasma PAF-AH gene. Genomic DNA was isolated from PAF-AH deficient individuals and used as a template for PCR reactions with PAF-AH gene specific primers. Each coding sequence exon was initially amplified and sequenced from one individual. A single nucleotide change was observed within exon 9 (with G instead of T at position 996 of SEQ ID NO: 7). The nucleotide change results in an amino acid substitution of valine with phenylalanine at position 279 of the PAF-AH sequence (V279F). Exon 9 was amplified from genomic DNA from an additional 11 PAF-AH deficient individuals found to have the same point mutation.
[356] To test whether this mutation renders the enzyme incapable, E. coli expression constructs containing the mutation were generated in a manner similar to the method described in Example 10. When introduced into E. coli, the expression construct lacked PAF-AH activity while the control construct lacking the mutation was sufficiently active. This amino acid substitution consequently represents a structural modification and results in a lack of immunoreactivity with the PAF-AH antibody of the invention and a lack of observed activity.
[357] Therefore, the PAF-AH specific antibody of the present invention can be used in a diagnostic method for detecting abnormal levels of serum PAF-AH (normal level is about 1-5 μ / ml) and progressing the treatment of pathological symptoms with PAF-AH. Can be. Moreover, the identification of the genetic lesions of the PAF-AH gene enables the genetic screening for PAF-AH deficiency seen in Japanese patients. This mutation makes it possible to obtain a restriction endonuclease site (Mae II), thus facilitating a fractional fragment length polymorphism (RFLP) analysis to distinguish between active and mutant alleles. Lewin, pp. See 136-141 in Genes V, Oxford University Press, New York, New York (1994).
[358] Genomic DNA from 12 PAF-AH deficient patients was screened by cutting DNA with Mae II, blotting Southern and hybridizing with exon 9 probe (nucleotides 1-396 of SEQ ID NO: 17). All patients were found to have RFLPs compatible with the mutant allele.
[359] Although the invention has been described in the form of specific embodiments, variations and modifications may occur by those skilled in the art. Therefore, the present invention should be evaluated only by the limitations indicated in the appended claims.
[360] Missing industrial availability
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ICOS CORPORATION
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Acetylhydrolase
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(C) TELEX: 25-3658
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala
1 5 10 15
Phe
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro
1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Lys Pro Leu Val Val Phe Val Leu Gly Gly
1 5 10
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME / KEY: Modified-site
(B) LOCATION: group (13, 21, 27)
(C) OTHER INFORMATION: / note = "The nucleotide at each of
these positions is an inosine. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
ACATGAATTC GGNATCYTTG NGTYTGNCCR AA 32
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TATTTCTAGA AGTGTGGTGG AACTCGCTGG 30
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CGATGAATTC AGCTTGCAGC AGCCATCAGT AC 32
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1520 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME / KEY: CDS
(B) LOCATION: 162..1484
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GCTGGTCGGA GGCTCGCAGT GCTGTCGGCG AGAAGCAGTC GGGTTTGGAG CGCTTGGGTC 60
GCGTTGGTGC GCGGTGGAAC GCGCCCAGGG ACCCCAGTTC CCGCGAGCAG CTCCGCGCCG 120
CGCCTGAGAG ACTAAGCTGA AACTGCTGCT CAGCTCCCAA G ATG GTG CCA CCC 173
Met val pro pro
One
AAA TTG CAT GTG CTT TTC TGC CTC TGC GGC TGC CTG GCT GTG GTT TAT 221
Lys Leu His Val Leu Phe Cys Leu Cys Gly Cys Leu Ala Val Val Tyr
5 10 15 20
CCT TTT GAC TGG CAA TAC ATA AAT CCT GTT GCC CAT ATG AAA TCA TCA 269
Pro Phe Asp Trp Gln Tyr Ile Asn Pro Val Ala His Met Lys Ser Ser
25 30 35
GCA TGG GTC AAC AAA ATA CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC 317
Ala Trp Val Asn Lys Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly
40 45 50
CAA ACT AAA ATC CCC CGG GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA 365
Gln Thr Lys Ile Pro Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr
55 60 65
GAC TTA ATG TTT GAT CAC ACT AAT AAG GGC ACC TTC TTG CGT TTA TAT 413
Asp Leu Met Phe Asp His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr
70 75 80
TAT CCA TCC CAA GAT AAT GAT CGC CTT GAC ACC CTT TGG ATC CCA AAT 461
Tyr Pro Ser Gln Asp Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn
85 90 95 100
AAA GAA TAT TTT TGG GGT CTT AGC AAA TTT CTT GGA ACA CAC TGG CTT 509
Lys Glu Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu
105 110 115
ATG GGC AAC ATT TTG AGG TTA CTC TTT GGT TCA ATG ACA ACT CCT GCA 557
Met Gly Asn Ile Leu Arg Leu Leu Phe Gly Ser Met Thr Thr Pro Ala
120 125 130
AAC TGG AAT TCC CCT CTG AGG CCT GGT GAA AAA TAT CCA CTT GTT GTT 605
Asn Trp Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val
135 140 145
TTT TCT CAT GGT CTT GGG GCA TTC AGG ACA CTT TAT TCT GCT ATT GGC 653
Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly
150 155 160
ATT GAC CTG GCA TCT CAT GGG TTT ATA GTT GCT GCT GTA GAA CAC AGA 701
Ile Asp Leu Ala Ser His Gly Phe Ile Val Ala Ala Val Glu His Arg
165 170 175 180
GAT AGA TCT GCA TCT GCA ACT TAC TAT TTC AAG GAC CAA TCT GCT GCA 749
Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala
185 190 195
GAA ATA GGG GAC AAG TCT TGG CTC TAC CTT AGA ACC CTG AAA CAA GAG 797
Glu Ile Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu
200 205 210
GAG GAG ACA CAT ATA CGA AAT GAG CAG GTA CGG CAA AGA GCA AAA GAA 845
Glu Glu Thr His Ile Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu
215 220 225
TGT TCC CAA GCT CTC AGT CTG ATT CTT GAC ATT GAT CAT GGA AAG CCA 893
Cys Ser Gln Ala Leu Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro
230 235 240
GTG AAG AAT GCA TTA GAT TTA AAG TTT GAT ATG GAA CAA CTG AAG GAC 941
Val Lys Asn Ala Leu Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp
245 250 255 260
TCT ATT GAT AGG GAA AAA ATA GCA GTA ATT GGA CAT TCT TTT GGT GGA 989
Ser Ile Asp Arg Glu Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly
265 270 275
GCA ACG GTT ATT CAG ACT CTT AGT GAA GAT CAG AGA TTC AGA TGT GGT 1037
Ala Thr Val Ile Gln Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly
280 285 290
ATT GCC CTG GAT GCA TGG ATG TTT CCA CTG GGT GAT GAA GTA TAT TCC 1085
Ile Ala Leu Asp Ala Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser
295 300 305
AGA ATT CCT CAG CCC CTC TTT TTT ATC AAC TCT GAA TAT TTC CAA TAT 1133
Arg Ile Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr
310 315 320
CCT GCT AAT ATC ATA AAA ATG AAA AAA TGC TAC TCA CCT GAT AAA GAA 1181
Pro Ala Asn Ile Ile Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu
325 330 335 340
AGA AAG ATG ATT ACA ATC AGG GGT TCA GTC CAC CAG AAT TTT GCT GAC 1229
Arg Lys Met Ile Thr Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp
345 350 355
TTC ACT TTT GCA ACT GGC AAA ATA ATT GGA CAC ATG CTC AAA TTA AAG 1277
Phe Thr Phe Ala Thr Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys
360 365 370
GGA GAC ATA GAT TCA AAT GTA GCT ATT GAT CTT AGC AAC AAA GCT TCA 1325
Gly Asp Ile Asp Ser Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser
375 380 385
TTA GCA TTC TTA CAA AAG CAT TTA GGA CTT CAT AAA GAT TTT GAT CAG 1373
Leu Ala Phe Leu Gln Lys His Leu Gly Leu His Lys Asp Phe Asp Gln
390 395 400
TGG GAC TGC TTG ATT GAA GGA GAT GAT GAG AAT CTT ATT CCA GGG ACC 1421
Trp Asp Cys Leu Ile Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr
405 410 415 420
AAC ATT AAC ACA ACC AAT CAA CAC ATC ATG TTA CAG AAC TCT TCA GGA 1469
Asn Ile Asn Thr Thr Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly
425 430 435
ATA GAG AAA TAC AAT TAGGATTAAA ATAGGTTTTT TAAAAAAAAA AAAAAA 1520
Ile Glu Lys Tyr Asn
440
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Val Pro Pro Lys Leu His Val Leu Phe Cys Leu Cys Gly Cys Leu
1 5 10 15
Ala Val Val Tyr Pro Phe Asp Trp Gln Tyr Ile Asn Pro Val Ala His
20 25 30
Met Lys Ser Ser Ala Trp Val Asn Lys Ile Gln Val Leu Met Ala Ala
35 40 45
Ala Ser Phe Gly Gln Thr Lys Ile Pro Arg Gly Asn Gly Pro Tyr Ser
50 55 60
Val Gly Cys Thr Asp Leu Met Phe Asp His Thr Asn Lys Gly Thr Phe
65 70 75 80
Leu Arg Leu Tyr Tyr Pro Ser Gln Asp Asn Asp Arg Leu Asp Thr Leu
85 90 95
Trp Ile Pro Asn Lys Glu Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly
100 105 110
Thr His Trp Leu Met Gly Asn Ile Leu Arg Leu Leu Phe Gly Ser Met
115 120 125
Thr Thr Pro Ala Asn Trp Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr
130 135 140
Pro Leu Val Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr
145 150 155 160
Ser Ala Ile Gly Ile Asp Leu Ala Ser His Gly Phe Ile Val Ala Ala
165 170 175
Val Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp
180 185 190
Gln Ser Ala Ala Glu Ile Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr
195 200 205
Leu Lys Gln Glu Glu Glu Thr His Ile Arg Asn Glu Gln Val Arg Gln
210 215 220
Arg Ala Lys Glu Cys Ser Gln Ala Leu Ser Leu Ile Leu Asp Ile Asp
225 230 235 240
His Gly Lys Pro Val Lys Asn Ala Leu Asp Leu Lys Phe Asp Met Glu
245 250 255
Gln Leu Lys Asp Ser Ile Asp Arg Glu Lys Ile Ala Val Ile Gly His
260 265 270
Ser Phe Gly Gly Ala Thr Val Ile Gln Thr Leu Ser Glu Asp Gln Arg
275 280 285
Phe Arg Cys Gly Ile Ala Leu Asp Ala Trp Met Phe Pro Leu Gly Asp
290 295 300
Glu Val Tyr Ser Arg Ile Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu
305 310 315 320
Tyr Phe Gln Tyr Pro Ala Asn Ile Ile Lys Met Lys Lys Cys Tyr Ser
325 330 335
Pro Asp Lys Glu Arg Lys Met Ile Thr Ile Arg Gly Ser Val His Gln
340 345 350
Asn Phe Ala Asp Phe Thr Phe Ala Thr Gly Lys Ile Ile Gly His Met
355 360 365
Leu Lys Leu Lys Gly Asp Ile Asp Ser Asn Val Ala Ile Asp Leu Ser
370 375 380
Asn Lys Ala Ser Leu Ala Phe Leu Gln Lys His Leu Gly Leu His Lys
385 390 395 400
Asp Phe Asp Gln Trp Asp Cys Leu Ile Glu Gly Asp Asp Glu Asn Leu
405 410 415
Ile Pro Gly Thr Asn Ile Asn Thr Thr Asn Gln His Ile Met Leu Gln
420 425 430
Asn Ser Ser Gly Ile Glu Lys Tyr Asn
435 440
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1123 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: Not Determined
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
AAATATAAAT TTTAATAACA CCACACATAA ATTTCAAACT ACTTTCCCTA AGTTTCTAGC 60
TGAAGTTTTA AATGAGTGTG TTTTTAATTT ATTAGAAAGT GGATTGAAGA GAAAACATTG 120
GAAGATGAAG GAAGGCGTTT CAGTTAAACC CCAAATAACT CTGTGTTACA CTGAGCTATG 180
AAACGGCTCC TTCTAGCTCC ATTTCTCCTC AGACCTAAGT GCTATTCCTG ATTGTCCTTC 240
ATTGTCATTT CCAGGGAGAA ATGACACCAG CACAGTGGCA GGCCTTCCAA TCTGGAGCAC 300
GGTCCACACA ACTTCCGAAT TGGTGTTCAG TGTAAAGTGT ATCGGAGTGC GGAAAATGCG 360
CAGGGCATTG CCAACTATAG ATGCTCGGAG TAATTCAGTG TATTCAGAGA ACACGGTGAA 420
ACAAGGAAAA CCGGCCTGAC TGGGGGGTGA ATTCAGCAGG GAGTAAATCT GATCGGCATC 480
AGGTCTGCGG AAAGGAGCTG GTGAGCACGA CACCACCAGG CATTGCCTGG CTCTCTCCGC 540
GGCGGGCTAA GTTAACCTCG GGTCCAGGTG CGGGCCATGG TCTTGGGGAG GGTGCTGGGT 600
GCGCTCGAGC AGGCTACGTC GGGAGCCGCC GCTGCTAGTG AGAGCCGGGC CACACACGCT 660
CCTCCCCGGT ACCTCCTCCA GCATCACCAG GGGAGGAGAG GGTCGGGCAC AAGGCGCGCT 720
AGGCGGACCC AGACACAGCC GCGCGCAGCC CACCCGCCCG CCGCCTGCCA GAGCTGCTCG 780
GCCCGCAGCC AGGGGGACAG CGGCTGGTCG GAGGCTCGCA GTGCTGTCGG CGAGAAGCAG 840
TCGGGTTTGG AGCGCTTGGG TCGCGTTGGT GCGCGGTGGA ACCCCCCAGG GACCCCAGTT 900
CCCGCGAGCA GCTCCGCGCC GCGCCTGAGT GAGGAGGGGC CCCGGGGGCG AGGCGGGAGT 960
GGGAGGAAGG GCACGGTCGC CGCGCTGGAG GTCGGGACCC CGGAGCGGCG ACCGGCCGGG 1020
GTGGGCTCGC TGAGTCGCAC CCGCTCTGCT GGCCGGTCCT GGGCTCACAG TCCCTGCAGC 1080
CCTCGGAAAC AGCGCTAGGA TCCTTCGGGA GAGGAGAGAT GAC 1123
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 145..287
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GTACCAATCT AAAACCCAGC ACAGAAAAAT ACATGTTTTA TTTTTTCCAA GTGTTACTAG 60
TACCTCAGCC TTTCTTGATT TGTCAGCTTA TTTAAGGCCT CTTCATTGCA TACTTCTTTT 120
TTCTTTTAAT CATCTGCTTC GAAGGAGACT AAGCTGAAAC TGCTGCTCAG CTCCCAAGAT 180
GGTGCCACCC AAATTGCATG TGCTTTTCTG CCTCTGCGGC TGCCTGGCTG TGGTTTATCC 240
TTTTGACTGG CAATACATAA ATCCTGTTGC CCATATGAAA TCATCAGGTA AGAGGTGTAT 300
TTGTTCAAGG TCTTGAGCAA CTGATCTGTC GCCATACTTC AAGTGGGCCC CAAGAAGTTG 360
CACATCTGCA CATCTAAACA AGTCCTATTT AAAGGCTTAT GGAGATCCTG TATTCTC 417
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 498 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 251..372
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CATTAGGAGG TAACAGTCCA AGGCAGCTGA GAGAAAGGCT ATGTCTACTT TCATCTCTTT 60
ACCCTCCAAA ACCCCTACAC AGTGTTTCAA ACAGAGAGAC CCTCAATAAT TGCATATCTT 120
ACTTGTTAGG TTGAGAAAGA AAGAAGGCCA GAAACTATGG GAAGTAACTT GATTCCGTTG 180
GAATTCTTTT GCATAATAAA ATCTGATATG TAATGGATGA CAAATGAGAT AATATTTACC 240
TGTTTTTCAG CATGGGTCAA CAAAATACAA GTACTGATGG CTGCTGCAAC GTTTGGCCAA 300
ACTAAAATCC CCCGGGGAAA TGGGCCTTAT TCCGTTGGTT GTACAGACTT AATGTTTGAT 360
CACACTAATA AGGTAATGCT TTGATTTATA CAACTTATCC TGATACTCTA ATATTGTCTG 420
TCGCTATGGA CCACTAGAAG GTGTTCAAAT GTGACCTTGC CCTCACCTGA GAATGACTCA 480
TTTTCGAATT TGTATTGT 498
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 433 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 130..274
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CAGCAGCCTA AAGTCTTAGA CTTTGTGAAC ACAGAGGTAT TGAGTCCCAC TAATTAATAT 60
CGAAAATAGC TGCTGGAATA TGTTTGAGAC ACAACTTCTC TAAAAGTGCA TTAATTTCTT 120
TCTTAACAGG GCACCTTCTT GCGTTTATAT TATCCATCCC AAGATAATGA TCACCTTGAC 180
ACCCTTTGGA TCCCAAATAA AGAATATTTT TGGGGTCTTA GCAAATTTCT TGGAACACAC 240
TGGCTTATGG GCAACATTTT GAGGTTACTC TTTGGTAAGA TTTCTGTTGA TCCTTCTTTG 300
TAGGCTCTTG CATGTATGAA AACCTTGAAA ACAACAAGAA CTTCAAGTAG TTAAGACCAA 360
AGTAGATTTT TCTTCAGTCC AAATAGCTCC TAAAATGATA AGGAAAGTAT TTCTTTAAAG 420
CCCAGGCAAC TAC 433
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 486 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 164..257
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
TTGGTGGGTA TCTAGTAGCA GTCTTTTTAA TGAATCTACT ATTCATCCAT AAAAAAGTAG 60
ATATAAATCA GATGGGTCTG CATTTTATGC TAATGAGATA TGAATTAAAT TCACTAGCAA 120
CACTCAGAGA AAACCTTAAC TATAACCTTC CATTGTTGTC TAGGTTCAAT GACAACTCCT 180
GCAAACTGGA ATTCCCCTCT GAGGCCTGGT GAAAAATATC CACTTGTTGT TTTTTCTCAT 240
GGTCTTGGGG CATTCAGGTA ATGTTTGAGA GGTTGAACAA TTTTGGCTTC CAGGAATAAA 300
TGACAATTTT TTTATTCAAG AAAGAAATAG CAGAGTTTGG AATGTCATGC AGGCCCTTGT 360
CTGGAGGAGT TGGGGTTCCT CAATAATTGG CTGTGGGTCT ATTGATCAGT CCTAGACCTG 420
TCTGGTCAAG TAGTTTTTTC CCTACTATCA GCTCATTGGG ATTAGCCTCA CAGCAGAGAA 480
GAAAGG 486
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 113..181
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CCCCAGGCTC TACTACAGGG TGTAATGGCC TCCATGTTCC CAGTTTTATT AGTGACTCAG 60
CCTTGTAATT CATGACTGGT AGTTGTAATT CTTCCCTCTT TTTGTTTTGA AGGACACTTT 120
ATTCTGCTAT TGGCATTGAC CTGGCATCTC ATGGGTTTAT AGTTGCTGCT GTAGAACACA 180
GGTATGTTAC CTGATATAAT TGGGCTCTTT GGCCAACTAC AGGGAATGTC AATGCTCATA 240
ACTATGTTTC TAATTTTCAT AAAAGTTTAT TTAAAATGTT GATGGAACTT TCAAGTATGG 300
TAACATCATG AGCAAAAAAG GAGATTGAGT TTTATCGACT TAAAAGACTT AAAAGCACCT 360
AAC 363
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 68..191
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GAACTGAGAA ACATGGTCAG ATGAGGAAGG GAAGGAGCAT GCATAAATAA TTTTGCTTGT 60
ATTATAGAGA TAGATCTGCA TCTGCAACTT ACTATTTCAA GGACCAATCT GCTGCAGAAA 120
TAGGGGACAA GTCTTGGCTC TACCTTAGAA CCCTGAAACA AGAGGAGGAG ACACATATAC 180
GAAATGAGCA GGTACATTGC AGTGAAAGGA GAGGTGGTTG GTGACCTAAA AGCATGTACA 240
AAAGGATGAC ATTTGTTAAT TTAATTTTAC ACCTGGCAAG TTATGCTCCT AGCTCTCCTA 300
TTTCCCATTC CCAAAAGATC TGTCAATAGA TTCCTGGAGC AGTAAAATTC CCTTAATGGA 360
ATATCTAGTT CATAGTAAAA ACAAAGGCAA ATACAAAAAT TTGGGAGATG ACAGTGAATA 420
TTCAGAATTC CTCGAGCCGG G 441
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 577 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 245..358
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGTTAAGTAA ATCGTCTGAA GTCACATAGT AGGTAAGGCA AAACAGAGCC AGGATTTGGA 60
CTAAGGCTAT ACCTATGTGC AAAGCTGGGG CCTGTGTCAT TATGGTAGCA AGTAATAGTC 120
ACTAATCAGA TTTCCAGTTT ATAACTGACC AACGATTTTT CCCAAATACA GCTTCTACCT 180
AAACTTTAAA ATAAGTGTTA TAACTTTTTA CTTTGTCATT TCCTTCTTCT AATAATTATA 240
TTAGGTACGG CAAAGAGCAA AAGAATGTTC CCAAGCTCTC AGTCTGATTC TTGACATTGA 300
TCATGGAAAG CCAGTGAAGA ATGCATTAGA TTTAAAGTTT GATATGGAAC AACTGAAGGT 360
AAGCTATAAA AAGTAATTTT TCTCTTGTCC TACAGTTCTT TATTGTTTTT TGTCATTTAA 420
TTTTCTGCCT ATATTGCAAG GTACAATATG ATAAAGGGCT GCAACCAGCC CCTCCCCAAT 480
GCGCACACAC AGACACACAA AGCAGTACAG GTAAAGTATT GCAGCAATGA AGAATGCATT 540
ATCTTGGACT AGATATGAAT GCAAAGTTAG TCAGTTT 577
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 396 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 108..199
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
ATCAATGTAT TTACCATCCC CATGAAATGA ACAATTATAT GATTGACAAA TCATTTCTTC 60
TAACACCACG AAATAGCTAT AAATTTATAT CATGCTTTTT CAAATAGGAC TCTATTGATA 120
GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT CAGACTCTTA 180
GTGAAGATCA GAGATTCAGG TAAGAAAATA AGATAGTAAA GCAAGAGAAT AGTAAATTAT 240
TGGAAGAAAT TATATTGTGA GATATAATTT TTATTCAAAT TCTTAGTGAA GGAAGGGGAT 300
CTCTTGGAGT TTATAAGGCT ATTCTTTTGC CCCCATAAAA TACTCTATAT ACATTTTCCT 360
AGGCTAAAAC ATCTCCTCTC CTGCTATTAA AATCTC 396
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 519 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 181..351
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
CTTACAAAGT TAATCATATC CCTTTCCCAC ATTGAAGTAT GATACCTCTT TATTCCAATC 60
AGATAACCCA TAATAAACTG GTATGGTGCG TGTCCACCAA TCCTAGCATT ATTAGGATGT 120
CCTCAATGTT GGCTAGTATG TAACCAGTTT AATTTCATCA TTGTCAACAA ATATCTACAG 180
ATGTGGTATT GCCCTGGATG CATGGATGTT TCCACTGGGT GATGAAGTAT ATTCCAGAAT 240
TCCTCAGCCC CTCTTTTTTA TCAACTCTGA ATATTTCCAA TATCCTGCTA ATATCATAAA 300
AATGAAAAAA TGCTACTCAC CTGATAAAGA AAGAAAGATG ATTACAATCA GGTAAGTATT 360
AGTGACTTAT TTCATTATGT GAAACAAACT TGAAGCTTGG GTAAATATCA ATCGATATCA 420
TTTGGTAACT ATTAAAGAAT TGCTGAATTG GTTGTTTAGA CTTTCAATAA GGAGAGAATT 480
AGATAATCTC AGTTTCTAAG TACATTTAGT CTACTCTTT 519
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 569 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 156..304
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
TGAAACACAT CTAAGTAGAT CAAATTACAA GTTTTATTTC TTCTTTGGTT TTCAGTAAAC 60
AGACCAACAA GACCAGTACC TTTCCTTACA CTCTAACTAA AAAAATAATA ATTTTATCAA 120
ACAATGTGAC TTTTAAATGT CTTGTTCTCT TTTAGGGGTT CAGTCCACCA GAATTTTGCT 180
GACTTCACTT TTGCAACTGG CAAAATAATT GGACACATGC TCAAATTAAA GGGAGACATA 240
GATTCAAATG TAGCTATTGA TCTTAGCAAC AAAGCTTCAT TAGCATTCTT ACAAAAGCAT 300
TTAGGTAAGA AACTATTTTT TTCATGACCT AAACCGAGAT GAATCTCGAG GACAAAGCTG 360
TCTATCTTAA TACAGCTTTA GTACTATTTA AACTATTTCC AGTTGGTTTA CAATGGAACA 420
AAGCAGTATA TCAATTTGAA AACAGAAATT TGAGAAAGTC AATTTTGCTG CTTTACATCT 480
CTATATCATA GAAAGCAAAT CAACTGTTAA AGGTAATATT CTTTGTATGA GCTAGAGTGA 540
CTCATGTGAG GATATCGAAC GACGGTGCT 569
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 469 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME / KEY: exon
(B) LOCATION: 137..253
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
GATACAGAGG CACATCGTCT CTACCATCCT AACGGAACTT GTGTAATTTG TAAATCTTTA 60
TTGCCACCTA GGGGCATCCA AACTGTTTAA TGCTCTCAAA AGTTTAATAT GTTGATTAAC 120
ACTTTATATT TTATAGGACT TCATAAAGAT TTTGATCAGT GGGACTGCTT GATTGAAGGA 180
GATGATGAGA ATCTTATTCC AGGGACCAAC ATTAACACAA CCAATCAACA CATCATGTTA 240
CAGAACTCTT CAGGAATAGA GAAATACAAT TAGGATTAAA ATAGGTTTTT TAAAAGTCTT 300
GTTTCAAAAC TGTCTAAAAT TATGTGTGTG TGTGTGTGTG TGTGTGTGTG AGAGAGAGAG 360
AGAGAGAGAG AGAGAGAATT TTAATGTATT TTCCCAAAGG ACTCATATTT TAAAATGTAG 420
GCTATACTGT AATCGTGATT GAAGCTTGGA CTAAGAATTT TTTCCCTTT 469
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1494 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME / KEY: CDS
(B) LOCATION: 117..1436
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
GGCACGAGCT AGGATCTGAC TCGCTCTGGT GGCATTGCTG CGCTCAGGGT TCTGGGTATC 60
CGGGAGTCAG TGCAGTGACC AGAACATCAA ACTGAAGCCA CTGCTCAGCT CCTAAG 116
ATG GTA CCA CTC AAA CTG CAG GCG CTT TTC TGC CTC CTC TGC TGC CTC 164
Met Val Pro Leu Lys Leu Gln Ala Leu Phe Cys Leu Leu Cys Cys Leu
1 5 10 15
CCA TGG GTC CAT CCT TTT CAC TGG CAA GAC ACA TCT TCT TTT GAC TTC 212
Pro Trp Val His Pro Phe His Trp Gln Asp Thr Ser Ser Phe Asp Phe
20 25 30
AGG CCG TCA GTA ATG TTT CAC AAG CTC CAA TCG GTG ATG TCT GCT GCC 260
Arg Pro Ser Val Met Phe His Lys Leu Gln Ser Val Met Ser Ala Ala
35 40 45
GGC TCT GGC CAT AGT AAA ATC CCC AAA GGA AAT GGA TCG TAC CCC GTC 308Gly Ser Gly His Ser Lys Ile Pro Lys Gly Asn Gly Ser Tyr Pro Val
50 55 60
GGT TGT ACA GAT CTG ATG TTC GGT TAT GGG AAT GAG AGC GTC TTC GTG 356
Gly Cys Thr Asp Leu Met Phe Gly Tyr Gly Asn Glu Ser Val Phe Val
65 70 75 80
CGT TTG TAC TAC CCA GCT CAA GAT CAA GGT CGC CTC GAC ACT GTT TGG 404
Arg Leu Tyr Tyr Pro Ala Gln Asp Gln Gly Arg Leu Asp Thr Val Trp
85 90 95
ATC CCA AAC AAA GAA TAT TTT TTG GGT CTT AGT ATA TTT CTT GGA ACA 452
Ile Pro Asn Lys Glu Tyr Phe Leu Gly Leu Ser Ile Phe Leu Gly Thr
100 105 110
CCC AGT ATT GTA GGC AAT ATT TTA CAC CTC TTA TAT GGT TCT CTG ACA 500
Pro Ser Ile Val Gly Asn Ile Leu His Leu Leu Tyr Gly Ser Leu Thr
115 120 125
ACT CCT GCA AGC TGG AAT TCT CCT TTA AGG ACT GGA GAA AAA TAC CCG 548
Thr Pro Ala Ser Trp Asn Ser Pro Leu Arg Thr Gly Glu Lys Tyr Pro
130 135 140
CTC ATT GTC TTT TCT CAT GGT CTC GGA GCC TTC AGG ACG ATT TAT TCT 596
Leu Ile Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr Ile Tyr Ser
145 150 155 160
GCT ATT GGC ATT GGC TTG GCA TCT AAT GGG TTT ATA GTG GCC ACT GTC 644
Ala Ile Gly Ile Gly Leu Ala Ser Asn Gly Phe Ile Val Ala Thr Val
165 170 175
GAA CAC AGA GAC AGA TCT GCA TCG GCA ACT TAC TTT TTT GAA GAC CAG 692
Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Phe Phe Glu Asp Gln
180 185 190
GTG GCT GCA AAA GTG GAA AAC AGG TCT TGG CTT TAC CTG AGA AAA GTA 740
Val Ala Ala Lys Val Glu Asn Arg Ser Trp Leu Tyr Leu Arg Lys Val
195 200 205
AAA CAA GAG GAG TCG GAA AGT GTC CGG AAA GAA CAG GTT CAG CAA AGA 788
Lys Gln Glu Glu Ser Glu Ser Val Arg Lys Glu Gln Val Gln Gln Arg
210 215 220
GCA ATA GAA TGT TCC CGG GCT CTC AGT GCG ATT CTT GAC ATT GAA CAT 836
Ala Ile Glu Cys Ser Arg Ala Leu Ser Ala Ile Leu Asp Ile Glu His
225 230 235 240
GGA GAC CCA AAA GAG AAT GTA CTA GGT TCA GCT TTT GAC ATG AAA CAG 884
Gly Asp Pro Lys Glu Asn Val Leu Gly Ser Ala Phe Asp Met Lys Gln
245 250 255
CTG AAG GAT GCT ATT GAT GAG ACT AAA ATA GCT TTG ATG GGA CAT TCT 932
Leu Lys Asp Ala Ile Asp Glu Thr Lys Ile Ala Leu Met Gly His Ser
260 265 270
TTT GGA GGA GCA ACA GTT CTT CAA GCC CTT AGT GAG GAC CAG AGA TTC 980
Phe Gly Gly Ala Thr Val Leu Gln Ala Leu Ser Glu Asp Gln Arg Phe
275 280 285
AGA TGT GGA GTT GCT CTT GAT CCA TGG ATG TAT CCG GTG AAC GAA GAG 1028
Arg Cys Gly Val Ala Leu Asp Pro Trp Met Tyr Pro Val Asn Glu Glu
290 295 300
CTG TAC TCC AGA ACC CTC CAG CCT CTC CTC TTT ATC AAC TCT GCC AAA 1076
Leu Tyr Ser Arg Thr Leu Gln Pro Leu Leu Phe Ile Asn Ser Ala Lys
305 310 315 320
TTC CAG ACT CCA AAG GAC ATC GCA AAA ATG AAA AAG TTC TAC CAG CCT 1124
Phe Gln Thr Pro Lys Asp Ile Ala Lys Met Lys Lys Phe Tyr Gln Pro
325 330 335
GAC AAG GAA AGG AAA AAT GAT TAC AAT CAA GGG CTC AGG CAC CAG AAC 1172
Asp Lys Glu Arg Lys Asn Asp Tyr Asn Gln Gly Leu Arg His Gln Asn
340 345 350
TTT GAC GAC TTT ACT TTT GTA ACT GGC AAA ATA ATT GGA AAC AAG CTG 1220
Phe Asp Asp Phe Thr Phe Val Thr Gly Lys Ile Ile Gly Asn Lys Leu
355 360 365
ACA CTG AAA GGA GAA ATC GAT TCC AGA GTA GCC ATC GAC CTC ACC AAC 1268
Thr Leu Lys Gly Glu Ile Asp Ser Arg Val Ala Ile Asp Leu Thr Asn
370 375 380
AAA GCT TCG ATG GCT TTC TTA CAA AAG CAT TTA GGG CTT CAG AAA GAC 1316
Lys Ala Ser Met Ala Phe Leu Gln Lys His Leu Gly Leu Gln Lys Asp
385 390 395 400
TTT GAT CAG TGG GAC CCT CTG GTG GAA GGA GAT GAT GAG AAC CTG ATT 1364
Phe Asp Gln Trp Asp Pro Leu Val Glu Gly Asp Asp Glu Asn Leu Ile
405 410 415
CCT GGG TCA CCC TTT GAC GCA GTC ACC CAG GCC CCG GCT CAG CAA CAC 1412
Pro Gly Ser Pro Phe Asp Ala Val Thr Gln Ala Pro Ala Gln Gln His
420 425 430
TCT CCA GGA TCA CAG ACC CAG AAT TAGAAGAACT TGCTTGTTAC ACAGTTGCCT 1466
Ser Pro Gly Ser Gln Thr Gln Asn
435 440
TTTAAAAGTA GAGTGACATG AGAGAGAG 1494
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2191 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME / KEY: CDS
(B) LOCATION: 92..1423
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
CCGCGCGCTC CGGCCGGGGG ACCCTGGTTC CGGCGAGCGG CTCAGCGCGG CGCCCGGAAG 60
TTTAAGCTGA AACCACTGCT CAGCTTCCAA G ATG TTG CCA CCC AAA CTG CAT 112
Met Leu Pro Pro Lys Leu His
1 5
GCG CTT TTC TGC CTC TGC AGC TGC CTC ACA CTG GTT CAT CCT ATT GAC 160
Ala Leu Phe Cys Leu Cys Ser Cys Leu Thr Leu Val His Pro Ile Asp
10 15 20
TGG CAA GAC CTA AAT CCT GTT GCC CAT ATT AGA TCA TCA GCA TGG GCC 208
Trp Gln Asp Leu Asn Pro Val Ala His Ile Arg Ser Ser Ala Trp Ala
25 30 35
AAT AAA ATA CAA GCT CTG ATG GCT GCT GCA AGT ATT AGG CAA AGT AGA 256
Asn Lys Ile Gln Ala Leu Met Ala Ala Ala Ser Ile Arg Gln Ser Arg
40 45 50 55
ATT CCC AAA GGA AAT GGA TCT TAT TCT GTC GGT TGT ACA GAT TTG ATG 304
Ile Pro Lys Gly Asn Gly Ser Tyr Ser Val Gly Cys Thr Asp Leu Met
60 65 70
TTT GAT TAT ACT AAT AAG GGC ACC TTT TTG CGT TTG TAT TAT CCA TCG 352
Phe Asp Tyr Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser
75 80 85
CAA GAG GAT GAC CAC TCT GAC ACG CTT TGG ATC CCA AAC AAA GAA TAT 400
Gln Glu Asp Asp His Ser Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr
90 95 100
TTT TTT GGT CTT AGT AAA TAT CTT GGA ACA CCC TGG CTT ATG GGC AAA 448
Phe Phe Gly Leu Ser Lys Tyr Leu Gly Thr Pro Trp Leu Met Gly Lys
105 110 115
ATA TTG AGC TTC TTT TTT GGT TCA GTG ACA ACT CCT GCG AAC TGG AAT 496
Ile Leu Ser Phe Phe Phe Gly Ser Val Thr Thr Pro Ala Asn Trp Asn
120 125 130 135
TCC CCT CTG AGG ACT GGT GAA AAA TAT CCA CTG ATT GTT TTT TCT CAT 544
Ser Pro Leu Arg Thr Gly Glu Lys Tyr Pro Leu Ile Val Phe Ser His
140 145 150
GGT CTT GGA GCA TTC CGG ACA ATT TAT TCT GCT ATT GGC ATT GAT CTA 592
Gly Leu Gly Ala Phe Arg Thr Ile Tyr Ser Ala Ile Gly Ile Asp Leu
155 160 165
GCA TCA CAT GGG TTC ATC GTT GCT GCT ATA GAA CAC AGA GAT GGA TCC 640
Ala Ser His Gly Phe Ile Val Ala Ala Ile Glu His Arg Asp Gly Ser
170 175 180
GCC TCT GCG ACT TAC TAT TTC AAG GAC CAG TCT GCT GCA GAA ATA GGG 688
Ala Ser Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly
185 190 195
AAC AAA TCT TGG TCT TAT CTT CAA GAA CTA AAA CCA GGG GAT GAG GAG 736
Asn Lys Ser Trp Ser Tyr Leu Gln Glu Leu Lys Pro Gly Asp Glu Glu
200 205 210 215
ATA CAT GTT CGA AAT GAG CAG GTA CAG AAA AGG GCA AAG GAG TGC TCC 784
Ile His Val Arg Asn Glu Gln Val Gln Lys Arg Ala Lys Glu Cys Ser
220 225 230
CAA GCT CTC AAC TTG ATT CTG GAC ATT GAT CAT GGA AGG CCA ATT AAG 832
Gln Ala Leu Asn Leu Ile Leu Asp Ile Asp His Gly Arg Pro Ile Lys
235 240 245
AAT GTA CTA GAC TTA GAG TTT GAT GTG GAA CAA CTG AAG GAC TCT ATT 880
Asn Val Leu Asp Leu Glu Phe Asp Val Glu Gln Leu Lys Asp Ser Ile
250 255 260
GAC AGG GAT AAA ATA GCA GTA ATT GGA CAT TCT TTT GGT GGA GCC ACA 928
Asp Arg Asp Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr
265 270 275
GTT CTT CAG GCT CTT AGT GAA GAC CAG AGA TTT AGG TGC GGG ATT GCC 976
Val Leu Gln Ala Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala
280 285 290 295
TTG GAT GCA TGG ATG CTT CCA CTG GAT GAT GCA ATA TAT TCC AGA ATC 1024
Leu Asp Ala Trp Met Leu Pro Leu Asp Asp Ala Ile Tyr Ser Arg Ile
300 305 310
CCT CAG CCC CTC TTT TTT ATT AAC TCG GAA CGG TTC CAA TTT CCT GAG 1072
Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu Arg Phe Gln Phe Pro Glu
315 320 325
AAT ATC AAA AAA ATG AAA AAA TGC TAC TCA CCT GAC AAA GAA AGA AAA 1120
Asn Ile Lys Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys
330 335 340
ATG ATT ACA ATC AGG GGT TCA GTC CAT CAG AAC TTT GCT GAT TTC ACT 1168
Met Ile Thr Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr
345 350 355
TTT ACA ACT GGC AAA ATA GTT GGA TAC ATA TTC ACA TTA AAA GGA GAT 1216
Phe Thr Thr Gly Lys Ile Val Gly Tyr Ile Phe Thr Leu Lys Gly Asp
360 365 370 375
ATA GAT TCA AAT GTA GCA ATT GAT CTT TGC AAC AAA GCT TCA TTG GCA 1264
Ile Asp Ser Asn Val Ala Ile Asp Leu Cys Asn Lys Ala Ser Leu Ala
380 385 390
TTT TTA CAA AAG CAT TTA GGA CTG CGG AAA GAT TTT GAT CAG TGG GAT 1312
Phe Leu Gln Lys His Leu Gly Leu Arg Lys Asp Phe Asp Gln Trp Asp
395 400 405
TCT TTG ATT GAA GGA AAA GAC GAA AAT CTT ATG CCA GGG ACC AAC ATT 1360
Ser Leu Ile Glu Gly Lys Asp Glu Asn Leu Met Pro Gly Thr Asn Ile
410 415 420
AAC ATC ACC AAC GAA CAT GAC ACT CTA CAG AAC TCT CCA GAA GCA GAG 1408
Asn Ile Thr Asn Glu His Asp Thr Leu Gln Asn Ser Pro Glu Ala Glu
425 430 435
AAA TCG AAT TTA GAT TAAAAGCACT TTTTTAAAGA TCTTGTTTAA AAACTGTCAA 1463
Lys Ser Asn Leu Asp
440
AAAATGTGTG TATGACTTTT AATATATTTT CTCAAATAAC TCATATTGGA AAATGTAGGC 1523
TATCCCATAA AAGTGATTGA AGCTTGGACT AGGAGGTTTT TTTCTTTAAA GAAAGATTGG 1583
TGTCTATCGA AATCATGCCA GCCTAAATTT TAATTTTACT AAAATGATGC TGTGTCAAAA 1643
TTAATAACTA CTTTTACATT CTTTAATGGA CAAGTATAAC AGGCACAAGG CTAATGAAAA 1703
CGTGTTGCAA TGACATAACA ATCCCTAAAA ATACAGATGT TCTTGCCTCT TTTTTCTATT 1763
ATAATTGAGT TTTAGCAACA TGTTATGCTA GGTAGAATTT GGAAGCACTT CCCTTTGACT 1823
TTTGGTCATG ATAAGAAAAA TTAGATCAAG CAAATGATAA AAGCAGTGTT TTACCAAGGA 1883
TTAGGGATAC TGAACAATTT CACTATGGTA ACTGAATGGG GAGTGACCAA GGGTAAAAAT 1943
ATTAAAGCCA AGGCAAAGGC AGCAGATTAG AATGGATTAA AGAGAGTTTA TAATTTGTTT 2003
GCATTTACTT GATGGTTTAT CTCATGGATT CATGAGTCAA GAAAGGTGCG TAGGACAGGC 2063
CAGGGATTCC AGTTATAACA CATTATTCAC CCAAAGGGTT CTTTAATTCT GTATGAGTAT 2123
TGGGAGTGGA TTAGCACAAT AGAGGCATAT GTTGCTTTAA AAAAAAAAAA AAAAAAAAAA 2183
AAAAAAAA 2191
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1533 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME / KEY: CDS
(B) LOCATION: 62..1394
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
CCGCGAGCAG TTCACCGCGG CGTCCGGAAG GTTAAGCTGA AACGGCAGCT CAGCTTCGGA 60
G ATG TTA CCG TCC AAA TTG CAT GCG CTT TTC TGC CTC TGC ACC TGC 106
Met Leu Pro Ser Lys Leu His Ala Leu Phe Cys Leu Cys Thr Cys
1 5 10 15
CTT GCA CTG GTT TAT CCT TTT GAC TGG CAA GAC CTG AAT CCA GTT GCC 154
Leu Ala Leu Val Tyr Pro Phe Asp Trp Gln Asp Leu Asn Pro Val Ala
20 25 30
TAT ATT GAA TCA CCA GCA TGG GTC AGT AAG ATA CAA GCT CTG ATG GCT 202
Tyr Ile Glu Ser Pro Ala Trp Val Ser Lys Ile Gln Ala Leu Met Ala
35 40 45
GCT GCA AAC ATT GGT CAA TCT AAA ATC CCC AGA GGA AAT GGA TCT TAT 250
Ala Ala Asn Ile Gly Gln Ser Lys Ile Pro Arg Gly Asn Gly Ser Tyr
50 55 60
TCC GTC GGT TGT ACA GAC TTG ATG TTT GAT TAC ACT AAT AAG GGC ACC 298
Ser Val Gly Cys Thr Asp Leu Met Phe Asp Tyr Thr Asn Lys Gly Thr
65 70 75
TTC TTG CGT TTG TAT TAT CCA TCT CAA GAT GAT GAT CAC TCC GAC ACC 346
Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp Asp Asp His Ser Asp Thr
80 85 90 95
CTT TGG ATC CCA AAC AAA GAA TAT TTT TTG GGT CTT AGT AAA TTT CTT 394
Leu Trp Ile Pro Asn Lys Glu Tyr Phe Leu Gly Leu Ser Lys Phe Leu
100 105 110
GGA ACA CAC TGG CTT GTG GGC AAA ATT ATG GGC TTA TTC TTC GGT TCA 442
Gly Thr His Trp Leu Val Gly Lys Ile Met Gly Leu Phe Phe Gly Ser
115 120 125
ATG ACA ACT CCT GCA GCC TGG AAT GCA CAT CTG AGG ACT GGG GAA AAA 490
Met Thr Thr Pro Ala Ala Trp Asn Ala His Leu Arg Thr Gly Glu Lys
130 135 140
TAC CCA CTA ATT ATT TTT TCT CAT GGT CTT GGA GCA TTC AGG ACG ATT 538
Tyr Pro Leu Ile Ile Phe Ser His Gly Leu Gly Ala Phe Arg Thr Ile
145 150 155
TAT TCT GCT ATT GGC ATT GAT CTG GCA TCC CAC GGG TTT ATA GTT GCT 586
Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser His Gly Phe Ile Val Ala
160 165 170 175
GCT GTA GAA CAC AGG GAT GGC TCT GCA TCC TCG ACA TAC TAT TTC AAG 634
Ala Val Glu His Arg Asp Gly Ser Ala Ser Ser Thr Tyr Tyr Phe Lys
180 185 190
GAC CAG TCT GCT GTA GAA ATA GGC AAC AAG TCT TGG CTC TAT CTC AGA 682
Asp Gln Ser Ala Val Glu Ile Gly Asn Lys Ser Trp Leu Tyr Leu Arg
195 200 205
ACC CTG AAG CGA GGA GAG GAG GAG TTT CCT TTA CGA AAT GAG CAG TTA 730
Thr Leu Lys Arg Gly Glu Glu Glu Phe Pro Leu Arg Asn Glu Gln Leu
210 215 220
CGG CAA CGA GCA AAG GAA TGT TCT CAA GCT CTC AGT TTG ATT CTG GAC 778
Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu Ser Leu Ile Leu Asp
225 230 235
ATT GAT CAC GGG AGG CCA GTG ACG AAT GTA CTA GAT TTA GAG TTT GAT 826
Ile Asp His Gly Arg Pro Val Thr Asn Val Leu Asp Leu Glu Phe Asp
240 245 250 255
GTG GAA CAG CTG AAG GAC TCT ATT GAT AGG GAT AAA ATA GCC ATT ATT 874
Val Glu Gln Leu Lys Asp Ser Ile Asp Arg Asp Lys Ile Ala Ile Ile
260 265 270
GGA CAT TCT TTT GGT GGA GCC ACA GTT ATT CAG ACT CTT AGT GAA GAC 922
Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln Thr Leu Ser Glu Asp
275 280 285
CAG AGA TTC AGG TGT GGC ATT GCT CTG GAT GCA TGG ATG TTT CCC GTG 970
Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala Trp Met Phe Pro Val
290 295 300
GGT GAT GAA GTA TAT TCC AGA ATT CCT CAA CCC CTC TTT TTT ATC AAC 1018
Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro Leu Phe Phe Ile Asn
305 310 315
TCG GAA CGA TTC CAA TAC CCT TCT AAT ATC ATA AGA ATG AAA AAA TGC 1066
Ser Glu Arg Phe Gln Tyr Pro Ser Asn Ile Ile Arg Met Lys Lys Cys
320 325 330 335
TTC TTA CCT GAT AGA GAA CGA AAA ATG ATT ACA ATC AGG GGT TCG GTC 1114
Phe Leu Pro Asp Arg Glu Arg Lys Met Ile Thr Ile Arg Gly Ser Val
340 345 350
CAT CAG AAT TTT GTT GAC TTC ACT TTT GCC ACT AGC AAA ATA ATT GGC 1162
His Gln Asn Phe Val Asp Phe Thr Phe Ala Thr Ser Lys Ile Ily Gly
355 360 365
TAC CTA TTC ACA CTG AAA GGA GAC ATC GAT TCC AAT GTA GCC ATC AGC 1210
Tyr Leu Phe Thr Leu Lys Gly Asp Ile Asp Ser Asn Val Ala Ile Ser
370 375 380
CTT AGC AAC AAA GCT TCC TTA GCG TTC TTA CAA AAA CAT TTA GGA CTT 1258
Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln Lys His Leu Gly Leu
385 390 395
CAG AAA GAT TTT GAT CAG TGG GAT TCT TTA GTT GAA GGC GAA GAT CAC 1306
Gln Lys Asp Phe Asp Gln Trp Asp Ser Leu Val Glu Gly Glu Asp His
400 405 410 415
AAT CTT ATT CCA GGG ACC AAC ATT AAC ACA ACC CAAC CAA GCC ATT 1354
Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr Asn His Gln Ala Ile
420 425 430
CTG CAG AAC TCC ACA GGA ATA GAG AGA CCA AAT TTA GAT T AAAAGAGCTT 1404
Leu Gln Asn Ser Thr Gly Ile Glu Arg Pro Asn Leu Asp
435 440
TTTAAAAAGT TTTGTTTACG AACTTGTCTA AAAGTGTGTG TGTGTATGAT TTAAATGTAT 1464
TTTCTCAAAT AGCTCATATT AAAAAATGTA GGCTATAGCA CAAAAAAAAA AAAAAAAAAA 1524
AAAAAAAAA 1533
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1876 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME / KEY: CDS
(B) LOCATION: 468..1734
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CGGCGGGCTG CTGGCCCTTC CCGGCTGTTC GTAGAGCCGG ATCCTGCAGC GCCCCTGAGA 60
CGAACCGCCC CGATGCGGTG CTCCTCAGCG CCACGGGACG CAGCCGGGGC CGGCCGTGTT 120
GGCGCAGCTC CCACGACGTA CGCTTCCTTT CCAGGCTCGA GGAAAGCCTC TCCCACAAAC 180
ACCGTCCCAG CTGGGAAGTG AGGCGGAGTT TTGGTCCCTC CCCTCCGGCA GCGCCCGGCA 240
TTCCGTCCGT CCGTCCGTCC GTCCGTGCGG CGCACGGCGC CCTGCAGAGC CGGGACACCG 300
CAGCAGGGTA GGAGGACCCG GAGGTGGTGT GCAGCCACAG GTTTCCATCC TGCCCCCACC 360
TCCCGGGGAG CAGCCCTGTG CTATACCCAA CCCCCCGCAC AGAGCACTGA GCCGGCTGCT 420
GCCTGCCTGC ACCCCGCCGT GGGACCTTCT GCTCTTCCCA ACAAGTG ATG GCA TCG 476
Met ala ser
One
CTG TGG GTG AGA GCC AGG AGG GTG TTC ATG AAA AGT CGT GCT TCA GGT 524
Leu Trp Val Arg Ala Arg Arg Val Phe Met Lys Ser Arg Ala Ser Gly
5 10 15
TTC TCG GCG AAG GCG GCG ACG GAG ATG GGG AGC GGC GGC GCG GAG AAG 572
Phe Ser Ala Lys Ala Ala Thr Glu Met Gly Ser Gly Gly Ala Glu Lys
20 25 30 35
GGC TAT CGG ATC CCC GCC GGG AAG GGC CCG CAC GCC GTG GGC TGC ACG 620
Gly Tyr Arg Ile Pro Ala Gly Lys Gly Pro His Ala Val Gly Cys Thr
40 45 50
GAT CTG ATG ACC GGC GAC GCG GCC GAG GGA AGC TTT TTG CGC CTG TAT 668
Asp Leu Met Thr Gly Asp Ala Ala Glu Gly Ser Phe Leu Arg Leu Tyr
55 60 65
TAC CTA TCG TGT GAC GAC ACA GAT ACT GAA GAG ACA CCC TGG ATT CCA 716
Tyr Leu Ser Cys Asp Asp Thr Asp Thr Glu Glu Thr Pro Trp Ile Pro
70 75 80
GAT AAA GAG TAC TAC CAG GGG CTG TCT GAC TTC CTC AAC GTG TAC CGG 764
Asp Lys Glu Tyr Tyr Gln Gly Leu Ser Asp Phe Leu Asn Val Tyr Arg
85 90 95
GCC CTG GGA GAA AGG CTT TTC CAG TAC TAC GTT GGC TCA GTG ACC TGT 812
Ala Leu Gly Glu Arg Leu Phe Gln Tyr Tyr Val Gly Ser Val Thr Cys
100 105 110 115
CCT GCA AAA TCA AAC GCT GCT TTT AAG CCA GGA GAG AAA TAC CCA CTG 860
Pro Ala Lys Ser Asn Ala Ala Phe Lys Pro Gly Glu Lys Tyr Pro Leu
120 125 130
CTC GTT TTT TCC CAT GGA CTT GGA GCT TTT CGG ACC ATC TAT TCT GCT 908
Leu Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr Ile Tyr Ser Ala
135 140 145
ATC TGC ATA GAG ATG GCT TCT CAA GGC TTT CTA GTG GCA GCT GTG GAG 956
Ile Cys Ile Glu Met Ala Ser Gln Gly Phe Leu Val Ala Ala Val Glu
150 155 160
CAC AGA GAT GAA TCG GCT TCA GCA ACG TAT TTC TGT AAA AAG AAG GCT 1004
His Arg Asp Glu Ser Ala Ser Ala Thr Tyr Phe Cys Lys Lys Lys Ala
165 170 175
GAT TCT GAG CCA GAG GAG GAT CAA ACA TCA GGC GTG GAG AAG GAG TGG 1052
Asp Ser Glu Pro Glu Glu Asp Gln Thr Ser Gly Val Glu Lys Glu Trp
180 185 190 195
ATC TAC TAC AGG AAG CTC AGA GCA GGA GAG GAG GAG CGC TGT CTG CGT 1100
Ile Tyr Tyr Arg Lys Leu Arg Ala Gly Glu Glu Glu Arg Cys Leu Arg
200 205 210
CAC AAG CAG GTA CAG CAG AGA GCA CAG GAG TGC ATC AAA GCG CTC AAC 1148
His Lys Gln Val Gln Gln Arg Ala Gln Glu Cys Ile Lys Ala Leu Asn
215 220 225
CTC ATT CTT AAG ATC AGT TCA GGA GAG GAA GTG ATG AAT GTG CTG AAC 1196
Leu Ile Leu Lys Ile Ser Ser Gly Glu Glu Val Met Asn Val Leu Asn
230 235 240
TCA GAC TTT GAC TGG AAC CAC CTG AAG GAT TCT GTT GAT ACT AGC AGA 1244
Ser Asp Phe Asp Trp Asn His Leu Lys Asp Ser Val Asp Thr Ser Arg
245 250 255
ATA GCT GTG ATG GGA CAC TCT TTT GGT GGT GCT ACA GTT ATT GAG AGC 1292
Ile Ala Val Met Gly His Ser Phe Gly Aly Thr Val Ile Glu Ser
260 265 270 275
CTC AGC AAA GAA ATT AGA TTT AGG TGT GGC ATT GCC CTT GAT GCG TGG 1340
Leu Ser Lys Glu Ile Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala Trp
280 285 290
ATG CTC CCG GTA GGC GAT GAC ACT TAC CAA AGC AGT GTG CAG CAA CCA 1388
Met Leu Pro Val Gly Asp Asp Thr Tyr Gln Ser Ser Val Gln Gln Pro
295 300 305
CTG CTC TTT ATT AAT TCC GAA AAA TTC CAG TGG GCT GCC AAT ATC TTA 1436
Leu Leu Phe Ile Asn Ser Glu Lys Phe Gln Trp Ala Ala Asn Ile Leu
310 315 320
AAG ATG AAG AAG CTT AGC TCC AAT GAT ACC AAC AAG AAA ATG ATC ACC 1484
Lys Met Lys Lys Leu Ser Ser Asn Asp Thr Asn Lys Lys Met Ile Thr
325 330 335
ATC AAA GGA TCG GTA CAT CAG AGC TTT CCT GAT TTT ACT TTT GTG AGT 1532
Ile Lys Gly Ser Val His Gln Ser Phe Pro Asp Phe Thr Phe Val Ser
340 345 350 355
GGA GAA ATC ATT GGA AAG TTT TTC AAG TTA AAA GGA GAA ATA GAC CCA 1580
Gly Glu Ile Ile Gly Lys Phe Phe Lys Leu Lys Gly Glu Ile Asp Pro
360 365 370
AAT GAA GCT ATT GAT ATA TGC AAC CAC GCT TCA TTG GCC TTC CTG CAG 1628
Asn Glu Ala Ile Asp Ile Cys Asn His Ala Ser Leu Ala Phe Leu Gln
375 380 385
AAA CAT CTG AGT CTT AAG AGA GAT TTT GAT AAG TGG GAT TCA CTC GTG 1676
Lys His Leu Ser Leu Lys Arg Asp Phe Asp Lys Trp Asp Ser Leu Val
390 395 400
GAT GGC ATA GGA CCC AAT GTT ATT TCT GGT ACC AAT ATC GAC TTA TCT 1724
Asp Gly Ile Gly Pro Asn Val Ile Ser Gly Thr Asn Ile Asp Leu Ser
405 410 415
CCA ACT GAG T AAGGAGTACA AGAAGTACTG CAAAGGCCAC CAGCAGCAGG 1774
Pro Thr Glu
420
ACACCAACGT TGGCCACACA TTGCTTGGAG CTGAGATAGC ACTGGCCTCC CACACAGCTT 1834
TTGGAGTGTG AAACAACAAA AAAAAAAATC ACAGGGGAGC CG 1876
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 517 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME / KEY: CDS
(B) LOCATION: 2..514
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
G GGG CAT TCT TTT GGA GGA GCA ACA GTT TTT CAA GCC CTA AGT GAA 46
Gly His Ser Phe Gly Gly Ala Thr Val Phe Gln Ala Leu Ser Glu
1 5 10 15
GAC CAG AGA TTC AGA TGT GGG ATT GCC CTT GAT CCG TGG ATG TTT CCC 94
Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Pro Trp Met Phe Pro
20 25 30
GTG AGT GAG GAG CTG TAC TCC AGA GTT CCT CAG CCT CTC TTC TTT ATC 142
Val Ser Glu Glu Leu Tyr Ser Arg Val Pro Gln Pro Leu Phe Phe Ile
35 40 45
AAC TCT GCC GAA TTC CAG ACT CCA AAG GAC ATT GCA AAA ATG AAA AAC 190
Asn Ser Ala Glu Phe Gln Thr Pro Lys Asp Ile Ala Lys Met Lys Asn
50 55 60
TTC TAC CAG CCT GAC AAG GAA AGG AAA ATG ATT ACG ATC AAG GGC TCA 238
Phe Tyr Gln Pro Asp Lys Glu Arg Lys Met Ile Thr Ile Lys Gly Ser
65 70 75
GTG CAC CAG AAT TTT GCT GAC GGG ACT TTT GTA ACT GGC AAA ATA ATT 286
Val His Gln Asn Phe Ala Asp Gly Thr Phe Val Thr Gly Lys Ile Ile
80 85 90 95
GGA AAC AAG CTG TCA CTG AAA GGA GAC ATA GAC TCC AGA GTT GCC ATA 334
Gly Asn Lys Leu Ser Leu Lys Gly Asp Ile Asp Ser Arg Val Ala Ile
100 105 110
GAC CTC ACC AAC AAG GCT TCC TTG GCT TTC TTA CAA AAA CAT TTA GGA 382
Asp Leu Thr Asn Lys Ala Ser Leu Ala Phe Leu Gln Lys His Leu Gly
115 120 125
CTT CAT AAA GAC TTT GAT CAG TGG GAC TGT CTG GTG GAG GGA GAG AAC 430
Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Val Glu Gly Glu Asn
130 135 140
GAG AAC CTC ATC CCG GGG TCA CCC TTT GAT GTA GTC ACC CAG TCC CCG 478
Glu Asn Leu Ile Pro Gly Ser Pro Phe Asp Val Val Thr Gln Ser Pro
145 150 155
GCT CTG CAG AGT TCT CCC GGA TCA CAC AAC CAG AAT TAG 517
Ala Leu Gln Ser Ser Pro Gly Ser His Asn Gln Asn
160 165 170
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 580 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME / KEY: CDS
(B) LOCATION: 1..580
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
CAA GTA CTG ATG GCT GCT GCA AGC TTT GGC GAA CGT AAA ATC CCT AAG 48
Gln Val Leu Met Ala Ala Ala Ser Phe Gly Glu Arg Lys Ile Pro Lys
1 5 10 15
GGA AAT GGG CCT TAT TCC GTT GGT TGT ACA GAC TTA ATG TTT GAT TAC 96
Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp Tyr
20 25 30
ACT AAA AAG GGC ACC TTC TTG CGT TTA TAT TAT CCA TCC CAA GAT GAT 144
Thr Lys Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp Asp
35 40 45
GAT CGC CTT GAC ACC CTT TGG ATC CCA AAT AAG GAG TAT TTT TGG GGT 192
Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp Gly
50 55 60
CTT AGC AAG TAT CTT GGA AAA CAC TGG CTT ATG GGC AAC ATT TTG AGT 240
Leu Ser Lys Tyr Leu Gly Lys His Trp Leu Met Gly Asn Ile Leu Ser
65 70 75 80
TTA CTC TTT GGT TCA GTG ACA ACT CCT GCA AAC TGG AAT TCC CCT CTG 288
Leu Leu Phe Gly Ser Val Thr Thr Pro Ala Asn Trp Asn Ser Pro Leu
85 90 95
AGG CCT GGT GAA AAA TAC CCA CTT GTT GTT TTT TCT CAT GGT CTT GGA 336
Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu Gly
100 105 110
GCA TTC AGG ACA ATT TAT TCT GCT ATT GGC ATT GAC CTG GCA TCT CAT 384
Ala Phe Arg Thr Ile Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser His
115 120 125
GGG TTT ATA GTT GCT GCT GTA GAA CAC AGA GAT AGA TCT GCA TCT GCA 432
Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser Ala
130 135 140
ACT TAC TAT TTC AAG AAC CAA TCT GCT GCA GAA ATA GGG AAA AAG TCT 480
Thr Tyr Tyr Phe Lys Asn Gln Ser Ala Ala Glu Ile Gly Lys Lys Ser
145 150 155 160
TGG CTC TAC CTT AGA ACC CTG AAA GAA GAG GAG GAG ATA CAT ATA CGA 528
Trp Leu Tyr Leu Arg Thr Leu Lys Glu Glu Glu Glu Ile His Ile Arg
165 170 175
AAT AAG CAG GTA CGA CAA AGA GCA AAA GAA TGT TCC CAA GCT CTC AGT 576
Asn Lys Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu Ser
180 185 190
CTG A 580
Leu
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
Gly Xaa Ser Xaa Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
TATTCTAGAA TTATGATACA AGTATTAATG GCTGCTGCAA G 41
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
ATTGATATCC TAATTGTATT TCTCTATTCC TG 32
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
ATGGTACCCC CAAAGCTGCA CGTCCTGTTT TGTCTGTGTG GATGTCTCGC CGTCGTGTAC 60
CCCTTCGATT GGCAGTATAT CAACCCCGTG GCTCACATGA AGAGCAGCGC CTGGGTGAAT 120
AAGATCCAGG TGCTCATGGC CGCACCAAGC TTCGGTCAGA CCAAGATTCC TAGAGGCAAC 180
GGCCCCTACA GCGTGGGCTG CACCGATCTG ATGTTCGACC ATACCAACAA AGGAACTTTT 240
CTGAGACTGT ACTACCCCAG CCAGGACAAC GACAGACTGG ATACTCTGTG GATCCCAAAT 300
AAAGAATATT TTTGGGGTCT TAGCAAATTT CTTGGAACAC ACTGGCTTAT GGGCAACATT 360
TTGAGGTTAC TCTTTGGTTC AATGACAACT CCTGCAAACT GGAATTCCCC TCTGAGGCCT 420
GGTGAAAAAT ATCCACTTGT TGTTTTTTCT CATGGTCTTG GGGCATTCAG GACACTTTAT 480
TCTGCTATTG GCATTGACCT GGCATCTCAT GGGTTTATAG TTGCTGCTGT AGAACACAGA 540
GATAGATCTG CATCTGCAAC TTACTATTTC AAGGACCAAT CTGCTGCAGA AATAGGGGAC 600
AAGTCTTGGC TCTACCTTAG AACCCTGAAA CAAGAGGAGG AGACACATAT ACGAAATGAG 660
CAGGTACGGC AAAGAGCAAA AGAATGTTCC CAAGCTCTCA GTCTGATTCT TGACATTGAT 720
CATGGAAAGC CAGTGAAGAA TGCATTAGAT TTAAAGTTTG ATATGGAACA ACTGAAGGAC 780
TCTATTGATA GGGAAAAAAT AGCAGTAATT GGACATTCTT TTGGTGGAGC AACGGTTATT 840
CAGACTCTTA GTGAAGATCA GAGATTCAGA TGTGGTATTG CCCTGGATGC ATGGATGTTT 900
CCACTGGGTG ATGAAGTATA TTCCAGAATT CCTCAGCCCC TCTTTTTTAT CAACTCTGAA 960
TATTTCCAAT ATCCTGCTAA TATCATAAAA ATGAAAAAAT GCTACTCACC TGATAAAGAA 1020
AGAAAGATGA TTACAATCAG GGGTTCAGTC CACCAGAATT TTGCTGACTT CACTTTTGCA 1080
ACTGGCAAAA TAATTGGACA CATGCTCAAA TTAAAGGGAG ACATAGATTC AAATGTAGCT 1140
ATTGATCTTA GCAACAAAGC TTCATTAGCA TTCTTACAAA AGCATTTAGG ACTTCATAAA 1200
GATTTTGATC AGTGGGACTG CTTGATTGAA GGAGATGATG AGAATCTTAT TCCAGGGACC 1260
AACATTAACA CAACCAATCA ACACATCATG TTACAGAACT CTTCAGGAAT AGAGAAATAC 1320
AATTAGGATT CTAGA 1335

权利要求:
Claims (16)
[1" claim-type="Currently amended] An isolated purified human plasma platelet-activating factor acetylhydrolase (PAF-AH) polypeptide fragment deleted from the initial 12 N-terminal amino acids of the mature human PAF-AH amino acid sequence set forth in SEQ ID NO: 8.
[2" claim-type="Currently amended] 2. The PAF-AH polypeptide fragment of claim 1, selected from the group consisting of:
(a) a polypeptide having Met ₄₆ of SEQ ID NO: 8 as an initial N-terminal amino acid;
(b) a polypeptide having Ala ₄₇ of SEQ ID NO: 8 as an initial N-terminal amino acid;
(c) a polypeptide having Ala ₄₈ of SEQ ID NO: 8 as the initial N-terminal amino acid.
[3" claim-type="Currently amended] 3. A PAF-AH polypeptide fragment according to claim 1 or 2, which lacks 30 C-terminal amino acids of the amino acid sequence of SEQ ID NO: 8.
[4" claim-type="Currently amended] 3. A PAF-AH polypeptide according to claim 1 or 2, having a residue of SEQ ID NO: 8 selected from the group consisting of as C-terminal residues:
(a) Ile ₄₂₉ ,
(b) Leu ₄₃₁ , and
(c) Asn ₄₄₁ .
[5" claim-type="Currently amended] Variants of the PAF-AH polypeptide fragment of claim 1, wherein the amino acid in the sequence of SEQ ID NO: 8 is selected from the group consisting of:
(a) S 108 A,
(b) S 273 A,
(c) D 286 A,
(d) D 286 N,
(e) D 296 A,
(f) D 304 A,
(g) D 338 A,
(h) H 351 A,
(i) H 395 A,
(j) H 399 A,
(k) C 67 A,
(l) C 229 S,
(m) C 291 S,
(n) C 334 S, and
(o) C 407 S.
[6" claim-type="Currently amended] Human PAF-AH polypeptide variant having an amino acid substituted in the sequence of SEQ ID NO: 8 selected from the group consisting of:
(a) D 286 A
(b) D 286 N
(C) D 304 A
[7" claim-type="Currently amended] An isolated polynucleotide encoding the PAF-AH polypeptide fragment, variant or variant fragment of claim 1.
[8" claim-type="Currently amended] An isolated polynucleotide encoding a human PAF-AH fragment or variant fragment having Met ₄₆ of SEQ ID NO: 8 as the N-terminal residue and Ile ₄₂₉ or Asn ₄₄₁ as the C-terminal residue.
[9" claim-type="Currently amended] The polynucleotide of claim 7 or 8, wherein the polynucleotide is DNA.
[10" claim-type="Currently amended] A DNA vector containing the DNA of claim 9.
[11" claim-type="Currently amended] A host cell stably transformed or transfected with the DNA of claim 9 so that a PAF-AH polypeptide fragment, variant or variant fragment is expressed in said host cell.
[12" claim-type="Currently amended] 12. A variant of the PAF-AH polypeptide fragment, plasma PAF-AH, comprising growing the host cell of claim 11 in suitable nutrition and isolating the PAF-AH fragment, variant or variant fragment from the cell or its growth medium. Method of Production of Variant Fragments.
[13" claim-type="Currently amended] A PAF-AH polypeptide fragment, variant or variant fragment produced by the method of claim 12.
[14" claim-type="Currently amended] A pharmaceutical composition comprising a pharmaceutically acceptable diluent, adjuvant or carrier with the PAF-AH fragment, variant or variant fragment of any one of claims 1, 6 or 13.
[15" claim-type="Currently amended] A mammal suffering from or susceptible to PAF-mediated pathological symptoms, comprising administering an amount of the pharmaceutical composition of claim 14 sufficient to add PAF-AH activity and inactivate the pathological effects of PAF in the mammal. Method of treatment.
[16" claim-type="Currently amended] The method of claim 15, wherein the pathological condition is pleurisy, asthma, rhinitis, necrotizing enterocolitis, acute respiratory distress syndrome, acute pancreatitis, or a neurological disease associated with HIV infection.

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同族专利:

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公开号 | 公开日

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1997-08-13|Application filed by 게리 엘. 윌콕스, 이코스 코포레이션

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1997-08-13|Priority to KR1019997003367A

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2000-11-25|Publication of KR20000068780A

优先权:

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申请号 | 申请日 | 专利标题

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KR1019997003367A|KR20000068780A|1999-04-13|1997-08-13|Truncated platelet-activating factor acetylhydrolase|