Novel polypeptide and dna thereof

专利摘要:
The present invention relates to a novel polypeptide and a DNA encoding the same, a medicament comprising the polypeptide or DNA, a compound or a salt for promoting or inhibiting the activity of the polypeptide or a method for screening / kit for screening, a compound obtained by the screening or a salt thereof The present invention relates to a medicine comprising the compound or a salt thereof. The polypeptide of the present invention and the DNA encoding the same can be used, for example, for the diagnosis, treatment, and prevention of bone and joint diseases and pathovascular neovascularization. In addition, the polypeptides of the invention are useful as reagents for the screening of compounds or salts thereof that promote or inhibit the activity of the polypeptides of the invention.
公开号:KR20020026465A
申请号:KR1020017016470
申请日:2000-06-29
公开日:2002-04-10
发明作者:이또야스아끼；니시가즈노리；오기가즈히로；오오꾸보쇼이찌；모기신이찌；노구찌유꼬；요시무라고지；다나까히데유끼
申请人:다케다 야쿠힌 고교 가부시키가이샤；
IPC主号:

专利说明:

New polypeptide and its DNA {NOVEL POLYPEPTIDE AND DNA THEREOF}
[2] Cells secrete multiple diverse proteins in their own mechanism, regardless of their prokaryotic or eukaryotic properties. In particular, multicellular organisms (living organisms) exchange information among cells in order to maintain their differentiation, proliferation, and homeostasis, but most of the various humoral factors, which play a central role, are secreted proteins or their maturants. In functional features, they are classified into hormones, neurotransmitters, cytokines, and growth factors. With the recent advances in recombinant DNA technology and cell alignment technology, the elucidation of genes and protein structures encoding these secreted proteins has been steadily progressing. On the other hand, such factor expression dramatically advances the interpretation of the receptor expressed on the cell surface, and further leads to clarification of the mechanism of information transmission in each cell, thereby characterizing its physiological function. In many diseases of humans or in various disease model animals, the unusual expression of any humoral factor that must maintain this inherent homeostasis is often caused, or consequently, worsens. There are also phenomena applicable in the field of diagnosis of various diseases, such as so-called tumor markers, in which hyperactivity is expressed, and the expression control mechanism is also an important target for drug discovery research.
[3] The melanoma inhibitory activity (MIA), a melanoma inhibitory activity (MIA) published in 1994 by Blesch et al., Is one of the secretory proteins in this category, and as its name suggests, melanoma is an indicator of antiproliferative activity against human melanoma cells. The gene was also isolated from the culture supernatant of species cells (Cancer Research 54, 5695-5701, 1994). Subsequently, in 1996, Sandell et al. Identified the homologous gene of the protein as a retinophosphate-sensitive protein (CD-RAP) produced by bovine chondrocytes. It has been suggested to function (The Journal of Biological Chemistry 271, 3311-3316, 1996). The MIA / CD-RAP gene has a high homology of 85% or more at the amino acid level among human, mouse, rat, and bovine species, but it has not been found to be homologous with the known proteins so far. It is believed that similar genes of this gene do not exist elsewhere in the historical interpretation (The Journal of Biological Chemistry 271, 3311-3316, 1996).
[4] On the other hand, the entire DNA, or genome, of a single organism is already finished in bacteria, and humans are expected to complete in a few years, but the expected number of genes is 100,000. Certainly, so far many genes encoding secretory proteins or secretory peptides have been isolated, but the numbers do not necessarily cover all of them in the whole genome. In order to understand the life-level phenomena at the individual level, the so-called exchange of information between cells should be explained, and in addition to these known genes, it is highly likely that unknown liquid molecules play an important physiological role. Such material expression was strongly required.
[5] The present invention provides a novel cell function secretion protein (hereinafter sometimes referred to as MLP protein or MLP), a partial peptide thereof or a salt thereof, DNA encoding the protein, a recombinant vector, a transformant, a method for producing the protein, and For the purpose of providing a drug containing a protein or DNA, an antibody against the protein, a method for screening and screening a receptor agonist / antagonist, a kit for screening, a receptor agonist / antagonist obtained by the screening, and a medicine thereof do.
[6] Isolation of new cytoregulatory secretory proteins gives new knowledge to mechanisms such as cell differentiation, proliferation, and cancer, and can further elucidate life phenomena such as oncogenesis and maintenance of homeostasis. It is possible to develop new medicines that exhibit inhibitory or promoting activity and are useful for the prevention, diagnosis and treatment of various diseases.
[1] The present invention relates to novel secretory cell function regulatory proteins and DNAs thereof.
[38] 1 shows amino acid sequences of human MLP precursor (hMLP), mouse MLP precursor (mMLP), human MIA precursor (hMIA), mouse MIA precursor (mMIA), rat MIA precursor (rMIA) and bovine MIA precursor (bMIA).
[39] 2 shows the results of the western blot analysis conducted in Example 6. FIG. Anti-FLAG antibodies are used as primary antibodies. Lane 1 is mouse MLP (no FLAG tag), lane 2 is mouse MLP-FLAG, lane 3 is mouse MIA (no FLAG tag), lane 4 is mouse MIA-FLAG, lane 5 is human MLP (no FLAG tag) Lane 6 shows lanes electrophoresed with the culture supernatant of COS-7 cells into which mouse MLP-FLAG was introduced.
[40] 3 shows the results of the western blot analysis conducted in Example 6. FIG. Anti-MLP antiserum is used as the primary antibody. Lane 1 is mouse MLP (no FLAG tag), lane 2 is mouse MLP-FLAG, lane 3 is mouse MIA (no FLAG tag), lane 4 is mouse MIA-FLAG, lane 5 is human MLP (no FLAG tag) Lane 6 shows lanes electrophoresed with the culture supernatant of COS-7 cells into which mouse MLP-FLAG was introduced.
[41] 4 shows the results of the immunostaining performed in Example 6. The left panel shows the results of the control experiment with pre-immune rabbit serum and the right panel shows the results of the experiment with anti-MLP antiserum.
[42] Best Mode for Carrying Out the Invention
[43] Polypeptide containing the amino acid sequence represented by SEQ ID NO: 24 of the present invention (hereinafter referred to as human-type polypeptide), Polypeptide containing the amino acid sequence represented by SEQ ID NO: 26 (hereinafter referred to as mouse polypeptide), SEQ ID NO: A polypeptide containing an amino acid sequence represented by: 49 (hereinafter referred to as a rat polypeptide) and a polypeptide containing an amino acid sequence substantially identical to a human type polypeptide (hereinafter, containing an amino acid sequence substantially identical to a human type polypeptide and a human type polypeptide). Polypeptides, collectively referred to as polypeptides of the present invention, refer to cells (eg, liver cells, spleen cells) of humans or warm-blooded animals (e.g. , Nerve cell, glea cell, pancreatic β cell, bone marrow cell, mesangium cell, Langelhans three , Epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibroblasts, myocytes, adipocytes, immune cells (eg macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, single cells Neutrophils, megakaryocytes, periosteal cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells or stromal cells or their progenitor cells, liver cells or cancer cells, etc.) or any tissue in which these cells are present For example, the brain, each part of the brain (e.g. posterior, amygdala, cerebral basal, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid gland , Gallbladder, bone marrow, adrenal gland, skin, muscle, lungs, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, salivary glands, peripheral blood, prostate, testes, ovaries, placenta, uterus, bones, cartilage, joints Or a polypeptide derived from skeletal muscle, or the like, or a recombinant polypeptide And also it may be a synthetic polypeptide.
[44] In addition, when the polypeptide of the present invention has a signal peptide, the polypeptide can be efficiently secreted out of the cell.
[45] An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24, is about 50% or more, preferably about 60% or more, more preferably about 70% or more, and the amino acid sequence represented by SEQ ID NO: 24, More preferably at least about 80%, particularly preferably at least about 90%, most preferably at least about 95% homology, and the like, specifically the amino acid sequence represented by SEQ ID NO: 26. Or an amino acid sequence represented by SEQ ID NO: 49.
[46] A polypeptide having an amino acid sequence represented by SEQ ID NO: 24 is selected from a human MLP or a human MLP protein, a polypeptide having an amino acid sequence represented by SEQ ID NO: 26 is selected from a mouse MLP or a mouse MLP protein and an amino acid sequence represented by SEQ ID NO: 49 The polypeptide having is referred to as rat MLP or rat MLP protein and these are collectively called MLP.
[47] As a polypeptide containing an amino acid sequence represented by SEQ ID NO: 24 or an amino acid sequence substantially identical thereto, specifically, for example, an amino acid sequence represented by SEQ ID NO: 6 or a polypeptide containing an amino acid sequence substantially identical thereto Can be.
[48] An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6, may be about 50% or more, preferably about 60% or more, more preferably about 70% or more of the amino acid sequence represented by SEQ ID NO: 6, More preferably at least about 80%, particularly preferably at least about 90%, most preferably at least about 95% homology, and the like, specifically the amino acid sequence represented by SEQ ID NO: 12. Or an amino acid sequence represented by SEQ ID NO: 47.
[49] A polypeptide having an amino acid sequence represented by SEQ ID NO: 6 is represented by a human MLP precursor or a human MLP precursor protein, a polypeptide having an amino acid sequence represented by SEQ ID NO: 12 is represented by a mouse MLP precursor or a mouse MLP precursor protein and SEQ ID NO: 47. Polypeptides having an amino acid sequence that is referred to as rat MLP precursors or rat MLP precursors are referred to collectively as MLP precursors.
[50] As a polypeptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 of the present invention, for example, an amino acid having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 and represented by SEQ ID NO: 24 Preferred are polypeptides having properties substantially homogeneous with the polypeptide having the sequence.
[51] As the polypeptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6, for example, an amino acid sequence having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 and represented by SEQ ID NO: 6 Preferred are polypeptides having properties substantially homogeneous with those having a polypeptide.
[52] Substantially homogeneous properties include, for example, secreted and act as a liquid factor. Substantially homogeneous indicates that these properties are qualitatively homogeneous. Therefore, although properties such as secretory action and solubility are equivalent (e.g., about 0.1 to 100 times, preferably about 0.5 to 10 times, more preferably 0.5 to 2 times), the degree of these properties, Quantitative factors such as molecular weight may be different.
[53] Further, a polypeptide containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO: 6, and more specifically, 1, for example, 1 of the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO: 6. Or an amino acid sequence in which two or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are deleted, ② SEQ ID NO: 24 or 1 or 2 or more (preferably about 1 to 40, more preferably about 1 to 30, more preferably about 1 to 10, particularly preferably, amino acid sequence represented by SEQ ID NO: 6 Amino acid sequence to which several (1 to 5) amino acids have been added, ③ one or two or more (preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO: 6 Degree, more preferably about 1 to 10, more preferably some (1 to 5) amino acid sequence inserted, ④ 1 or the amino acid sequence represented by SEQ ID NO: 24 or SEQ ID NO: 6 or An amino acid sequence in which two or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5) amino acids are substituted with other amino acids or So-called muteins, such as polypeptides containing amino acid sequences, are also included.
[54] When the amino acid sequence is inserted, deleted, or substituted as described above, the position at which the amino acid sequence is inserted, deleted, or substituted is not particularly limited, but is represented by SEQ ID NO: 24, SEQ ID NO: 26, and SEQ ID NO: 49, respectively. Positions other than the amino acid sequence common to the amino acid sequence to become, position other than the amino acid sequence common to the amino acid sequence shown by sequence number of SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 47 are mentioned.
[55] In the present specification, a polypeptide has an N terminus (amino terminus) and a C terminus (carboxyl terminus) at the left end according to the convention of peptide notation. SEQ ID NO: The polypeptides of the present invention including the polypeptide containing the amino acid sequence shown in 24 is a C-terminal is normally carboxyl group (-COOH) or carboxylate (-COO ^-), but, the C-terminus may be an amide (-CONH ₂₎ or It may also be an ester (-COOR).
[56] Here, as the R of the ester, for example, C _1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl or n-butyl, such as C _3-8 cycloalkyl groups such as cyclopentyl, cyclohexyl and the like, such as phenyl, α C _6-12 aryl groups such as naphthyl, such as C _7-14 such as phenyl-C _1-2 alkyl groups such as benzyl, phenethyl, or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl, etc. In addition to the aralkyl group, a pivaloyloxymethyl group or the like which is commonly used as an oral ester is used.
[57] When the polypeptide of the present invention has a carboxyl group (or carboxylate) in addition to the C terminus, the polypeptide of the present invention includes amidated or esterified carboxyl group. In this case, as the ester, for example, the above-mentioned ester of the C terminal is used.
[58] In addition, the amino acid residues of the N-terminal polypeptide of the invention, the protective group of amino group (e. G., Methionine residue), protected (e.g., formyl group, C _1-6 acyl group such as such as C _1-6 alkanoyl such as acetyl group) Pyroglutaminated by N-terminal glutamine residues produced by cleavage in vivo, and substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanies) gavel, etc.) a suitable protecting group (e.g., formyl, acetyl, C _1-6 alkanoyl group such as C _1-6 acyl, etc.) to the composite, such as would have been protected, or a sugar chain binding polypeptides, such as so-called sugar Polypeptides and the like.
[59] As the polypeptide of the present invention or a salt thereof, salts with physiologically acceptable acids (e.g., inorganic acids, organic acids), bases (e.g., alkali metal salts) and the like are used, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, Salts with methanesulfonic acid, benzenesulfonic acid) and the like.
[60] The polypeptide of the present invention or a salt thereof may be prepared by a method for purifying a known polypeptide (protein) from the cells or tissues of the human or warm-blooded animals described above, and cultured a transformant containing DNA encoding the polypeptide described below. It can also manufacture by doing. Moreover, it can also manufacture according to the peptide synthesis method mentioned later.
[61] In the case of manufacturing from human or mammalian tissues or cells, homogenize human or mammalian tissues or cells, extract them with acid, etc., and combine the extracts with chromatography such as reversed phase chromatography, ion exchange chromatography, or the like. It can isolate | separate by tablet.
[62] In the synthesis of the polypeptide of the present invention or salts thereof or amides thereof, commercially available resins for synthesizing polypeptides (proteins) can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylaceto Amide methyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resin Etc. can be mentioned. Such resins are used to condense the amino acids on which the α-amino group and the side chain functional group are appropriately protected on the resin by various condensation methods known per se according to the sequence of the desired polypeptide. At the end of the reaction, the polypeptide is cleaved from the resin, various protective groups are removed, and intramolecular disulfide bond formation reaction is performed in a high dilution solution to obtain the desired polypeptide or these amides.
[63] Regarding the condensation of the above-mentioned protective amino acids, various activating reagents that can be used for polypeptide synthesis can be used, but carbodiimides are particularly preferred. As carbodiimide, DCC, N, N'- diisopropyl carbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide, etc. are used. Activation by these may be performed by adding a protective amino acid directly to the resin together with a racemization inhibitor (eg, HOBt, HOOBt), or after activation of the protective amino acid as a symmetric acid anhydride or HOBt ester or HOOBt ester in advance. have.
[64] As a catalyst used for activation of a protective amino acid or condensation with resin, what can be used for a polypeptide (protein) condensation reaction can be suitably selected from the known solvent. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetoamide, N-methylpyrrolidone and the like, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide and the like Sulfoxides such as seeds, ethers such as pyridine, dioxane, tetrahydrofuran and the like, nitriles such as acetonitrile, propionitrile and the like, esters such as methyl acetate, ethyl acetate and the like, and suitable mixtures thereof. The reaction temperature is appropriately selected from the ranges known to be used in the polypeptide (protein) bond formation reaction, and is usually appropriately selected in the range of about -20 to 50 ° C. Activated amino acid derivatives are usually used in 1.5 to 4 times excess. As a result of the test using the ninhydrin reaction, when condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even if the reaction is repeated, acetylation of the unreacted amino acid with acetic anhydride or acetylamidazole can be performed so as not to affect subsequent reactions.
[65] Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl and tri Fluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc and the like are used.
[66] Carboxyl groups are, for example, alkyl esterifications (e.g., linear, branched or cyclic alkylesters such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.). Aralkyl esterification (e.g., benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide Protection, t-butoxycarbonylhydrazide, tritylhydrazide, and the like.
[67] The hydroxyl groups of serine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl groups and alloyl groups such as benzoyl groups, groups derived from carbonic acid such as benzyloxycarbonyl groups, ethoxycarbonyl groups and the like. Moreover, suitable groups for etherification are, for example, benzyl group, tetrahydropyranyl group, t-butyl group and the like.
[68] As the protecting group of the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.
[69] As the protecting group for the imidazole of histidine, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
[70] Activated carboxyl groups of the starting materials include, for example, the corresponding acid anhydrides, azides, active esters [alcohols (eg pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl). Alcohol, paranitrophenol, HONB, N-hydroxysuccimid, N-hydroxyphthalimide, ester with HOBt)] and the like. As the amino group of the starting material is activated, for example, the corresponding phosphate amide is used.
[71] The removal (desorption) method of the protecting group includes, for example, catalytic reduction in hydrogen groups in the presence of a catalyst such as Pd-black or Pd-carbon, or also hydrogen fluoride anhydrous, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or the like. Acid treatment with a mixed solution, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine and the like, and reduction with sodium in liquid ammonia are also used. The desorption reaction by the acid treatment is generally carried out at a temperature of about -20 to 40 ° C., but in the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanediti Addition of cationic moisturizers such as ol, 1,2-ethanedithiol and the like is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group for histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group for tryptophan is 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol and the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[72] The protection and protecting group of the functional group which should not be involved in the reaction of the raw materials, the detachment of the protecting group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
[73] As another method of obtaining the amide of the polypeptide, for example, the α-carboxyl group of the carboxy terminal amino acid is protected by amidation, and then the peptide (polypeptide) chain is stretched to the desired chain length on the amino group side, and then the N terminal of the peptide chain. A polypeptide obtained by removing only the deprotection of the α-amino group of and a protecting group of the carboxyl group of the C terminus is prepared, and the two polypeptides are condensed in a mixed solution as described above. The details of the condensation reaction are the same as described above. After purification of the protective polypeptide obtained by condensation, all of the protecting groups can be removed in this manner to obtain the desired crude polypeptide. The crude polypeptide can be purified using a variety of known purification means and lyophilized major fractions to obtain the amide of the desired polypeptide.
[74] In order to obtain an ester of a polypeptide, for example, an α-carboxyl group of a carboxy terminal amino acid can be condensed with desired alcohols to be subjected to amino acid esterification, and then the ester of a desired polypeptide can be obtained in the same manner as the amide of the polypeptide.
[75] Polypeptides or salts thereof of the invention can be prepared according to the synthesis of peptides known per se. As a method for synthesizing the peptide, it can be produced by any of a solid phase synthesis method and a liquid phase synthesis method, for example. In other words, the desired peptide can be prepared by condensing the partial peptide or amino acid and the remaining moiety which can constitute the partial peptide of the present invention, and when the product has a protecting group, by removing the protecting group. As a known condensation method or desorption of a protecting group, the method as described in (1)-(5) below is mentioned, for example.
[76] M. Bodanszky and M.A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
[77] Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
[78] ③ Nozuo Izumiya et al., Fundamentals and Experiments of Peptide Synthesis, Maruzen Corporation (1975)
[79] ④ Harajaki Yajima and Sakakibara Sunpei, Biochemistry Experiment Lecture 1, Protein Chemistry IV, 205, (1977)
[80] ⑤ Supervision of Harajuku Yajima, Development of Genus Medicine, Vol. 14, Peptide Synthesis, Hirogawa Sho 뗑
[81] After the reaction, the polypeptide peptide of the present invention can be purified and isolated by combining conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and the like. When the polypeptide obtained by the above method is a vitreous body, it can be converted into a suitable salt by a known method or a similar method, and when obtained as a salt, it can be converted into a vitreous or another salt by a known method or a similar method. .
[82] The DNA encoding the polypeptide of the present invention may be any type as long as it contains the above-described base sequence encoding the polypeptide of the present invention. In addition, any of genomic DNA, cDNA derived from said cell and tissue, and synthetic DNA may be sufficient.
[83] The vector used for the library may be any of bacteriophage, plasmid, cosmid, and phagemid. In addition, a total RNA or mRNA fraction prepared from the cells and tissues described above can be used and directly amplified by Reverse Transcriptase Polymerase Chain Reaction (abbreviated as RT-PCR method).
[84] As the DNA encoding the polypeptide of the present invention, for example, DNA having a nucleotide sequence represented by SEQ ID NO: 23 or a base sequence represented by SEQ ID NO: 23 and a nucleotide sequence hybridized under stringent conditions, Any DNA having a DNA encoding a polypeptide having a substantially homogeneous property (eg, immunogenicity, etc.) with the polypeptide of the present invention, and encoding a polypeptide having a property substantially homogeneous with the polypeptide of the present invention. You can do it.
[85] As the DNA containing the nucleotide sequence represented by SEQ ID NO: 23, a DNA or the like containing the nucleotide sequence represented by SEQ ID NO: 4 is used.
[86] As the DNA which can be hybridized under the nucleotide sequence represented by SEQ ID NO: 23 and under high stringent conditions, for example, the base sequence represented by SEQ ID NO: 23 is about 60% or more, preferably about 70% or more, more Preferably, DNA containing a nucleotide sequence having a homology of about 80% or more is used.
[87] Further, the DNA can be hybridized under the nucleotide sequence represented by SEQ ID NO: 23 and under highly stringent conditions, specifically, the DNA containing the nucleotide sequence represented by SEQ ID NO: 25 or represented by SEQ ID NO: 25. Has a base sequence that hybridizes under the same conditions as that of the nucleotide sequence, and has a DNA encoding a polypeptide having a property (e.g., immunogenicity) substantially homogeneous with the polypeptide of the present invention. And DNAs encoding polypeptides having substantially homogeneous properties with the polypeptides.
[88] As the DNA which can be hybridized under the nucleotide sequence represented by SEQ ID NO: 25 and under high stringent conditions, for example, the base sequence represented by SEQ ID NO: 25 is about 60% or more, preferably about 70% or more, more Preferably, a DNA containing a nucleotide sequence having a homology of about 80% or more is used, and specifically, a DNA containing a nucleotide sequence represented by SEQ ID NO: 10 is used.
[89] Further, the DNA can be hybridized under the nucleotide sequence represented by SEQ ID NO: 23 and under highly stringent conditions, specifically, the DNA containing the nucleotide sequence represented by SEQ ID NO: 48 or represented by SEQ ID NO: 48. A DNA sequence encoding a polypeptide having a nucleotide sequence which is hybridized under the same conditions as that of the polypeptide of the present invention and substantially homogeneous with the polypeptide of the present invention (eg, immunogenicity), and the like. And DNAs encoding polypeptides having substantially homogeneous properties with the polypeptides.
[90] As the DNA which can be hybridized under the stringent conditions and the stringent conditions represented by SEQ ID NO: 48, for example, at least about 60%, preferably at least about 70%, more preferably the base sequence represented by SEQ ID NO: 48 Preferably, a DNA containing a nucleotide sequence having a homology of about 80% or more is used, and specifically, a DNA containing a nucleotide sequence represented by SEQ ID NO: 41 or SEQ ID NO: 46 is used.
[91] Hybridization can be carried out according to a method known per se or a method corresponding thereto, such as the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). In addition, when using a commercially available library, it can carry out according to the method described in the attached instruction manual. More preferably, it can carry out according to high stringent conditions.
[92] Hystergent conditions refer to conditions of, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C.
[93] As DNA encoding the polypeptide of the present invention having an amino acid sequence represented by SEQ ID NO: 24, a DNA having a nucleotide sequence represented by SEQ ID NO: 23, or the like, a polypeptide of the present invention having an amino acid sequence represented by SEQ ID NO: 6 As DNA encoding the DNA, DNA having a nucleotide sequence represented by SEQ ID NO: 4 and the like, and DNA encoding the polypeptide of the present invention having an amino acid sequence represented by SEQ ID NO: 26 are represented by SEQ ID NO: 25. As a DNA which has the DNA etc. which encode the polypeptide of this invention which has an amino acid sequence shown by SEQ ID NO: 12, DNA etc. which have a base sequence shown by SEQ ID NO: 10 replace the amino acid sequence shown by SEQ ID NO: 49, and the like. As DNA which codes the polypeptide of this invention which has, the DNA which has a nucleotide sequence represented by SEQ ID NO: 48, etc. is represented by SEQ ID NO: 47. Include DNA having the nucleotide sequence shown in 46 is used: SEQ ID NO: As the DNA encoding the polypeptide of the invention having the amino acid sequence.
[94] As a means of cloning DNA which completely encodes the polypeptide of the present invention, a part of the polypeptide of the present invention is obtained by using a synthetic DNA primer having a partial sequence of the polypeptide of the present invention and amplifying by PCR or combining a suitable vector with a suitable vector. Or by hybridization with those labeled using DNA fragments or synthetic DNA encoding the entire region. The hybridization method can be performed, for example, according to the method described in Molecular Cloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method described in the attached instruction manual.
[95] DNA sequence conversion is performed using PCR or a known kit such as Mutan ^TM -G (Takara Shuzo Co., Ltd.), Mutan ^TM -K (Takara Shuzo Co., Ltd.), etc. It can carry out according to the method known per se or the method according to these.
[96] The DNA encoding the cloned polypeptide can be digested with restriction enzymes or added with a linker as desired or as desired. This DNA may have ATG as a translation start codon at the 5 'end side, and may have TAA, TGA or TAG as a translation end codon at the 3' end side. These translation start codons and translation end codons can also be added using a suitable synthetic DNA adapter.
[97] Expression vectors of the polypeptides of the invention can be prepared, for example, by (a) cutting out the desired DNA fragment from the DNA encoding the polypeptide of the invention and (b) linking the DNA fragment downstream of the promoter in a suitable expression vector. .
[98] As vectors, bacteriophages such as E. coli-derived plasmids (e.g. pBR322, pBR325, pUC12, pUC13), Bacillus plasmids (e.g. pUB110, pTP5, pC194), plasmids derived from yeast (e.g. pSH19, pSH15), lambda phage, PA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used in addition to animal viruses such as retrovirus, vaccinia virus and baculovirus.
[99] As the promoter used in the present invention, any promoter can be used as long as it is a promoter appropriate for the host used for gene expression. For example, when an animal cell is used as a host, an SRα promoter, an SV40 promoter, an LTR promoter, a CMV promoter, an HSV-TK promoter, β-actin, and the like can be given.
[100] Of these, it is preferable to use a CMV (cytomegalovirus) promoter, an SRα promoter, and the like. If the host is E. coli, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, etc., if the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. In the case of PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.
[101] In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter abbreviated as SV40ori) and the like can be used as the expression vector. As selection markers, for example, dihydrofolate reductase (hereinafter abbreviated dhfr) gene [mesotrexate (MTX) resistance], ampicillin resistance gene (hereinafter abbreviated Amp ^r ), neomycin resistance gene (hereinafter Neo ^r) Abbreviation, Geneticin resistance), etc. are mentioned. In particular, when dhfr gene deletion Chinese hamster cells are used and the dhfr gene is used as a selection marker, recombinant cells can also be selected by a medium that does not contain thymidine.
[102] If necessary, a signal sequence suitable for the host is added to the N terminal side of the polypeptide of the present invention. If the host is E. coli, PhoA, signal sequence, OmpA, signal sequence, etc. If the host is Bacillus, α-amylase, signal sequence, subtilisin, signal sequence, etc., if the host is yeast MFα, signal sequence, SUC2, signal sequence, and the like, when the host is an animal cell, insulin, signal sequence, α-interferon, signal sequence, antibody molecule, signal sequence, or the like can be used, respectively.
[103] A transformant can be produced using a vector containing a DNA encoding the polypeptide of the present invention thus constructed.
[104] As the host, for example, E. coli, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
[105] Specific examples of the genus Escherichia coli include, for example, Escherichia coli K12.DH1 [Proc. Natl. Acad. Sci, USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 ( 1981), JA221 (Journal of Molecular Biology), Vol. 120, 517 (1978), HB101 [Journal of Molecular Biology, Vol. 41, 459 (1969)], C600 [Genetics, Vol. 39, 440 (1954)]. Etc. are used.
[106] As Bacillus genus, Bacillus subtilis MI114 [Gene, Vol. 24, 255 (1983)], 207-21 [Journal of Biochemistry, Vol. 95, 87 (1984)] and the like are used.
[107] As the yeast, for example Saccharomyces cerevisiae AH22, AH22R ^- this, NA87-11A, DKD-5D, 20B -12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastrois KM71 and the like are used.
[108] If the Examples of the insect cells, for example the virus is AcNPV, the larvae derived from the coin cell of beet armyworm (Spodoptera frugiperda cell; Sf cells), Trichoplusia ni midgut derived MGI cells, Trichoplusia ni egg-derived High Five ^TM in the cells, Mamestra brassicae cells derived from Or cells derived from Estigmena acrea. When the virus is BmNPV, silkworm-derived chemotactic cells (Bombyx mori N cells; BmN cells) and the like are used. As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf21 cells (above, Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used.
[109] Examples of insects include silkworm larvae and the like (Maeda et al., Nature, 315, 592 (1985)).
[110] Animal cells as e.g. monkey cell COS-7, Vero, difference varnish hamster cell CHO (hereinafter, CHO cells, abbreviated), dhfr gene-deficient difference varnish hamster cell CHO (hereinafter, CHO (dhfr ^-) cell abbreviated), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.
[111] To transform Escherichia coli, for example, Proc. Natl. Acad.Sci. USA, 69, 2110 (1972) or Gene, 17, 107 (1982) can be carried out according to the method described.
[112] To transform Bacillus bacteria, for example, it can be carried out according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979) and the like.
[113] To transform yeast, see, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) etc. can be implemented.
[114] In order to transform an insect cell or an insect, it can carry out according to the method described, for example in Bio / Technology, 6, 47-55 (1988) etc.
[115] In order to transform animal cells, for example, cell engineering annex 8 renal cell engineering protocol. 263-267 (1995) (issued by Shusheng), Virology, Vol. 52, 456 (1973) and the like.
[116] In this way, a transformant transformed with an expression vector containing DNA encoding the polypeptide can be obtained.
[117] When the host cultures transformants of Escherichia spp. And Bacillus spp., A medium used for the cultivation is a liquid medium, and among them, a carbon source, a nitrogen source, an inorganic material and the like necessary for the growth of the transformant are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose and the like, as a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor peptone, casein, yeast extract, meat extract, soybean bag, potato extract, etc. Calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
[118] As a medium for culturing Escherichia spp., For example, M9 medium containing glucose and kazamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. To this, a drug such as 3β-indolylacrylic acid may be added, for example, to efficiently operate the promoter.
[119] When the host is Escherichia spp., The culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and aeration or agitation may be added if necessary.
[120] When the host is Bacillus bacteria, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration or agitation may be added if necessary.
[121] When cultivating a transformant in which the host is a yeast, as a medium, for example, Burkholder minimal medium [Bostian, K.L. et al., Proc. Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)] or SD medium containing 0.5% kazamino acid [Bitter, G.A. et al., Proc. Natl. Acad. Sci. USA, Vol. 81, 5330 (1984). The pH of the medium is preferably adjusted to about 5-8. Cultivation is normally performed at about 20-35 degreeC for about 24 to 72 hours, and aeration or stirring is added as needed.
[122] When the host cultures insect cells or transformants that are insects, as a medium, additives such as 10% bovine serum immobilized to Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) are appropriately added. Thing is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Cultivation is normally performed at about 27 degreeC for about 3 to 5 days, and aeration and stirring are added as needed.
[123] When cultivating a transformant in which the host is an animal cell, as a medium, for example, MEM medium containing about 5-20% of fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396] (1959)], RPMI 1640 badge [The Journal of the American Medical Association] Volume 199, 519 (1967), 199 badge [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)], and the like. . The pH is preferably about 6-8. Cultivation is normally performed at about 30-40 degreeC for about 15 to 60 hours, and aeration and stirring are added as needed.
[124] As described above, the polypeptide of the present invention can be produced in the cell extracellular or in the cell.
[125] In order to isolate | separate and purify the polypeptide of this invention from the said culture, it can carry out by the following method, for example.
[126] When the polypeptide of the present invention is extracted from the cultured cells or cells, after the culture, the cells or cells are collected by a known method and suspended in a suitable buffer, and the cells or cells are destroyed by ultrasonication, lyozyme and / or lyolysis. For example, a method of obtaining a crude extract of a polypeptide by centrifugation or filtration is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100TM. When the polypeptide is secreted in the culture, the supernatant is collected by separating the cells or cells and the supernatant by a method known per se after the end of the culture.
[127] Purification of the polytetide contained in the culture supernatant or extract obtained in this way can be carried out by appropriately combining the separation and purification methods known per se. These known separation and purification methods use a solubility such as salting out or solvent precipitation, a method using mainly molecular weight differences such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, and ion exchange. Method using the difference of load such as chromatography, Method using specific affinity such as affinity chromatography, Method using the hydrophobic difference such as reversed phase high performance liquid chromatography, Method using the difference of isoelectric point such as isoelectric electrophoresis Etc. are used.
[128] When the thus obtained polypeptide is obtained as a vitreous body, it can be converted into a salt by a method known per se or a method similar thereto, and conversely, when it is obtained as a salt, it can be converted into a vitreous or another salt by a method known per se or a method equivalent thereto. have.
[129] In addition, by modifying the polypeptide produced by the recombinant before or after purification, a suitable protein modification enzyme or protease can be optionally added to modify or partially remove the polypeptide. As these enzymes, for example, trypsin, chymotrypsin, arginyl end peptidase, protein kinase, glycoxidase and the like are used.
[130] The presence of the polypeptide of the present invention or a salt thereof produced in this way can be measured by enzyme immunoassay or Western blot analysis using a specific antibody.
[131] The antibody to the polypeptide of the present invention or a salt thereof may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the polypeptide of the present invention or a salt thereof.
[132] Antibodies to the polypeptide of the present invention or salts thereof can be prepared by using the polypeptide of the present invention as an antigen and according to a method for producing an antibody or antiserum known per se.
[133] [Production of Monoclonal Antibody]
[134] (a) Preparation of monoclonal antibody producing cells
[135] The polypeptide of the present invention or a salt thereof is administered on its own or together with a carrier and a diluent at a site capable of producing an antibody by administration to a warm blooded animal. In order to improve antibody production ability at the time of administration, you may administer a complete Freund's adjuvant or an incomplete Freund's adjuvant. Administration is usually about once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animals used include monkeys, rabbits, dogs, morphs, mice, rats, sheep, goats, and chickens, but mice and rats are preferably used.
[136] In the preparation of monoclonal antibody-producing cells, an individual whose antibody value is known from an antigen-warmed animal, such as a mouse, is selected to collect spleen or lymph nodes 2 to 5 days after the final immunization, and the antibody contained therein. Monoclonal antibody producing hybridomas can be prepared by fusing the production cells with myeloma cells from homologous or heterologous animals. The measurement of the antibody titer in antiserum can be performed, for example, by reacting a labeling polypeptide described below with an antiserum and then measuring the activity of the labeling agent bound to the antibody. Fusion operations can be carried out according to known methods, such as Keller and Milstein's method [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG), Sendai virus, and the like, but PEG is preferably used.
[137] Myeloma cells include, for example, myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, AP-1, and the like. P3U1 is preferably used. The preferred ratio of the number of antibody producing cells (splenocytes) to the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably at about 30 to 37 ° C. for about 1 to 10 minutes.
[138] Several methods can be used for screening monoclonal antibody-producing hybridomas, for example, by adding the hybridoma culture supernatant to a solid phase (e.g., microplate) on which the polypeptide antigen is adsorbed directly or with a carrier, followed by radioactive substances or enzymes. Anti-immunoglobulin antibody labeled with the back (anti-mouse immunoglobulin antibody is used when the cell used for cell fusion is mouse) or protein A to detect monoclonal antibody bound to the solid phase, anti-immunoglobulin antibody Or a method of detecting a monoclonal antibody bound to a solid phase by adding a hybridoma culture supernatant to a solid phase onto which Protein A is adsorbed, and adding a polypeptide labeled with a radioactive substance or an enzyme.
[139] The selection of monoclonal antibodies can be carried out according to a method known per se or equivalent thereto. Usually, it can carry out in the medium for animal cells which added HAT (hippoxanthin, aminopterin, thymidine). As a medium for selection and breeding, any medium can be used as long as it can grow hybridomas. For example, RPMI 1640 medium containing about 1-20%, preferably about 10-20% fetal bovine serum, GIT medium containing about 1-10% fetal bovine serum (Wakoyan Yakuo Kogyo Co., Ltd.) or Serum-free medium for hybridoma culture (SFM-101, Nissui Seiyaku Co., Ltd.) may be used. The culture temperature is usually about 20 to 40 ℃, preferably about 37 ℃. Incubation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Cultivation can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the antibody titer.
[140] (b) Purification of Monoclonal Antibodies
[141] Separation and purification of monoclonal antibodies is known per se, such as separation and purification of immunoglobulins (e.g., salting out, alcohol precipitation, isoelectric point precipitation, electrophoresis, desorption by ion exchangers (e.g. DEAE)). , Ultracentrifugation method, gel filtration method, antigen-binding solid phase or a specific purification method in which only an antibody is collected by an active adsorbent such as Protein A or Protein G, and the binding is dissociated to obtain the antibody.
[142] [Production of Polyclonal Antibody]
[143] The polyclonal antibodies of the present invention can be prepared according to methods known per se or equivalent thereto. For example, an immune antigen (polypeptide antigen) itself or a complex of a carrier protein thereof is prepared and immunized to a warm-blooded animal in the same manner as the preparation of the monoclonal antibody, and the antibody against the polypeptide of the present invention or a salt thereof It can manufacture by extracting a content and performing separation purification of an antibody.
[144] Regarding the complex of immune antigen and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio of carrier and hapten may be any ratio at any rate as long as the antibody can be efficiently immunized against the hapten immunized by crosslinking to the carrier. Although crosslinking may be carried out, for example, a method of coupling bovine serum albumin, bovine silo globulin, hemocyanin and the like in a weight ratio of about 0.1 to 20, preferably about 1 to 5 with respect to hapten 1 is used.
[145] In addition, various condensates may be used for coupling the hapten and the carrier, but an active ester reagent containing glutalaldehyde, carbodiimide, maleimide active ester, thiol group, and dithiolpyridyl group is used.
[146] The condensation product is administered by itself or with a carrier or diluent at the site where the antibody can be produced for warm-blooded animals. In order to improve antibody production ability at the time of administration, you may administer a complete Freund's adjuvant or an incomplete Freund's adjuvant. Administration is usually about once every 2 to 6 weeks, in total about 3 to 10 times.
[147] The polyclonal antibody can be obtained from blood, ascites, preferably blood of a warm-blooded animal immunized by the above method.
[148] The measurement of the polyclonal antibody titer in antiserum can be measured similarly to the measurement of the antibody titer in the said antiserum. Isolation and purification of the polyclonal antibody can be carried out according to the isolation and purification method of the immunoglobulin the same as the isolation and purification of the monoclonal antibody.
[149] Antisense DNA having a nucleotide sequence complementary or substantially complementary to a DNA encoding a polypeptide of the present invention (hereinafter also referred to as the DNA of the present invention) may be a sequence complementary or substantially complementary to the DNA of the present invention. And any antisense DNA may be used as long as it has a function of inhibiting the expression of this DNA.
[150] The base sequence substantially complementary to the DNA of the present invention is at least about 70%, preferably at least about the entire or partial sequence of the base sequence complementary to the DNA of the present invention (ie, the complementary chain of the DNA of the present invention). Base sequences having at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology, and the like. In particular, at least about 70% of the complementary chain of the nucleotide sequence of the portion encoding the N-terminal region of the polypeptide of the present invention (e.g., the nucleotide sequence near the start codon) among the entire nucleotide sequence of the complementary chain of the DNA of the present invention is preferred. Preferably, antisense DNA having a homology of at least about 80%, more preferably at least about 90%, most preferably at least about 95%. These antisense DNA can be manufactured by a well-known DNA synthesis apparatus.
[151] When the polypeptide of the present invention has a signal peptide, it is efficiently secreted out of the cell and exhibits important biological activities for signal transduction, self-defense, and the like as a liquid factor.
[152] Hereinafter, a polypeptide of the present invention or a salt thereof (hereinafter abbreviated as a polypeptide of the present invention), a DNA encoding a polypeptide of the present invention (hereinafter abbreviated as a DNA of the present invention), a polypeptide of the present invention or a salt thereof The use of an antibody (hereinafter abbreviated as an antibody of the present invention) and antisense DNA will be described.
[153] (1) The polypeptide of the present invention is specifically expressed in cartilage tissue and can be used as a tissue marker. In other words, it is useful as a marker for detection of tissue differentiation, pathology, cancer metastasis, and the like. It can also be used for fractionation of corresponding receptors, ligands, binding polypeptides, and the like. In addition, panels for high throughput screening known per se can be used to investigate biological activity. In addition, chromosomal mapping can be used to study genetic diseases.
[154] (2) Treatment and prevention of various diseases involving the polypeptide of the present invention
[155] Since the polypeptide of the present invention exists as a humoral factor in vivo (especially cartilage tissue) and has a function of inhibiting cartilage differentiation, the abnormality or deletion of the polypeptide of the present invention or the DNA of the present invention, or the amount of expression is abnormally reduced. Many diseases develop when they do or are hyperactive.
[156] When the DNA or the like of the invention is defective or the amount of expression is abnormally reduced, various diseases such as osteoarthritis, chronic arthrosis, marble and joint disease such as marble disease, pathological angiogenesis, and the like develop.
[157] Therefore, the polypeptide of the present invention and the DNA of the present invention can be used, for example, as a medicine for treating and preventing various diseases such as osteoarthritis, chronic arthritis, marble disease such as marble disease, and pathogenesis of neovascularization.
[158] For example, if there is a patient whose information transmission in a cell is not sufficiently or accurately exerted due to the decrease or deletion of the polypeptide of the present invention in vivo, (a) the DNA of the present invention is administered to the patient to Expressing the polypeptide of the present invention by (b) inserting the DNA of the present invention into the cell to express the polypeptide of the present invention, and then transplanting the cell into the patient or (c) administering the polypeptide of the present invention to the patient By doing so, the patient can fully or accurately play the role of the polypeptide of the present invention.
[159] In the case of using the DNA of the present invention as the therapeutic and prophylactic agent, the DNA is inserted alone or into a suitable vector such as a retroviral vector, an adenovirus vector, an adenovirus-associated viral vector, or the like, and then human Or to a warm-blooded animal. The DNA of the present invention may be administered as it is, or may be formulated with a physiologically acceptable carrier such as an adjuvant for ingestion and administered by a catheter such as a gene gun or a hydrogel catheter.
[160] When the polypeptide of the present invention is used as the therapeutic / prophylactic agent, it is preferable to use a purified product of 90% or more, preferably 95% or more, more preferably 98% or more, and more preferably 99% or more.
[161] Polypeptides of the present invention are, for example, aseptic tablets, capsules, elixirs, microcapsules, etc., if necessary, aseptic solutions or suspensions with orally or water or other pharmaceutically acceptable liquids, etc. It can be used parenterally in the form of injections. For example, the polypeptides of the present invention can be prepared by blending with the physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like into unit dosage forms required for the practice of generally accepted formulations. The active ingredient in these formulations is such that a suitable dose in the range indicated is obtained.
[162] Additives that can be blended into tablets, capsules, etc. include, for example, gelatin, corn starch, tragant, binders such as gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, arginic acid, magnesium stearate and the like. Lubricants, sweeteners such as sucrose, lactose or saccharin, peppermint, acamono (Gaultheria ovatifolia ssp. Adenothrix) oil or flavoring agents such as cherries are used. When the dosage form is a capsule, the material of this type may further contain a liquid carrier such as oil or fat. Sterile compositions for injection may be prescribed in accordance with conventional formulation practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, citron oil and the like.
[163] Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose or other auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable dissolution aids such as alcohol (e.g., ethanol, etc.). ), Polyalcohol (for example, propylene glycol, polyethylene glycol, etc.), nonionic surfactant (for example, polysorbate 80 ^TM , HCO-50, etc.), etc. may be used together. Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a dissolution aid. In addition, buffers (e.g., phosphate buffers, sodium acetate buffers, etc.), analgesics (e.g., benzalkonium chloride, procaine, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives (e.g., benzyl alcohol, Phenol, etc.), antioxidant, etc. may be combined. The prepared injection solution is usually filled in a suitable ampoule.
[164] The vector into which the DNA of the present invention is inserted is also formulated in the same manner as above, and is usually used parenterally.
[165] Because the preparations thus obtained are safe and low toxicity, they can be administered, for example, to humans or warm-blooded animals (rats, mice, morphotes, rabbits, birds, sheep, pigs, cattle, horses, cats, dogs, monkeys, etc.).
[166] The dosage of the polypeptide of the present invention varies depending on the target disease, the subject to be administered, the administration route, and the like. However, when the polypeptide of the present invention is orally administered, for example, for the purpose of treating bone and joint diseases, it is generally administered to an adult (60 kg) a day. The polypeptide of the present invention is administered about 1 mg to 1000 mg, preferably about 10 to 500 mg, more preferably 10 to 200 mg. In the case of parenteral administration, the single dose of the polypeptide of the present invention depends on the subject and the target disease, but for example, the polypeptide of the present invention is administered to an adult (60 kg) in the form of an injection for the purpose of treating bone and joint diseases. In this case, it is preferable to administer the polypeptide by injection of about 1 to 1000 mg, preferably about 1 to 200 mg, more preferably about 10 to 100 mg per day to the affected area. For other animals, the amount converted to 60 kg can be administered.
[167] (2) Screening of Pharmaceutical Candidate Compounds for Disease
[168] Since the polypeptide of the present invention exists as a liquid factor in vivo (especially in cartilage tissue and the like) and has a function of inhibiting cartilage differentiation, a compound or a salt thereof for promoting the function of the polypeptide of the present invention is for example deformed arthrosis, chronic It can be used as a medicine for treatment and prevention of osteoarthritis, bone and joint diseases such as marble disease, pathological neovascularization and the like.
[169] On the other hand, a compound or a salt thereof that inhibits the function of the polypeptide of the present invention is a disease resulting from the excessive production of the polypeptide of the present invention, such as deformed arthrosis, chronic arthrosis, osteoplasia, osteoporosis, fracture, femoral head necrosis, cartilage formation It can be used as a medicament for the treatment and prevention of bone and joint diseases such as insufficiency and the disease of neovascularization.
[170] Thus, the polypeptides of the invention are useful as reagents for the screening of compounds or salts thereof that promote or inhibit the function of the polypeptides of the invention.
[171] That is, the present invention
[172] (1) A compound which promotes the function of the polypeptide of the present invention or a salt thereof, or a salt thereof (hereinafter abbreviated as an accelerator) or a polypeptide of the present invention or a salt thereof, characterized by using the polypeptide of the present invention or a salt thereof Provided are a method for screening a compound to inhibit (hereinafter abbreviated as an inhibitor).
[173] The screening kit of the present invention contains the polypeptide of the present invention or a salt thereof.
[174] Compounds or salts thereof obtained using the screening method or kit for screening of the present invention are selected from, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. Compounds that promote or inhibit the function of the polypeptides of the invention.
[175] As a salt of this compound, the same salt as the salt of the polypeptide of the present invention described above is used.
[176] When the compound obtained using the screening method of the present invention or the kit for screening is used as the treatment / prophylactic agent described above, it can be carried out according to conventional means. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used in the same manner as the medicines containing the polypeptide of the present invention described above.
[177] Because the preparations thus obtained are safe and low toxicity, they can be administered orally or parenterally, for example, to humans or warm-blooded animals (mouse, rats, rabbits, sheep, pigs, cattle, horses, birds, cats, dogs, monkeys, etc.). Can be.
[178] The dosage of the compound or its salt varies depending on its action, target disease, target to be administered, administration route, and the like, but when orally administering a compound that promotes the function of the polypeptide of the present invention, for example, for the treatment of bone and joint diseases, Generally, an adult (60 kg) is administered about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably 1.0 to 20 mg of the compound per day. In the case of parenteral administration, the single dose of this compound varies depending on the subject to be administered and the target disease. For example, a compound which promotes the function of the polypeptide of the present invention for the purpose of treating bone and joint diseases is usually injected in the form of an adult. When administered to (60 kg), it is preferable to administer about 0.01 to 30 mg of the compound per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg intravenously. Other animals may be administered in an amount equivalent to 60 kg.
[179] On the other hand, in the case of oral administration of a compound that inhibits the function of the polypeptide of the present invention, it is generally about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably 1.0 to 1 day to an adult (60 kg) per day. 20 mg is administered. In the case of parenteral administration, the single dose of this compound varies depending on the subject to be treated and the disease to be treated.However, when a compound that inhibits the function of the polypeptide of the present invention is administered to an adult (60 kg) in the form of an injection, It is preferable to administer about 0.01 to 30 mg of the compound, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg by intravenous injection. Other animals may be administered in an amount equivalent to 60 kg.
[180] (3) Quantification of the polypeptide or salt thereof of the present invention
[181] Since the antibody against the polypeptide of the present invention (hereinafter abbreviated as the antibody of the present invention) can specifically recognize the polypeptide of the present invention, the quantification of the polypeptide of the present invention in a test solution, in particular, by sandwich immunoassay. It can be used.
[182] That is, the present invention
[183] (Iii) a competitive reaction between the antibody of the present invention and the test solution and the labeled polypeptide of the present invention to determine the proportion of the labeled polypeptide of the present invention bound to the antibody to determine the ratio of the polypeptide of the present invention to the test solution. Assay,
[184] (Ii) a test solution characterized by measuring the activity of a labeling agent on an insoluble carrier after reacting the test solution with the antibody of the present invention insolubilized on a carrier and the other antibody of the present invention labeled or simultaneously. It provides a quantification method of the polypeptide of the present invention in.
[185] In addition, the monoclonal antibody to the polypeptide of the present invention (hereinafter referred to as the monoclonal antibody of the present invention) can be used to quantify the polypeptide of the present invention, and detection by tissue staining or the like can also be performed. have. For these purposes, the antibody molecule itself may be used or the F (ab ') ₂ , Fab' or Fab fraction of the antibody molecule may also be used.
[186] Assay of the polypeptide of the present invention using the antibody of the present invention is not particularly limited, but the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (e.g., the amount of polypeptide of the present invention) in the solution to be measured is determined. Or as long as it is a measurement method which detects by a physical means and computes it from the standard curve manufactured using the standard liquid containing a known amount of antigen, you may use what kind of measurement method. For example, the nephrometry, the contention method, the immunometric method and the sandwich method are preferably used, but it is particularly preferable to use the sandwich method described later in view of sensitivity and specificity.
[187] As a labeling agent used in the measurement method using a labeling substance, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, etc. are used, for example. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable and highly inactive one is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluoresceen isothiocyanate and the like are used. As the light emitting material, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. In addition, a biotin-avidin system can also be used for binding an antibody or antigen to a labeling agent.
[188] In the insolubilization of an antigen or an antibody, physical adsorption may be used, or the method of using the chemical bond normally used for insolubilizing and immobilizing a polypeptide or an enzyme or the like may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicone, and glass.
[189] In the sandwich method, the test solution is reacted with the insoluble monoclonal antibody of the present invention (first reaction), and further reacted with the other labeled monoclonal antibody of the present invention (second reaction), and then on an insoluble carrier. By measuring the activity of the labeling agent, the amount of polypeptide of the present invention in the test liquid can be quantified. The primary reaction and the secondary reaction may be carried out in the reverse order, at the same time, or may be performed at a time offset. Labeling agents and insolubilization methods can be carried out in accordance with the above. In the sandwich immunoassay, the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies may be used for the purpose of improving the measurement sensitivity.
[190] In the measurement method of the polypeptide of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used for the first reaction and the second reaction is preferably an antibody having a different site to which the polypeptide of the present invention is bound. That is, the antibody used in the first reaction and the second reaction, for example, when the antibody used in the second reaction recognizes the C end of the polypeptide of the present invention, the antibody used in the first reaction is preferably, in addition to the C end, For example, antibodies that recognize the N end are used.
[191] The monoclonal antibodies of the present invention can be used for measurement systems other than the sandwich method, such as competition method, immunometric method, or neprometry.
[192] In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted label antigen (F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), B, The labeled amount of any one of F is measured to quantify the amount of antigen in the test solution. In this reaction method, B / F separation is performed using soluble antibody as an antibody, a liquid phase method using polyethylene glycol, a second antibody against the antibody, and a solidified antibody is used as a first antibody, or the first antibody is soluble. The solidification method using the solidification antibody as a 2nd antibody is used.
[193] In the immunometric method, the antigen and the solidified antigen in the test solution are competitively reacted with a predetermined amount of the labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen and the excess of the labeled antibody in the test solution are reacted. After adding the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Subsequently, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid.
[194] In addition, neprometry measures the amount of insoluble precipitates generated as a result of antigen antibody reactions in the gel or in solution. Even when the amount of antigen in the test liquid is so small that only a small amount of precipitates can be obtained, a laser nephrometry using scattering of the laser is preferably used.
[195] In applying these individual immunoassay methods to the quantitative method of the present invention, setting of special conditions, operations, and the like is not necessary. What is necessary is just to build the measurement system of the polypeptide of this invention, adding the usual technical consideration of a person skilled in the art to the normal conditions and operation method in each method. For details of these general technical means, reference may be made to general articles, books, and the like.
[196] For example, Irie Hiroshi, `` Radio Immuno Assay '' (Kodan, 1974), Irie Hiroshi, `` Sok Radio Immuno Assay '' (Kodan, 1979), Ishikawa Ei et al. Enzyme Immunoassay "(Igaku Shoing, 1978), Ishikawa Eiji Enzyme Immunoassay (2nd Edition) (Igaku Shoing, 1982), Ishikawa Eiji" Enzyme Immunoassay " (3rd edition) (Igakusho, 1987), `` Methods in ENZYMOLOGY '' Vol. 70 (Immunochemical Techniques (Part A)), East-West Vol.73 (Immunochemical Techniques (Part B)), East-West Vol.74 (Immunochemical Techniques (Part C)), East-West Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), East-West Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), East-West Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) Can refer to the like line).
[197] By using the antibody of the present invention as described above, the polypeptide of the present invention can be quantitatively sensitized.
[198] In addition, by quantifying the concentration of the polypeptide of the present invention using the antibody of the present invention, (1) when an increase in the concentration of the polypeptide of the present invention is detected, for example, bone and joint disease (e.g., osteoarthritis, chronic arthrosis, bone) Dysplasia, osteoporosis, fracture, femoral head necrosis, cartilage insufficiency, etc.), pathological neovascularization (e.g., neovascular angiogenesis, etc.) or a high probability of future disease. And (2) when a decrease in the polypeptide concentration of the present invention is detected, for example, a disease of bone and joint disease (e.g., degenerative arthrosis, chronic arthritis, marble disease, etc.), pathological neovascularization (e.g., tumor angiogenesis, etc.). Or you can be diagnosed with a higher chance of getting sick in the future.
[199] Moreover, the antibody of this invention can be used in order to detect the polypeptide of this invention existing in the subject, such as a bodily fluid or a tissue. Moreover, it can use for manufacture of the antibody column used for refine | purifying the polypeptide of this invention, detection of the polypeptide of this invention in each fraction at the time of purification, analysis of the behavior of the polypeptide of this invention in a test cell, etc.
[200] (4) gene diagnostics
[201] The DNA of the present invention can be used as a probe, for example, to encode a polypeptide of the present invention in humans or warm-blooded animals (rats, mice, morphotes, rabbits, birds, sheep, pigs, cattle, horses, cats, dogs, monkeys, etc.). Alternatively, since abnormalities (genetic abnormalities) of mRNA can be detected, they are useful as gene diagnostic agents, such as damage, mutation or expression of the DNA or mRNA, increase or excessive expression of the DNA or mRNA, and the like.
[202] The genetic diagnosis using the DNA of the present invention can be carried out using, for example, known Northern hybridization or PCR-SSCP methods (Genomics, Vol. 5, p. 874 to 879 (1989), Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, p. 2766 to 2770 (1989)), DNA microarrays and the like.
[203] For example, when a decrease in expression is detected by Northern hybridization or DNA microarray, or when a mutation of DNA is detected by PCR-SSCP method or DNA microarray, for example, bone and joint disease (eg, osteoarthritis, chronic It can be diagnosed that the disease is likely to be a disease of aneurysm, marble disease), pathological neovascularization (eg, neovascularization).
[204] (5) Medicine containing antisense DNA
[205] Since antisense DNA capable of complementarily binding to the DNA of the present invention and inhibiting the expression of the DNA can inhibit the function of the polypeptide of the present invention or the DNA of the present invention, for example, expression of the polypeptide of the present invention. Treatment and prevention of diseases caused by excessive (e.g., osteoarthritis, chronic arthrosis, osteoarthritis, osteoplastic insufficiency, osteoporosis, fracture, femoral head necrosis, cartilage insufficiency, pathological angiogenesis such as tumor angiogenesis) Can be used as
[206] The antisense DNA can be used as the therapeutic / prophylactic agent for the various diseases containing the DNA of the present invention described above as the therapeutic / prophylactic agent.
[207] For example, the antisense DNA may be administered alone or in a suitable vector such as a retroviral vector, adenovirus vector, adenovirus associated virus vector and the like, and then administered according to conventional means. This antisense DNA can be administered as it is, or with a physiologically acceptable carrier such as an adjuvant to promote uptake, and administered by a catheter such as a gene gun or a hydrogel catheter.
[208] The antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of DNA of the present invention in tissues or cells.
[209] (6) A medicine containing the antibody of the present invention
[210] Antibodies of the present invention having the action of neutralizing the activity of the polypeptides of the present invention include, for example, diseases caused by overexpression of the polypeptides of the present invention (e.g., deformed arthrosis, chronic arthrosis, osteogenic insufficiency, osteoporosis, fractures, femoral head necrosis) And pathological angiogenesis such as bone and joint diseases such as cartilage insufficiency and neovascularization).
[211] Treatment and prevention of the above-mentioned diseases containing the antibody of the present invention, as a liquid or as a pharmaceutical composition of a suitable formulation, for humans or mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) It can be administered orally or parenterally. The dosage may vary depending on the subject to be administered, the target disease, the symptoms, the route of administration, and the like. For example, for the purpose of treating bone and joint diseases, the dosage of the antibody of the present invention is usually about 0.01 mg to 20 mg / kg, preferably 0.1 It is preferable to administer about 10 mg / kg body weight, more preferably about 0.1 mg to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day, by intravenous injection. Other parenteral and oral administrations may also be administered in an equivalent amount. If your symptoms are particularly heavy, you can increase them according to your symptoms.
[212] The antibody of the present invention can be administered by itself or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as formulations suitable for oral or parenteral administration.
[213] That is, for example, compositions for oral administration include solid or liquid formulations, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions And suspending agents. Such compositions are prepared by methods known per se and contain carriers, diluents or excipients commonly used in the formulation art. For example, lactose, starch, sucrose, magnesium stearate and the like are used as the carrier for purification and excipient.
[214] As the composition for parenteral administration, for example, injections, suppositories, and the like are used, and injections include formulations such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drop injections. Such injections are prepared by dissolving, suspending or emulsifying the antibody or salts thereof in sterile aqueous or oily solutions usually used in injections, according to methods known per se. As aqueous solutions for injection, for example, physiological saline, isotonic solutions containing glucose or other auxiliaries are used, and suitable dissolution aids such as alcohols (e.g. ethanol), polyalcohols (e.g. propylene glycol, polyethylene glycol), nonionic surfactants ( For example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As an oily liquid, sesame oil, soybean oil, etc. are used, for example, benzyl benzoate, benzyl alcohol, etc. are used as a dissolution aid. The prepared injection solution is usually filled in a suitable ampoule A suppository used for rectal administration is prepared by mixing the antibody or its salt with a conventional suppository base.
[215] The oral or parenteral pharmaceutical composition is preferably formulated in dosage unit dosage forms suitable for dosage of the active ingredient. Such dosage unit dosage forms include tablets, pills, capsules, injectables (ampoules), suppositories, etc., typically about 5 to 500 mg, in particular about 5 to 100 mg for injectables, 10 for other dosage forms per dosage form. It is preferable that the said antibody of about 250 mg is contained.
[216] In addition, each of the above-mentioned compositions may contain other active ingredients so long as they do not cause undesirable interaction with the antibody.
[217] (7) DNA transfer animals
[218] The present invention provides a non-human mammal having a DNA encoding an exogenous polypeptide of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a variant DNA thereof (abbreviated as the exogenous displacement DNA of the present invention).
[219] That is, the present invention
[220] (1) a non-human mammal having the exogenous DNA or variant DNA thereof of the present invention,
[221] (2) the animal according to (1), wherein the non-human mammal is a rodent;
[222] (3) the animal according to item (2), wherein the rodent is mouse or rat; and
[223] (4) It provides a recombinant vector which contains the foreign DNA or the mutant DNA of this invention, and can be expressed in a mammal.
[224] The non-human mammal having the exogenous DNA or the mutant DNA of the present invention (hereinafter abbreviated as DNA transfer animal of the present invention) is preferably a non-human mammal for embryos containing embryos, fertilized eggs, sperm and embryonic cells, etc. Calcium phosphate method, electropulse method, lipofection method, agglomeration method, micro injection method, particle drying method, at the stage of embryonic development (more preferably at the stage of single cell or fertilized egg cell and generally before 8 cell stage) It can produce by transferring the DNA of interest by DEAE-dextran method. In addition, the DNA transfer method can be used to transfer foreign DNA of the present invention for somatic cells, organs, tissue cells, etc. of the present invention to be used for cell culture, tissue culture, and the like. The DNA transfer animal of the present invention can also be produced by fusing with a method.
[225] As non-human mammals, for example, cows, pigs, sheep, goats, rabbits, dogs, cats, mormots, hamsters, mice, rats and the like are used. Among them, rodents, which have a relatively short onset and biological cycle in the development of pathological animal model systems, and which are easy to breed, especially mice (e.g., C57BL / 6 strains as pure strains, DBA2 strains such as B6C3F ₁ strains, BDFs as mating strains) ₁ line, B6D2F ₁ line, BALB / C line, ICR line, etc.) or rats (e.g., Wistar, SD, etc.) and the like.
[226] Examples of the "mammal" in the recombinant vector which can be expressed in mammals include humans in addition to the above non-human mammals.
[227] The exogenous DNA of the present invention refers to the DNA of the present invention isolated from the mammal once, not the DNA of the present invention originally possessed by the non-human mammal.
[228] As the mutated DNA of the present invention, a mutant (for example, a mutation, etc.) originally occurred in the DNA nucleotide sequence of the present invention, specifically, a DNA in which base addition, a deletion, substitution with another base, etc. are used, and strange DNA is also used. Included.
[229] As this abnormal DNA, it means the DNA which expresses the abnormal polypeptide of this invention, For example, the DNA which expresses the polypeptide which suppresses normal polypeptide function of this invention, etc. are used.
[230] The foreign DNA of the present invention may be derived from any mammal of the same kind or different types as the target animal. In transferring the DNA of the present invention to a subject animal, it is generally advantageous to use this DNA as a DNA construct bound downstream of a promoter capable of expressing in animal cells. For example, when transferring the human DNA of the present invention, it expresses DNA from various mammals (for example, rabbit, dog, cat, morph, hamster, rat, mouse, etc.) having the DNA of the present invention having high homology with this. DNA transfer mammals that express high DNA of the present invention by microinjecting DNA constructs (e.g., vectors, etc.) bound to the human DNA of the present invention downstream of various promoters that can be introduced into a fertilized egg, such as a mouse fertilized egg, of a target mammal. Animals can be harvested.
[231] Expression vectors of the polypeptide of the present invention include plasmids derived from E. coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as lambda phage, retroviruses such as moronic leukemia virus, animal viruses such as vaccinia virus or baculovirus, and the like. This is used. Especially, the plasmid derived from E. coli, the plasmid derived from Bacillus subtilis, the plasmid derived from yeast, etc. are used preferably.
[232] Examples of promoters that regulate DNA expression include (1) promoters of DNA derived from (1) viruses (e.g., Simian virus, cytomegalovirus, moronic leukemia virus, JC virus, breast cancer virus, polio virus, etc.) and (2) various mammals ( Promoters derived from humans, rabbits, dogs, cats, morphotes, hamsters, rats, mice, etc.) such as albumin, insulin II, uroprakin II, elastase, erythropoietin, endocelin, muscle creatine kinase, Glea Sequestrian Acid Protein, Glutathione S-Transferase, Platelet-derived Growth Factors β, Keratin K1, K10 and K14, Collagen Types I and II, Cyclic AMP-dependent Protein Kinase βI Subunits, Dystrophin, Tartaric Acid Resistant Alkali Phosphata Enzymes, atrial sodium diuretic factor, endothelial receptor tyrosine kinase (commonly called Tie2), sodium potassium Nosine trikinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, Dopamineβ-hydroxylase, thyroid peroxidase (TPO), polypeptide chain extenders 1α (EF-1α), βactin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin base protein, tyroglobulin, Thy-1, Immunoglobulins, H chain variable regions (VNPs), serum amyloid P components, myoglobin, troponin C, smooth muscle α actin, preproenkeparin A, vasopressin and the like are used. Among them, cytomegalovirus promoters, promoters of human polypeptide chain extension factor 1α (EF-1α), human and chicken β-actin promoters, which can be highly expressed systemically, are preferred.
[233] The vector preferably has a sequence (generally called a terminator) for terminating transcription of a messenger RNA of interest in a DNA transgenic mammal, and for example, each DNA sequence derived from a virus and various mammals can be used. Preferably, the SV40 terminator of Simian virus is used.
[234] In addition, the splicing signal of each DNA, an enhancer region, a part of an intron of eukaryotic DNA, etc. may be 5 'upstream of the promoter region, between the promoter region and the translation region, or 3 'Connecting downstream is also possible depending on the purpose.
[235] This translational region can be prepared by conventional DNA engineering techniques, which are DNA constructs that can be expressed in transition animals, downstream of the promoter and upstream of the transcription termination site as desired.
[236] In the fertilized egg cell stage, the transfer of the exogenous DNA of the present invention is ensured to exist in all embryonic and somatic cells of the mammal of interest. The presence of the exogenous DNA of the present invention in the embryonic cells of the harvested animal after DNA transfer means that all of the later generations of the harvested animal maintain the exogenous DNA of the present invention in all of the embryonic cells and somatic cells. An animal progeny of this kind that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in its embryonic and somatic cells as a whole.
[237] Non-human mammals that have transferred the foreign normal DNA of the present invention have been confirmed to stably maintain the foreign DNA by breeding, and can be passaged in a normal breeding environment as the DNA-bearing animal.
[238] The transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be excessively present in all embryonic and somatic cells of the mammal of interest. Excess of the exogenous DNA of the present invention in the embryonic cells of the harvested animals after DNA transfer means that all of the progeny of the harvested animals have an excess of the exogenous DNA of the present invention in all of their embryonic cells and somatic cells. Animal progeny of this kind which inherits the foreign DNA of the present invention has the foreign DNA of the present invention in its embryonic and somatic cells as a whole.
[239] Homozygote animals having introduced DNA on both homologous chromosomes can be obtained, and the male and female animals can be crossed to breed and passage so that all progeny have an excess of this DNA.
[240] The non-human mammal having the normal DNA of the present invention is a model animal of the condition because the normal DNA of the present invention is highly expressed, and thus, the function of endogenous normal DNA is promoted to finally develop hyperfunctionality of the polypeptide of the present invention. It is available. For example, the normal DNA transfer animal of the present invention can be used to elucidate the pathophysiology of the hyperfunction of the polypeptide of the present invention, the disease associated with the polypeptide of the present invention, and to examine methods for treating these diseases.
[241] In addition, since the mammal which has transferred the foreign normal DNA of the present invention has an increased symptom of the free polypeptide of the present invention, the mammal can be used for screening tests of therapeutic drugs for diseases related to the polypeptide of the present invention.
[242] On the other hand, the non-human mammal having the foreign abnormal DNA of the present invention can be safely subcultured in the normal breeding environment as the DNA-retaining animal by confirming that the foreign DNA is safely maintained by mating. In addition, the desired exogenous DNA can be used as a raw material in combination with the plasmid described above. DNA constructs with promoters can be prepared by conventional DNA engineering techniques. Metastasis of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present throughout the embryonic and somatic cells of the mammal of interest. The presence of abnormal DNA of the present invention in embryonic cells of crops after DNA transfer means that all of the progeny of the crops have the abnormal DNA of the present invention in all of their embryos and somatic cells. This kind of animal progeny inheriting the foreign DNA of the present invention has the abnormal DNA of the present invention in all of these embryonic cells and somatic cells. Homozygote animals having both introduced DNAs on both homologous chromosomes can be obtained, and the male and female animals can be bred so that all progeny can be propagated to have this DNA.
[243] In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and the function of endogenous normal DNA may be inhibited, resulting in functional inactivation-induced non-compliance of the polypeptide of the present invention. It can be used as a condition model animal. For example, the abnormal DNA transfer animal of the present invention can be used to clarify the condition of the function-inactivated non-compliance of the polypeptide of the present invention and to examine a method for treating the disease.
[244] In addition, as a specific applicability, the abnormal DNA high expression animal of the present invention is a model for elucidating the dominant negative action of the normal polypeptide by the abnormal polypeptide of the present invention in the functional inactivation of the polypeptide of the present invention. do.
[245] In addition, since the mammal which has transferred the foreign abnormal DNA of the present invention has an increased symptom of the increased polypeptide of the present invention, the mammal can be used for a therapeutic drug screening test for a functional inactive inferiority of the polypeptide of the present invention.
[246] And, as other possibilities of the two kinds of DNA transfer animal of the present invention, for example
[247] ① use as a cell source for tissue culture,
[248] (2) an interpretation of the association of the polypeptide specifically expressed or activated by the polypeptide of the present invention by directly analyzing DNA or RNA in the tissue of the DNA transfer animal of the present invention or by analyzing the polypeptide tissue expressed by the DNA,
[249] (3) cultivate tissue cells with DNA by standard tissue culture techniques, and use them to study cell functions from tissues that are generally difficult to culture;
[250] ④ Screening of drugs for enhancing cellular function by using the cells described in ③ above
[251] (5) It is possible to isolate and purify the mutant polypeptide of the present invention and to prepare antibodies thereof.
[252] Moreover, the clinical symptoms of the disease related to the polypeptide of the present invention can be investigated using the DNA transgenic animal of the present invention, including the functional inactivation of the polypeptide of the present invention, and the like. Detailed pathological findings in each organ can be obtained and contribute to the development of new therapeutic methods and the study and treatment of secondary diseases caused by this disease.
[253] In addition, each organ is taken out from the DNA transfer animal of the present invention and cut, and then, DNA transfer cells liberated by polypeptide (protein) degradation enzymes such as trypsin can be obtained, cultured or cultured of the cultured cells. In addition, the abnormalities and the like can be investigated by examining the specificity, apoptosis, differentiation, or proliferation of the polypeptide-producing cells of the present invention, or their signal transduction mechanisms, and are effective for clarifying the polypeptide of the present invention and its function. It becomes research material.
[254] In order to carry out the development of a therapeutic drug for a disease related to the polypeptide of the present invention, which uses the DNA transfer animal of the present invention and includes the functional inactivation of the polypeptide of the present invention, it is effective and rapid by the above-described test method and quantitative method. It is possible to provide a method for screening a disease therapeutic drug. In addition, the DNA transfer animal of the present invention or the exogenous DNA expression vector of the present invention can be used to examine and develop DNA therapy for diseases involving the polypeptide of the present invention.
[255] (8) knockout animals
[256] The present invention provides a non-human mammal embryonic cell in which the DNA of the present invention is inactivated and a non-human mammal lacking the DNA expression of the present invention.
[257] That is, the present invention
[258] (1) a non-human mammal embryonic cell in which the DNA of the present invention is inactivated,
[259] (2) the embryonic cells according to item (1), wherein the DNA is inactivated by introducing a reporter gene (e.g., a β-galactosidase gene derived from E. coli);
[260] (3) the hepatic stem cell according to item (1) which is neomycin resistant,
[261] (4) the liver cells according to (1), wherein the non-human mammal is a rodent;
[262] (5) the liver cells according to (4), wherein the rodent is a mouse;
[263] (6) this DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated,
[264] (7) This DNA is inactivated by introducing a reporter gene (e.g., β-galactosidase gene from E. coli), and the reporter gene can be expressed under the control of the promoter for the DNA of the present invention (6 The non-human mammal described in the above),
[265] (8) the non-human mammal according to item (6), wherein the non-human mammal is a rodent;
[266] (9) the non-human mammal according to (8), wherein the rodent is a mouse; and
[267] (10) A method for screening a compound or a salt thereof that promotes or inhibits a promoter activity against the DNA of the present invention, which comprises administering a test compound to an animal according to (7) and detecting expression of a reporter gene. do.
[268] Non-human mammalian stem cells in which the DNA of the present invention is inactivated means that the expression of the DNA is inhibited or the polypeptide activity of the present invention is encoded by artificially adding a mutation to the DNA of the present invention of the non-human mammal. Refers to the embryonic cells of non-human mammals (hereinafter abbreviated as ES cells) in which the DNA is substantially free of the expression ability of the polypeptide of the present invention (hereinafter referred to as knockout DNA of the present invention) by substantially losing.
[269] As a non-human mammal, the same thing as the above is used.
[270] The method of artificially adding a mutation to the DNA of the present invention can be carried out by, for example, deleting some or all of the DNA sequence or inserting or replacing other DNAs by genetic engineering techniques. According to these variations, the knock-out DNA of the present invention may be prepared by shifting the codon read structure or disrupting the promoter or exon function.
[271] As a specific example of the non-human mammalian embryonic cells (hereinafter, abbreviated as DNA-inactivated ES cells of the present invention or knock-out ES cells of the present invention) in which the DNA of the present invention is inactivated, for example, the present invention possessed by the desired non-human mammal Isolated DNA and drug resistance gene representing the neomycin resistance gene, hygromycin resistance gene or lacZ (β-galactosidase gene), cat (kuramphenicol acetyl transferase gene) By inserting a reporter gene or the like, the DNA sequence (for example, polyA addition signal, etc.) that destroys exon function or terminates transcription of genes in the intron portion between exons cannot be synthesized. Thereby resulting in a DNA chain having a DNA sequence constructed to destroy the gene (hereinafter abbreviated as targeting vector). For example, a homologous recombination method is introduced into a chromosome of the animal, and the resulting ES cell is used for Southern hybridization analysis or DNA vector on a targeting vector or a targeting vector prepared using a DNA sequence on or near the DNA of the present invention as a probe. It can obtain by interpreting the PCR sequence which used the DNA sequence of the vicinity region other than the DNA of this invention used as a primer, and selecting the knockout ES cell of this invention.
[272] As the original ES cells which inactivate the DNA of the present invention by homologous recombination or the like, for example, those already established as described above may be used, or newly established according to known Evans and Kaufma methods. . For example, ES cells of mouse are generally used in the 129 series of ES cells. However, since the immunological background is not clear, ES cells having a clear immunological genetic background are obtained from the pure strains instead. in the object, for example C57BL / 6 also to the established using the BDF ₁ mice (F ₁ of C57BL / 6 and DBA / 2) improved by the low of the mouse or can chaeran of C57BL / 6, crossing with DBA / 2 It can be used favorably. BDF ₁ mice have a large number of eggs and a healthy egg, and in addition to C57BL / 6 mice in the background, ES cells obtained using these cells were recrossed with C57BL / 6 mice when the condition model mice were harvested (back cross). This can be advantageously used in that the genetic background can be replaced by C57BL / 6 mice.
[273] In general, when ES cells are established, blastocysts at 3.5 days after fertilization are used, but a large number of initial embryos can be efficiently obtained by culturing and using 8-cell embryos and culturing up to blastocysts.
[274] In addition, either ES cells may be used, but male ES cells are generally preferred for producing germline chimeras. In addition, it is desirable to discriminate between males and females as soon as possible in order to reduce the trouble of complicated culture.
[275] As an example of the method for determining the sexes of ES cells, a method of amplifying and detecting a gene of the sex determining region on the Y chromosome by the PCR method is mentioned as an example. Using this method requires about 10 ⁶ cells for conventional karyotyping, while only one colony of ES cells (about 50) is sufficient, so the first order of ES cells at the beginning of culture is sufficient. The selection can be made by male and female discrimination, and the selection of male cells at an early stage allows the labor in the early stages of culture to be greatly reduced.
[276] The second selection can be carried out, for example, by confirming the number of chromosomes according to the G-banding method. The chromosome number of the obtained ES cells is preferably 100% of the normal number. However, in the case where it is difficult due to physical manipulation during establishment, the knockout gene of the ES cell is knocked out, and then the normal cell (for example, the mouse has a chromosome number of 2n = 40). Cloning back to the cells).
[277] The germ cell line thus obtained is usually very proliferative, but it is easy to lose the ability to generate an individual, and it is necessary to pass-through deeply. For example, in a carbon dioxide incubator (preferably about 5% carbon dioxide, about 95% air or about 5% oxygen, about 5% carbon dioxide, in the presence of LIF (1-10000 U / ml) on feeder cells such as STO fibroblasts, Incubation at about 37 ° C. in a passage of about 90% air), and when passaged, for example, trypsin / EDTA solution (typically about 0.001-0.5% trypsin / about 0.1-5 mM EDTA, preferably about 0.1% trypsin / And a method of unicellularizing with about 1 mM EDTA) and seeding on newly prepared feeder cells. Such passage is usually performed every 1-3 days, but when the cells are observed at this time and morphologically abnormal cells are seen, the cultured cells are required to be abandoned.
[278] ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle and myocardium by monolayer culture until high density under appropriate conditions or by floating culture until cell aggregation is formed. [MJEvans and MHKaufman , Nature Vol. 292, p. 154, 1981; G.R.Martin Proc. Natl. Acad. Sci. U.S.A Vol. 78, p.7634, 1981; TCDoetschman et al., Journal of Emblemology and Experimental Morphology, Vol. 87, p. 27, 1985]. The DNA-deficient cells of the present invention obtained by differentiating ES cells of the present invention are in vitro. Is useful in the cytobiological review of polypeptides of the invention.
[279] DNA-deficient non-human mammals of the present invention can be distinguished from normal animals by measuring the amount of mRNA of the animal by a known method and indirectly comparing the expression level.
[280] As said non-human mammal, the same thing as the above is used.
[281] The DNA expression-deficient non-human mammal of the present invention introduces, for example, a targeting vector prepared as described above into a mouse embryonic cell or a mouse egg cell, and the DNA sequence in which the DNA of the present invention of the targeting vector is inactivated is introduced by gene introduction. The DNA of the present invention can be knocked out by homologous recombination which is replaced by DNA of the present invention on chromosomes of mouse embryonic cells or mouse egg cells by recombination.
[282] The cells of the DNA knocked out of the present invention are DNA sequences of Southern hybridization analysis or targeting vectors using DNA sequences on or near the DNA of the present invention as probes, and DNAs of the present invention derived from mice used in the targeting vector. It can judge by the analysis by the PCR method which made the DNA sequence of other nearby region into a primer. In the case of using non-human mammalian embryonic stem cells, a chimera prepared by cloning a cell line in which DNA of the present invention is inactivated by homologous recombination and injecting the cells into a non-human mammal embryo or blastocyst at an appropriate time, for example, 8 cell phase. The abdomen is implanted into the womb of this non-human mammal, which is a fake pregnancy. The resulting animal is a chimeric animal composed of both cells having the DNA site of the present invention and cells having the DNA site of the present invention which are artificially mutated.
[283] When a part of the germ cells of the chimeric animal has a DNA site of the present invention, all tissues in the population obtained by crossing the chimeric individual and a normal individual are composed of cells having the DNA site of the present invention artificially added to the mutation. The individual can be obtained by, for example, judging by the determination of the coat color. The individuals thus obtained are usually heteroexpressed individuals of the polypeptides of the present invention, and the heteroexpressed individuals of the polypeptides of the present invention can be crossed with each other to obtain homo-expressed individuals of the polypeptides of the present invention from these offspring.
[284] In the case of using egg cells, transgenic non-human mammals into which the targeting vector is introduced into the chromosome can be obtained by injecting a DNA solution into the egg cell nucleus, for example, by micro-injection. It can obtain by selecting the thing which has a mutation in the DNA site of this invention.
[285] In this way, the individual in which the DNA of the present invention is knocked out can check that the DNA is also knocked out by crossbreeding, and can breed in the normal breeding environment.
[286] In addition, the acquisition and maintenance of the germline may be carried out according to a conventional method. That is, homozygote animals having the inactive DNA on both homologous chromosomes can be obtained by crossing the male and female animals held by the inactive DNA. The obtained homozygote animal can be efficiently obtained by rearing the mother animal in the state of being a normal individual 1 or homozygote plural. By breeding the male and female of heterozygote animals, homozygotes and heterozygote animals carrying this inactivated DNA are passaged for breeding.
[287] Non-human mammalian stem cells in which the DNA of the present invention is inactivated are very useful for constructing a non-human mammal that lacks the DNA expression of the present invention.
[288] In addition, since the DNA expression-deficient non-human mammal of the present invention lacks various biological activities that can be induced by the polypeptide of the present invention, it can be a model of a disease that causes inactivation of the biological activity of the polypeptide of the present invention. This is useful for determining the cause of these diseases and reviewing the treatment.
[289] (8a) A method for screening a compound having therapeutic and prophylactic effects against diseases caused by defects or damage to the DNA of the present invention
[290] Non-human mammals lacking DNA expression of the present invention include diseases caused by defects or damage to the DNA of the present invention (bone and joint diseases (e.g., degenerative arthrosis, chronic arthritis marble), pathological neovascularization (e.g., neovascularization). And the like, and can be used for screening compounds having therapeutic and prophylactic effects.
[291] That is, the present invention treats and prevents diseases caused by deficiency or damage to the DNA of the present invention, which comprises administering a test compound to a non-human mammal having a lack of DNA expression of the present invention and observing and measuring changes in the animal. Provided are methods for screening compounds or salts thereof that have an effect.
[292] Examples of the DNA expression-deficient non-human mammal of the present invention used in this screening method include the same ones described above.
[293] Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermented products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like. These compounds may be novel compounds or known compounds. have.
[294] Specifically, non-human mammals in which the DNA expression-deficient non-human mammal of the present invention is treated with a test compound are compared with control animals that are not treated. The preventive effect can be tested.
[295] As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection, etc. may be used, and the test animal may be appropriately selected according to the symptoms of the test animal and the properties of the test compound. In addition, the dosage of a test compound can be suitably selected according to the administration method, the nature of the test compound, and the like.
[296] For example, in the case of screening a compound having a therapeutic and prophylactic effect on tumor angiogenesis, cancer cells are transplanted into a DNA-deficient non-human mammal of the present invention, and a test compound is administered before or after the transplantation of cancer cells to administer a tumor marker of the animal. Value and tumor size are measured over time.
[297] The compound obtained by the screening method of the present invention is a compound selected from the above-described test compounds, and diseases caused by damage or breakage of the polypeptide of the present invention (bone and joint diseases (e.g., deformed arthrosis, chronic arthritis marble disease, etc.), It has therapeutic and prophylactic effects on pathological neovascularization (eg, neovascularization) and can be used as a medicine such as safe and low-toxic therapeutics and preventive agents against this disease. Moreover, the compound derived from the compound obtained by the said screening can also be used simultaneously.
[298] Compounds obtained by this screening method may form salts, and salts of such compounds are salts with physiologically acceptable acids (e.g., inorganic acids, organic acids) or bases (e.g., alkali metals), and the like. Acid addition salts which are acceptable are preferred. Such salts include, for example, salts with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, taric acid, malic acid, oxalic acid, benzoic acid). And salts with methanesulfonic acid, benzenesulfonic acid).
[299] The medicine containing the compound obtained by this screening method or its salt can be manufactured similarly to the medicine containing the polypeptide of this invention mentioned above.
[300] Because the preparations thus obtained are safe and low toxicity, they can be administered, for example, to humans or mammals (eg, rats, mice, morphs, rabbits, sheep, pigs, cattle, horses, cats, dogs, monkeys, etc.).
[301] The dosage of the compound or its salts varies depending on the target disease, the subject to be administered, the route of administration, and the like, but when orally administered to the compound, for example, for the treatment of bone and joint diseases, it is generally administered to adults (60 kg body weight) a day. The compound is administered about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose of the compound varies depending on the administration target, the target disease, and the like, but when the compound is usually administered to an adult (60 kg) in the form of an injection for the purpose of treating bone and joint diseases, for example. It is preferable to administer about 0.01 to 30 mg of the compound per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. For other animals, the amount converted to 60 kg can be administered.
[302] (8b) A method for screening a compound that promotes or inhibits promoter activity against the DNA of the present invention
[303] The present invention provides a method for screening a compound or a salt thereof that promotes or inhibits promoter activity against a DNA of the present invention, wherein the DNA expression-deficient non-human mammal is administered to a test compound to detect expression of a reporter gene. to provide.
[304] In the above screening method, the DNA expression-immune non-human mammal of the present invention is inactivated by introducing a reporter gene among the above-described DNA expression-deficient non-human mammals of the present invention, and the reporter gene is inactivated by the DNA of the present invention. Those that can be expressed under the control of a promoter are used.
[305] The same thing as the above-mentioned as a test compound is mentioned.
[306] As the reporter gene, the same ones as described above are used, and β-galactosidase gene (lacZ), soluble alkaline phosphatase gene, luciferase gene and the like are preferable.
[307] In non-human mammals in which the DNA of the present invention is substituted with a reporter gene, since the reporter gene is present under the promoter for the DNA of the present invention, promoter activity is traced by tracing expression of a substance encoded by the reporter gene. Can be detected.
[308] For example, when a part of the DNA region encoding a polypeptide of the present invention is replaced with a β-galactosidase gene (lacZ) derived from E. coli, β-gal instead of the polypeptide of the present invention in a tissue in which the polypeptide of the present invention is expressed. The lactosidase is expressed. Thus, for example, it is a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). By staining with a reagent, the expression state in the animal living body of the polypeptide of the present invention can be easily observed. Specifically, the polypeptide-deficient mouse or tissue fragment thereof of the present invention is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal containing dye at room temperature or near 37 ° C. After 30 minutes to 1 hour of reaction, tissue samples are washed with 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the banding color. Moreover, you may detect mRNA which codes lacZ by a conventional method.
[309] The compound obtained by the above screening method or a salt thereof is a compound selected from the above-described test compounds and is a compound that promotes or inhibits the promoter activity against the DNA of the present invention.
[310] The compound obtained by the above screening method or a salt thereof is a compound selected from the above-described test compounds and is a compound that promotes or inhibits the promoter activity against the DNA of the present invention.
[311] The compounds obtained by this screening method may form salts, and salts with physiologically acceptable acids (e.g., inorganic acids) or bases (e.g., organic acids) and the like are used as the salts of the compounds, in particular physiologically acceptable Acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, taric acid, malic acid, oxalic acid, benzoic acid). And salts with methanesulfonic acid, benzenesulfonic acid).
[312] Compounds or salts thereof that promote the promoter activity against the DNA of the present invention can promote the expression of the polypeptide of the present invention and promote the function of the polypeptide, such as bone and joint diseases (e.g., degenerative arthrosis, chronic arthritis marble) ), And is useful as a medicament for safe and low-toxic treatment and prevention of diseases such as pathological neovascularization (eg, neovascularization).
[313] Moreover, the compound derived from the compound obtained by the said screening can be used similarly.
[314] The drug containing the compound obtained by this screening method or its salt can be manufactured similarly to the above-mentioned medicine containing the polypeptide of this invention or its salt.
[315] Because the preparations thus obtained are safe and low toxicity, they can be administered, for example, to humans or mammals (such as rats, mice, morphotes, rabbits, sheep, pigs, cattle, horses, cats, dogs, monkeys, etc.).
[316] The dosage of this compound or salt thereof varies depending on the target disease, the subject to be administered, the route of administration, and the like. However, when orally administering a compound that promotes promoter activity to the DNA of the present invention, for example, in the treatment of bone and joint diseases, Is administered about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound per day to an adult (60 kg body weight). In the case of parenteral administration, the single dose of the compound varies depending on the administration target, target disease, and the like, but for example, injectable form of the compound which promotes the promoter activity against the DNA of the present invention for the purpose of treating bone and joint diseases. In general, when administered to an adult (60 kg body weight), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered intravenously per day. It is preferable. For other animals, the amount converted to 60 kg can be administered.
[317] On the other hand, for example, when orally administering a compound that inhibits the promoter activity against the DNA of the present invention, the adult (body weight 60 kg) is generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, More preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose of this compound varies depending on the subject to be administered, the target disease, and the like.However, when a compound that inhibits the promoter activity on the DNA of the present invention is administered to an adult (60 kg) in the form of an injection, It is preferable to administer about 0.01 to 30 mg of the compound per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. For other animals, the amount converted to 60 kg can be administered.
[318] As such, the non-human DNA expression dysfunction of the present invention is very useful for screening a compound or a salt thereof that promotes or inhibits the promoter activity against the DNA of the present invention, and is effective for screening various diseases resulting from the DNA expression dysfunction of the present invention. It can greatly contribute to the cause identification or the development of preventive and therapeutic drugs. In addition, since the gene encoding the polypeptide of the present invention is expressed in very large quantities in mice and humans, particularly in cartilage tissues, the promoter sequence of this gene is used in cartilage of non-human warm-blooded animals to express the target protein (any useful gene product, etc.). It is preferred as a promoter for expression in large quantities in tissues. Examples of the non-human warm-blooded animal include those similar to those exemplified as the warm-blooded animal described above.
[319] That is, the present invention comprises the target protein downstream of the promoter region of the gene encoding the polypeptide characterized by containing the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 47. A method of expressing a target protein (optional useful gene product, etc.) by linking (any useful gene product, etc.) and introducing into a non-human warm blood animal, is superior to the cartilage tissue of a non-human warm blood animal.
[320] Examples of the target protein (any useful gene product, etc.) include, for example, cytokines (e.g., interleukins, interferons, chemokines, hematopoietic factors), growth factors (e.g., epidermal growth factor (EGF) or substances having substantially the same activity) For example, EGF, halegrin (HER2 ligand, etc.), insulin or a substance having substantially the same activity (eg, insulin, insulin-liki growth factor (IGF-1), IGF-2, etc.), fibroblast growth factor (FGF) ) Or having substantially the same activity (e.g., aFGF, bFGF, Keratindcyte Growth Factor (KGF), Hepatocyte Growth Factor (HGF), FGF-10, etc.), other cell proliferation factors (e.g., colony stimulating factor (CSF) , EPO (erythropoietin), IL-2 (interleukin-2), NGF (nerve growth factor), PDGF (platelet-derived growth factor), TGFβ (transforming growth factorβ), etc.), hormones (e.g., luteinizing hormone-releasing hormone (LH-RH), growth hormone, growth hormone release Hormone (GH-RH), prolactin, melanocyte stimulating hormone, thyroid hormone releasing hormone, thyroid stimulating hormone, luteinizing hormone, progesterone, follicle stimulating hormone, gastrin, motiline, somatostatin, secretin, glucagon, PACAP, VIP, etc. Digestive enzymes (e.g., amylases, pepsinogens, lipases, etc.), antibodies against pathogens (e.g., antibodies against pathogenic cells such as pathogenic Salmonella), antibodies against pathogenic viruses such as influenza, antibodies against parasites such as Echinococcus, etc. ), And useful gene products such as antimicrobial polypeptides (e.g., cecropin, hisstatin, indolisidine, protegrin, defensin, lycheechi, etc.).
[321] Among the target proteins
[322] ① by expressing cytokines specifically to cartilage, for example, it is possible to achieve enhancement and regulation of immune activity of non-human warm-blooded animals,
[323] ② cartilage-specific expression of proliferation factors, for example, to achieve protection of cartilage tissue of non-human warm-blooded animals.
[324] Hereinafter, the target protein (optionally) is located downstream of the promoter region (3 'end side) of the gene encoding the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 47. The method of expressing cartilage specific expression of non-human warm-blooded animals by linking DNAs or RNAs encoding useful gene products, etc.), and introducing them into non-human animals (any useful gene product, etc.) do.
[325] First, a promoter of a gene encoding a polypeptide characterized by containing an amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 12 or SEQ ID NO: 47 has its own such as colony hybridization, plaque hybridization or PCR. It can be obtained by a known method (eg, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) and the like). In addition, identification of the region having promoter activity can be obtained by a method known per se such as a reporter assay (e.g., a method described in Analytical Biochemistry, Vol. 188, p.245 (1990)).
[326] In order to link the desired protein (any useful gene product, etc.) downstream of the promoter obtained by the above method (3 'end side), a self-known method (eg, Molecular Cloning) for constructing a plasmid using T4DNA ligase 2nd (methods as described in J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) and the like.
[327] In order to introduce into a non-human warm-blooded animal which connects DNA which codes the target protein (any useful gene product etc.) downstream of the promoter region (3 'terminal side), the method using an electroporation and a gene gun are used Methods, methods of using retroviral vectors (e.g., Blood Cells, Volume 17, methods described in p.407 (1991), etc.), methods of using adenovirus vectors (e.g., Pathology, Volume 30, p.335 ( 1998) and the like.
[328] In the present specification and drawings, bases, amino acids, and the like are abbreviated as IUPAC-IUB Commision on Biochemical Nomenclature or based on abbreviations commonly used in the art, and examples thereof will be described below. In the case where an optical isomer may exist with respect to an amino acid, the L form is assumed unless otherwise specified.
[329] DNA: deoxyribonucleic acid
[330] cDNA: complementary deoxyribonucleic acid
[331] A: Adenine
[332] T: Thymine
[333] G: guanine
[334] C: cytosine
[335] RNA: ribonucleic acid
[336] mRNA: messenger ribonucleic acid
[337] dATP: deoxyadenosine triphosphate
[338] dTTP: deoxythymidine triphosphate
[339] dGTP: deoxyguanosine triphosphate
[340] dCTP: deoxycytidine triphosphate
[341] ATP: Adenosine Triphosphate
[342] EDTA: Ethylenediaminetetraacetic acid
[343] SDS: Sodium Dodecyl Sulfate
[344] Gly: Glycine
[345] Ala: Alanine
[346] Val: Ballin
[347] Leu: leucine
[348] Ile: Isoleucine
[349] Ser: Serine
[350] Thr: Threonine
[351] Cys: Cysteine
[352] Met: Methionine
[353] Glu: glutamic acid
[354] Asp: Aspartic Acid
[355] Lys: Lysine
[356] Arg: Arginine
[357] His: histidine
[358] Phe: Phenylalanine
[359] Tyr: tyrosine
[360] Trp: tryptophan
[361] Pro: Proline
[362] Asn: Asparagine
[363] Gln: Glutamine
[364] pGlu: pyroglutamic acid
[365] In addition, substituents, protecting groups, and reagents frequently used in the present specification are denoted by the following symbols.
[366] Me: methyl
[367] Et: ethyl group
[368] Bu: Butyl
[369] Ph: phenyl group
[370] TC: thiazolidine-4 (R) -carboxamide group
[371] Tos: p-toluenesulfonyl
[372] CHO: Formyl
[373] Bzl: Benzyl
[374] Cl ₂ -Bzl: 2,6-dichlorobenzyl
[375] Bom: Benzyloxymethyl
[376] Z: benzyloxycarbonyl
[377] Cl-Z: 2-chlorobenzyloxycarbonyl
[378] Br-Z: 2-bromobenzyloxycarbonyl
[379] Boc: t-butoxycarbonyl
[380] DNP: dinitrophenyl
[381] Trt: Trityl
[382] Bum: t-butoxymethyl
[383] Fmoc: N-9-fluorenylmethoxycarbonyl
[384] HOBt: 1-hydroxybenztriazole
[385] HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
[386] HONB: 1-hydroxy-5-norbornene-2,3-dicarboxyimide
[387] DCC: N, N'-dicyclohexylcarbodiimide
[388] The sequence numbers in the sequence table of the present specification represent the following sequences.
[389] [SEQ ID NO: 1]
[390] The base sequence of the antisense chain primer used in Example 1 is shown.
[391] [SEQ ID NO: 2]
[392] The base sequence of the sense chain primer used in Example 1 is shown.
[393] [SEQ ID NO: 4]
[394] The base sequence of the DNA encoding the human MLP precursor having the amino acid sequence shown by SEQ ID NO: 6 is shown.
[395] [SEQ ID NO: 5]
[396] The amino acid sequence of the signal sequence contained in the human MLP precursor which has an amino acid sequence shown by SEQ ID NO: 6 is shown.
[397] [SEQ ID NO: 6]
[398] The amino acid sequence of the human MLP precursor is shown.
[399] [SEQ ID NO: 7]
[400] The base sequence of the antisense chain primer used in Example 2 is shown.
[401] [SEQ ID NO: 8]
[402] The base sequence of the sense chain primer used in Example 2 is shown.
[403] [SEQ ID NO: 10]
[404] The base sequence of DNA which codes the mouse MLP precursor which has the amino acid sequence shown by SEQ ID NO: 12 is shown.
[405] [SEQ ID NO: 11]
[406] The amino acid sequence of the signal sequence contained in the mouse MLP precursor which has the amino acid sequence shown by SEQ ID NO: 12 is shown.
[407] [SEQ ID NO: 12]
[408] The amino acid sequence of the mouse MLP precursor is shown.
[409] [SEQ ID NO: 13]
[410] The base sequence of the G3PDH specific oligo DNA used in Example 3 is shown.
[411] [SEQ ID NO: 14]
[412] The base sequence of the G3PDH specific oligo DNA used in Example 3 is shown.
[413] [SEQ ID NO: 15]
[414] The base sequence of the aggrecan specific oligo DNA used in Example 3 is shown.
[415] [SEQ ID NO: 16]
[416] The base sequence of the aggrecan specific oligo DNA used in Example 3 is shown.
[417] [SEQ ID NO: 17]
[418] The base sequence of type II collagen specific oligo DNA used in Example 3 is shown.
[419] [SEQ ID NO: 18]
[420] The base sequence of type II collagen specific oligo DNA used in Example 3 is shown.
[421] [SEQ ID NO: 19]
[422] The base sequence of the type V collagen specific oligo DNA used in Example 3 is shown.
[423] [SEQ ID NO: 20]
[424] The base sequence of the type V collagen specific oligo DNA used in Example 3 is shown.
[425] [SEQ ID NO: 21]
[426] The base sequence of the mouse MLP specific oligo DNA used in Example 3 is shown.
[427] [SEQ ID NO: 22]
[428] The base sequence of the mouse MLP specific oligo DNA used in Example 3 is shown.
[429] [SEQ ID NO: 23]
[430] The base sequence of the DNA encoding human MLP having the amino acid sequence shown by SEQ ID NO: 24 is shown.
[431] [SEQ ID NO: 24]
[432] The amino acid sequence of human MLP is shown.
[433] [SEQ ID NO: 25]
[434] The base sequence of DNA which codes mouse MLP which has an amino acid sequence shown by SEQ ID NO: 26 is shown.
[435] [SEQ ID NO: 26]
[436] The amino acid sequence of mouse MLP is shown.
[437] [SEQ ID NO: 27]
[438] The base sequences of the primers used in Examples 4 and 6 are shown.
[439] [SEQ ID NO: 28]
[440] The base sequence of the primer used in Example 4 is shown.
[441] [SEQ ID NO: 29]
[442] The base sequence of the cDNA fragment obtained in Example 1 is shown.
[443] [SEQ ID NO: 30]
[444] The base sequence of the cDNA fragment obtained in Example 2 is shown.
[445] [SEQ ID NO: 31]
[446] The amino acid sequence of the synthetic peptide used in Example 6 is shown.
[447] [SEQ ID NO: 32]
[448] The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[449] [SEQ ID NO: 33]
[450] The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[451] [SEQ ID NO: 34]
[452] The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[453] [SEQ ID NO: 35]
[454] The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[455] [SEQ ID NO: 36]
[456] The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[457] [SEQ ID NO: 37]
[458] The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[459] [SEQ ID NO: 38]
[460] The base sequence of the oligo DNA used as a PCR primer in Example 6 is shown.
[461] [SEQ ID NO: 39]
[462] The partial amino acid sequence of the rat MLP precursor encoded in the DNA obtained in Example 9 is shown.
[463] [SEQ ID NO: 40]
[464] The base sequence of the DNA encoding a part of the rat MLP precursor obtained in Example 9 is shown.
[465] [SEQ ID NO: 41]
[466] The nucleotide sequence of DNA containing DNA which codes a part of rat MLP precursor obtained in Example 9 is shown.
[467] [SEQ ID NO: 42]
[468] The base sequence of the oligo DNA used as a PCR primer in Example 9 is shown.
[469] [SEQ ID NO: 43]
[470] The base sequence of the oligo DNA used as a PCR primer in Example 9 is shown.
[471] [SEQ ID NO: 44]
[472] The base sequence of the oligo DNA used as a PCR primer in Example 9 is shown.
[473] [SEQ ID NO: 45]
[474] The base sequence of the oligo DNA used as a PCR primer in Example 9 is shown.
[475] [SEQ ID NO: 46]
[476] The base sequence of the DNA encoding the rat MLP precursor having the amino acid sequence represented by SEQ ID NO: 47 is shown.
[477] [SEQ ID NO: 47]
[478] The amino acid sequence of the rat MLP precursor is shown.
[479] [SEQ ID NO: 48]
[480] The base sequence of the DNA encoding the rat MLP precursor having the amino acid sequence represented by SEQ ID NO: 49 is shown.
[481] [SEQ ID NO: 49]
[482] The amino acid sequence of rat MLP is shown.
[483] [SEQ ID NO: 50]
[484] The amino acid sequence of the signal sequence contained in the rat MLP precursor which has the amino acid sequence shown by SEQ ID NO: 47 is shown.
[485] The transformant Escherichia coli XL10-Gold / pDRL128vH obtained in Example 1, which will be described later, was manufactured from June 11, 1999, 1-1-3 Higashi, Tsukubashi, Ibaragi-ken, Japan. NIBH) has been deposited as the Accession No. IFO 16292 to the Fermentation Research Institute (IFO), 2-17-85, Yodogawa-ku, Yodogawa-ku, Osaka, Japan, on June 25, 1999 as Accession No. FERM BP-6750.
[486] The transformant Escherichia coli XL10-Gold / pDRL128vM obtained in Example 2 to be described later was deposited with the FERM BP-6747, deposited with the Institute of Biotechnology and Technology (NIBH), June 9, 1999, 1999. It has been deposited with IFO 16293 at the Foundation's Fermentation Research Institute (IFO) on 25 June.
[487] The transformant Escherichia coli XL10-Gold / pDRL128vR obtained in Example 9 to be described later was deposited with the FERM BP-7167, deposited with the Institute of Biotechnology and Industrial Technology (NIBH), on May 19, 2000. It has been deposited as IFO 16439 with the Foundation Fermentation Research Institute (IFO) on 26 May.
[7] Disclosure of the Invention
[8] As a result of intensive studies, the present inventors have succeeded in cloning cDNAs having new sequences in the cDNA libraries derived from human fetal brain and mouse fetuses. The present inventors have found that the protein encoded in the obtained cDNA is a precursor protein of the MIA / CD-RAP-shaped protein MLP having useful cell function regulating activity, and the MLP produced by cleaving a signal sequence from the MLP precursor is a secreted protein. As a result of further studies in accordance with these findings, the present invention has been completed.
[9] That is, the present invention
[10] (1) a polypeptide, an amide or ester thereof or a salt thereof, which contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 24;
[11] (2) the polypeptide, amide or ester thereof or salt thereof according to (1), which contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 6;
[12] (3) the polypeptide according to (1), amide or ester thereof or salt thereof, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 is an amino acid sequence represented by SEQ ID NO: 26;
[13] (4) the polypeptide according to (2), amide or ester thereof or salt thereof, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 is an amino acid sequence represented by SEQ ID NO: 12;
[14] (5) the polypeptide according to (1), amide or ester thereof or salt thereof, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 is an amino acid sequence represented by SEQ ID NO: 49;
[15] (6) the polypeptide according to (2) above, the amide or ester thereof or a salt thereof, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 is an amino acid sequence represented by SEQ ID NO: 47;
[16] (7) DNA containing DNA having a nucleotide sequence encoding the polypeptide according to (1) above;
[17] (8) the DNA according to the above (6), wherein the nucleotide sequence encoding the polypeptide according to the above (1) is a nucleotide sequence represented by SEQ ID NO: 23;
[18] (9) the DNA according to the above (6), wherein the nucleotide sequence encoding the polypeptide according to the above (1) is a nucleotide sequence represented by SEQ ID NO: 4;
[19] (10) the DNA according to the above (6), wherein the nucleotide sequence encoding the polypeptide according to the above (1) is a nucleotide sequence represented by SEQ ID NO: 25;
[20] (11) the DNA according to the above (6), wherein the nucleotide sequence encoding the polypeptide according to the above (1) is a nucleotide sequence represented by SEQ ID NO: 10;
[21] (12) the DNA according to the above (6), wherein the nucleotide sequence encoding the polypeptide according to the above (1) is a nucleotide sequence represented by SEQ ID NO: 48;
[22] (13) the DNA according to the above (6), wherein the nucleotide sequence encoding the polypeptide according to the above (1) is a nucleotide sequence represented by SEQ ID NO: 46;
[23] (14) a recombinant vector containing the DNA according to the above (6),
[24] (15) a transformant transformed with the recombinant vector according to (14),
[25] (16) A method for producing a polypeptide, an amide or an ester thereof, or a salt thereof according to (1), wherein the transformant according to (15) is cultured to produce the polypeptide.
[26] (17) an antibody against the polypeptide according to (1), an amide thereof, or an ester thereof or a salt thereof;
[27] (18) A method for screening a compound or salt thereof that promotes or inhibits the activity of the polypeptide or salt thereof according to (1), characterized by using the polypeptide as described in (1), an amide or an ester thereof or a salt thereof.
[28] (19) A kit for screening a compound or a salt thereof that promotes or inhibits the activity of the polypeptide, the amide or the ester thereof or the salt thereof according to (1), comprising the polypeptide or the salt thereof according to (1) above,
[29] (20) A compound which promotes or inhibits the activity of the polypeptide, amide or ester thereof or salt thereof according to (1) obtained using the screening method according to (18) or the screening kit according to (19) above, or His salt,
[30] (21) A compound which promotes or inhibits the activity of the polypeptide, its amide or its ester or its salt according to (1) obtained using the screening method according to (18) or the screening kit according to (19) above, or Medicines containing their salts,
[31] (22) A medicament comprising the polypeptide, amide or ester thereof or salt thereof according to (1) above,
[32] (23) A prophylactic / treatment agent for bone, joint disease or pathological neovascularization, comprising the polypeptide according to (1), an amide or an ester thereof, or a salt thereof;
[33] (24) It relates to the diagnostic agent etc. which contain the antibody as described in said (17).
[34] In addition, the present invention
[35] (25) at least about 50% (preferably at least about 60%, more preferably at least about 70%) of the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24; , More preferably at least 80%, particularly preferably at least about 90%, most preferably at least 95%), the polypeptide as described in (1) above, its amide or its ester or salt thereof ,
[36] (26) An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 is deleted in one or two or more (preferably about 1 to 30) amino acids of the amino acid sequence represented by SEQ ID NO: 24. Amino acid sequence, ② an amino acid sequence in which one or two or more amino acids (preferably about 1 to 40, more preferably about 1 to 30) are added to the amino acid sequence represented by SEQ ID NO: 24, ③ SEQ ID NO: The polypeptide according to the above (1), wherein the amino acid sequence of 1 or 2 or more (preferably about 1 to 30) amino acids in the amino acid sequence represented by: 24 is an amino acid sequence substituted with another amino acid, or ④ an amino acid sequence combining them; Amides or esters thereof or salts thereof.
[37] In addition, the DNA and polypeptide of the present invention, amide or ester thereof or salt thereof may be used for basic research such as molecular weight markers, tissue markers, chromosome mapping, identification of genetic diseases, design of primers, probes, and the like.
[488] Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these. In addition, genetic engineering using E. coli is in accordance with the method described in Molecular cloning.
[489] Example 1 Cloning of cDNA Encoding Human MLP Precursor Protein
[490] Cloning of the cDNA encoding the human MLP precursor protein is performed by performing 5'RACE (Rapid Amplification of cDNA End) and 3'RACE in the following regions using poly (A ⁺ ) RNA derived from human fetal brain. 1st of the human fetal brain-derived poly (A ⁺ ) RNA (Clontech) using an anchored primer having a poly (T) following the restriction site and Superscript II MMLV reverse transcriptase (Gibco BRL) were used. After synthesizing the strand cDNA, using RNA ligase (Takarashuzo Co., Ltd.), the 1st. Add anchored primer to the 3 'end of strand cDNA. Subsequently, 5'RACE is used as the antisense chain primer as the oligo DNA represented by SEQ ID NO: 1, and 3'RACE is performed as the sense chain primer as the oligo DNA represented by SEQ ID NO: 2, respectively. Obtain an 'upstream sequence, 3' a downstream sequence. As a result of determining the nucleotide sequence of each of the two chain DNAs obtained here, it can be seen that since the overlapping consensus sequences exist, the two sequences are derived from the same gene. Thus, each cDNA fragment obtained from this 5'RACE, 3'RACE is conjugated in this consensus sequence part and finally a full length 923 base pair (bp) cDNA fragment represented by SEQ ID NO: 29 including a poly (A ⁺ ) chain. Get This cDNA fragment encodes a novel human MLP precursor protein comprising a typical signal sequence of 18 amino acid residues represented by SEQ ID NO: 5 and consisting of 128 amino acids represented by SEQ ID NO: 6. This human MLP precursor protein has the highest homology with the human MIA precursor protein, and the positions of the four cysteine residues present are consistent (Fig. 1), but the homology between them is only 23.4% at the amino acid level.
[491] The plasmid pDRL128vH holding the DNA encoding the human MLP precursor protein obtained in this Example was introduced into Escherichia coli (E. coli) XL10-Gold to obtain a transformant: Escherichia coli (E. coli) XL10-Gold / pDRL128vH. .
[492] Example 2 Cloning of cDNA Encoding Mouse MLP Precursor Protein
[493] Cloning of the cDNA encoding the mouse MLP precursor protein was performed using 5'RACE (Rapid Amplification of) as in the case of cloning the cDNA encoding the human MLP precursor protein using 17.5 days old mouse embryo derived poly (A ⁺ ) RNA. cDNA End) and 3'RACE. The total RNA is fractionated from the 17.5-day-old mouse fetus by the guanidine thiocyanate method, and then the total RNA is raised and poly (A ⁺ ) RNA is prepared using (dT) span column (palmacia). A 17.5-day-old mouse embryo-derived poly (A ⁺ ) RNA was synthesized using 1st. Mutant using an anchored primer having a poly (T) following the restriction site and a Superscript II MMLV reverse transcriptase (Gibco BRL). strand cDNA was synthesized, and then synthesized using RNA ligase (Takarashuzo Co., Ltd.). Add anchored primer to the 3 'end of strand cDNA. The primer sequences used for 5'RACE and 3'RACE were mouse MLP precursor proteins found in a public EST (Expressed Sequence Tag) database by querying the nucleotide sequence of the cDNA encoding the human MLP precursor protein obtained in Example 1. The sequence of AA222797 is prepared based on the only EST that is thought to include the region on the 3 'side of the cDNA that encodes. 5'RACE of the oligo DNA represented by SEQ ID NO: 7 as an antisense chain primer and 3'RACE of the oligo DNA represented by SEQ ID NO: 8 as the sense chain primer, respectively. Sequence, the sequence of 3 'downstream side is obtained. As a result of determining the nucleotide sequence of the two-chain DNA obtained here, it can be seen that the two sequences are derived from the same gene because overlapping consensus sequences exist. Thus, each cDNA fragment obtained from this 5'RACE, 3'RACE is conjugated in its consensus sequence and finally a 947 bp full length cDNA fragment including the poly (A ⁺ ) chain is obtained (SEQ ID NO: 30). This cDNA fragment, like the human MLP precursor protein, encodes a novel mouse MLP precursor protein comprising a typical signal sequence of 18 amino acid residues represented by SEQ ID NO: 11 and consisting of 128 amino acids represented by SEQ ID NO: 12. This mouse MLP precursor protein coincides with the positions of the human and mouse MIA precursor proteins and the cysteine residues present in four positions of the human MLP precursor protein (FIG. 1). In addition, homology at the amino acid level of the mouse MLP precursor protein and the human MLP precursor protein reaches 84.3%, while the homology of the mouse MIA precursor protein and the mouse MLP precursor protein is only 22.6%.
[494] The plasmid pDRL128vM, which retains the DNA encoding the mouse MLP precursor protein obtained in this Example, was introduced into Escherichia coli (E. coli) XL10-Gold to obtain a transformant: Escherichia coli (E. coli) XL10-Gold / pDRL128vM. .
[495] Example 3 Expression of MLP in in vitro Cartilage Differentiation Model
[496] Since the mouse embryonic tumor-derived cell line ATDC5 maintains very good properties of progenitor chondrocytes and also induces cartilage differentiation at a high rate in culture in the presence of insulin, it can then simulate all stages of cartilage differentiation seen in bone formation. Used as an in vitro cartilage differentiation model (Cell Diff. Dev., 30: 109-116, 1990, J. Cell Biol., 133: 457-468, 1996, J.Bone Min.Res., 10: S234, 1995 ). Therefore, the change of expression of various genes in each differentiation step is examined by performing the RT-PCR method in the following areas. First, in the ATDC5 cells of each stage of the differentiation model culture system, the cells were dissolved in a homogeneous liquid ISOGEN (Nippon Gin) containing phenol and thiocyanate guanidine and centrifuged by adding chloroform to obtain an aqueous phase fraction containing RNA. The purified total RNA is obtained by collecting and isopropanol is added and stirred, followed by centrifugation and precipitation again. Subsequently, the total RNA was reverse transcribed using AMV Reverse Transcriptase XL and Random 9 mers in the TAKARA RNA PCR Kit (AMV) Ver.2.1 (Takarashuzo Co., Ltd.) to obtain cDNA, which was then used as template DNA. And a G3PDH (glycelaldehyde-3-phosphate dehydrogenase) specific oligo DNA (SEQ ID NO: 13, SEQ ID NO: 14) or a cartilage differentiation marker gene specific oligo DNA (Aggrecan (SEQ ID NO: 15)) as a house keeping gene. , SEQ ID NO: 16), type II collagen (SEQ ID NO: 17, SEQ ID NO: 18), and type V collagen (SEQ ID NO: 19, SEQ ID NO: 20) as primer DNA, using TaKaRaEx Taq ^TM (Takarashuzo ( The reaction product is separated by agarose gel electrophoresis, and the production amount is compared, and as a result, each gene is shown in Table 1 corresponding to the differentiation stage of chondrocytes. Expression patterns can be detected. Therefore, the oligo DNA (SEQ ID NO: 21, SEQ ID NO: 22) specific for the cDNA encoding the mouse MLP precursor protein was used as the primer DNA and RT-PCR was carried out under the same reaction conditions. There is a marked increase in the amount of mouse MLP precursor mRNA from stage 2 to stage 4. From this point, it can be seen that the gene of MLP precursor is a gene whose expression is increased in the early stage of cartilage differentiation.
[497] stepOne234567 MLP+++++++++++++ Aggrecan+++++++++ Type II collagen+++++++++++++ Type collagen+++++++++++++ G3PDH+++++++ In the table, the number of + indicates the difference in the expression amount of each gene at each differentiation stage, and the larger the number, the higher the expression amount.
[498] Example 4 Expression and Detection of Mouse MLP-FLAG Fusion Protein in COS-7 Cells
[499] To ensure that MLP is a secreted protein, mouse MLP is expressed and detected in mouse MLP-FLAG fusion protein in COS-7 cells in the following regions. First, two kinds of primer DNAs are chemically synthesized according to the nucleotide sequence of cDNA encoding the mouse MLP precursor polypeptide obtained in Example 2. One is 5'-CGAATTCCCACCATGGCAAGGATATTGATTCTTTTGCTTG-3 '(SEQ ID NO: 27), which is from +1 to +28 having the anchor sequence at the 5' end, including the restriction enzyme EcoRI recognition site, with the translation initiation site as +1. Oligo DNA containing the sense sequence of the. Another is 5'-GTACAGTCGACTTCACAGAAGAAGTCAATATCCGTGGTTG-3 '(SEQ ID NO: 28), which is an oligo DNA having a sequence of +355 to +378 antisense sequence on the 3' side of the anchor sequence containing the restriction enzyme SalI recognition site. . Example 2 First the plasmid pDRL128vM using as a template (Takara Shuzo Co., Ltd.) The two types of DNA primers, and TaKaRa LA Taq ^TM use and thermal between cluster GeneAmp ^TM PCR system9700 (Perkin Elmer), obtained in 98 ℃ After 30 seconds at, a 25 cycle amplification reaction was repeated with 10 seconds at 98 ° C., 20 seconds at 55 ° C., 2 minutes at 72 ° C. as a reaction cycle, and finally an extension reaction at 72 ° C. for 5 minutes. The obtained DNA fragment is purified, terminally digested with restriction enzymes EcoRI and SalI, and then rearranged and inserted and linked to the EcoRI and SalI sites of the expression vector pCAN618FLAG for animal cells. pCAN618FLAG is derived from the plasmid vector pCAN618, has a neomycin resistance gene as a selection marker, and simultaneously inserts a DNA fragment encoding a target protein into its cloning site, EcoRI, SalI site, and the cytomegalovirus early gene enhancer. Not only can this protein be expressed under the control of the downstream β-actin promoter but also encodes the FLAG epitope sequence of the 8 amino acid (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) immediately after the SalI site. The target protein can also be expressed as a FLAG fusion protein by tailoring the reading frame to the nucleotide sequence and the stop codon. Insertion of the above-described PCR cloning DNA fragment into pCAN618FLAG was also carried out for the purpose of expressing the full length of the mouse MLP precursor and the fusion protein of FLAG epitope (inserting one residue between Val), thereby obtaining the expression vector plasmid pMMLP-F. .
[500] Subsequently, COS-7 cells 1.2 × 10 ⁵ cells were incubated for 24 hours in Dalbecco's minimal media (DMEM) using 6 well plates and containing 10% fetal bovine serum (FBS), and the expression plasmid pMMLP -F (0.4 μg per well) is introduced using lipofectamine (Gibco BRL). After 24 hours of introduction, the culture medium was replaced with the fresh medium, and after 5 hours, the resultant was changed to FBS-free Opti-MEM (Gibco BRL), followed by 36 hours of culture, to obtain the culture supernatant and cell extract. Cell extracts were washed twice with physiological saline-containing phosphate buffer (PBS) and then lysed with Tris SDS sample buffer, while the culture supernatant was appropriately concentrated in ultrafiltration (molecular weight 3000 cuts) and then Mix with Tris SDS sample buffer. These samples are subjected to heat treatment and then electrophoresed with 15% -25% SDS-polyacrylamide gels, which in turn are transferred onto PVDF membranes (Amersham pharmacia biotech). Subsequently, this PVDF membrane was blocked for 1 hour in a block ace (Kyujirushi Kyukyo Co., Ltd.) and anti-FLAG monoclonal antibody (10 µg / ml; Kodak Corporation) in PBS (PBS-T) containing 0.05% Tween 20. React for 2 hours. After washing three times in PBS-T, it was reacted with horseradish peroxidase-labeled anti-mouse IgG goat antibody (Amersham pharmacia biotech; 5000-fold dilution) in PBS-T for 1 hour. After washing five times in PBS-T, chemiluminescence is detected using ECLplus color development kit (Amersham pharmacia biotech) and ECL film (Amersham pharmacia biotech). As a result, it was found that about 14 kDa gene product was detected in the culture supernatant together with the cell extract, thereby expressing and secreting mouse MLP-FLAG fusion protein in COS-7 cells.
[501] Thus, the N terminal amino acid sequence of this mouse MLP-FLAG fusion protein is then determined. First, acidic elution fraction (Glycine-HCl) by affinity chromatography using Anti-FLAG ^™ M2-Agarose Affinity Gel (Sigma) in COS-7 cell culture supernatant containing mouse MLP-FLAG expressed according to the method described above. To the buffer (pH3.5). After concentrating this fraction, SDS-polyacrylamide gel electrophoresis and subsequent CBB staining as described in the present example showed only a single band corresponding to mouse MLP-FLAG as the target protein. Then, the concentrated sample of the same fraction was electrophoresed in the same manner, and after transfer of this protein from the gel onto the PVDF membrane, the N-terminal amino acid sequence was purified using a pulsed liquid amino acid sequencer Procise CLC491 (manufactured by PE Biosystems). Decide As a result, each amino acid residue of 1.histidine, 2.glycine, 3.valine, and 4.phenylalanine is detected in sequence at the N-terminal, and coincides with the N-terminal sequence of the mouse MLP represented by SEQ ID NO: 26. In the above results, the mouse MLP-FLAG protein is a mouse MLP-FLAG mature protein derived from histidine residues at the 19th residue by cleaving a signal sequence of 18 amino acid residues at the N-terminus of the mouse MLP-FLAG precursor protein in COS-7 cells. It can be seen that secreted into the medium.
[502] Example 5 Establishment of Mouse MLP-FLAG Fusion Protein Expressing CHO-K1 Cell Line
[503] The mouse MLP-FLAG fusion protein expressing CHO-K1 cell line is established in the following areas. CHO-K1 cells 3.3 × 10 ⁴ cells were incubated for 24 h in F-12 medium (Gibco BRL) containing 10 cm diameter plastic chalets on 10% fetal bovine serum (FBS) and in these cells in Example 4 The obtained expression plasmid pMMLP-F (1.5 µg per chalet) was introduced using the calcium phosphate method (Cell Phect Transfection Kit (Amersham Pharmacia Biotech)). After 12 hours of introduction, cells were washed twice in F-12 medium without FBS, and then glycerol shock was added for 3 minutes using 3 ml of an isotonic HEPES solution (pH7.5) containing 15% glycerol. Then, washed twice in F-12 medium without FBS, incubated for 12 hours in F-12 medium containing FBS, and then containing 500 mg / L Geneticin (Gibco BRL) and 10% FBS. Exchange with F-12 selection medium (hereinafter, selection medium). After 10 days, Geneticin resistant colonies formed in the chalets are transferred to 24-well plates each and incubated for 3 days in selection medium. Subsequently, the cells proliferated in the selection medium were transferred to 6-well plates, and again cultured in the selection medium for 4 days, followed by Opti-MEM containing 0.02% CHAPS and 0.1mMp-ABSF (Wako Pure Chemical Industries, Ltd.). (Gibco BRL Co., Ltd.) It is exchanged with 1 ml of medium, the culture supernatant is recovered after incubation for 48 hours. The obtained culture supernatant is concentrated using Centricon YM-3 ultrafiltration membrane (Amicon), and then mixed with isotropic Tris SDS sample buffer. The sample is subjected to heat treatment at 95 ° C. for 5 minutes, followed by electrophoresis on an 18% SDS-polyacrylamide gel, from which the electrophoretic protein is transferred onto a nylon membrane. Subsequently, the nylon membrane was blocked for 1 hour with a block ace (Yukijirushi Newkyo Co., Ltd.), followed by anti-FLAG antibody (1/2000 dilution, SIGMA) and 1 in PBS (PBS-T) containing 0.05% Tween 20. React time. After 5 washes in PBS-T, the cells were reacted with HRP-labeled anti mouse IgG positive antibody (1/2000 dilution, Amersham Pharmacia Biotech) in PBS-T for 1 hour. After washing five times in PBS-T, chemiluminescence is detected using ECL color development kit (Amersham Pharmacia Biotech) and Hyperfilm ECL (Amersham Pharmacia Biotech). As a result, since a large amount of about 16 kDa of the target gene product was detected in the cell culture supernatant derived from CHO-Kl / mMLP.FLAG # 2-1 strain, this cell line was selected as the mouse MLP-FLAG fusion protein expressing CHO-K1 cell line.
[504] Example 6 Preparation of MLP Antisera and Detection of Recombinant MLP Protein Using the Antisera
[505] Anti-MLP antiserum is prepared in the following areas. First, a synthetic peptide represented by SEQ ID NO: 31 in which a cysteine was additionally added to the C terminus of the peptide chain corresponding to the amino acid sequence from the valine at the 105th residue of the mouse MLP precursor protein to the aspartic acid at the 124th residue was used (Val- Lys-Glu-Gln-Arg-Val-Tyr-Gln-Glu-Ala-Thr-Lys-Glu-Ile-Pro-Thr-Thr-Asp-Ile-Asp-Cys) is chemically synthesized by known methods. This peptide was immunized to rabbits (SPF Japanese White Rabbit) by binding to KLH (Keyhole Limpet Hemocyanin) as a carrier. After a total of seven immunizations, the whole blood was collected and serum fractions were obtained by a known method, and sodium azide (final concentration 0.1%) was added as a preservative, and this standard was used as anti-MLP antiserum.
[506] Subsequently, the reactivity with respect to various recombinant proteins of this antiserum is investigated according to Western blot analysis. First, in addition to the mouse MLP-FLAG fusion protein described in Example 4, mouse MLP protein (no FLAG tag), human MLP-FLAG fusion protein, human MLP protein (no FLAG tag), mouse MIA-FLAG fusion protein, mouse MIA Each expression vector plasmid of the protein (no FLAG tag) is constructed. Constructing was carried out by inserting the target DNA fragment previously PCR cloned into the EcoRI, SalI site of the cloning site of pCAN618FLAG in the same manner as in Example 4. Each base sequence of the primer DNA set used in each PCR reaction is SEQ ID NO: 27 and SEQ ID NO: 32 for mouse MLP protein (no FLAG tag), SEQ ID NO: 33 and SEQ ID NO: for human MLP-FLAG fusion protein 34, SEQ ID NO: 33 and SEQ ID NO: 35 for human MLP protein (no FLAG tag), SEQ ID NO: 36 and SEQ ID NO: 37 for mouse MIA-FLAG fusion protein, mouse MIA protein (no FLAG tag) SEQ ID NO: 36 and SEQ ID NO: 38. In addition, as a template DNA, mouse MLP protein (no FLAG tag) used pDRL128vM in the same manner as in Example 4, human MLP-FLAG fusion protein and human MLP protein (no FLAG tag) used pDRL128vH obtained in Example 1, and mouse MIA-FLAG fusion protein and mouse MIA protein (no FLAG tag) use cDNA prepared in mouse melanomer cell line B16. Introduction of each expression vector plasmid thus obtained into COS-7 cells and Western blotting analysis using an anti-FLAG antibody to each culture supernatant obtained are carried out in accordance with the method of Example 4. In addition, Western blot analysis using anti-MLP antiserum uses this antiserum (1000-fold dilution) as the primary antibody and horseradish peroxidase-labeled anti-rabbit IgG antibody (Amersham pharmacia biotech; 5000-fold dilution) as the secondary antibody. Except for doing this, it carries out based on the method at the time of western blot analysis using anti-FLAG antibody.
[507] The result of the western blot analysis using anti-FLAG antibody in FIG. 2, and the western blot analysis using anti-MLP antiserum in FIG. The anti-MLP antiserum exhibits cross-reactivity with human MLP along with mouse MLP, which is a protein derived from an antigenic peptide, and its reactivity is hardly changed. In addition, since the mouse MLP-FLAG fusion protein and the human MLP-FLAG fusion protein also respond to this antiserum, the presence of the FLAG tag is not affected. On the other hand, since no signal is detected for the mouse MIA-FLAG fusion protein and the mouse MIA protein (no FLAG tag), it can be seen that the antiserum is specific for the MLP molecular species.
[508] 4 shows the results of immunostaining for the mouse MLP-FLAG expressing CHO cell line obtained in Example 5 using this antiserum, according to a known method. In control experiments with pre-immune rabbit serum, each cell is not stained, but all cells are stained with this antiserum, indicating that the antiserum also reacts with unmodified MLP protein.
[509] Example 7 Expression of MLP Proteins in Cartilage Tissue
[510] The anti-MLP antiserum obtained in Example 6 was used to detect the MLP protein in cartilage tissue. As the sample to be tested, mice (BALB / c) femoral head cartilage were frozen and crushed with liquid nitrogen, extracted with TRIS SDS βME SAMPLE BUFFER (Central Daiichi Chemical Co., Ltd.), and centrifuged to remove the residue. A considerable amount of samples derived from one mouse per lane were electrophoresed by SDS-PAGE (15-25%), followed by Western blotting analysis by anti-MLP antiserum in the same manner as in Example 6. As a result, it can be seen that the MLP protein is expressed in the cartilage tissue because the signal is detected at the same kinetic position as the mouse MLP recombinant protein.
[511] Further, in order to examine the expression of human MLP mRNA in various human tissues other than cartilage tissue, Example 1 DNA fragment encoding human MLP precursor protein obtained in the ^{[α- 32 P] dCTP (Amersham} pharmacia biotech Company: 6000 Ci / mmol) and a probe was prepared by the method of Multiprime DNA labeling system (Amersham pharmacia biotech: RPN. 1601Y), using this probe (inactive 1.3 × 10 ¹⁰ cpm / μg) and using a Human MTE ^TM Array (CLONTECH Company: Perform hybridization for # 7775-1). The conditions for hybridization are carried out at 0.1 x SSC and 0.1% SDS 55 DEG C according to the manual attached to this array film, and detection is performed using BAS-2000 (Fuji Film). As a result, the signal obtained on this array was a little black spot, fetal brain spot with human chromosomal DNA of control (100ng, 500ng), but any signal was near the detection limit, and its expression level was very low at the transcriptional stage. It seems to be. That is, it is strongly suggested that MLP protein is specifically expressed in cartilage tissue.
[512] Example 8 Effect of Addition of MLP Recombinant Protein on Various Gene Expression Variation in in vitro Cartilage Differentiation Model
[513] The effect of the addition of MLP recombinant protein on various gene expression variations in an in vitro cartilage differentiation model using ATDC5 cells described in Example 3 is investigated. MLP recombinant protein was purified using affinity column chromatography by anti-FLAG antibody in a COS-7 cell culture supernatant expressing a mouse MLP-FLAG fusion protein in the same manner as in Example 4 using a purified concentrate. do. The protein is added to ATDC5 cells every two days from the first day of model system setting, and after culturing for 10 days, the cells are recovered, and the expression of each gene is examined by the same RT-PCR method as in Example 3. As a result, in this protein-added cell group, expression inhibition of each marker gene whose expression amount increases with differentiation such as aggrecan, type II collagen, and type V collagen is observed. On the other hand, there is no significant effect on the fluctuation of the PTH / PTHr receptor expression that inhibits the differentiation to cartilage. In this respect, it can be seen that MLP protein inhibits cartilage differentiation in this model system using ATDC5.
[514] Example 9 Interpretation of Genes Encoding Rat MLP Precursor Protein (rMLP)
[515] First, a DNA fragment encoding a part of rMLP is obtained by the following PCR method. That is, 4 pmol of oligo DNA represented by SEQ ID NO: 21 as a sense chain primer and oligo DNA represented by SEQ ID NO: 22 as an antisense chain primer are each contained, and further 10 x Advantage ^TM 2 PCR Buffer (Clontech Co., Ltd.) ) 2 μl, 50 × dNTP mix (Clontech Co., Ltd.) 0.4 μl, 50 × Advantage 2 Polymerase Mix (Clontech Co., Ltd.), 2 μl of cDNA solution derived from SD (IGS) rat sewerage as template DNA 20 μl was prepared, using a thermal cycler (GeneAmp ^™ PCR system model 9700 (Perkin Elmer)), 95 ° C., 1 minute, then 95 ° C., 10 seconds → 54 ° C., 10 seconds → 72 ° C., 1 minute The cycle was repeated for 35 cycles, followed by PCR reaction in a program in which the reaction was elongated at 72 ° C. for 3 minutes. After the reaction solution was electrophoresed using a 2.0% agarose gel, the gel was stained with SYBR ^™ Green I nucleic acid gel stain (Morcular Probe), and amplified by PCR at a position near 300bp in terms of molecular weight marker. Check the band for the DNA. This DNA fragment was recovered using the Kiaquigel Extra Cushion Kit (Kiagen) and TA cloned using pCR ^™ 2.1-Topo (Invitrogen) to determine the sequencing and the plasmid was isolated from Escherichia coli. It is introduced into a competent cell of Epicurian Coli XL10-Gold ^™ strain (stratter cinnabar). From the colonies of ampicillin resistant transformants appearing on the ampicillin-containing LB agar medium, a clone holding the plasmid into which the foreign DNA fragment was inserted is selected and the plasmid DNA, pDRL128vR is prepared from the clone. To determine the base sequence of the inserted DNA, pDRL128vR is used as a template DNA and a commercial primer DNA (Bca BEST Primer RV-P (Takara Shuzo Co., Ltd.)) as a sequence primer, and ABI PRISM ^TM BigDye Terminator Cycle Sequencing FS Ready Reaction Kit ( The sequence reaction using Perkin Elmer Co.) was carried out using a thermal cycler (GeneAmp ^™ PCR system model 9700 (Perkin Elmer Co.)) according to the conditions of the attached document, and the reaction sample was subjected to the DNA sequencer ABI PRISM ^™ 377 (Perkin Elmer Co.). Analyze with
[516] As a result, pDRL128vR contains a DNA fragment of 261 base pairs represented by SEQ ID NO: 40 which encodes a part of the novel rat MLP precursor protein consisting of 87 amino acids represented by SEQ ID NO: 39 and 307 represented by SEQ ID NO: 41. DNA fragments of base pairs are contained.
[517] The gene encoding this protein is then subjected to genome walking to further investigate the structures of the 5 'upstream side and the 3' downstream side from the base sequence. Rat GenomeWalker ^™ Kit (Clontech Co., Ltd.) was used as the test material, and the method was followed according to the attached data of the kit except that TaKaRaEx Taq ^™ (Takara Shuzo Co., Ltd.) was used as the enzyme for PCR reaction. First, as a gene specific primer, two kinds of oligo DNA (rMLPGWF1 (SEQ ID NO: 42), rMLPGWF2 (SEQ ID NO: 43)) corresponding to a part of the nucleotide sequence represented by SEQ ID NO: 40 and complementary to a part of this sequence Two kinds of oligo DNAs (rMLPGWR1 (SEQ ID NO: 44) and rMLPGWR2 (SEQ ID NO: 45)) of the sequence are chemically synthesized, respectively. They use rMLPGWF1 for 1st PCR reaction for 3 'downstream DNA acquisition, rMLPGWF2 for subsequent nested PCR reaction, rMLPGWR1 for 1st PCR reaction for 5' upstream DNA acquisition, and rMLPGWR2 for subsequent nested PCR reaction. For each of the amplified DNA fragments obtained by these reactions, each base sequence is analyzed while comparing the homology with the base sequences of the cDNA encoding the human and mouse MLP precursor proteins starting from the primer sequence region. As a result, it can be seen that in the primary structure of the identified genome, the rat MLP precursor protein is a protein consisting of 128 amino acid residues represented by SEQ ID NO: 47 encoded by DNA of 384 bases represented by SEQ ID NO: 46. The homology at the amino acid level of rat MLP precursor protein, human MLP precursor protein and mouse MLP precursor protein reached 84.3% and 96.0%, respectively, but homology between rat MIA precursor protein and rat MLP precursor protein was only 26.5%. Rat MLP is a protein consisting of 110 amino acid residues represented by SEQ ID NO: 49 encoding 330 bases of DNA represented by SEQ ID NO: 48, the same as mouse MLP represented by SEQ ID NO: 50 from rat MLP precursor protein It is believed that a signal peptide consisting of 18 amino acid residues has been processed and produced.
[518] The plasmid pDRL128vR, which retains the DNA encoding a portion of the rat MLP precursor protein obtained in this example, is introduced into Escherichia coli XL10-Gold to obtain transformant: Escherichia coli XL10-Gold / pDRL128vR.
[519] The polypeptide of the present invention and the DNA encoding the same can be used, for example, for the diagnosis, treatment, and prevention of bone and joint diseases and pathovascular neovascularization. In addition, the polypeptides of the invention are useful as reagents for the screening of compounds or salts thereof that promote or inhibit the activity of the polypeptides of the invention. Moreover, since the antibody to the polypeptide of this invention can recognize the polypeptide of this invention specifically, it can be used for detection, quantification, neutralization, etc. of the polypeptide of this invention in test liquid.
[520] In addition, by using the promoter of the present invention, proteins (any useful gene product, etc.) can be expressed predominantly (preferably specifically) and in large quantities in cartilage of non-human warm-blooded animals, contributing to the field of gene therapy. Can be.

权利要求:
Claims (24)
[1" claim-type="Currently amended] A polypeptide, an amide thereof, or an ester thereof, or a salt thereof, which contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 24.
[2" claim-type="Currently amended] A polypeptide, an amide thereof, or an ester thereof, or a salt thereof, according to claim 1, which contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 6.
[3" claim-type="Currently amended] The polypeptide, amide or ester thereof or salt thereof according to claim 1, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 is an amino acid sequence represented by SEQ ID NO: 26.
[4" claim-type="Currently amended] The polypeptide, amide thereof or ester thereof or salt thereof according to claim 2, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 is an amino acid sequence represented by SEQ ID NO: 12.
[5" claim-type="Currently amended] The polypeptide, its amide or ester thereof or salt thereof according to claim 1, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 24 is an amino acid sequence represented by SEQ ID NO: 49.
[6" claim-type="Currently amended] The polypeptide, amide thereof or ester thereof or salt thereof according to claim 2, wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 6 is an amino acid sequence represented by SEQ ID NO: 47.
[7" claim-type="Currently amended] DNA containing DNA which has a base sequence which codes the polypeptide of Claim 1.
[8" claim-type="Currently amended] The DNA according to claim 6, wherein the nucleotide sequence encoding the polypeptide of claim 1 is a nucleotide sequence represented by SEQ ID NO: 23.
[9" claim-type="Currently amended] The DNA according to claim 6, wherein the nucleotide sequence encoding the polypeptide of claim 1 is a nucleotide sequence represented by SEQ ID NO: 4.
[10" claim-type="Currently amended] The DNA according to claim 6, wherein the nucleotide sequence encoding the polypeptide of claim 1 is a nucleotide sequence represented by SEQ ID NO: 25.
[11" claim-type="Currently amended] The DNA according to claim 6, wherein the nucleotide sequence encoding the polypeptide of claim 1 is a nucleotide sequence represented by SEQ ID NO: 10.
[12" claim-type="Currently amended] The DNA according to claim 6, wherein the nucleotide sequence encoding the polypeptide of claim 1 is a nucleotide sequence represented by SEQ ID NO: 48.
[13" claim-type="Currently amended] The DNA according to claim 6, wherein the nucleotide sequence encoding the polypeptide of claim 1 is a nucleotide sequence represented by SEQ ID NO: 46.
[14" claim-type="Currently amended] A recombinant vector containing the DNA according to claim 6.
[15" claim-type="Currently amended] A transformant transformed with the recombinant vector according to claim 14.
[16" claim-type="Currently amended] A method for producing a polypeptide according to claim 1, an amide thereof, or an ester thereof, or a salt thereof, wherein the transformant according to claim 15 is cultured to produce the polypeptide.
[17" claim-type="Currently amended] The antibody against the polypeptide of Claim 1, its amide, or its ester, or its salt.
[18" claim-type="Currently amended] A method for screening a compound or a salt thereof, which promotes or inhibits the activity of the polypeptide or a salt thereof according to claim 1, using the polypeptide according to claim 1, an amide or an ester thereof, or a salt thereof.
[19" claim-type="Currently amended] A kit for screening a compound or salt thereof, which promotes or inhibits the activity of the polypeptide of claim 1, an amide thereof, or an ester thereof or a salt thereof, comprising the polypeptide of claim 1 or a salt thereof.
[20" claim-type="Currently amended] A compound or a salt thereof which promotes or inhibits the activity of the polypeptide of claim 1, an amide thereof, or an ester thereof or a salt thereof obtained by using the screening method according to claim 18 or the kit for screening according to claim 19.
[21" claim-type="Currently amended] A compound or a salt thereof, which promotes or inhibits the activity of the polypeptide of claim 1, an amide thereof, or an ester thereof or a salt thereof obtained by using the screening method according to claim 18 or the kit for screening according to claim 19. medicine.
[22" claim-type="Currently amended] A pharmaceutical comprising the polypeptide according to claim 1, an amide thereof, or an ester thereof or a salt thereof.
[23" claim-type="Currently amended] An agent for the prevention and treatment of bone, joint disease, or pathological neovascularization, comprising the polypeptide of claim 1, an amide thereof, or an ester thereof, or a salt thereof.
[24" claim-type="Currently amended] The diagnostic agent containing the antibody of Claim 17.

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同族专利:

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公开号 | 公开日

---

EP1191099B1|2009-10-07|

---

HU0201660A2|2002-08-28|

---

AU5705600A|2001-01-22|

---

NO20016344D0|2001-12-21|

---

EP1191099A1|2002-03-27|

---

CA2377791A1|2001-01-11|

---

CN1359421A|2002-07-17|

---

US6797483B1|2004-09-28|

---

WO2001002564A1|2001-01-11|

---

PL353065A1|2003-10-06|

---

AT445013T|2009-10-15|

---

NO20016344L|2002-02-19|

---

DE60043106D1|2009-11-19|

---

EP1191099A4|2002-08-07|

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:
1999-06-30|Priority to JPJP-P-1999-00186718

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1999-06-30|Priority to JP18671899

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2000-06-29|Application filed by 다케다 야쿠힌 고교 가부시키가이샤

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2000-06-29|Priority to PCT/JP2000/004278

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2002-04-10|Publication of KR20020026465A

优先权:

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申请号 | 申请日 | 专利标题

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JPJP-P-1999-00186718|1999-06-30|

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JP18671899|1999-06-30|

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PCT/JP2000/004278|WO2001002564A1|1999-06-30|2000-06-29|Novel polypeptide and dna thereof|