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    • 专利摘要:
    A hybrid plasminogen activator-like polypeptide (I) comprising a polypeptide region responsible for an affinity to fibrin derived from tissue plasminogen activator (TPA) polypeptide and a polypeptide region responsible for an enzyme activity derived from prourokinase polypeptide is new. The polypeptide region responsible for the affinity to fibrin may consist of 2 cringles from N-terminal first serine to 219th glycine of human TPA, 1 cringle from 128th serine to 219th glycine or half or a cringle from 161th methionine to 219th glycine. (I) may contain as a C-terminal half, an amino acid sequence from 150th glutamine to C-terminal 411th leucine of prourokinase. Also claimed are DNA segments coding for (I), plasmids contg. the DNA segment and microorganisms transformed with the plasmids.
    公开号:SU1732814A3
    申请号:SU874028997
    申请日:1987-01-30
    公开日:1992-05-07
    发明作者:Тагава Митито；Вада Масакацу；Ямада Масаюки
    申请人:Сагами Кемикал Рисерч Сентр (Фирма)；Сентрал Гласс Компани, Лимитед (Фирма)；Ходогая Кемикал Ко., Лтд (Фирма)；Ниппон Сода Компани, Лимитед (Фирма)；Ниссан Кемикал Индастриз, Лимитед (Фирма)；Тоио Сода Мануфакчуринг Ко., Лтд (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to medicine and can be used for the prevention and treatment of various types of disorders or diseases of the cardiovascular system.
    The aim of the invention is to obtain a hybrid plasminogen activator containing a region responsible for affinity with tissue plasminogen activator fibrin and a region responsible for the enzyme activity of a pro-urokinase polypeptide.
    PRI me R 1 (comparative). Isolation - poly (A) + RNA.
    Human throat cancer cell line Det rolt 5.62 was cultured in Eagle modified medium with 10% fetal calf serum, 0.1 mM non-essential amino acids, 1 mM sodium peat and 0.1% lactic acid albumin on a plastic cup in the presence of carbon dioxide at a concentration of 5%. The medium is separated and 2 ml of a denaturing solution (6 M guanidine thiocyanate, 5 mM sodium citrate, 0.5% sarcosine and 0.1 M 2-mercaptoethanol) are added to a 9 cm diameter plastic cup to denature the cell material to obtain an extract. The resulting extract
    4 SA) S 00
     Ca
    5.7 M CsCI is introduced into the solution and centrifuged. From 60 cups, 12 mg of RNA is isolated. Then poly (A) RNA is purified by oligo-T-cellulose chromatography and approximately 600 μg of RNA is obtained.
    PRI me R 2 (comparative). Getting the library to DNA.
    2 μg of the vector primer and 3 μg of poly (A) + RNA are mixed and 12 units are incubated. reverse transcriptase. Then oligo dC is inserted, hydrolyzed with Hind Ml, annealed and cyclized. The reaction mixture obtained in this way is stored as a cDNA library at -20 ° C. About 100,000 transformants were obtained from the reaction mixture, using E. coli phage x 1776 as competent cells.
    PrimerZ (comparative). Screen ning.
    To isolate clones that contain a plasmid carrying a gene encoding a plasminogen activator-like protein, the DNA of these transformants is hybridized. The probe uses synthetic DNA fragments of the following nucleotide sequence:
    Probe i
    5 3
    AATCGGGCACGATTTCCTG,
    Probe II
    5 3
    GCCCCCGCACAGGAACCG
    These sequences are complementary with respect to partial sequences to the DNA that encodes the plasminogen activator melanoma, (Probe I: nucleotide number 154-173; probe II: nucleotide number 1099-1116).
    Fragments are labeled with 32P - y - ATP at the 5th end. Labeled probes are hybridized on a filter in a hybridization buffer solution consisting of 900 mM NaCI, 90 mM sodium citrate, Denhart at 45 ° C for 20 hours with E. coli transformants grown on a nitrocellulose filter, denatured with alkali, Nitrocellulose filter washed in a solution of 300 mM NaCl and 30 mM sodium citrate, covered with an x-ray film to obtain an autoradiogram, and clones positive for the hybridization were determined. When using a mixture of probes I and II, 10 clones positive for hybridization were isolated from 5,000 clones. Among these clones positive for hybridization, one probe hybridized one clone of phage x 1776 E. coli (pD / TIME). From the specified clone receive plasmid RORAZ, x 1776 E. coli (pD / TIME). the plasmid containing rORAZ is deposited in F.R.I. eight
    but br 1984 in the depository number FERM-P-7391.
    The cDNA sequence consists of 2,459 base pairs, except for the poly (A) + - sequence located at the 3-terminus. Among these base pairs, 1548 base pairs encode 516 amino acids. This coding region additionally contains a sequence encoding
    0 ripened plasminogen activator (number of nucleotides 261-1703). There is a 5-non-coding region (nucleotide number 1-155), located in front of the coding region, and a 3-non-coding region (Nuk5 leotide number 1704-2459), located beyond the coding region.
    P 4 (comparative). Obtaining mRNA.
    20 g of kidney tissue cells frozen at -80 ° C are crushed under a liquid nitrogen atmosphere, suspended in 80 ml. 5M guanidine thiocyanate, 0.5% N sodium lauroylsarcosine, 25 mM sodium tartrate, 0.1 M mercaptoethanol and 0.1% anti-foam (solution A). The suspension is homogenized and the nucleic acid is separated by a 20G injection needle. 24 ml of the solution is distributed into 12 ml of 5.7 M CsCl, and after centrifugation, the crude RNA is isolated.
    All RNA is dissolved in a 2% potassium acetate solution, two volumes of ethanol are added, incubated overnight at -20 ° C and centrifuged.
    5 Poly (A) 1 RNA is isolated and purified by chromatography on a column of oligo (LT) cellulose. 10 g of kidney tissue cells give about 3 mg of total RNA, from 2 to 3% of which is poly (A) + RNA.
    0 example (comparative). Construction of cDNA library (1).
    The synthesis of cDNA was carried out using 40 µg of poly (A) + RNA obtained in comparative example 4.
    5 When using as a primer 40 µg of oligo (dT) 12-18, for the synthesis of the first chain, it is reacted with 40 units. reverse transcriptase for 2 h at 42 ° C. Pattern RNA is removed by
    0 alkalis and the synthesis of the second chain with 100 units, the Klenow fragment of E.coli DNA polymerase 1 is carried out.
    After removing the hairpin loop with the S1 nuclease chain (DP), the o-20 is connected 5 to the 3-terminus of the double-stranded cDNA using terminal deoxynucleotide-transferase and about 400 mg of dC-terminal cDNA is obtained.
    This DNA is stitched with 800 mg of the pBR322 vector DNA at the Pstl site. transform into
    E. coll XI 776. Thus, a cDNA library (1) is obtained, consisting of about 2x10 tetracycline-resistant and ampicillin-sensitive transformants.
    PRI me R 6 (comparative). Preparation of synthetic DNA oligomers for screening a cDNA library.
    The following 16 different DNA oligomers consisting of 14 nucleotides complementary to mRNA, which corresponds to the amino acid sequence of human urokinase (CC), are synthesized by the phosphotriester ester method.
    AACCAAGGTTGATT,
    AACCAAGGTTGGTT,
    AACCAGGGTTGATT.
    AACCACGGTTGATT, AACCACGGTTGGTT,
    AACCATGGATTGATT,
    AACCATGGTTGGTT.
    AACCAAGGCTGATT,
    AACCAAGGCTGGTT,
    AACCAGGGCTGATT,
    AACCAGGGCTGGTT,
    AACCAGGGCTGATT,
    AACCACGGCTGGTT,
    AACCATGGCTGATT,
    AACCATGGCTGATT,
    AACCAGGCTTGGTT.
    These 16 oligomers hereinafter referred to as the UK probe I.
    For use as test probes, the following 8 different DNA oligomers consisting of 14 nucleotides complementary to mRNA, which correspond to Met238, Try, Asn, Asp, Pro287, are synthesized in the same way:
    5 GGATCATTATACAT 3
    GGATCGTTATACAT,
    GGATCGTTGTACAT,
    GGATCATTGTACAT,
    GGGTCATTATACAT,
    GGGTCGTTATACAT,
    GGGTCGTTGTACAT,
    GGGTCATTGTACAT.
    These 8 oligomers are designated as UK probe II.
    To obtain probes used for colony hybridization, probes I and II have a radioactive label inserted at the 5th end of the probe.
    Example (comparative). Screening cDNA library0).
    1. Screening.
    The autoclaved nitrocellulose filter is placed on an LB agar medium containing 15 μg / ml of tetracycline, and the medium is inoculated with transformants prepared in comparative example 5 so as to provide approximately 2000 colonies of transformants. After 8 hours of incubation at 37 ° C, colonies replicate onto two nitrocellulose filters, which are incubated for another 3 hours at 37 ° C. The initial nitrocellulose filter is used as a reference filter, and two secondary filters are transferred to an LB agar medium containing 15 µg / ml tetracycline and 100 mg / ml chloramphenicol, incubated overnight at 37 ° C, then placed over 0, 5M NaOH
    0 and 1.5 m NaCI for 3 min to induce lysis of the colonies and DNA denaturation, and after neutralization with 0.5 M tris-HCl, pH 7.6 and 1.5 M NaCI, dried in air for 2 hours at 80 ° s
    5 After washing the filters in 4xSSC for 30 minutes at 60 ° C, prehybridization was performed in 4xSSC, 10 x Denhardt and 50 µg / ml of denatured E.coll DNA, for 1 hour at 60 ° C. After adding 0.1
    0 mM of ATP and the UK of probe I with a radioactive label are hybridized for 16 hours at 37 ° C, washed 6-8 times in 4xSSC filters at 39 ° C, dried in air and screened for transformants, hybridized with a UK probe I, 21 candidate clones from 8x10 colonies are obtained. The plasmids of these clones are hereinafter referred to as plasmids pKYU1 -PKYU21.
    The obtained 21 candidate clones are treated in the same way as described, and clones hybridizing with the AC probe II are obtained. The plasmids in these clones are hereinafter referred to as plasmids pKYU and 21.
    2. Plasmid DNA Characteristics
    5 pKYU21.
    After DNA cleavage of the plasmid pKYU21 by various restriction endonucleases and their subcloning in M13tp8, the nucleotide sequence is determined. When comparing this sequence with the well-known amino acid sequence of the CC, it is confirmed that, although the cDNA contains the complete coding region of the low molecular weight CC, but about 100 bp is missing. DNA at the 5'-end of the high molecular weight coding region.
    Example (comparative). Construction of cDNA library.
    0 Based on the nucleotide sequence as defined in paragraph 2 of Example 7, the following DNA oligomer is synthesized by the phosphotriester method:
    55 STSAASSASSATSA 3,
    consisting of 15 nucleotides, complementary to the mRNA sequence, which corresponds to Ser, Asp, Ala. Leu, Glu.
    Using 100 μg of poly (A) + RNA as a template, the first strand of cDNA is synthesized with 100 units. reverse transcriptase together with 1 μg of 5 -32p-labeled primer, followed by the synthesis of the second strand using the Klenow fragment of DNA polymerase with 1 E. col. The single-stranded DNA was digested with S1 nuclease and the dC-chain was added to the 3-terminus using a terminal deoxynucleotidyl transferase. After co-renaturation of this dC-tail insert and the dG-tail vector pBR-322, E.coll x 1776 is transformed into a cDNA library (I) consisting of approximately 5x10 4 transformants.
    PRI me R 9 (comparative). Screening of cDNA library (N).
    The transformants obtained in Example 8 are hybridized according to the procedure described in claim 1 of Example 7. In this case, a 150 bp cleavage fragment is used as a probe. Pst l-Bgl II 5, from pKYU 21 with a radioactive label, introduced by the translation method. Hybridization is carried out at 60 ° C.
    The filter is air dried after washing two or three times with 2xSSC at 60 ° C and clones hybridizing with the probe are detected by autoradiography. Get eight positive clones. The plasmids in these clones are hereinafter referred to as plasmids ppe1-ppe8. By digesting the DNA of these plasmids with the restriction enzyme Pstl, it was confirmed that the PRE3 plasmid contains a cDNA insert with a length of approximately 420 bp.
    The fragments obtained by cleaving the DNA of the plasmid PRE3 endonuclease restriction Pstl, are subcloned into M13 pt8 and the sequence of the bases is determined. The results confirm that KDN K contains not only the coding region of sufficient length on the 5-terminal side of the prouroclinase gene, but also the 5-untranslated region of length 66 bp. up from the translational initiation codon.
    Example 10 (comparative). Constructing a prourokinase gene.
    5 µg of the plasmid pKYU 21 DNA containing the complete urokinase coding region are digested with restriction endonucleases Bgl II and Hind III (no 10 units) and DNA fragments of about 5.7 kb are isolated. By digesting the DNA of the indicated plasmid pKYU 21 with restrictases Bgl II and Nco I (10 units), a DNA fragment of 66 bp is obtained. 5 µg of the plasmid pEPP3 obtained in Example 6 digested 10 units. restriction Nco I and Hind III, obtaining a DNA fragment
    about 1.1 kb These three different DNA fragments are re-extracted with phenol / chloroform, precipitated 2 vol. ethanol for purification and extraction of DNA. These fragments
    DNA is fused together with T4 DNA ligases and transformed into E.coll x 1776. The resulting transformants are screened by rapid isolation using an alkaline lysis procedure, and
    0 get a clone carrying the plasmid pKYU 22, which contains the complete pro-Urokinase gene.
    PrimerI (comparative). Determination of nucleotide sequence
    5 insert site plasmid pKYU 22.
    The nucleotide sequence of the insertion site of the plasmid pKYU 22 is determined by the MaxamTilbert method and the dideoxy chain termination method before subcloning in M13tp8.
    The insert consists of a 5 bp 66-bp region, the ATG (Met) -GGC (Cly) main sequence, the AGC (Ser) -CTC (Leu) coding region of the prourokinase,
    5 codon of the termination of translation of TCA (XXX) and 3-untranslated codon for the stop codon.
    PRI me R 12 (comparative), the synthesis of the gene prourokinase, modulated in
    0 5 end area.
    The codons of the 5-terminal region of the native cDNA encoding prourokinase are replaced so as to ensure efficient expression of the prourokinase gene in E. coli
    5, the saSD sequence of the C230 gene obtained by H3 Pceudomonas putide, the translation start codon ATG (Met) is inserted before the codon of the first amino acid (Ser) so that purokinase can be directly expressed; The restriction enzyme recognition restriction enzyme is linked to the coding region next to the SD sequence of the expression vector. For this, double-stranded DNA oligomer is synthesized
    5 phosphotriester method. This two-stranded synthetic DNA has an Aat II site at one end for introducing it into a vector and at the other end a Tag I site for attaching it to the prourokinase gene.
    0 The following single-stranded oligomers of DNA are synthesized using the Phosphothryester method, containing respectively 29.15 and 20 nucleotides;
    5 CATGAGCAACGAGCTCCACCAGGTTCCGT 3 53 TGCAGTACTCGTTGC 5,
    3 TCGAGGTCCTCCAAGGCAGC 5.
    1 μg of each of the synthetic oligomers of DNA is heated for 2 minutes at 90 ° C, phosphorylated at the 5th end with T4 polynucleotide kinase and purified. After drying, the purified material is dissolved in 50 µl of 20 mM Tris-HCl, pH 7.6 and 10 mM MgCl2, renatured by heating for 2 minutes at 95 ° C, cooled slowly to room temperature and kept at room temperature overnight. 12 ° C, obtaining the following double-stranded DNA
    5 CATGAGCAACGACCTCCACCAGGTTCCGT 3, 3 TGCAGTACTCGTTGCTCGAGGTGGTCCAAGGCAGC.
    Aatll Sstl Bstl Tag I
    5 μg of plasmid pKYU22 DNA was digested with restriction endonucleases Bglll and Aatll, a 5.7 kb DNA fragment. Another 5 µg of the same plasmid pKYU22 DNA was digested with the restriction endonucleases Pst I and Bgl II and a DNA fragment of about 400 bp was obtained. This fragment is cleaved again with the restriction enzyme Tag I and the 260 bp DNA fragment is extracted. The DNA fragments are recovered, purified by extraction with phenol / chloroform and precipitated with 2% ethanol.
    These two DNA fragments and the indicated double-stranded synthetic oligomer of DNA are stitched together using T4 DNA ligase and the cross-linking product is used to transform E.coll x 1776. Transformants are screened by rapid isolation using an alkaline lysing procedure and a clone of E.coll x 1776 / pKMUI carrying the plasmid cKMSh, which contains a modified pro-urokinase gene. The clone E.coll x 1776-pKMUI was deposited in F.R.I on January 11, 1985, as FERM-P-8040.
    Example 13 (comparative). Construction of plasmid pMuT4L.
    A pMuT4L expression plasmid containing a human prourokinase gene without a point mutation is constructed from the indicated pKMUl plasmid and the pTCMI expression vector. E.coll IM103 / pTCMI, deposited from F.R.1.17 August 1984, as PERM P-7779.
    5 µg of the pKMUl plasmid from Example 12 was cleaved with 10 units of the Aatll restriction enzyme and the digest was isolated after phosphate treatment of the calf intestine (FCT). Another 5 µg of the plasmid pTCMI cleaves 10 units. restriction endonucleases Aatll and electrically eluted a 500 bp DNA fragment. These two DNA fragments are purified by re-extraction with phenol / chloroform and ethanol precipitation.
    Both DNA fragments are bound by T4 DNA ligase and transformed into E.colle M103. Transformants are screened and receive a clone carrying the plasmid pMiT11, in which tac promoter {operator)
    and the C230 SD sequence have the correct orientation towards the prourokinase gene.
    Next, 5 µg of the plasmid pKK223-3 cleaves 10 units. Hind III restriction endonuclease and digest are treated with calf intestine phosphatase.
    1 µg of the plasmid pMu T1, digested 4 units. restriction endonuclease Dral. The cleavage fragment and 1 μg of the 5-phosphorylated Hlndlll linker (dSAACSTTS) are crosslinked with T4 DNA ligase. The solution is extracted with an equal volume of the phenol / chloroform mixture and the DNA is precipitated by adding 2 vol. ethanol. The precipitate is collected at 16000 rpm and 4 ° C and dried.
    The resulting pM and TIL cleavage fragment and the Hlndlll cleavage fragment of the indicated pKK233-3 were stitched using T4 DNA ligase and transformed into E. coll M103. Transformants are screened and clone E.coll M103 / pM T21 is obtained. containing plasmid pH and T21.
    5 µg of the pMiT21 plasmid cleaves 10 units. the restriction endonucleases Sphl and Tth III and after extraction with a mixture of phenol / chloroform DNA precipitated with ethanol. The DNA thus extracted is supplied with a blunt end using T4 polymerase in the presence of 0.1 mM dGTP, dCTP and TTP and recycled T4 DNA ligase. The DNA is transformed into E.coll I M103 and form colonies on LB agar medium containing 50 μg / ml ampicillin. Transformants are screened and a clone of E. coli IM103 / pMuT4 is obtained.
    Example 14 (comparative). The introduction of specific base substitution mutations into the codon for amino acid 157 using phage M13.
    1. Getting single-stranded matrix
    DNA
    1 micron of each of the pKYu 22 plasmid and the double-stranded DNA of phage M13 tr8 was split into 20 µl of a solution containing 10 mM Tris-HCl, pH 7.5 7 mM MgCl2, 7 mM / J-mercaptoethanol and 50 mM NaCI, for 1 hour at 37 ° C when using 5 units. Pstl. The corresponding DNA fragments are extracted after treatment with phenol and precipitation with ethanol. DNA fragments are stitched in 20 μl of a solution containing 66 mM Tris-HCl, pH 7.5, 5 mM MgCIa, 5 mM DTT and 1 mM ATP for 16 hours at 12 ° C using T4 DNA ligases and transformed into E.coll IM103, Transformants are placed on plates of soft agar containing 0.02% X-gal and 1 mM IPTG, incubated overnight at 37 ° C. Single-stranded DNA is obtained from white spots formed by a recombinant.
    When using some of the forming single stranded DNA as a template, the base sequence is determined. The coding chain and the anti-coding chain were obtained.
    2. Introduction of a specific base substitution mutation.
    When using the resulting recombinant single-stranded DNA of phage M13 (the anticoding sequence of the CC gene as a matrix, the site-directed mutation is introduced using an oligonucleotide mutagen,
    5 GGCCCGCCGATAACATTA 3; .
    Although this 18-base oligonucleotide is complementary to the CC gene in single-stranded template DNA, the two bases are changed, with the TTT code, which defines phenylalanine, replaced by a CAT codon, which determines aspartic acid.
    When using the indicated oligonucleotide as a primer, two-strand DNA is synthesized in vitro. At the same time, 2 mmol of 5-phosphorylated primer is added to 0.5 mmol of matrix single-stranded DNA and the mixture is incubated in 10 µl of a solution containing 7 mm of Tris-HCl (pH 7.5) with 0.1 mM EDTA, 20 mM NaCI and 7 mM MgCI for 20 min at 60 ° C, followed by incubation for 20 min at 23 ° C. 0.5 mM dATP, dGTP, dTTP, dCTP are added to the reaction mixture to a volume of 20 µl and 2 units. fragment maple DNA polymerase. The mixture was incubated for 20 minutes at 23 ° C and after adding 2 µl of 10 mM ATP and 1 U. T4 DNA l-jazy overnight at 12 ° C. The mixture is directly transformed into E. coli M103. About 10,000 phage patches are obtained per microliter of the indicated reaction mixture.
    After transferring the spots from the soft agar medium to the nitrocellulose filter, the filter is dried in vacuum for 2 at 80 ° C, hybridized with the oligonucleotide primer as a probe labeled with 32P. The filter is washed in GxSSC at 52 ° C and the spots of the mutant phage, giving positive signals, are recovered by autoradiography. DNA of mutant phage of double-stranded type (rH3) is obtained from mutant phage. When mutant phage DNA is used as a template, the nucleotide sequence of the mutant DNA is determined and the appearance of a single base mutation is confirmed.
    In addition, it is possible to replace the TTT code, which determines the phenyl / cumin and 137 position, to the Gin-determining codon of the ALA using the following oligonucleotide:
    5 GGCCCCGCGAAAACATTA 3 as a mutagen.
    Example 15 (comparative). Construction of plasmid pMu T4L rtZ,
    0 After complete cleavage of 10 double DNA DNA. mutant M13 (rgpZ) using Pstl isolate a fragment of about 1.2 kb in length. 10 μg of plasmid pMu T41. Pst is partially cleaved and a fragment of about 4.6 kb in length is isolated, from which a fragment of about 1.2 kb in length is removed.
    Each of these two fragments is isolated by treatment with phenol and precipitation with ethanol, the precipitates are mixed.
    0 are ligated overnight at 12 ° C. T 4 with DNA ligase and transformed into E. coli HB101. Transformants are screened and a clone containing the plasmid pMu T4L rhpZ E. coli x 1776 / pMu T41 rH3, carrying the plasmidu pMu T41 rhpl, deposited on July 11, 1985, p. F.R.I, as FERM-P-8341 and placed from the international diplomacy on January 22, 1986, as PERM BP-971 in accordance with the provisions of the Budapest Treaty.
    0P rime 16 (comparative). Construction of plasmid pMu T41 pmS.
    Mutamic double-stranded phage M13RFpml, containing DNA fragment, is obtained. wherein the AAA codon for the 135th lysine is mutaro5 van to the CAA codon for glutamine, following the same procedure as described in Example 14, except that the following synthetic oligonucleotide is used: 5 GATGGACAAAAGAG. 3
    0 Then from phage M13 RF prnl and plasmid
    pMu T41 design PMu T4L prnl no the same method as described in example 15,
    Example 17 (comparative). Construction of plasmid pMu T4L pt4.
    .5 A mutant double-stranded phage containing a DNA fragment in which the AAA codon for the 135th lysine is replaced by the glutamine codon is obtained according to the procedure described in Example 14, except that
    0 using synthetic oligonucleotide
    5 GATGGACAAAAGCCC 3 From the mutant phage thus obtained, single-stranded phage are obtained according to the method described in Example 14. When using single-phage as the matrix, the TTT codon for 157th phenylalanine is then mutated to the GAT codon for asparzginic acid according to the described procedure. Except that the following synthetic oligonucleotide is used:
    5 GGCCCCGCGATAAGATTA 3 to obtain mutant double-stranded Phage M13 RFpm4 containing the DNA fragment. in which the AAA code for the 135th lysine is replaced by the codon CAA for glutamine and the codon for the 157th phenylalanine is mutated to the codon for aspartic acid.
    Then from the thus obtained mutant phage M13RFpm4 and the plasmid pMu T41L construct plasmid pMu T41L pt4 according to the method described in example 15.
    Example 18 (comparative). Construction of plasmid pMu T91 pt1.
    5 µg of plasmid pMu T41 cleaves 50 units. Asa I and 100 units. Aatll in 100 μl of buffer containing 10 mM Tris-HCl, pH 7.5, 7 mM MgCJ2, 125 mM NaCI, 7 mM / -mercaptoethanol at 37 ° C for 6 hours. The reaction mixture was subjected to 0.7% electrophoresis. - nom agarose gel and extract the DNA fragment about 5500 p. Another 10 µg of the same plasmid pMu T4L cleaves 50 units. Aatll and 50 units. Smal. The reaction mixture was subjected to electrophoresis on a 2.0% agarose gel and the 55 bp DNA fragment was extracted. Thus obtained two DNA fragments sew 5 units. T4 DN K-ligase. The reaction mixture is used to transform E.coll. Ampicillin-resistant transformants are screened for colonies containing the pMu T8L plasmid, and the pMu T8L plasmid is isolated from the selected colonies.
    5 µg of the PMu T84 plasmid thus obtained splits 50 units. HindiII. After extraction with phenol and precipitation with ethanol, the precipitate is treated with 1 unit. fragment maple in 20 μl of buffer containing 50 mm Tris-HCI. pH 7.2, 10 mM MgCl 2, 0.1 mM DTT and 80 μM dN IPS at 22 ° C for 30 minutes. After extraction with phenol and precipitation with ethanol, the precipitate is treated with 5 units of T4 DN K-ligase in 20 µl of buffer containing 1 µg of pSs T1 linker and 66 mM Tris-HCl, pH 7.6, 6.6 mM MgCl2, 10 mM DTT and 1 mM ATP at 12 ° C for 2 h, extracted with phenol and precipitated with ethanol. The pellet decomposes 50 units. Pstl in 100 µl of a solution containing 20 mM Tris-HCl, pH 7.5.10 mM MgCl2 and 100 mM NaCl at 37 ° C for 2 h. The reaction mixture was subjected to electrophoresis on a 0.7% agarose gel in a fragment DNA size 4300 p.
    5 µg of the pMu T4L plasmid pml cleaves 50 units. Pstl. The reaction mixture is then
    subjected to electrophoresis in a 0.7% agarose gel and extract the DNA fragment about 1200 p. O.
    DNA fragments are mixed and stitched using 5 units. T4 DNA ligase. The reaction mixture is transformed into E.coll IM103. Ampicillin resistant transformants are screened for colonies containing a plasmid pMu T9L pml and the plasmid pMi
    0 T9 pml selected.
    PRI me R 19. Construction of plasmids pD PAT2.
    50 µg of pD plasmid 75 times digested Bgl II in 200 μl of buffer containing
    5 10 mM Tris-HCl, pH 7.5.7 MM, 100 mM NaCI and 7mm / 5-mercaptoethanol at 37 ° C for 4 h.
    After ethanol precipitation, the precipitate is incubated with 5 units. fragment maple in 50 μl
    0 buffer containing 50 mM Tris-HCl, pH 7.2, 10 mM MgCl2, 0.1 mM DTT and 80 μM at 22 ° C for 30 minutes. The reaction mixture is subjected to electrophoresis on a 0.7% agarose gel and the DNA fragment is extracted.
    5 size 1800 p. This DNA fragment is referred to as DNA fragment (A).
    10 µg of plasmid pYTU3 cleaves 20 units. Sail, precipitated with ethanol, treated with 2 units. fragment maple. After precipitation
    0 ethanol precipitate is treated with alkaline phosphatase, phenol and ethanol. The precipitate is dissolved in 20 µl of a solution containing 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. 5 μg of this DNA and 5 μg of the DNA fragment (A) process 10 units. T4 DNA ligase. After precipitation with ethanol, the precipitate is cleaved with 15 units of C1a I. After precipitation with ethanol, the precipitate is cleaved with 15 units. BamHI. After DNA precipitation with ethanol, the precipitate is treated with 2 units.
    0 fragment maple. The reaction mixture was subjected to electrophoresis on a 0.7% agarose gel, and a 2000 bp DNA fragment was recovered. This DNA fragment is designated as DNA fragment (B).
    5 2 µg of the plasmid pK12 cleaves 10 units, Sal I. After precipitation with ethanol, the precipitate is treated with 20 units. fragment maple, precipitated with ethanol, the precipitate is treated with alkaline phosphatase. After treatment with phenol, ethanol precipitation is carried out. The precipitate is dissolved in 20 µl of a solution containing 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. 1 μg of this DNA and 1 μg of DNA fragment (B) are ligated. Reactionary
    5, the mixture is used to transform E.coll IM103. Ampicillin resistant transformants are screened from colonies containing plasmid pDPAT2 and DNA is isolated.
    PRI me R 20. Construction of plasmids RNAOO.
    5 µg of the PREZ plasmid (same as T4L PRMU) split 10 units. PVU I. After precipitation with ethanol, the precipitate is cleaved by 10 units. EcoR I. The reaction mixture is subjected to electrophoresis in a 1.2% agarose gel and a DNA fragment of about 2000 bp is recovered, which is designated as DNA fragment (A).
    10 µg of plasmid digested 15 units. PVUI, and then 15 units. BamPI. The reaction mixture was subjected to electrophoresis on a 1.2% agarose gel and DNA fragments of about 2,300 bp were recovered. and about 1346 p. A DNA fragment of about 2300 p. O, is designated as a DNA fragment (B). 2 μg of the DNA fragment 1346 p. split 5 units. BamHI and subjected to electrophoresis in a 1.2% agarose gel, extract the DNA fragment of about 75 O. o., and is designated as a DNA fragment (B).
    10 µg of the plasmid pDPAT2 constructed in Example 19 digested 15 units. EcoRI, subjected to electrophoresis in a 1.2% agarose gel, and extracting the DNA fragment of about 472 p.o, 2 μl of the fragment is cleaved with 1 U-Hindlll, subjected to electrophoresis in 5% PAAG, and removing the DNA fragment of about 180 bp. . 1 μg of this fragment and 1 μg of the DNA fragment (B) are ligated and a DNA fragment of about 931 bp is extracted by electrophoresis in a 1.2% agarose gel. 1 μg of this DNA fragment, 1 μg of DNA fragment (A) and 1 μg of DNA fragment (B) are stitched 2 units. T4 DNA ligase. The reaction mixture is transformed into E.coll IM103. Ampicillin resistant transformants are screened for colonies containing the pNAO plasmid, and the plasmid is isolated.
    PRI me R 21. Construction of plasmids rnaoz.
    The pNAOZ plasmid is constructed according to the procedure described in Example 20, except that the plasmid PRE3 rH3 is used instead of the plasmid PRE3 (same as pMu T4Lpm3).
    PRI me R 22. Construction of plasmids RNA.
    5 μg of the PHAOO plasmid, hydrolyzed with Pstl restriction enzyme, precipitated with ethanol. The pellet was digested with 10 units. Seal by electrophoresis in a 1.2% agarose gel and extract the DNA fragment of about 1100 p. This DNA fragment is designated as DNA fragment (A).
    5 µg of the pNAOO plasmid digested 7 units. pstl, precipitated with ethanol. The pellet was digested with 10 units. EcoRI, precipitated with ethanol, the precipitate subjected to electrophoresis in a 0.7% agarose gel and extract the DNA fragment about 3500 p. O. This DNA fragment is designated as DNA fragment (B).
    15 µg of the plasmid pDPAT2, constructed in Example 19, digested 20 units. Bgl II. After precipitation with ethanol, the precipitate was digested 25 units of Zsa), subjected to electrophoresis on a 1.2% agarose gel, and a DNA fragment of about 625 bp was recovered. This DNA fragment is designated as DNA fragment (B).
    10 µg of the plasmid pDPAT2 is cleaved with 15 units of Bglll, and after precipitation with ethanol, the residue is cleaved with 10 units of EcoRI. The reaction mixture was subjected to electrophoresis in 5% PAAG, and a fragment of DN K of about 150 bp was extracted. This DNA fragment is designated as a DNA fragment (G).
    5 1 μg of the DNA fragment (A), 1 μg of the DNA fragment (B), 1 μg of the DNA fragment (C) and 1 μg of the DNA fragment (G) are stitched with 4 units. T4 DNA ligase. The reaction mixture is used to transform E.coll IM103,
    0 Ampicillin resistant transformants are screened for colonies containing the plasmid pNA20. The plasmid is isolated.
    PRI me R 23. Construction of plasm5 of pNA23.
    The plasmid pNA23 is constructed according to the procedure described in Example 22, except that the pNAOZ plasmid constructed in Example 21 is used instead of
    0 pNAOO plasmid used to construct pNA20 plasmid.
    PRI me R 24. Construction of plasmids RNA.
    25 µg of plasmid pNA23 cleaved
    5 30 units. Bgl II. After ethanol precipitation, the precipitate is treated with 4 units. fragment Klenov, treated with phenol and precipitated with ethanol. The pellet decomposes 30 units. Pst by electrophoresis on a 0.7% agar gel and extract the DNA fragment K about 3500 bp, designated as the DNA fragment (A), and the DNA fragment about 1700 bp, designated as the DNA fragment (B ).
    7 µg of DNA fragment (B) cleaved 10
    5 items RSal. The reaction mixture was subjected to 1.2% agarose gel electrophoresis, and a DNA fragment of about 1100 bp, designated as a DNA fragment (B), and a DNA fragment of about 626 bp, designated
    0 as a DNA fragment (G).
    2 µg of the DNA fragment (D) was digested with 5 units of Ddel in 20 µl of buffer containing 100 mm Tris-HCl (pH 7.5), 5 mm MgCk, 100 mM NaCI and 7 mM / 3-mercaptozanol at 37 ° C.
    5 for 8 hours. After precipitation with ethanol, the precipitate is treated with 1 unit of Klenow fragment, treated with phenol and precipitated with ethanol. Using electrophoresis in a 5% polyacrylamide gel, a DNA fragment of 241 bp is extracted. 1 mcg of this.
    DNA fragment 0.5 wr DNA fragment (B) crosslinked with 3 units, T4 DNA ligase. The reaction mixture is used to transform E. coli IM103. Ampicillin resistant transformants are screened for colonies containing plasmid pHA13. Plasmid selection.,
    Example 25. Expression and extraction of the gene product,
    a. E. coli IM103 / pDPAT2, IM103 / PHAOO, SHO3 / pNAOZ, IM103 / pHA20, 1M103 / pHA23 m 1M103 / pHA13, obtained in examples 19-23, -l E. coli IM 103 / pMU T4L, obtained in example 15, cultured in 100 ml of LB-cultures with the addition of 100 µg / ml of ampicillin at 37 ° C with lysis. When the optical density at 600 im of the culture medium reaches 0.6, medium isopropyl-D-thiogalcopyranoside is added to the final concentration of 1 mM and the culture is continued for another 5 hours. The broth broth is then transferred to an ice bath.
    The cooled bouton is centrifuged to collect the cells, which are then suspended in 50 ml of buffer containing 50 mM Tris-HCl (pH 7.5) and 100 ml of NaC. The cells are collected by centrifugation, resuspended in 10 ml of the same buffer. After that, the cells are sonicated and centrifuged at 15000 rpm for 10 minutes at 4 ° C.
    5. The sediments are suspended in 10 ml of buffer containing 7.5 M solution of guanidine hydrochloride and 50 mM Tris-HCl, pH 7.5, and set aside at room temperature for 90 minutes, centrifuged at 10,000 rpm for 10 min, and the supernatant diluted to 5 ml of a solution containing 1M guapidine hydrochloride, 0.05 M tris-HCl pH 7.5, 2 mM glutathione of reduced hypo 0.2 mM glutathione of oxidized hypa, and incubated overnight at room temperature . Then the solution is dialyzed against 100 vol. a solution containing 10 mM Tris-HCl, pH 7.4 and 0.4 mM, at 4 ° C for 4 hours, and then against a 100 v solution containing 10 mM Tris-HCl, pH 7.4 and 0.1 m NaCI, for 2 hours to obtain the extract
    Example 26. Each precipitate obtained from each transformant in Example 25a, in cc / quality corresponding to 1 ml of cupral broth, was supplemented with 20 µl of a solution containing 3 M urea solution, 0.08 M dithiothreitol solution and 1% sodium dodecyl sulfate. The mixture is heated at 95 ° C for 5 minutes, centrifuged, the supernatant layer (8 µl) is layered on a sodium dodecyl sulfate - polyacrylamide gel gradient and electrophoresis is performed. After electrophoresis, the gel is stained with 1% Coomasie dye and washed. A sample of an expression product from 1M103 / pNAOZ, E. coli, which produces a plasminogen hybrid activator, a similar NAOPH 1 polypeptide, is compared with a sample of an expression product from E. coli IM103 / pDPAT2, which produces a native human plasminogen activator (APT), with a sample an expression product from SHOZ (pREZ / pMi T4L) E.coll, which produces the native (human) human, using electrophoresis.
    Each sample obtained in the manner described is subjected to gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and the separated proteins are transferred to a nitrocellulose filter, which is then subjected to an immunoassay. For this, the nitrocellulose filter is impregnated in a solution of 3% gelatin in TBS (20 mM tris -HCI, pH 7.5, and 500 mM NaCI), after which the filter is impregnated for 2 hours in a solution containing antiserum of the antisyntin plasminogen activator obtained from a rabbit immunized with tissue activator pl azminogen. The filter is washed with a solution of TBS and then impregnated in a solution containing an anti-rabbit IgG antibody labeled with peroxidase horsetail. After thorough washing, the filter is impregnated in a solution containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCI, 0.06% 4-chloro-1-naphthol, and 0.015% Na02 for 5 minutes. The results show that the hybrid polypeptide is immunoreactive to both serum against tissue plasminogen activator and antiserum against urokinase.
    Example 27. Preparation of a sepha rose column related to fibrin.
    3 g of fibrinogen is dissolved in 100 ml of binding buffer containing 0.1 M NaHCOs, pH 8.3 and 0.5 M NaCl, centrifuged and the supernatant is used for the subsequent reaction.
    2 g of CNBr-activated Sepharose 4B (Pharmacia) are saturated with 30 ml of 1 mM HCl to obtain about 7 ml of carrier. The carrier is loaded onto a C3 glass filter and washed 4 times with 25 ml of 1 mM HCl. In addition, the carrier is washed with 20 ml of binding buffer. The washed carrier is suspended in 20 ml of the same buffer. The suspension is added to the fibrinogen solution and incubated at room temperature for 2 hours with stirring. The supernatant is then removed. The residual material is replenished with 50 ml of a 0.2 M solution of glycine.
    (pH 8.0), stirred at room temperature for 2 hours in order to inactivate residual active groups. After removing the supernatant, the residual material is washed alternately five times with 20 ml of each 0.2 M solution of glycine (pH 8.0} and acetate buffer containing 0.1 M sodium acetate (pH 5.0) and 0.5 M NaCl, glass filter so as to finally obtain about 7 ml of carrier associated with fibrinogen.This carrier is introduced into a column with a diameter of 16 x 70 mm.
    300 units bovine thrombin is dissolved in 3 ml of distilled water, and the solution is gradually passed through a column for 2 hours in order to convert the fibrinogen associated with the carrier into fibrin. The column is then equilibrated with 100 μl of a buffer containing 0.02 M Tris-HCl, pH 7.5, 0.15 m NaCl and 0.05% Triton X-100.
    PRI me R 28. The extract obtained in example 256, in an amount corresponding to 5 μg of the expression product, is adjusted to 100 μl of the final liquid volume containing 0.02 m Tris-HCl, pH 7.5, 0.15 M NaCl and 0.05% Triton X-100, the solution is passed through a column. The column was washed with 40 ml of buffer containing 0.02 M Tris-HCl, pH 7.4, 0.15 M NaCl and 0.05% Triton X-100, eluted with elution buffer containing 2 M KSCN, 0.2 M Tris-HCl, pH 7.4 and 0.05% Triton X-100 with a fraction collector content. Each fraction was measured for plasminogen activator activity using fibrinolytic activity on a plasminogen-containing fibrin plate.
    Fibrinolytic activity is measured as follows. 0.1 g of type I fibrinogen is dissolved in 5 ml of 0.06 M phosphate buffer solution and mixed with 0.025 g of agarose dissolved in 5 ml of the same buffer. Bovine thrombin was added to the mixture to a final concentration of 1 mM, and after stirring the mixture was poured onto a 8.5-cm Petri dish. 10. µl of each sample was stained with a fibrin cup, which was then incubated at 37 ° C for 14 hours. The diameters of the developed lysis circles are measured. From the measurement of a commercial CM, a graduation curve of activity is obtained depending on the diameter of the lysis circle, on the basis of which the activity of the test sample is obtained.
    From these data, it can be seen that, if a commercial CM is not adsorbed on fibrin-sepharose, then the hybrid polypeptide that exhibits the activity of plasminogen activator, which contains complete or
    the part of the kringle region responsible for the fibrin affinity of the human tissue plasminogen activator, as well as the recombinant tissue plasminogen activator,
    produced in E.coll and a commercial tissue plasminogen activator are bound by fibrin-sepharose.
    PRI me R 29. Determination of Km using synthetic substrate S 2288.
    S-2288 is dissolved in reaction buffer containing 0.1 M Tris-HCl, pH 8.07, 0.01% Triton X-100 and 0.5 M NaCl, and solutions are obtained containing 0.1.0.25, 0,5,0,75
    and 1 mM S-2288.
    Extracts of NAOZ and HA20, obtained in Example 256, and a solution of a commercial CM (10 µl each) are dissolved by reaction buffer
    to a final volume of 400 μl. To these samples, 2 µl of solution 1 of plasmin was added and incubated at 37 ° C for 1 h, transferred to ice-cold water, and 2 µl of a solution of 5 mg / ml soybean trypsin inhibitor was added to them to complete the reaction with plasmin. Five reaction mixtures were prepared for each test sample, and a solution of S-2288 with different concentrations was added to each reaction mixture.
    concentrations and incubated at 37 ° C for 1 h, transferred to an ice-water bath, added 50 ml of a 10% aqueous solution of acetic acid. As a control, use 400 μl of the reaction
    buffer instead of 400 μl of the test sample.
    Absorbance at 405 nm was measured for each reaction mixture, with the absorbance of the control sample minus (value A).
    The reaction rate is V A / 60.
    The results show that the NAOZ hybrid polypeptide consisting of the polypeptide region from 161st Met to 219th Gly, corresponding to approximately half of the kringle region of human tissue plasminogen activator, and the polypeptide region from 150th Gin to 411th Leu of human prourokinase where 157-Phe is substituted with Asp, and this hybrid HA20 polypeptide,
    consisting of the polypeptide region from the first to the 219th Gly, including the full kringle region of human tissue plasminogen activator and the polypeptide region from the 150th Gin to the 411th Leu of human
    prourokinase have the same Km values as the commercial CM.
    This means that the region responsible for the enzymatic activity of the hybrid polypeptide has the same characteristics as the corresponding part of the native
    MC, regardless of the length of the region responsible for fibrin affinity, originating from tissue plasminogen activator, and regardless of the presence or absence of a point mutation in the serine protease region, i.e. at the 157th amino acid,
    PRI me R 30. Construction of plasmid pHA21L
    10 μg of the plasmid pNA20 constructed in Example 22 are hydrolyzed with 100 units. Bgl II and 96 units. Pstl, was subjected to electrophoresis on a 0.7% agarose gel and a DNA fragment of 3400 bp in size was isolated. This DNA fragment is designated as DNA fragment (E).
    In addition, 4 μg of the same plasmid pNA20 digest 100 units, Bgl II and 120 units. Seal was subjected to electrophoresis on a 0.7% agarose gel and a DNA fragment of 630 bp was isolated. This DNA fragment is designated as a DNA fragment (F).
    5 μg of the plasmid pM UT9IL pml obtained in Example 18 was digested 30 units. Pstl, was subjected to electrophoresis on a 0.7% agarose gel and a 1200 bp DNA fragment was isolated. This DNA fragment is partially digested 40 units. and isolating the DNA fragment at about 1100 bp. This DNA fragment is designated as DNA fragment (C).
    The following two oligonucleotides are synthesized:
    5 ACTGTGATGTGCCCTCCTGCACAGGAA 3, 3 TGACACTACACGGGACCACGTGTCC 5.
    1 μg of each of these oligonucleotides is mixed in 30 μl of a solution containing 50 μM Tris-HCl, pH 7.6, 10 mM MgCl and 10 mM / -mercaptoethanol, heated at 70 ° C for 5 minutes and then cooled on ice, added are 20 units. T4 polynucleotide kinase and incubated at 37 ° C for 1 hour. The reaction mixture is heated at 70 ° C for 2 minutes and held at room temperature for 5 minutes. After precipitation with ethanol, the precipitate is mixed with a DNA fragment (F) and 5 units. T4 DNA ligases, incubated, extracted with phenol and precipitated with ethanol. Sediment digested 10 units. FSpl and 20 units. Bgl II, was subjected to electrophoresis on a 1.0% agarose gel, and a DNA fragment of about 650 bp was isolated. This DNA fragment is designated as DNA fragment (H).
    DNA fragments (E), (G) and (H) are mixed and ligated. The reaction mixture transform IM103 E.cotl. Ampicillin resistant transformants are screened for colonies containing the pHA21L plasmid. The plasmid pHA21L is isolated.
    P mIme p31. Construction of plasmid pHA24L
    5 μg of the pMUT4pm4 plasmid (Example 17) are digested 50 units. Pstl, were subjected to electrophoresis on a 0.7% agarose gel and a 1200 bp DNA fragment was isolated, which was then partially digested with 40 units. FSpl, isolate a DNA fragment of about 1100 bp. and is referred to as DNA fragment (I).
    DNA fragments (E) and (G) are prepared according to the procedure described in Example 29. These DNA fragments (E), (G) and (I) are mixed and ligated. The ligase mixture is used to transform IM 103 E. coli. Ampicillin resistant transformants are screened for colonies containing the plasmid pHA24L. Plasmid pHA24L was isolated.
    Example 32. Construction of plasmid pHA11L
    5 μg of the plasmid pH A21L (Example 29) are digested 50 units. Seal and 50 units. Pstl was subjected to electrophoresis on a 1.0% agarose gel; a DNA fragment of about 1100 bp was isolated. and designate it as a DNA fragment (I).
    10 mkg of plazmida rna211 are digested
    100 units, subjected to electrophoresis in
    4.0% agarose gel, isolate a DNA fragment of about 330 bp. and denote it
    as a DNA fragment (K).
    Two oligonucleotides are synthesized:
    5 ATGAAGAGGTGACGTCATGTC 3, 3 TACTTCTCCACTGCAGTTAGAGACT 5.
    2 μg of each of the oligonucleotides are mixed, phosphorylated 5-ends and annealed. After ethanol precipitation annealed
    oligonucleotides sediment mixed with DNA fragment (K), and sew. After precipitation with ethanol, the precipitate is digested 100 units of Zsa and 100 units. Aatll, isolate the DNA fragment at about 250 bp. This DNA fragment
    denoted as DNA fragment (L).
    5 μg of the plasmid pMUTQpml (Example 18) are digested 50 units. Aatll and 50 units. Pstl, subjected to electrophoresis in a 0.7% agarose gel, isolate a DNA fragment of about
    3000 bp and designate it as a DNA fragment (M), DNA fragments (I), (L) and (M) are mixed and stitched. . The reaction mixture is transformed with E.coli IM103. Ampicillin resistant transformants are screened for colonies containing the plasmid pNA11L. The plasmid pHA11L is isolated according to the conventional method.
    PRI me R 33. Construction of plasmid pHA14L
    5 μg of the pHA24L plasmid (Example 31) are digested 50 units. Sea and 50 units. Pstl, subjected to electrophoresis in a 1.0% agarose gel and a DNA fragment of about 1100 p. O. are highlighted. This DNA fragment is designated as a DNA fragment (N).
    DNA fragments (L) and (M) are prepared according to the procedure of Example 32. These DNA fragments (L), (M) and (N) are mixed and stitched with T4 DNA ligase. The ligase mixture is transformed with IM103 E. col. Ampicillin-resistant transformants are screened for plasmid pHA14L content. The plasmid pNA141 is isolated.
    PRI me R 34. Construction of plasmid pHAOIL.
    5 μg of the RNA21 plasmid (Example 30) digest 50 units. EcoR I and 50 units. Pst I,, was subjected to electrophoresis on a 0.7% agarose gel, and a DNA fragment of about 330 bp was isolated. This DNA fragment is designated as DNA fragment (O).
    5 μg of the plasmid pNA211 are digested 50 units. Seal and 50 units. Pstl, subjected to electrophoresis in a 0.7% agarose gel, a DNA fragment of about 100 p. isolated and designated as a DNA fragment (P).
    10 μg of the same plasmid pHA21L is digested 50 units. EcoRI and 50 units. Seal, subjected to electrophoresis in a 2.0% agarose gel, and a DNA fragment of about 150 p. isolated and designated as DNA fragment (Q).
    The DNA fragments (O), (P) and (Q) are mixed, stitched with the T4 DNA ligase mixture, transformed with IM103 E. coli. Ampicillin resistant transformants are screened for the content of the plasmid pNA01. Plasmid pNA01 was isolated.
    PRI me R 35. Construction of plasmid pHA04L
    The plasmid pNA041 was constructed according to the procedure of example 34, except that the plasmid pNA241 constructed in example 31 was used instead of the plasmid pHA21L
    Example 36. Expression and extraction of a hybrid HPA24L polypeptide exhibiting activity of a plasminogen activator.
    Plasmid pHA24L (Example 31) was used to transform KU 1436 E coli, transformants were cultivated in 5 ml of medium L with 50 μg / ml of ampicillin in a test tube at 30 ° C overnight with stirring 2 ml of culture broth, inoculated to 1 l of medium L with 50 µg ml of ampicillin in a 5-liter conical flask, and cultured at 30 ° C in an air shaker at 260 rpm, transferred to an incubator at 37 ° C and cultivated
    5 hours with shaking in order to express the HPA24L gene of a hybrid plasminogen activator-like polypeptide. The culture broth is transferred, centrifuged. The cells are suspended in 1.0 ml of a buffer containing 0.1 M NaCl and 50 mM Tris-HCl, pH 8.0, sonicated and centrifuged. The precipitate is subjected to polyacrylamide gel electrophoresis in
    the presence of sodium dodecyl sulfate.
    A portion of the insoluble fraction was suspended in 160 µl of a solution containing 6M guanidine hydrochloride and 25 mM Tris-HCl, pH 8.0, incubated at room temperature for 30 minutes, adjusted to a final concentration of 50 mM Tris-HCl, pH 8.0 1 M guanidine hydrochloride, 2 mM glutathione of the reduced type, 0.2 mM of the oxidized glutathione, 1 mM of EDTA and 0.1%
    Tween-80 is incubated for 15 hours at room temperature to obtain a crude extract.
    PRI me R 37. Determination of the activity of the crude extract of HPA24L using synthetic substrate S - 2444.
    10 µl of HPA24L crude extract is added to a buffer solution containing 0.1 M Tris-HCl pH 8.0 and 0.01% Triton
    X-100 to a volume of 99 μl. 1 µl (1 µg / ml) of plasmin solution was added to the mixture, incubated at 37 ° C for 15 min, 1 µl of soybean trypsin inhibitor solution was added, mixed thoroughly, and 0.7 ml of a buffer solution containing 2 mM substrate S - 2444 and 0.1 m Tris-HCl, pH 8.0 and 0.01% Triton X-100, and incubated at 37 ° C for 30 min, after which 100 µl is added
    glacial xyluy acid.
    The absorbance of the reaction mixture is measured at 405 nm. Enzyme activity in the reaction mixture is calculated according to the formula: 1 international
    unit (ME) (OD405 / 0.395) x6.5, and approximately 120 enzyme activity is obtained.
    Example 38: Expression and Extraction of Other Hybrid Polypeptides Showing Plasminogen Activator Activity. The procedure described in examples 36 and 37 is repeated for the plasmids pHA21L, pHA11L, pHA14L, pHAOIL and pHA04L, and results are obtained similar to those described
    in examples 36 and 37.
    权利要求:
    Claims (1)
    [1]
    The invention of the method for producing a hybrid plasminogen activator. containing the region responsible for affinity with
    25 173281426
    tissue plasminogen activator fibrin, irHA21C or pHA24L, or pHA11C or
    the region responsible for the enzyme ak-pHA14L, or pHAOIL. cultivating
    the potency of a pro-urokinase polypeptide, transformed cells into a feeder in transformation of the cellular medium at 25-45 ° C, the collection of cultivirubacteria Escherchla collas recombinant-5
    mi plasmid DNA pNA20 and pNA13, or shagging, suspending. centrifugal 23, or RNAOO, or pnaoz, or the line and the selection of the target product.
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    同族专利:
    公开号 | 公开日
    DK50987D0|1987-01-30|
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    DK50987A|1987-08-01|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    IL68561A|1982-05-05|1991-01-31|Genentech Inc|Preparation of polypeptide with human tissue plasminogen activator function,processes for making it,and dna and transformed cell intermediates thereof|
    GB8314362D0|1983-05-24|1983-06-29|Celltech Ltd|Polypeptide and protein products|
    JPH0365151B2|1983-12-23|1991-10-09|
    GB8334498D0|1983-12-24|1984-02-01|Beecham Group Plc|Compounds|US4916071A|1985-08-14|1990-04-10|American Home Products Corporation|Poly-kringle plasminogen activator|
    FI100106B|1986-12-05|1997-09-30|Novartis Ag|Process for producing a single-stranded hybrid plasminogen activator|
    US5580559A|1986-12-05|1996-12-03|Ciba-Geigy Corporation|Hybrid plasminogen activator|
    US5242819A|1986-12-05|1993-09-07|Ciba-Geigy Corporation|DNA molecules encoding hybrid proteins of tissue plasminogen activator and urokinase|
    ZA879286B|1986-12-16|1988-10-26|Smith Kline Rit|New plasminogen activators|
    WO1988005081A2|1986-12-30|1988-07-14|Cetus Corporation|Novel plasminogen activator|
    NL8700013A|1987-01-06|1988-08-01|Leuven Res & Dev Vzw|HYBRID PLASMINOGEN ACTIVATORS WITH IMPROVED THROMBOLYTIC PROPERTIES AND MEDICINAL PRODUCTS CONTAINING THESE PLASMINOGEN ACTIVATORS.|
    NL8701021A|1987-04-29|1988-11-16|Stichting Centraal Lab|HUMANE T-PASUBSTITUTION-MUTANT PROTEINS, CODING RECOMBINANT DNA, TRANSFECTED HOST CELLS, PREPARATION OF THE MUTANT PROTEINS, AND PHARMACEUTICAL PREPARATIONS.|
    JP2561122B2|1988-04-13|1996-12-04|寳酒造株式会社|Functional polypeptide|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    JP1773486|1986-01-31|
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