RU2107727C1 - Recombinant tissue activator of plasminogen and method for its production - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: in order to produce tissue activator of plasminogen (perfected TAP) host cells are cultured and transformed by recombinant DNA comprising specific DNA sequence encoding TAP analog. Preparation is effective for use as inflammation inhibitor around site where thrombus is formed. EFFECT: prolonged biological life half-period; higher resistance to heat and acids. 2 cl, 29 dwg 3 tbl

Description

 The invention relates to a new improved tissue active plasminogen (improved APT), having a prolonged half-life in the body and increased stability to heat and acids, which can be used to suppress ignition around the area of thrombosis.

 The invention also relates to a method for producing the named tissue plasminogen activator using recombinant DNA technology and agents used for its implementation.

 It is known that human tissue plasminogen activator (APT) has useful fibrinolytic activity and is extremely effective against fibrin-bound plasminogen, whereas plasminogen in the free circulation phase in the body does not activate as efficiently as conventional thrombolytic agents, streptokinase (SC) and urokinase (UK) . The amino acid sequence of human APT and the nucleotide sequence of a cDNA encoding human APT are known (Pennica. D., et al., Nature, 301, 214-221, 1983). It is also known that human APT dissolves clots of venous and arterial blood. In large-scale clinical trials, it is noted that human APT administered intravenously is effective in re-perfusing a clogged coronary artery in a patient with acute myocardial infarction.

 However, the drawback of using this drug in the treatment of a disease associated with thrombosis is the extremely short half-life of its enzymatic activity in the blood (Rijken, DC, et al., Thromb. Heamost. 54 (1), 61, 1985, Hubert, EF, et al., Blood, 65, 539, 1985). When used for treatment, human APT has to be used as a continuous high-dose intravenous injection.

 It is known that naturally occurring human APT has a domain structure, starting from the N-terminus of the molecule, follow the fingerdomain, the EDF domain (epidermal growth factor), the two Kringle 1 and Kringle 2 domains and the serine protease domain. In the work of Rijken et al., It is noted (Rijken DC, et al., Thromb. Heamost., 54 (1), 61, 1985) that the short duration of the biological half-life of human APT can be related to all domains of human APT, except for the serine domain proteases. In the work of Zonneveld et al. (Zonneveld, AJV, et al, Proc. Natl. Acad. USA., 83, 4670, 1986) it is also noted that fingerdomain, EED domain and Kringle 2 domain may be important for the fibrin-binding activity of naturally occurring human APT, as well as to maintain fibrin-dependent activation of APT. However, no specific measures have yet been developed to maintain the fibrin-binding activity possessed by naturally occurring human APT and its fibrin-dependent activity, as well as to prolong the biological half-life.

 Japanese Published Patent Application Laid-Open No. 48378/1987 describes an APT obtained by a deletion of 87-175 amino acids of a naturally occurring human APT in which kringle 1 has been deleted. This APT is characterized by an additional induced point mutation in the region of epidermal growth factor. A Japanese patent application discloses that a modified APT has the ability to bind to fibrin, but the interaction with a tissue plasminogen activator inhibitor is weakened.

 Europatent N 241208 describes APT obtained by deletion of 92-179 amino acids of naturally occurring human APT, in which “kringle 1” is also deleted. This work mentions that this APT has fibrinolytic activity.

 In addition, Europatent N 231624 discloses a modified APT with a prolonged half-life. A modified APT having the F-EGFK2-A sequence is devoid of the kringle 1 domain, however, no specific method of its preparation has been shown. In light of the above cited, it is understood that the modified APT in accordance with the invention should differ from the naturally occurring APT in the amino acid sequence in the region of internal domains.

 As a result of extensive research, the applicant received an improved APT that contains a finger domain, EDF domain, Kringle 2 domain and Serine protease domain, but the first “Kringle 1” domain was deleted in a specific site and in the site linking domains mutation was introduced in Kringla 2 and serine protease, as a result of which an improved APT was obtained, exhibiting excellent resistance to heat and acids, having a noticeably prolonged biological half-life and pronounced anti-inflammatory activity even that retains the desirable properties of naturally occurring human t-PA.

 The invention relates to an improved APT. APT in accordance with the invention differs markedly in its chemical structure from naturally occurring human APT and exhibits better properties.

An improved APT in accordance with the invention is a polypeptide having the amino acid sequence represented by the general formula shown in FIGS. 28-29, where R is a direct bond, Y is A-Ile-B (A is Arg or Glu, and B is lys or Ile), preferably Glu-Jle-Lys. H ₂ N is an amino terminus and —COOH is a carboxy terminus).

In the invention, the term “advanced APT” is used to mean an analog of APT, in which A and B are the amino acids described below, respectively:
Advanced APT (II): Arg, Lys;
Advanced APT (V): Arg, Ile;
Advanced APT (VI): Glu, Lys;
Advanced APT (VIII): Glu, Ile.

 The invention is also directed to the expression of the proposed APT analogue using recombinant DNA techniques. Associated with this are new DNAs encoding an improved APT and recombinant DNA expression vectors.

 Figure 1, 2 shows the sequence of 16 oligodeoxynucleotides used to construct a fragment of a synthetic gene encoding an improved APT (II); figure 3 - 4 is a fragment of a synthetic gene for constructing improved APT (II) of the invention, containing the ends of the restriction enzymes Bge 11 and Eco R1, which is constructed using 16 oligodeoxynucleotides shown in figures 1 - 2; in Fig.5 is a methodology for constructing an improved APT (II) (in the figure, the black portion, the shaded portion and the unpainted portion indicate the region encoding mature APT protein, the propeptide encoding region, and the untranslated region, respectively; Fig.6 is a method for testing a synthetic gene fragment block IV by determining the sequence of DNA bases with a dideoxymethod and 7-DEAZA method; in Fig. 7, a method for constructing the pVY1 expression vector in animal cells and the integration of the improved APT DNA in pVY1; in Figs. 8-13, DNA sequences encoding an improved APT (II) and an improved APT (V); Figs. 14-19 show the amino acid sequences derived from DNA sequences encoding an improved APT (II) and an improved APT (V); in Fig. 20, restriction enzymes and functional map of the plasmid pTPA 2 having the Eco R1-Xho fragment (about 1000 base pairs) of the natural APT gene integrated into the pBR322 vector at Eco R1 and Bam H1 cleavage sites; in Fig.21 - mp9 (advanced APT (II), having a fragment of BgL11-Xho 11 (about 1500 base pairs) of the gene, advanced APT (II) integrated into the double-stranded DNA M13 mp9 in the BamH1 cleavage site; in Fig.22 - dependence " dose-effect "for the activity of APT of advanced APT (VI) and naturally occurring APT by S-2251 method in the presence of (+ Fb) and the absence of (-Fb) fibrin substituent; in Fig.23 - change in activity of advanced APT (VI) and native APT in rabbit blood over time; in Fig.24 - change in the residual activity of the improved about APT (VI) after heat treatment; in Fig. 25 - inhibition of advanced lymphocyte activating factor (LAF) by improved APT (VI); in Fig. 26 - activation by using a denatured protein of improved APT (VI); in Fig. 27 - degradation denatured protein under the action of improved APT (VI).

 Below is described in detail a method for producing recombinant DNA and transformed cells.

 A method of obtaining an improved APT.

A gene encoding a natural APT based on which the APT of the present invention is obtained is isolated from a cDNA bank made from Bowes human melanoma cells. A ⁺ RNA polyes are isolated from Bowes human melanoma cells and fractionated by centrifugation in a sucrose density gradient. A small amount of fractionated poly (A) ⁺ RNA is then selected and the mRNA fraction encoding the APT gene is identified by a subhybridization method using an oligonucleotide probe capable of recognizing a specific APT mRNA sequence. Using this fraction rich in APT mRNA as the starting material, a cDNA bank is obtained and screened with a probe to identify APT mRNA described above. Since not a single clone having the full sequence of the APT gene has been isolated, the missing base sequence is synthesized by a DNA synthesizer to obtain the desired gene. The desired gene is then constructed by induction of a site-specific mutation.

 The Eco R1-Xho 11 fragment is found in the nature of the APT gene (about 1000 base pairs), part of which is deleted at the N = terminus, introduced into the pBR332 vector at the Eco R1 and BamH1 cleavage sites, and pTPA2 was obtained. The strain (E. coli HB 101 / pTPA2) obtained by transformation of E. coli with this plasmid was deposited at the Institute of Fermentation Studies of the Agency for Industrial Science and Technology of Japan under registration number P-9649 (FERM BP-2107). Restriction and functional map of the plasmid pTPA2 shown in Fig.20.

 The improved APT gene is inserted into the plasmid pVY1.

 The plasmid pVY1 is obtained by ligation of the BamH1-Kpn1 fragment (about 2900 base pairs) of the pRSV10 plasmid (manufactured by Fain Chemical) with the fragment from the Eco R1 cleavage of the plasmid pAdD26SV (A) N 3 (N) (obtained from Dr. Hiroshi Hand from the University of Tokyo (after receiving at both blunt ends. Accordingly, this vector contains the mouse dihydrofolate reductase gene cDNA under transcriptional control of the main late promoter of the adenovirus (Ad2), early SV 40 promoter up from the site of insertion of the improved APT and intron gene and sequence polyadenylation downstream of the gene The gene of the present invention can be inserted into another suitable expression vector The expression vector is further introduced into a suitable host cell to obtain transformants Prokaryotic cells such as E. coli, Bacillus subtilis and etc., eukaryotic microorganisms, such as yeast, etc., as well as cells of higher animals. Strain JM109, strain W3110, Q, etc., belonging to strain K12, are usually used as representative of E. coli, strain BD170, strain BR151, etc. are used as representative of Bacillus subtilis. From yeast, strain RH218, strain SHY1, etc. can be used. Saccharomyces cerevisiae yeast.

 For expression, a plasmid vector or phage vector containing a replicon derived from species compatible with the host cells and a regulatory sequence is typically used. Examples of a vector for E. coli are, for example, plasmids pBR322, pUC18, pUC19, etc., λ is a phage, for example, λqt, Charon 4A, etc., phage M13, etc. As a vector for Bacillus subtilis, you can use pUB110, pSA2100, etc., and as a vector for yeast, you can use YRp7, YEp61, etc.

 The vector must carry a promoter capable of expressing the desired protein. As a promoter for the E. coli gene or phage gene, for example, Lae, trp, tac, trc, pL, etc. can be used.

 As a host, cultured animal cells can be used, such as rhesus macaque kidney cells, mosquito larva cells, African green monkey kidney cells, mouse fetal fibroblast, Chinese hamster ovary cells, human fetal kidney cells, butterfly egg tissue cells, human cervical epithelium-like cells, human myeloma cells, murine fibroblasts and so on. As a vector, the early promoter SV40, the late promoter SV40, SV40, bearing the promoter from the eukaryotic gene (for example, the estrogen-induced avian ovalbumin gene, interferon gene, glucocorticoid-induced tyrosine aminotransferase gene, thymidine kinase gene, early and late genes, can be used) , phosphoglycerate kinase gene, α-factor gene, etc.), cow papilloma virus or vectors derived from them.

 In addition, it is known that APT secreted and produced by cells have different N-terminations depending on the difference in cleavage sites.

 In the case of secreting and producing APT using culture cells as a host, the method for cleaving with a signal peptidase or protease varies depending on the type of cells, so that types of APT having different N-terminations can also be obtained.

 This phenomenon is not only suitable for the case of secretion and production using culture cells, since it is believed that a similar phenomenon can also occur when receiving APT by E. coli, Bacillus sublitis, yeast and other cells subjected to special modification.

 Hanahan, Hanahan, D.J. Mol can be used to transform the host using an expression vector with an improved APT gene integrated into it in the case of using E. coli. Biol., 166, 557, 1983), in the case of manipulation of animal cells, the calcium phosphate method (Vander Eb, A.J. and Graham, F.L., Method in Enrymoloqy, 65, 826, 1980, Academic Press) and so on can be used.

 As described above, an improved APT is useful in treating a variety of acquired diseases, including vascular coagulation (even deep vein), pulmonary embolism, peripheral arterial thrombosis, embolism due to damage to the heart or peripheral artery, acute myocardial infarction and thrombotic attack.

 Like the naturally occurring human APT, the advanced APT is particularly suitable for the treatment of acute myocardial infarction. It has recently been proven that naturally occurring human APT is effective in dissolving a clot clogging the coronary artery, regenerating myocardial perfusion, and recovering most of the parts in the ischemic myocardial layer when administered intravenously at a dosage of 30 to 70 mg for 1-3 hours. The improved APT is characterized by a prolonged biological half-life in the blood and therefore is effective in the same cases as the naturally occurring human APT. An improved APT is expected to produce a clinical effect similar to that of natural human APT at a dose of about 10% of the dose that is recommended when using naturally occurring human APT, even with a single administration.

 In addition, the improved APT of the present invention exhibit the following valuable properties that have not yet been known with respect to native human APT and modified APT.

 a) Anti-inflammatory activity.

 On the thrombus site, not only the formation of the thrombus itself is detected, but also the formation of fibrin degradation products or trace amounts of kinin. It is known that these substances have inflammation-inducing activity and thus cause inflammation in the thrombus area. For this reason, it is desirable that the agent used to treat thrombosis possess not only thrombolytic activity, but also anti-inflammatory activity.

 As a result of the studies, the applicant was able to give the improved APT anti-inflammatory activity based on two functions.

 One of them is that the improved APT inhibits the biological activity of interleukin 1 (IL-1), which is one of the mediators of the inflammatory response. The macrophage-produced IL-1 is thought to be involved in the inflammatory response through hyperthermia, accelerated fibroblast growth, collagenase production in the synovial cell membrane, and so on, or by accelerated prostacyclin synthesis in vascular endothelial cells. It is also known that IL-1 acts on liver cells, accelerating the production of proteins (serum amyloid protein, fibrinogen, etc.) in the acute phase, which increases with inflammation. The Applicant has determined that an improved APT inhibits activity (LAF activity) by increasing the mitogenic reactivity of mouse thymocyte, which is one of the biological activities of IL-1.

 Another function is that an improved APT has an affinity for a denatured protein (denatured immunoglobulin G, denatured albumin, and so on) resulting from inflammation in the thrombus area, and additionally has the property of being activated by this denatured protein.

 Due to this activity, an improved APT decomposes only denatured protein in the area of inflammation, and inflammation can temporarily weaken. The applicant has confirmed by gel electrophoresis in sodium dodecyl sulfate that the improved APT decomposes only the denatured protein.

 As shown in FIG. 26, the activation and selectivity of an improved APT under the action of a denatured protein are obvious. With immunoglobulin G treated with HCl, and at several times lower concentration, the same activity is shown as with fibrinogen treated with BrCN. On the other hand, normal immunoglobulin C does not show an activating effect with respect to the improved APT even at a concentration of 500 μg / ml.

 Prevention of reocclusion after restoration of perfusion of a clogged blood vessel.

 It is known that in the treatment of thrombosis with natural APT, repeated occlusion is observed with a high frequency after the restoration of blood flow in a blocked blood vessel. For this reason, combined therapy with a platelet coagulation inhibitor or anticoagulant is performed. However, combination therapy involves the problems of drug interactions, dosage control, similar effects, and so on. Preferably, the APT itself additionally has the activity of preventing reocclusion.

 The improved APT of the present invention has the ability to prevent cases of re-occlusion due to two types of activity.

 The first type is the prevention of a rapid decrease in the concentration of APT after the introduction of an improved APT due to the prolonged duration of action, which leads to the elimination of the Stuart-Holmes symptom and thereby prevents the occurrence of repeated occlusion.

 The second type is that by preventing damage to vascular endothelial cells caused by IL-1, platelet coagulation is indirectly inhibited, which prevents the occurrence of reocclusion.

 c) Increased stability.

 Protein preparations are usually unstable, therefore it is advisable to store the preparations in a frozen, dry state or at low temperatures in the form of a solution. With the introduction of plasminogen activator to a patient with acute myocardial infarction, there is a need to carry out the procedure within a few hours after the onset of the attack in order to reduce the mortality rate. In this case, stable preparations that can be stored at room temperature are desirable.

 In addition, the increased stability allows heat treatment, acid treatment, etc. during preparation. In particular, with respect to the improved APT of the present invention, which is produced by cell cultures, it becomes possible to remove a retrovirus of cell origin, which is known to be unstable to heat.

 Below the invention is described more specifically with reference to examples, but it is not limited to them. Unless otherwise indicated, recombinant DNA is produced in accordance with laboratory guidelines.

 Maniatis T et al., Molecular Cloning: A Laboratory Manual, Could Spring Harbor Laboratories, Could Spring Harbor, New York (1982).

 Example 1. Cloning to DNA of APT.

Human Bowes melanoma cells (purchased from Dr. Roblin, R. at the National Cancer Research Institute, USA) were cultured according to the method of Opdenakker et al. (Opdenakker, G., et al., Eur. J. Biochem, 131, 481-487 (1983)). In order to induce APT mRNA, TFA (12-O-tetradecanoylphorbol-13-acetate) was added to the culture mixture at a final concentration of 100 ng / ml, followed by culturing for 16 hours. Then, total cellular RNA is extracted from cultured cells in accordance with a modified method of Freeman et al. ((Okayama) Berqa DNA Manual, p. 3, 1985, Pharmacy Chemicals). Using a column of oligo-dT cellulose (manufactured by Pharmacy Fine Chemicals), poly (A) ⁺ RNA was separated from all cellular RNA. As a result, about 400 μg of poly (A) ⁺ RNA is obtained from about 10 ^o cells.

This poly (A) ⁺ RNA is fractionated by centrifugation in a sucrose density gradient in a conventional manner. A portion of the fractionated poly (A) ⁺ RNA was selected and dot blot hybridization was performed (Perbal, B., Apractical Gube to Molecular Cloninq, 410, 1984, John Wiley and Sons, Inc) using an oligonucleotide probe specific for APT mRNA. The probe (probe Y) used in this case has a 5'-GCNNGGCAAAGATGGCA-3 'base sequence, which is complementary to the mRNA region encoding amino acid residues from +291 to +297 in the APT sequence described by Pennicaetal and synthesized by the β-cyanophosphamidate method, using a DNA synthesizer, model 380A (manufactured by Applied Biosystems). Synthesis of a DNA oligomer, cleavage of the protective group, cleavage from the resin and purification are carried out in accordance with the operating instructions for the DNA synthesizer, Model 380A. Labeling with the radioactive isotope of the Y probe at the 5'-end is carried out in accordance with laboratory guidelines using T4 polynucleotide kinase (manufactured by Taka-Ra Shuzo Co., Ltd.) t and γ- ( ³² P) ATP.

Probe Y strongly hybridizes mainly with 20-30S poly (A) ⁺ RNA (this fraction is called the M fraction).

Using a matrix, 10 μg of poly (A) ⁺ RNA is obtained from fraction M; 3 μg of double-stranded cDNA is synthesized using reverse transcriptase (manufactured by Biochemical Industry Co., Ltd.) according to the Gubler-Hoffman method (Gubler, U. and Hoffman, BJ, Gene 25, 263, 1983), and added to double-stranded cDNA in 3 the 'ends of the deoxy C chain according to the Denq-Wu method (Denq, GR and Wu, R., Nucleic Acids Res., 9, 4173, 1981). Then, double-stranded cDNA extended with a deoxy C-chain is subjected to gel filtration on CL 4B Sepharose (manufactured by Fine Chemicals Pharmacy) to remove low molecular weight nucleic acids having less than 500 base pairs. After that, the cDNA was annealed using pBR322 (manufactured by Bethesda Research) containing a deoxy G chain at the P _st 1 site using a traditional technique. The mixture obtained after annealing transforms the competent E. coli HB101 cells (manufactured by Takara Shuzo Co., Ltd.). The result is a cDNA bank consisting of approximately 4,000 independent transformants.

This cDNA is subjected to colony hybridization using the Y probe described above according to the Woods method (Woods, D., Focus, 6 (3), 1, 1984), manufactured by Bethesda Research Lab.) To obtain clones interacting with the Y probe Among the clones, the pTPA1 clone containing the longest APT cDNA was detected. A dideoxymethod is then carried out (Carlson, J., et al., J. Biotechnoloqy, 1, 253, 1984) using the M13 phage vector and 7-DEAZA method (Mizusawa S., et al., Nucleis Acids Res., 14, 1319 , 1986). As a result, it was found that the plasmid pTPA1 contains a base sequence from T _{y + 441} to A _{y + 2544} for the APT gene described by Pennicaetal.

 Example 2. Construction of an improved APT (II).

 In the plasmid pTPA1 shown in Example 1, the N-terminal region is insufficient to construct an improved APT (II) that lacks 1-domain kringle. Therefore, an insufficient DNA segment is synthesized as described above using a 380A DNA synthesizer (manufactured by Applied Biosystems). The base sequence of the synthesized oligomer and the complete synthesized sequence are shown in FIG. 1-4. Specific techniques for constructing advanced APT (II) using these oligomers are shown in FIG. 5-6.

 2-1). Block IV construction (Bql II-Eco R1 fragment, about 480 bp).

 A fragment of block IV in FIG. 5 is obtained as follows.

First, in accordance with laboratory guidelines, 40 pmol of each of the synthetic oligonucleotides 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 shown in FIG. 1-2, phosphorylate with 10 units of T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) at a temperature of 37 ^° C. for one hour in 50 μl of the reaction solution for each of them. The reaction solution is treated with phenol. After precipitation with ethanol, the precipitates are dried under reduced pressure and dissolved in sterile distilled water. After settling 40 pmol of each oligomer in 150 μl of a solution containing 6 mm Tris-HCl (pH 7.5), 20 mm NaCl, 7 mm MgCl ₂ and 0.1 mm EDTA, at a temperature of 80 ^o C for 5 minutes, at at a temperature of 60 ^° C. for 5 minutes and at room temperature for one hour, in the corresponding blocks of block I (oligomers 1, 2, 3 and 4), block II (oligomers 5, 6, 7, 8, 9 and 10) and block III (oligomers 11, 12, 13, 14, 15 and 16) carry out precipitation with ethanol and drying under reduced pressure. The residue was dissolved in 40 μl of sterile distilled water. The reaction is carried out in 400 μl of the reaction solution at a temperature of 4 ^° C. for 15 hours using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.). After precipitation with ethanol and drying under reduced pressure, the precipitate is dissolved with teryl distilled water: in case of block I (1), gel electrophoresis is carried out in 5% polyacrylamide (laboratory manual), separated and purified in the traditional way (laboratory manual), a fragment of about 100 base pairs and in the case of block II (2) and block III (3), gel electrophoresis is carried out in a 3% agarose gel (LMP agarose, manufactured by BRL) (laboratory manual) and fragments of about 190 pairs are isolated and purified by electroelution (laboratory manual) about dreams.

Then 0.1 μg, 0.2 μg and 0.2 μg of fragments of block I, block II and block III are respectively ligated using the above DNA ligation kit. Carry out gel electrophoresis at an agarose concentration of 1.5% in order to isolate a fragment of Bgl II-Eco R1 (block IV) of about 480 base pairs. DNA is then isolated from the agarose gel by electroelution. This DNA is then phosphorylated in 100 μl of the reaction solution at 37 ^° C. for one hour using 10 units of the above T4 polynucleotide kinase, after which it is treated with phenol, precipitated with ethanol and dried under reduced pressure.

 This fragment of the synthetic gene and the base sequence of block IV are confirmed by determining the base sequence in accordance with the dideoxymethod using the phage vector M13. Specific techniques are shown in FIG. 6. After ligation of the above fragment of Bgl II-Eco R1 of block IV with M13 mp18 DNA (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd.) digested with restriction enzymes BamH1 (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd.) and Eco R1 ( manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd.) determine the sequence of its bases using the sequencing kit M13 (manufactured by Taraka Shuzo K., Ltd.) and sequencing kit 7 - DEAZA (manufactured by Takara Shuzo Co., Ltd.).

 The cleavage site of the restriction enzyme Bgl11 and the cleavage site of the restriction enzyme BamH1 are ligated isochosomerically (BamH1 is the cleaved end of the Bgl11 cleavage site), and the ligation fragment can be cleaved with the restriction enzyme Xho 11, resulting in natural cleavage ends of Bgl 11 and Bamh1, respectively.

 To more accurately determine the base sequence, phage M 13mp18 (including a fragment of block IV) infects E. cjli strain JM109 according to Messing / Messing J., Methods in Enzymology 101, 20-78 (1983)), after which double-stranded DNA is obtained (replicative type). After cleavage of this DNA (50 μg) with restriction enzymes Xho 11 (manufactured by Boehringer Mannheim-Yamanouchi Co) and Eco R1, gel electrophoresis was carried out on a 1.5% agarose gel to isolate a fragment (block IV) of about 480 base pairs. This DNA is extracted by electroelution. After ligation of the extracted DNA with M13mp19 DNA (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd) digested with restriction enzymes Eco R1 and BamH1 in the same manner as described above, using the DNA ligation kit, the base sequence was determined. As described above, this sequence can be verified more accurately by determining the sequence of both DNA using M13mP18 and M13mp19. In addition, double-stranded replicative DNA M13mp19 (with block IV) is obtained by the described method. After cleavage of this DNA (50 μg) with restriction enzymes Eco R1 and Xho 11, gel electrophoresis was carried out in 1.5% agarose, while isolating a fragment (block IV) of about 480 base pairs.

 2-2). Isolation of block V (fragment Eco R1-Bal1, about 1250 base pairs).

 Large amounts of plasmid DNA were isolated from the pTRA1 clone obtained in Example 1 in accordance with the method described in the laboratory manual, as shown in FIG. 5. After cleavage of 70 μg of this DNA with restriction enzymes Bal1 (manufactured by Takara Shuzo Co., Ltd.) and Nar1 (manufactured by Nirro Gen Co., Ltd.), electrophoresis is performed on a 0.8% agarose gel, thereby isolating the Nar1-Bal1 fragment (about 1540 base pairs). DNA is isolated by electroelution. After further partial digestion of this DNA with the restriction enzyme Eco R1, electrophoresis is performed on a 0.7% agarose gel, isolating the Eco R1-Bal1 fragment (about 1250 base pairs). DNA is isolated by electroelution.

 2-3). Construction of the improved APT (II) gene from block IV and block V.

 As shown in FIG. 5, the improved APT gene is obtained as follows.

 After doping of block IV (fragment Bgl11-Eco R1, about 480 base pairs) obtained in Example 2-1, with block V (fragment of Eco R1-Bal1, about 1250 base pairs) obtained in Example 2-2, using the kit for the DNA doping described above, the doped product is precipitated with ethanol. After drying under reduced pressure, the precipitate is cleaved with the restriction enzyme Xho 11 in a conventional manner. Then electrophoresis is carried out on a 0.8% agarose gel in order to isolate a fragment of Bgl 11-Xho 11 (about 1500 base pairs, contains an improved APT gene). Then DNA is isolated by electroelution. The complete base sequence of the improved APT (II) gene thus obtained is shown in FIG. 8-13. The deduced amino acid sequence is also shown in FIG. 14-19.

 Example 3. Construction of the improved APT gene V, VI and VIII.

 The construction of the improved APT gene V, VI or VIII is carried out on the basis of the improved APT (II) gene with reference to the following publications.

 Genetic conversion is carried out by induction of a site-specific mutation.

 Publications: Zoller, M.J. and Smith, M., Method in Fermentology, 100, pp. 468-500 (1983), Zoller, M.J. and Smith. M. DNA, 3, pp. 479-488 (1984), Morinaga J. et al. Biotechnology, pp. 636-630 (July 1984), Adelman J.P. et al., DNA, 2, pp. 183- 193 (1983), 6. M13 Sequencing Guide (puC) published by Gene Sayens Room Co., Ltd.).

 3-1). Construction of the improved APT (V) gene.

 A) Create M13mp19 (APT / P /) for mutation.

 The advanced APT (II) gene fragment described in detail in Example 2, 2-3) is ligated with double-stranded DNA M13mp9 treated with the restriction enzyme BamH1 and alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.). Competent E. cjli JM109 cells (manufactured by Takara Shuzo Co., Ltd.) are transfected with the ligation product. Each clone giving a colorless sterile spot is used to infect E.Coli JM109. Single-stranded DNA was isolated from the culture supernatant, and double-stranded (replicative) DNA was isolated from E. cjli cells in accordance with the Messing method (J. Messing, Methods in Fermentology, 101, pp. 20-78, 1983). When analyzing the nature of these double-stranded DNAs after cleavage with the restriction enzyme Pst1 by agarose gel electrophoresis, clone mp9 (improved APT (II), in which the APT (II) gene is inserted into the mp9 DNA in the desired direction, as shown in Fig. 21, is obtained.

 After cleavage of some of these DNAs with Pst restriction enzyme, electrophoresis was performed on a 0.8% agarose gel, where clone mp9 (advanced APT (II) shows a simple strip at 7300 base pairs, 840 base pairs, 430 base pairs and 80 base pairs, approximately The single-stranded DNA of this clone is used in a subsequent experiment to induce a site-specific mutation.

 B) Synthesis of a primer capable of inducing a site-specific mutation.

 The synthetic oligonucleotide used to induce a site-specific mutation in the improved APT (II) gene is synthesized by the β-cyanoethylphosphoamidate method using a DNA synthesizer, model 380 A (manufactured by Applied Biosystems). Synthesis of a DNA oligomer, removal of the protective group, cleavage from the resin and purification is carried out in accordance with the operating instructions for a 380 A DNA synthesizer. To induce a mutation at a specific site, a primer (1) capable of inducing a site-specific mutation and a primer (2) is obtained for sequencing with a dideoxymethod using a phage vector M13 (Carlson, J. et al., Journal of Biotechnology, 1, p. 253, 1984).

 The amino acid and nucleotide sequences for advanced APT (II) are given. A primer (1) capable of inducing a mutation differs in the underlined base from the gene sequence of the improved APT (II) (see Table 1).

 C) Induction of a site-specific mutation.

 The following is a method for creating a clone containing the base sequence of a primer (1) capable of producing a mutation, namely, an improved APT (IV) gene. After annealing (renaturation) of the single-stranded DNA described in example 3.3-1), A) clone mp9 (advanced APT (II) and primer (1), the renaturation product is converted into double-stranded DNA, which is then transformed with E. coli JM109. Then, using the primer for sequencing, DNA sequences are screened to isolate a phage clone carrying the mutated improved APT (II) gene, namely the improved APT (V) gene. From this clone, double-stranded (replicative) phage DNA is extracted and the improved APT gene (V )

 5'-terminal phosphorylation of a synthetic oligomer.

 Primer DNA (1) to induce a site-specific mutation is phosphorylated by the method described in example 2.2-1).

 Obtaining heteroduplex DNE.

0.5 μg of single-stranded DNA M13mp9 (advanced APT (II)) and 1.5 μg of double-stranded DNA M13mp9 digested with restriction enzyme BamH1 are heated in 30 μg of a solution containing 2 pmol of phosphorylated primer (1) 10 mM Tris-HCl (pH 7 , 5), 0.1 mM EDTA and 50 mM NaCl, at a temperature of 90 ^o C (2 min), 50 ^o C (5 min), 37 ^o C (5 min) and at room temperature (10 min). 36 μl of a solution of 50 mM Tris-HCl (pH 8.0) containing 4 units of Klenov enzyme, 7 units of T4 phage DNA ligase, 0.1 mM EDTA, 12 mM MgCl ₂ , 10 mM dithiothreitol, 0.7 mt was added to the solution mM ATP, 0.07 dATP and 0.2 mM each of dHTF, dTTP and dTTP, in order to stimulate primer elongation. The mixture is reacted at a temperature of 20 ^° C. for 2 hours and at a temperature of 4 ^° C. for 15 hours.

 Transformation is carried out using the solution described above and competent E. coli JM109 cells (manufactured by Takara Shuzo Co., Ltd.) until lysis spots form. After separation of the colorless spot, phage infect E. coli JN109 for proliferation. The template single-stranded DNA is then obtained from the culture supernatant relative to each clone. These single-stranded DNAs are only subjected to the “T” reaction (reaction “A” and “T” in Example 3-2) of the dideoxymethod using a primer (2) for sequencing, followed by polyacrylamide gel electrophoresis. After drying, the gel is analyzed by autograph. Based on the results, a clone is identified having the desired mutant sequence. The clone culture supernatant is used to infect E. coli JM109 cells and is again inoculated onto a plate in order to isolate a single spot. From the obtained single spot, single-stranded DNA is isolated in accordance with the above method. Using these DNAs, firstly, the DNA base sequence is determined by a dideoxymethod using a primer (2) for sequencing to obtain a clone mutated into the desired base sequence. After infection of this phage clone with E. coli JM-109 cells using the Messing method described in Example 2, double-stranded DNA is obtained. This double-stranded DNA is digested with the restriction enzyme Xho 11, electrophoresis is carried out on a 0.8% agarose gel to isolate a fragment (the improved APT (V) gene of about 1500 base pairs containing the advanced APT gene. DNA is then extracted by electroelution.

 In addition, the complete base sequence for the DNA thus obtained is determined by a dideoxymethod, and as a result, the DNA is found to be an improved APT (V) gene. The complete base sequence of the improved APT (V) gene thus obtained (however, containing a signal peptide of −35 to −1) is shown in FIG. 11 to 13. The derived amino acid sequence is also shown in FIG. 17-19.

 3-2) Design of advanced APT (VI) and (VIII).

 The methods are similar to those described in example 3, 3-1). First, M13mp3 (Advanced APT (II)) is designed, and then primers are synthesized to induce a site-specific mutation. The base sequence of these primers is described above, however, to construct the improved APT (VI) gene and the improved APT (VIII) gene, primers (3) phosphorylated from the 5'-end and primers (5) phosphorylated from the 5'-end are used (see table . 2). Following the induction of a site-specific mutation, the complete base sequence is determined with a dideoxymethod. It has been confirmed that they have the desired base sequence. Thus, the genes of improved APT (VI) and improved APT (VIII) are obtained.

 Then these genes are integrated into the vector pVY1 in accordance with the procedure described in examples 4 and 5.

 Example 4. Integration of the improved APT (II) gene into the pVY1 vector.

 4-1) Construction of the vector pVY1.

 The vector pVY1 is obtained as shown in FIG. 7.

 A) Construction of pAdD26SV (A) N3 (N) and blunting of Eco R1 cleavage site.

 First, pAdD26SV (A) N3 DNA (purchased from Dr. Hiroshi Hand at the University of Tokyo, is known by theses Mo1, Ce 11. Biol, 2 (11, (1982)) is disrupted by the Bgl11 restriction enzyme (manufactured by Boehringer Mannheim-Yamanouchi Co ., Ltd.) in the traditional way. Then the DNA is made blunt in the traditional way using the Klenov enzyme (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd.). After phenol treatment, ethanol precipitation and drying under reduced pressure, the precipitates are dissolved in sterile distilled water. ligation transform reaction th mixture, competent cells of HB101 E.coli (manufactured TAKR Shuzo, Co., Ltd.). Plasmid DNA was prepared from the transformants exhibiting tetracycline resistance in a conventional manner.

 After cleavage of some of these DNAs with the restriction enzyme BgL 1, 0.7% agarose electrophoresis is carried out. The result is a clone carrying DNA that is not cleaved with the restriction enzyme BgL 11. After digesting (pAdD26SV (A) N3 (N)) the DNA of this clone with the restriction enzyme Eco R1 in the traditional way, the DNA is made blunt using the Klenov enzyme as described above. After treatment with phenol, precipitation with ethanol and drying under reduced pressure, the precipitates are dissolved in distilled sterile water.

 B) Isolation of the Kpn 1-BamH1 fragment (about 2900 base pairs) from pKSV10 and the formation of blunt ends.

After cleavage of pKSV10 DNA (manufactured by Fine Chemicals Pharmacy) with the restriction enzymes Kpn1 and BamH1, DNA is made blunt using the T4 DNA polymerase in the traditional way (laboratory manual, pp. 114 - 121). Then carry out gel electrophoresis of 0.7% agarose with the release of a fragment of about 2900 base pairs. Then the fragment is subjected to electrolysis for DNA extraction
C) Construction of pVY1.

 After ligation of the DNA fragment obtained in A) and the DNA fragment obtained in B), the competent E. coli HB101 cells are transformed (described above).

 From transformants exhibiting resistance to tetracycline, plasmid DNAs are prepared in a conventional manner. After digestion of a portion of these plasmid DNAs with the restriction enzyme Pst1 (manufactured by Boehringer Mannheim-Yamanouchi Co., Ltd), 1.0% agarose gel electrophoresis was performed. The result is a clone (plasmid pVY1), characterized by bands of about 3400 base pairs, about 3200 base pairs and about 1400 base pairs. This clone is E / coli HB101 (pVY1, deposited at the Japan Institute of Industrial Science and Technology's Fermentation Research Institute under registration number P-9625 (FEPM BP 2106).

 4-2) Integration of the improved APT (II) gene into the pVY1 vector.

 After DNA digestion of the plasmid pVY1 obtained in Example 4-1) with the restriction enzyme BgL 11 in the traditional manner, dephosphorylation using alkaline phosphatase (manufactured by Takara Shuzo. Co. Ltd.) is carried out. Then carry out the treatment with phenol three times. And after precipitation with ethanol and drying under reduced pressure, the precipitates are dissolved in sterile distilled water.

 After ligation of this DNA with the BgL 11-Xho 11 fragment (about 1500 base pairs) obtained in Examples 3, 3-1), the E. coli competent HB101 cells were transformed with the ligation product in accordance with the method described above. Plasmid DNAs are prepared in a conventional manner from transmigrants having tetracycline resistance. After cleavage of these DNAs with restriction enzymes (BqL 11, Pst 1), a clone is selected that has the improved APT (II) gene in the pVY1 vector and is integrated in the desired direction, and selection is based on analysis of agarose gel electrophoresis. Firstly, part of these DNAs is digested with the restriction enzyme BqL 11, after which electrophoresis is performed on a 0.8% agarose gel to obtain a clone having a fragment band of about 1500 base pairs when the BqL 11-Xho 11 fragment is ligated with the BqL fragment 11 of plasmid pVY1, the ligated portion of Xho 11 and BqL 11 can be cut with the restriction enzyme BqL 11.

 Part of the plasmid DNA of these clones is further digested with the restriction enzyme Pst1, and the DNA is subjected to electrophoresis in a 0.8% agarose gel to obtain a clone having one band of about 3400 base pairs, two bands of about 2300 base pairs, one band of about 1400 base pairs and one strip of about 80 base pairs. Using this clone (plasmid pVY1-APT (II), plasmid DNAs are prepared according to laboratory guidelines.

 Example 5. Integration of improved APT genes (V), (VI) and (VIII) into the pVY1 vector.

 After DNA digestion of the plasmid pVY1 obtained in Example 4-1), the restriction enzyme BqL 11 is conventionally dephosphorylated using alkaline phosphatase (manufactured by Takara Shuzo, Co., Ltd.), followed by treatment (3 times) with phenol, precipitation with ethanol and drying with reduced pressure. Then the precipitate is dissolved in sterile distilled water.

 After ligation of this DNA with a BqLII-Xho 11 fragment of about 1,500 base pairs obtained in Examples 2, 2-3), the above competent E. coli HB101 cells are transformed with the ligation product. Plasmid DNAs are obtained from transformants exhibiting tetracycline resistance in accordance with a conventional method. After cleavage of these DNAs with restriction enzymes BqL11 and Pstl, agarose gel electrophoresis was performed. By analyzing the nature of the separation in the agarose gel, clones are selected in which the enhanced APT (V) gene is inserted into the pVYI vector in the desired direction. First, after cleavage of a portion of these DNAs with the BqL11 restriction enzyme, electrophoresis is carried out on a 0.8% agarose gel to give clones and a band of about 1,500 base pairs is obtained. When the BqL11-Xholl fragment is linked to the BqL11 fragment of the pVYI vector, a portion of Xholl and BqL11 can be cleaved with the restriction enzyme BqL11 due to the aforementioned isoshizomer configuration.

 After further cleavage of a portion of the plasma DNA of these clones with the restriction enzyme Pstl, electrophoresis is carried out at an agarose gel concentration of 0.8% to obtain a clone giving a band of about 3400 base pairs, a band of about 2300 base pairs, two bands of about 1400 base pairs , one strip of about 800 base pairs and one strip of about 80 base pairs. Using a clone (plasmid pVYI-APT (V)), plasmid DNA is obtained in large quantities, based on laboratory guidance.

 Similarly, the genes of improved APT (VI) and (VIII) are integrated into the pVYI vector.

 Example 6. Expression of improved APT in CHO cells.

 Plasmid pVYI - Advanced APT (VI), APT (II), APT (V) or APT (VIII) transfects DHF-deficient CHO cells (Urlaub, et al., Proc.Natl., Acad. Sci. USA, 77 (7 ), 4216-4224, 1980) by the calcium phosphate method (Graham, et al., Viroloqy, 52, 456, 1973). It was found that the transformer clone obtained on selective medium (MEM A LPHA (-), GIBCO) in the presence of methotrexate (MTX) showed APT activity at a level of 50 to 100 units / ml (the value determined by the fibrin / agarose cup method described below). This clone is used for subsequent studies. As a production medium, a GIT medium (manufactured by Huaco Pué Chemical Industry Co., Ltd.) enriched with 20 international units / ml (SIGMA) of aprotinin is used.

 Example 7. Purification of the improved APT from the culture supernatant of CHO cells.

 The culture supernatant obtained in example 6, partially purified on an affinity column with anti-APT monoclonal antibody. A hybrid monoclonal antibody-producing antibody is prepared for APT derived from human melanoma cells in a conventional manner. Antibody-producing hybridomas are inoculated to mice, and a monoclonal antibody (subclass: IgGM1) that develops in ascites is extracted and purified using Cellulofin Protein A (manufactured by Biochemical Industry Co., Ltd.) and MAPS buffer system for purification of the monoclonal antibody manufactured by Biorad. The antibody is coupled to CN3r-activated Sepharose (manufactured by Pharmacy Fine Chemicals) in a ratio of 4 mg per ml of gel in a conventional manner.

The antibody gel (24 ml) was mixed with four liters of culture supernatant. After shaking gently overnight at 4 ^° C, the gel was loaded onto a column (diameter 1.5 cm x 20 cm). Then the gel is washed successively with 125 ml of each of the following solutions of (1) Tris-HCl buffer, pH 7.4 (buffer A), containing 25 international units / ml of aprotinin (manufactured by SIGMA) and 0.01% (weight / volume) Tween 80, (2) buffer A containing 0.5 M NaCl, (3) buffer A containing 4 M urea, and (4) buffer A. The enhanced gel-bound APT was eluted with 0.2 M glycine-HCl pH 2 buffer, 5 containing 25 international units / ml aprotinin and 0.01% (w / v) Tween 80. The active fractions are reduced and combined. After dialysis against 10 mM Tris-HCl buffer, pH 7.4, containing 25 international units / ml aprotinin and 0.01% (mass / volume) Tween 80, the dialysate is concentrated 20-30 times overnight with a vacuum centrifugal concentrate (Speed VAC, manufactured by SAVANT Inc.). The concentrate was redialized again against 10 mM Tris-HCl buffer, pH 7.4, containing 0.15 M NaCl, 25 international units / ml aprotinin and 0.01% (w / v) Tween 80, overnight, and used for subsequent in vitro and in vivo assessments. Finally, the specific activity increases by 3700-5000 times, and the yield is from 36 to 42% of the activity of APT (determined by fibrin / agarose cup method).

This active fraction is analyzed by electrophoresis with sodium dodecyl sulfate and silver staining. Under reducing conditions at a level of 54 kilodaltons, a very strong band is noted along with several other bands. Next, the gel after electrophoresis is treated with 2.5% (mass / volume) Triton X-100 and placed on a fibrin / agarose plate in order to autograph fibrin at a temperature of 37 ^o C, due to which the dissolved band is detected at about 50 kilodaltons. On the same cup, natural APT appears at about 60 kilodaltons. The results indicate that APT absorbed on an antibody affinity column and eluted by this method corresponds to an improved APT having a molecular weight that is about 10,000 less than the molecular weight of a naturally occurring type.

 Example 8. Measurement of the specific activity of the improved APT.

 The amount of protein in a partially purified advanced APT is determined by measuring total protein according to the BradFord method (Bradford, Anal.Bochem. 72, 248 (1976)) using bovine serum albumin as a reference protein. The amount of antigen APT is measured by enzyme immunoassay (ELISA).

Fibrinolytic activity is determined by fibrin / agarose cup method and dissolution method of a ¹²⁵ 1-labeled fibrin film. A fibrin / agarose plate is prepared by adding agar to 95% coagulated fibrinogen. The method of dissolving the film ¹²⁵ 1-labeled fibrin is carried out as described by Hoyraeerts et al. (J. Biol. Chem. 257, 2912, 1982) using Bioscott Inc. as a reference for human anti-melanoma cells. and standardized in accordance with the International Standard APT (Gaffuey and Curtis, Thromb. Haemostas, 53, 34, 1985).

The specific activity value calculated from the activity value determined by dissolving the ¹²⁵ 1-fibrin film and the amount of antigen determined by enzyme-linked immunosorbent assay (ELISA) ranged from 300,000 to 420,000 units / mg of antigen.

Example 9. The affinity of the improved APT for fibrin and activation by fibrin
In accordance with the work of Verheijen, et al. / EMBOJ, 5, 3525, 1986), the affinity of the improved APT for fibrin is investigated. An improved or naturally occurring APT (1000 units / ml) is added to fibrinogen at various concentrations, after which one unit of trombone is added, followed by reaction at room temperature for 3 minutes. The formed fibrin clot is precipitated by centrifugation at a rotation speed of 16,000 rpm for 8 minutes and the amount of APT that is not associated with fibrin is determined by measuring the activity of the fibrin / agarose plate method. As a result, it was found that the improved APT (VI) exhibits the same affinity for fibrin as the natural form. In order to investigate the degree of plasminogen activation by an improved APT in the presence or absence of fibrin, the following experiment is carried out. Using a titration tablet, a naturally occurring or advanced APT is added to 0.1 M Tris-HCl buffer, pH 7.5, containing 0.3 mM synthetic substrate p-niroanilide-tripeptide S-2251 (HD-Val-leulys-pNA HCl, manufactured by Kaby Inc.), 0.13 μM plasminogen without plasmin, 120 μg / ml DESAFIB ^™ (manufactured by American Diagnostics Inc.) and 0.1% Tween 80, to give a total volume of 200 μl. The system is maintained at a temperature of 37 ^° C. After a certain period of time, absorbance (optical density) is measured at a wavelength of 405 nm using a Titertech Multiscan 310 Model.

The dose-response curve for the amidolytic activity of the improved APT (VI) and naturally occurring APT is shown in FIG. 22. The shift of the dose-response curve due to the addition of DESAFIB ^™ for naturally occurring APT corresponds to a value of 158 times, while for advanced APT it reaches 100 times. This is due to the fact that the activity of improved APT (VI) in the absence of DESAFIB ^{™ is} lower, approximately 1/20, than the activity of natural APT.

 Example 10. Analysis of advanced APT for fibrinolytic activity in the bloodstream of a rabbit.

 Pharmacokinetics by comparing the activity of naturally occurring APT (n-APT) and the improved APT of the present invention in a rabbit. As can be seen from FIG. 23, the improved APT shows a noticeable prolongation of the biological half-life of the active state (natural APT shows the half-life of 1-2 minutes, whereas the improved APT is biologically active for 8-15 minutes). In addition, it is obvious that an activity value of 5% (the value 30 s after administration is 100%) still remains in the improved APT even 60 minutes after its administration (the natural APT exhibits an activity of 0.1 in 60 minutes % of initial).

This experiment is as follows
A Japanese white rabbit weighing 2.4 kg was selected for testing. Under anesthesia with pentobarbital, APT is administered through the peripheral vein of the ear. The dose is 15,400 units (0.8 ml) of advanced APT per rabbit and 5400 units (0.8 ml) of n-APT per rabbit (values determined by fibrin-plate method). Then 2.5 ml of blood is collected from the femoral artery using a catheter at various time intervals (from 0.5 to 60 minutes) and added to 1/9 of the volume of citric acid sodium (3.8%). Within 30 minutes after blood collection, centrifugation is carried out at low speed, separating the plasma. Using separated plasma, APT activity in the blood is measured.

 (1) Measurement of APT activity.

After diluting 0.2 ml of plasma with 3 mM glacial acetic acid 16 times, the diluted product is centrifuged at a low rotation speed to obtain precipitates. The precipitates dissolve 20 mM Tris-HCl, pH 7.4, with 140 mM NaCl in a volume equivalent to the volume of the plasma, to obtain a fraction of euglobulin. APT activity is determined by adding this euglobulin fraction to a fibrin / agarose plate. After the plates were incubated at 37 ^° C. for 16 hours, APT activity was observed as a plaque. The standard curve for the fibrin / agarose cup method is obtained by diluting the APT used to administer to the animal to 0.1-10000 units / ml. The blood APT activity determined in this way is expressed as a percentage using the APT activity obtained by blood collection 30 s after administration, taken as 100%.

 Example 11. The resistance of the improved APT (VI) to the effects of heat and acids.

To determine the resistance to heat, the improved APT (VI) and natural APT are diluted with 50 mM Tris buffer containing 100 mM NaCl and 0.01% Tween 80, pH 7.4, to a concentration of 100 μg / ml, respectively. Each solution is kept in boiling water (temperature 98 ^o C) for 2-60 minutes. After cooling, the residual activity is determined by the fibrin plate method. As shown in FIG. 24, the decrease in activity of the improved APT (VI) is insignificant in comparison with the decrease in the activity of natural APT. For example, after heat treatment for 2 minutes, the activity of natural APT is reduced to 25%, while the advanced APT (VI) still maintains activity at 71%.

 To study acid resistance, an improved APT (VI) and natural APT are dissolved in 0.5N. HCl solution at a concentration of 100 μg / ml, followed by settling at room temperature for 30 minutes. After neutralization, fibrin-plate activity is determined. Advanced APT does not detect any change in activity, while the activity of natural APT is reduced by 50%.

Example 12. Inhibition of the active factor stimulating lymphocytes, improved APT (VI)
Advanced APT (VI) and natural APT are suitably diluted with PPM1 1640 tissue culture medium containing 7% amniotic calf serum and 58 μM 2-mercaptoethanol. 100 μl of the dilution is loaded into a 96-well tissue culture plate, after which 50 μl of a cell suspension containing thymocytes (2 × 10 ⁷ cells / ml) from male C3H / He ^J mice from 4 to 6 weeks old, concanavalin are added. A (1.2 μg / ml), as well as 50 μl of IL-1 (4 units / ml, Aenzyme Inc), followed by cultivation for 48 hours in an incubator at a temperature of 37 ^o C containing 5% carbon dioxide. Then add H ³ -thymidine at a concentration of 0.5 microns. cube inch / 20 μl / well. After culturing for 18 hours, the cells are collected on a glass fiber filter and the amount of ³ H-thymidine introduced into the cells is measured by a liquid scintillation counter to determine the activity of the lymphocyte stimulating factor.

 As shown in Fig.25, natural APT does not inhibit the activity of the factor stimulating lymphocytes, and an improved APT significantly suppresses it. When tested with solvent only, no effect was observed.

 Example 13. Anti-inflammatory activity based on action against a denatured protein.

 1) Obtaining denatured protein.

After incubation of the protein solution (5 mg / ml) in 0.1 N. HCl solution or 0.1 N NaOH solution at a temperature of 37 ^o C for 2-3 hours, the protein solution is neutralized with the same amount of NaOH or HCl.

 2) The affinity of the improved APT (VI) to the denatured protein.

 Method: In accordance with the procedure below, the denatured protein is “linked” to a nitrocellulose film. Then measure the amount of improved APT associated with the processing of the protein and nitrocellulose film, thereby assessing the affinity of the improved APT for the denatured protein.

 A piece of nitrocellulose film was immersed in 20 mM Tris-HCl buffer solution, pH 7.5, containing 140 mM NaCl.

 Drying. The denatured protein (50 μg / 10 μl) is released dropwise onto a piece of nitrocellulose film.

 Drying. Blocking with 3% gelatin solution.

 Flushing. A piece of nitrocellulose film is immersed in a solution of advanced APT / 1 μg / ml /.

Flushing. Add plasminogen and a synthetic substrate S-2251, and then incubate at a temperature of 37 ^o C (quantitative analysis of absorbed advanced APT).

 Absorption measurement at 405 nm.

 Results: as shown in Table 3, the improved APT shows affinity for HCl-treated immunoglobulin G, HCl-treated albumin, and NaOH-treated albumin. On the other hand, the improved APT does not show affinity for intact immunoglobulin G and albumin.

 3) Activation of advanced APT (VI) by denatured protein.

 Method: plasminogen (0.0078 units in 10 μl), 100 μl of 3 mM S-2251 synthetic substrate and various amounts of TBS buffer are added to the reaction solution of the enhanced APT activator (denatured protein, BrCN-treated fibrinogen, etc.) at various concentrations, receiving 0.275 ml of the reaction solution. Advanced APT (2.5 n / g in 25 μl) is added to the reaction solution to initiate the reaction. After reaction over a period of time, 2% sodium dodecyl sulfate (equimolar amount) is added to the reaction solution in order to stop the reaction. By measuring the optical density (OD 405), the activity of the improved APT is determined.

 Results: as shown in FIG. 26, NaOH-treated albumin and HCl-treated immunoglobulin G show a pronounced activating effect of the improved APT. In particular, in HCl-treated immunoglobulin G, activation is strong, and the activity of HCl-treated immunoglobulin G is approximately equal to the activity of BrCN-treated fibrinogen, and at a concentration that is several times lower. Intact albumin and immunoglobulin G do not show activation.

 4) Degradation of denatured protein under the influence of an improved APT (VI).

 Method: after the interaction of the denatured protein with the improved APT under the same conditions as in the method described in the previous subparagraph, except that the synthetic substrate S - 2251 is not added to the reaction system and the amount of the denatured protein is 133 μg / ml, carry out polyacrylamide gel electrophoresis with sodium dodecyl sulfate in the presence of β-mercaptoethanol.

 Results: as shown in Fig. 27, a protein denatured by NaOH or HCL treatment leads to the disappearance of the eff-protein bands in the picture and the formation of degradation products, which indicates its decomposition. On the other hand, when using intact albumin, no change in the ef-picture was found after interaction with the improved APT, and therefore, no degradation of the denatured protein was detected.

Claims (2)

 1. Recombinant tissue plasminogen activator having the amino acid sequence shown in p. where Y is Glu-Ile-Lys;
H ₂ N is amino terminus;
-COOH - carboxy terminus;
R is a direct link or a sequence similar to it, containing substitutions and / or deletions and / or inserts not associated with changes in activity,
and having the following properties: fibrinolytic activity, determined by the method of dissolving a ^{1 2 5} I-fibrin film, the ability to be activated by fibrin, and improved tpA activity in the absence of fibrin, which is lower than the natural tpA activity, increased compared to the natural form, half-life, increased, in comparison with natural tpA, resistance to acids and heat, the ability to inhibit the activation factor of lymphocytes, the ability to be activated by a denatured protein.  2. A method of obtaining a recombinant tissue plasminogen activator, comprising cultivating host cells transformed with a recombinant DNA containing a sequence encoding a tpA analog, and subsequent purification of the target product, characterized in that cultivating host cells transformed with a recombinant vector containing a recombinant vector containing a DNA sequence encoding tpA by item 1.

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