JPH07163369A - DNA encoding a peptide having an action of promoting activation of protein C by thrombin - Google Patents

Abstract

(57) [Summary] (Modified) [Structure] A novel DNA encoding a peptide having an action of promoting activation of protein C by thrombin or a homologous variant thereof. [Effect] The peptide encoded by the DNA of the present invention, which has an action of promoting activation of protein C by thrombin, has anti-coagulant action, platelet aggregation inhibitory action, thrombolytic action, and cardiovascular diseases with few side effects. It is a very useful substance as a therapeutic drug. Further, the present peptide is bound to a medical artificial material such as an artificial blood vessel, an artificial organ, or a catheter, in addition to such medical use,
It can be used as a drug for preventing the formation of blood clots.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel deoxyribonucleic acid (hereinafter referred to as "DNA") encoding a peptide having an action of promoting protein C activation of thrombin. More specifically, the present invention relates to a DNA encoding a peptide having a thrombolytic action, an anticoagulant action and a platelet aggregation inhibitory action, which is useful for the treatment of cardiovascular diseases. The present invention also provides a DNA encoding the novel peptide, a replicable recombinant DN containing the DNA.
A, a microorganism or cell transformed with the recombinant DNA and a method for producing the peptide by recombinant DNA technology. The present invention also relates to a pharmaceutical composition containing the peptide or a homologous variant thereof and containing no other human-derived peptide, in particular, a pharmaceutical composition for blood circulation.

In the present specification, amino acids and peptides refer to the IUPAC-IUB Biochemical Nomenclature Committee (C
BN). When amino isomers may have optical isomers, L form is shown unless otherwise specified. Further, unless otherwise specified, the left and right ends of the amino acid sequence of a peptide are N-terminal and C-terminal, respectively. Gln: Glutamine residue Asp: Aspartic acid residue Pro: Proline residue Tyr: Tyrosine residue Val: Valine residue Lys: Lysine residue Glu: Glutamic acid residue Ala: Alanine residue Asn: Asparagine residue Leu: Leucine residue Group Phe: Phenylalanine residue Gly: Glycine residue His: Histidine residue Ser: Serine residue Thr: Threonine residue Ile: Isoleucine residue Trp: Tryptophan residue Arg: Arginine residue Met: Methionine residue Cys: Cysteine residue Basis

Polydeoxyribonucleotides and oligonucleotides are represented by the deoxyribonucleotide sequences represented by the following abbreviations. A: 2'-deoxyadenylic acid residue C: 2'-deoxycytidylic acid residue G: 2'-deoxyguanylic acid residue T: thymidylic acid residue Unless otherwise specified, the left and right ends of the deoxyribonucleotide sequence are 5'end and 3'end respectively.

[0004]

2. Description of the Related Art Streptokinase and urokinase are currently used as thrombolytic agents. Heparin and warfarin are used as anticoagulants. Furthermore, aspirin, sulfinpyrazone, dipyridamole and the like are used as platelet aggregation inhibitors.

Currently, these thrombolytic agents, anticoagulants and platelet aggregation inhibitors are used individually or in combination, for example, myocardial infarction, thrombosis, embolism, peripheral vascular occlusion, arteriosclerosis obliterans. , Intravascular blood coagulation syndrome (D
IC), angina, transient cerebral ischemic attack, pregnancy toxemia and the like. However, these thrombolytic agents, anticoagulants and platelet aggregation inhibitors act on only a small part of the blood coagulation / fibrinolysis system, which has a very complicated mechanism. Therefore, there has been a demand for a drug that widely acts on the coagulation / fibrinolysis system of blood and exhibits an excellent inhibitory effect on blood coagulation.

Meanwhile, protein C is known as a vitamin K-dependent protein that plays an important role in the blood coagulation mechanism. In recent years, a substance that promotes the activation of protein C and suppresses platelet activation and fibrin formation by the action of thrombin exists in rabbit lung, bovine lung, human lung, human placenta, etc. It has been reported to have an excellent blood coagulation inhibitory action as compared with the above-mentioned drugs.

Regarding substances present in the lungs of rabbits, for example, CT Esmon
, Proceeding of National Academy of Sciences USA (Proc. Nat
l. Acad. Sci. USA, 78, 2249 (1981); NL Esmon (NL Es).
mon) et al., The Journal of Biological
Chemistry (J. Biol Chem.), 257
Vol. 859 (1982); CT Esmon et al., The Journal of Biological Chemistry (J. Biol. Che.
m. ), 257, p. 7944 (1982); N
L. Esmon et al., The Journal of Biological Chemistry (J. Bi.
ol. Chem. ), 258, 12238 (198)
2 years) can be referred to.

For substances present in bovine lung, for example, Kusumoto et al., Biochemistry, 56, 890 (1984) can be referred to. Regarding substances existing in human placenta, for example, JP-A-60-199819; Kurosawa et al., Journal of Japanese Society of Hematology, Vol. 47, p. 632 (1984).
Year); Etch. Etch. Salem. (H.S. Sale
m) et al., Journal of Biological Chemistry (J. Biol. Chem.), 259, 122.
46 pages (1984); S. Kuro
Sawa et al., Thrombosis Research (Thromb
sis Research), 37, 353 (1
1985). Regarding substances existing in human lungs, for example, Kusumoto et al., Biochemistry, 57
Vol. 1102 (1985).

The above mentioned prior art documents describe the general properties of the above substances. However, the structure of the substance, such as the amino acid sequence, has not been elucidated, and the substance has not yet been identified. Therefore, whether the substance reported in the above-mentioned prior art documents is a single substance,
Further, it is completely unknown whether the same substance can be repeatedly obtained according to the description of these prior art documents.

[0010]

In view of the above technical background, the inventors of the present invention not only suppress blood coagulation by activating protein C, which is one factor of the blood coagulation / fibrinolysis system, but also As a result of repeated studies to find a substance that enhances the lytic action, surprisingly, as will be described later, a peptide having a specific amino acid sequence was identified as protein C by thrombin.
It has been found that not only can activation be promoted to control blood coagulation, but also fibrinolysis can be promoted, which is useful as a drug for controlling blood coagulation. Then, the DNA encoding the protein was isolated, and it was further found that the peptide can be easily produced in a large amount in a pure form containing no other human-derived protein by recombinant DNA technology. The present invention has been completed based on these findings.

That is, an object of the present invention is to provide the peptide or a homologous variant thereof having an action of promoting activation of protein C by thrombin in a pure form containing no other human-derived protein. is there. Another object of the present invention is to provide DNA encoding the above peptide or its complementary DNA. Furthermore, another object of the present invention is the DN encoding the peptide.
To provide a replicable recombinant DNA containing A. Still another object of the present invention is to provide a microorganism or cell transformed with the recombinant DNA as described above. Still another object of the present invention is to provide a method for producing the above peptide.
Furthermore, another object of the present invention is to provide a pharmaceutical composition containing a peptide having an action of promoting activation of protein C by thrombin in a form not containing other human-derived peptides, more specifically, a pharmaceutical composition for blood circulation. To provide things.

[0012]

That is, according to the present invention,
At least an amino acid sequence represented by the following formula (I): Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Ser Gln Cys Val Glu His Asp Cys Phe Ala Leu yr Gyr His Gyr Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Gly Pro Gary Gly Pro Gly Gly Pro Gly Gly Pro Gly Gly Pro Gly Gly Gly Gly Gly Gly Gly Gly Gly Leu Met Thr Val Arg Ser Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro Val Gly Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His Ser Gly Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu Val Val Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala Ala Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val Val LeuGlnHisValArgThrGluArgThrProGlnArgLeu, characterized by containing the amino acid sequence represented by:
A large amount of a peptide having a function of promoting activation of protein C by thrombin or a homologous variant thereof is provided in a pure form containing no other human-derived protein.

The DNA of the present invention encodes the amino acid sequence represented by the above formula (I). The DNA of the present invention has the formula (I
I): GCACCCGCAG AGCCGCAGCC GGGTGGCAGC CAGTGCGTCG AGCACGACTG CTTCGCGCTC TACCCGGGCC CCGCGACCTT CCTCAATGCC AGTCAGATCT GCGACGGACT GCGGGGCCAC CTAATGACAG TGCGCTCCTC GGTGGCTGCC GATGTCATTT CCTTGCTACT GAACGGCGAC GGCGGCGTTG GCCGCCGGCG CCTCTGGATC GGCCTGCAGC TGCCACCCGG CTGCGGCGAC CCCAAGCGCC TCGGGCCCCT GCGCGGCTTC CAGTGGGTTA CGGGAGACAA CAACACCAGC TATAGCAGGT GGGCACGGCT CGACCTCAAT GGGGCTCCCC TCTGCGGCCC GTTGTGCGTC GCTGTCTCCG CTGCTGAGGC CACTGTGCCC AGCGAGCCGA TCTGGGAGGA GCAGCAGTGC GAAGTGAAGG CCGATGGCTT CCTCTGCGAG TTCCACTTCC CAGCCACCTG CAGGCCACTG GCTGTGGAGC CCGGCGCCGC GGCTGCCGCC GTCTCGATCA CCTACGGCAC CCCGTTCGCG GCCCGCGGAG CGGACTTCCA GGCGCTGCCG GTGGGCAGCT CCGCCGCGGT GGCTCCCCTC GGCTTACAGC TAATGTGCAC CGCGCCGCCC GGAGCGGTCC AG GGCACTG GGCCAGGGAG GCGCCGGGCG CTTGGGACTG CAGCGTGGAG AACGGCGGCT GCGAGCACGC GTGCAATGCG ATCCCTGGGG CTCCCCGCTG CCAGTGCCCA GCCGGCGCCG CCCTGCAGGC AGACGGGCGC TCCTGCACCG CATCCGCGAC GCAGTCCTGC AACGACCTCT GCGAGCACTT CTGCGTTCCC AACCCCGACC AGCCGGGCTC CTACTCGTGC ATGTGCGAGA CCGGCTACCG GCTGGCGGCC GACCAACACC GGTGCGAGGA CGTGGATGAC TGCATACTGG AGCCCAGTCC GTGTCCGCAG CGCTG GTCA ACACACAGGG TGGCTTCGAG TGCCACTGCT ACCCTAACTA CGACCTGGTG GACGGCGAGT GTGTGGAGCC CGTGGACCCG TGCTTCAGAG CCAACTGCGA GTACCAGTGC CAGCCCCTGA ACCAAACTAG CTACCTCTGC GTCTGCGCCG AGGGCTTCGC GCCCATTCCC CACGAGCCGC ACAGGTGCCA GATGTTTTGC AACCAGACTG CCTGTCCAGC CGACTGCGAC CCCAACACCC AGGCTAGCTG TGAGTGCCCT GAAGGCTACA TCCTGGACGA CGGTTTCATC TGCACGGACA TCGACGAGTG CGAAAACG GC GGCTTCTGCT CCGGGGTGTG CCACAACCTC CCCGGTACCT TCGAGTGCAT CTGCGGGCCC GACTCGGCCC TTGTCCGCCA CATTGGCACC GACTGTGACT CCGGCAAGGT GGACGGTGGC GACAGCGGCT CTGGCGAGCC CCCGCCCAGC CCGACGCCCG GCTCCACCTT GACTCCTCCG GCCGTGGGGC TCGTGCATTC GGGCTTGCTC ATAGGCATCT CCATCGCGAG CCTGTGCCTG GTGGTGGCGC TTTTGGCGCT CCTCTGCCAC CTGCGCAAGA AGCAGGGCGC CGCCAGGGCC AAGATGGAGT ACAAGTGCGC It GCCCCTTCC AAGGAGGTAG TGCTGCAGCA CGTGCGGACC GAGCGGACGC CGCAGAGACT may consist C represented by DNA sequences, also,
It may encode the amino acid sequence of a homologous variant peptide having a composition substantially homologous to the amino acid sequence represented by formula (I).

It is possible to alter part of the structure of a peptide by natural mutations or by artificial mutations without significantly altering the activity of the peptide. It is also possible to artificially produce these peptides using the DNA of the present invention. Therefore, the DNA of the present invention is a homologous variant of a peptide having the above amino acid sequence (Homo).
It also includes a DNA encoding a peptide having a structure corresponding to a logvariant).

The peptide encoded by the DNA of the present invention may or may not contain at least one sugar residue. That is, the present invention shows that the amino acid sequence is at least the sequence described in the present specification, and is not particularly limited by the sugar residue. According to the degeneracy of the genetic code, at least one base in the base sequence of the gene can be replaced with another type of base without changing the amino acid sequence of the polypeptide produced from the gene. Therefore, the DNA of the present invention can also contain a nucleotide sequence changed by substitution based on the degeneracy of the genetic code. In this case, the amino acid sequence deduced from the base sequence obtained by the above substitution matches the previously defined amino acid sequence.

The DNA of the present invention can be used to produce the aforementioned peptides using recombinant DNA technology.
The DNA of the present invention can be obtained as follows. (1) Using a rabbit-specific antibody specific for a human lung-derived peptide capable of promoting protein C activation of thrombin, a peptide that binds to the antibody was prepared from a cDNA library prepared from human lung. The encoding cDNA fragment is isolated, and the base sequence of the isolated cDNA fragment is analyzed. The resulting cDNA fragment encodes a portion of a human lung-derived peptide capable of promoting protein C activation of thrombin. That portion includes the C-terminus but not the N-terminus of the peptide.

(2) As described above, the obtained cDNA fragment does not encode the entire amino acid sequence of the peptide derived from human lung, and lacks the base sequence corresponding to the N-terminal amino acid sequence of the peptide. The cDNA fragment encoding the N-terminal amino acid sequence is cD obtained in the above step (1).
The NA fragment is used to obtain by the following known ordinary primer extension method. First, a part of the cDNA fragment corresponding to the amino acid sequence on the N-terminal side of the peptide encoded by the cDNA fragment obtained in the above step (1) is chemically synthesized. Next, using the synthesized DNA as a primer, the cDNA obtained in the above step (1) from poly (A) ⁺ RNA prepared from human umbilical cord endothelial cells by a commonly known primer extension method.
A cDNA fragment having a nucleotide sequence upstream of the 5'end of the fragment is obtained. By repeating the above primer extension, a cDNA fragment encoding the N-terminal amino acid sequence of a peptide derived from human lung is obtained.

(3) Next, the cDNA fragments obtained in steps (1) and (2) above are ligated so as to encode the entire amino acid sequence of the desired peptide, starting from the codon of the N-terminal amino acid 1671. Base pairs (hereinafter “bp”)
(Abbreviated as) is obtained. The base sequence of this open reading frame is the same as the base sequence represented by the above formula (II). The nucleotide sequence of the cDNA thus obtained is analyzed by a known method to confirm that it matches the nucleotide sequence represented by the formula (II).

The DNA of the present invention can also be obtained by organic chemical synthesis. Further, the DNA of the present invention can be prepared from the precursor DNA without performing the above-mentioned primer extension. The precursor DNA is
DNA fragment obtained in the step (1) or its DNA
It can be obtained from a human chromosomal DNA library by an ordinary hybridization method using a synthetic DNA prepared based on the nucleotide sequence of the fragment as a probe. DN of the present invention containing the base sequence of the above formula (II)
A further contains, as a 5'terminal base sequence, a base sequence encoding a leader sequence represented by, for example, the following formula: Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu Ala Gly Leu Gly Phe Pro. Good.

According to the present invention, D complementary to the above DNA is used.
NA is also provided. According to the present invention, the above DNA and its complementary DNA may be bound to each other complementarily to form a double-stranded DNA. Furthermore, according to the present invention, a replicable recombinant DNA comprising the above-mentioned DNA of the present invention and a replicable expression vector is provided. The recombinant DNA is capable of expressing the peptide of the present invention in the microorganism or cell transformed with it. An example of a suitable vector is the plasmid pBR3.
22, pBR327, YRp7, pSV2-dhfr
(ATCC 37146), pBPV-1 (9-1)
(ATCC 37111) and the like. It is necessary to select an expression vector suitable for the microorganism or cell used as the host.

Furthermore, the present invention also relates to a microorganism or cell transformed with the above-mentioned replicable recombinant DNA. Examples of microorganisms include Escherichia coli ( Esc
strains of herichia coli), for example, E. coli (E. coli) K12 strain 294 (ATCC 314
46), E. coli ( E. coli ) B, E. coli ( E. coli ) X1776 (ATCC 3153)
7), E. coli (E coli) C600 and E. coli (E coli) C600 hfl and E. coli ^{(E coli) W3110 (F -} , λ -, prototropic Fick, ATCC27375);... Bacillus subtilis (Bacillus subtilis) Strains of the genus Bacillus such as Salmonella typhimuu
rium) or Serratia Masesansu (Serra
Various strains of shoe Domo eggplant (Pseudomonas) genus;; tia marcesans) intestinal bacteria other than E. coli, such as and Saccharomyces cerevisiae (Sac
charomyces cerevisiae ) and the like. Examples of cells include VERO (ATCC
CCL-81) cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, CO
Animal cells such as S-7 and MDCK cell lines are mentioned.

According to another aspect of the present invention, (a) a replicable expression comprising the DNA and the replicable expression vector linked to a DNA encoding the above-mentioned peptide and a replicable expression vector. Obtaining recombinant DNA, (b) transforming a microorganism or cell with the replicable recombinant DNA to form a transformant, and (c) transferring the transformant from a parent cell of the microorganism or cell. Selecting, (d) culturing the transformant, expressing the DNA in the transformant to produce the peptide, and (e) isolating the peptide from the cultured transformant. Including D of the present invention
A method for producing a peptide encoded by NA is provided.

According to the method of the present invention, the above-mentioned D of the present invention is used.
A replicable recombinant having the DNA of the present invention incorporated downstream of a DNA region such as a promoter of a replicable expression vector so that NA is correctly transcribed and translation from the resulting mRNA is correctly carried out. Body D
NA is obtained, and a microorganism or cell is transformed with the obtained recombinant DNA to obtain a transformant containing the recombinant DNA. The obtained transformant is the recombinant DNA
Isolated from the parent cell of the microorganism or cultured cell according to the phenotype conferred on. The transformant thus obtained is cultured to express the genetic information contained in the DNA to produce the peptide of the present invention.

In order to clone the DNA sequences necessary for constructing the DNA of the present invention and the recombinant DNA, such as promoter and origin of replication, a host-vector system using prokaryotic cells as a host is used. Is preferred. Examples of prokaryotic cells include Escherichia coli ( Esc
strains of herichia coli), for example Ikori (E. coli) K12 strain 294 (ATCC 3144
6), E. coli (E. Coli) B, E coli (E. Coli) X1776 (ATCC 3153
7), E. coli (E coli) C600 and E. coli (E coli) C600 hfl and E. coli ^{(E coli) W3110 (F -} , λ -, prototropic Fick, ATCC 27375);... B.
Strains of the genus Bacillus, such as Bacillus subtilis ; Salmonella typhimurium
imurium ) or Serratia Mercases ( Se
Enterobacteria other than Escherichia coli such as Rratia marcesans ; Pseudomona
s) Various strains of the genus; and Saccharomyces cerevisiae (Saccharomyces cerevisia
e ) and the like.

Among these bacteria, E. coli K12 strain 294 is the most preferable. When the above microorganisms are used as hosts, plasmid vectors suitable for these microorganisms are generally used as expression vectors capable of replicating recombinant DNA. For example, plasmids pBR322 and pBR327 can be used as a plasmid vector for transforming E. coli. A plasmid vector usually contains an origin of replication, a promoter, and a marker gene which gives the recombinant DNA a phenotype useful for selecting cells transformed with the recombinant DNA. An example of a promoter is β
-Lactamase and lactose promoters, tryptophan promoters and the like. Examples of marker genes include ampicillin resistance gene and tetracycline resistance gene.

On the other hand, in order to express the DNA of the present invention to produce the peptide of the present invention, a host-vector system using the above-mentioned prokaryotic cells as a host and eukaryotic cells such as vertebrate cells are used as host cells. The host-vector system used as can be used. Examples of eukaryotic cells include cells such as the aforementioned animal cell lines. DN of the present invention
In order to express A in the aforementioned eukaryotic cells, the recombinant DNA of the present invention generally has a functional sequence for controlling gene expression, such as an origin of replication, a promoter to be located upstream of the DNA of the present invention, a ribosome. It contains binding sites, polyadenylation sites and transcription termination sequences. DNA of the present invention
Such functional sequences that can be used to express E. coli in eukaryotic cells can be obtained from viruses and viral agents.

For example, the promoter which can be used in the present invention can be obtained from adenovirus 2, polyoma virus, simian virus 40 (SV40) and the like. In particular, the adenovirus 2 major late promoter and S
The early and late promoters of V40 are preferred. In addition, if a promoter originally present at a position upstream of a gene encoding a peptide derived from human lung, which has an action of promoting protein C activation of thrombin, is suitable for use in the above-mentioned host-vector system. Can be used. Regarding the origin of replication, there are foreign origins such as adenovirus, polyoma, SV40, vesicular stomatitis virus (VSV), bovine papilloma virus (BP).
Replication origins derived from viruses such as V) can be used. When a vector having a property of being integrated into the host chromosome is used as the expression vector, the origin of replication of the host chromosome can be used.

The microorganism or cell transformed with the replicable recombinant DNA of the present invention is a parental plant which remains untransformed by at least one phenotype imparted to the recombinant DNA as described above. Sorted from cells. The phenotype can be conferred by inserting at least one marker gene into recombinant DNA. Alternatively, the marker gene originally possessed by the replicable expression vector can be used. Examples of marker genes include drug resistance genes such as neomycin resistance and genes encoding dihydrofolate reductase (hereinafter referred to as “DHFR”). In this regard, DHF
When using the R gene as a marker gene, DHFR
Since there are various types of DHFR, the host to be used must be selected depending on the type of DHFR encoded by the marker gene used.

For example, as a marker gene, wild type DH is used.
When a gene encoding FR is used, the host is D
It is preferable to use an HFR-deficient strain. Since the DHFR-deficient strain requires hypoxanthine, glycine and thymidine, it cannot grow in a medium free of hypoxanthine, glycine and thymidine. However, when a DHFR-deficient strain is transformed with a recombinant DNA containing the DHFR gene, the strain no longer requires hypoxanthine, glycine and thymidine, and can grow in a medium free of hypoxanthine, glycine and thymidine. it can. Therefore, the transformed cells can be easily selected from the cells remaining untransformed based on the auxotrophy for hypoxanthine, glycine and thymidine as criteria.

On the other hand, when a gene encoding a mutant DHFR having a low affinity for methotrexate (MTX) (hereinafter referred to as “MTX-resistant DHFR gene”) is used as a marker gene, the host cell is a normal DHF.
It does not need to be deficient in DHFR as long as it has a gene encoding R. The reason is as follows. Since normal DHFR is inhibited by MTX, the host cell containing the gene encoding normal DHFR is M
It requires hypoxanthine, glycine and thymidine in the presence of TX. However, when the host cells are transformed with recombinant DNA containing the MTX-resistant DHFR gene, the transformed cells no longer require hypoxanthine, glycine and thymidine in the presence of MTX. Therefore, transformed cells can be selected from non-transformed cells using the auxotrophy for hypoxanthine, glycine and thymidine in the presence of MTX as a criterion. In this regard, the MTX-resistant DHFR gene is convenient to use as a marker gene because the majority of eukaryotic cells are MTX-sensitive.

[0031] Saccharomyces cerevisiae (Sacch
Yeast such as A. cerevisiae ) can also be used as a host for expressing the DNA of the present invention. For expressing the DNA of the present invention in yeast, a replicable expression vector such as plasmid YRp can be used.
7 can be used. The plasmid YRp7 is trp
Since it contains 1 gene, this trpl gene can be used as a marker gene.

Examples of promoters for expression vectors for yeast cells include 3-phosphoglycerate kinase or enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6.
-Promoters of genes for enzymes involved in glycolysis such as phosphate isomerase and glucokinase, alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, enzymes involved in nitrogen metabolism, enzymes involved in the use of maltose and lactose The promoter of the gene is mentioned. Of these, the promoters of alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, enzymes involved in nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in the utilization of maltose and lactose are It is advantageous that the transcription by the promoter can be controlled by changing the culture conditions of the host.

As the origin of replication, stop codon and other DNA sequences for controlling transcription and translation in yeast cells, known DNA sequences which are generally known and suitable for yeast cells can be used. The transformed microorganism or cell can be cultured in an ordinary nutrient medium by an ordinary known method to express the DNA encoding the peptide of the present invention to produce the peptide of the present invention. After culturing, the peptide of the present invention can be isolated from the culture of the transformant by using a commonly known method such as column chromatography. The peptide thus obtained may contain at least one sugar chain of various types and lengths. Whether or not the obtained peptide contains a sugar chain depends on the type of host cell used. In addition, the type and length of the sugar chain when the peptide contains the sugar chain also differs depending on the type of host cell used.

It is generally known that a peptide translated from the translation initiation signal ATG undergoes processing to become a mature protein when secreted from a host cell. The peptide provided by the present invention may be subjected to such processing. The site where the peptide is processed may vary depending on the host or culture conditions. For example, the peptide encoded by the DNA of the present invention is a peptide represented by the formula (I) and N
When the unprocessed immature form containing the aforementioned 18 amino acid leader sequence as a terminal amino acid sequence is produced in a transformed cell, the immature form peptide is processed and the leader sequence is deleted. It may be matured. However, as described above, the position where the immature peptide is processed varies depending on the type of host used and the culture conditions of the host, and thus the above processing does not always occur.

As described above, the peptide having the action of promoting activation of protein C by thrombin can be produced by a method using recombinant DNA technology.
Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation / fibrinolysis mechanism, and is activated by the action of thrombin. Active protein C is
It is known to inactivate active factor V and active factor VIII of blood coagulation coenzyme in vivo, and to be involved in the production of plasminogen activator having a thrombolytic action. . [Koji Suzuki, Medical History, 12th
5: 901, (1983)]. The peptide is an active protein C that promotes the activation of protein C by this thrombin and exhibits anticoagulant action and thrombolytic action.
Is produced in large quantities. Therefore, the peptide greatly contributes to anticoagulation and thrombolysis in the living body.

As described above, since the peptide has an anticoagulant action, a platelet aggregation inhibitory action and a thrombolytic action, for example, myocardial infarction, thrombosis, embolism, peripheral vascular occlusion,
Arteriosclerosis obliterans, intravascular coagulation (DIC),
It can be used for treatment and prevention of diseases such as angina, transient cerebral ischemic attack, toxemia of pregnancy. However, in order to produce a pharmaceutical composition containing the peptide, it is essential to produce the highly pure peptide with good reproducibility and in a large amount. As such a method, for example, a genetic engineering method using the DNA of the present invention can be considered. According to this method, the peptide can be obtained in a high-purity form free from other human-derived proteins.

As described above, according to the prior art, the peptides derived from tissues have been studied by the extraction method for rabbit lung, bovine lung, human placenta, and human lung.
Its general nature has been investigated. However, the structure of the substance, such as the amino acid sequence, has not been elucidated, and the substance has not yet been identified. Therefore, whether the substances reported in the above-mentioned prior art documents are single substances, whether they are of sufficient purity to produce a pharmaceutical composition, and also the description of these prior art documents. According to the method of (1), it is completely unknown whether the same substance can be repeatedly obtained.

When the peptide is used for treating the above-mentioned diseases, it can be mixed with a pharmaceutically usable carrier. That is, by mixing an amount of the peptide provided by the present invention effective for treating or preventing the above-mentioned diseases with a suitable amount of a carrier, a pharmaceutical composition suitable for effective administration to a patient,
Preferably, a pharmaceutical composition for cardiovascular disease can be prepared. The peptide of the present invention can be used as an injection or the like. When used as an injection, a thickener such as sucrose, glycerin, methylcellulose, carboxymethylcellulose, a pH adjusting agent for various inorganic salts and the like can be added as additives. Adult 1 of the bioactive substance of the present invention
The dose per administration varies depending on age, sex, body weight, symptoms, etc., but is generally about 0.1 to 200 mg, and is administered once a day or several times as needed.

The present invention will be described in more detail with reference to Examples and Examples, but the scope of the present invention is not limited to these Examples.

【Example】

Reference Example 1 (Measurement of action promoting protein C activation) The action of promoting the protein C activation of the peptide of the present invention was measured by the synthetic substrate Boc-Leu-Ser-Thr-Arg-MC.
A (Boc and MCA are the abbreviations for t-butoxycarbonyl group and 4-methylcoumaryl-7-amide, respectively) known method for protein C [Y. Ohno et al., The Journal of Biochemistry (J. Biochem.) 90 volumes, 1387.
Page (1981)]. That is, 5 μl of an aqueous solution containing protein C (final concentration 0.5 μM) and thrombin (final concentration 80 nM) contained 5 μl of an aqueous solution containing the peptide obtained in the present invention (0 to 0.01 A).
₂₈₀ / ml), and NaCl, CaCl ₂ , serum albumin and Tris-HCl buffer (pH 7.4) were added to the final concentrations of 0.15 M, 2.5 mM and 1 mg / ml, respectively.
And 20 mM, and a total volume of 30 μl.

The resulting mixture was reacted at 37 ° C. for 15 minutes to activate protein C, 10 μl of 2 μM antithrombin III and 10 μl of an aqueous solution containing 10 units / ml heparin were added, and the mixture was incubated at 37 ° C. for 15 minutes. The reaction was stopped by warming. The obtained reaction mixture was added to the above-mentioned synthetic substrate Boc-Leu-Ser-Thr-Arg-M.
CA [Protein Research Foundation Peptide Study Group (Pe
ptide Institute) (Japan)] 200
20 mM Tris-HCl buffer (pH 7.4) containing μM 2
After adding 50 μl and reacting at 37 ° C. for 10 minutes, 20
% 0.5% acetic acid was added to stop the reaction, and the released concentration of AMC (7-amino-7-methyl-coumarin) was measured with an excitation wavelength of 380 nm and an emission wavelength of 440 nm using a fluorescence spectrophotometer RF-540 type (Shimadzu Corporation Manufactured by Japan). The amount of liberated AMC was determined by comparing the obtained fluorescence intensity with that of AMC of known concentration. The value is represented by the amount of AMC produced per minute. The value obtained by subtracting the AMC amount when the peptide-free aqueous solution obtained in the present invention was added from this AMC amount indicates the strength of promoting protein C activation by thrombin in the sample.

Here, protein C is derived from human plasma by the method of Suzuki et al. [Suzuki et al., The Journal of Biological Chemistry (J. Bio).
l. Chem. ), 258, 1914, (1983)
Etc.)]. In addition, human thrombin can be prepared by the method of Lundblad et al. [Lundblad et al., Biochemical and Biophysical Research Communication (Bi
ochem. Biophys. Res. Commun. )
66, p. 482, (1975)].

Reference Example 2 (1): (Obtaining Human Lung cDNA Library) A bacteriophage λgt ₁₁ cDNA library prepared from human lung poly (A) ⁺ RNA was prepared by Clontech Laboratories, USA.
ies, Inc. , 922 Industrial, A
ve. Palo Alto, CA 94303) (catalog number HL1004).

(2): (Protein C by thrombin
Purification of Glycopeptide Having Action of Promoting Activation Glycopeptide having action of promoting protein C activation was obtained by extracting from human lung as follows. About 1 g of human lung specimen provided by public hospital
After being cut into pieces each having a size of about cm cm, 1 mM DFP (Diisopropyl fluo) was added to the obtained tissue pieces.
Rhophosphate) -cooled to 4 ° C 500
After adding ml of physiological saline, the mixture was homogenized at 4 ° C. for 5 minutes using Ace Homogenizer AM-1 type (manufactured by Nippon Seiki Co., Ltd.) as a Waring blender. After homogenization, the mixture was cooled in ice for 5 minutes.

The mixture was then further homogenized for 5 minutes at 4 ° C. and cooled in ice for 5 minutes. The above homogenization and cooling operations were repeated three more times. The homogenate obtained is centrifuged at 12,000 g for 30 minutes at 4 ° C. to separate the supernatant and pellets and the pellets are collected. 0.5% (v / v) Triton X-100, 0.25
M sucrose, 1 mM benzamidine hydrochloride, 0.5 mM Ca
0.02 M Tris-HCl buffer containing Cl ₂ (pH 7.
5) The cells were suspended in 100 ml and homogenized 5 times at 4 ° C. for 5 minutes using a Waring blender to obtain a cell extract.

35,000 g of the obtained extract, 10 ° C.
The supernatant was collected by centrifugation at 60 minutes. NL Esmon et al. [The Journal of Biological Chemistry (J. Bio
l. Chem. ), 257, 859 (1982)
, DIP-thrombin [diisopropylphosphorothrombin (diisopro
pylphosphoro-thrombin)],
P. Cuatrecasas
Method [The Journal of Biological Chemistry (J. Biol. Chem.), 245, 3
DIP-thrombin-agarose by ligation to bromocyanated agarose according to pp. 059 (1970)].

Next, DIP-thrombin-agarose was packed in a column having a size of 2.5 cmφ × 10 cm, and D
An IP-thrombin-agarose column was prepared, and 0.1 M NaCl, 0.5 mM CaCl ₂ , 1 mM was prepared at room temperature.
Benzamidine hydrochloride, 0.5% (v / v) Lubrol
The column was equilibrated with 0.02 M Tris-hydrochloric acid buffer solution (pH 7.5) containing PX (manufactured by Hanai Kagaku Yakuhin). Then, the above extraction supernatant was applied to the column. Column is 0.3M NaCl, 0.5mM CaCl ₂ , 1mM
Benzamidine hydrochloride, 0.5% (v / v) Lubrol
0.02 M Tris-HCl buffer containing PX (pH 7.
After washing with 5), 1M NaCl, 0.1 mM ED
TA, 1 mM benzamidine hydrochloride 0.5% (v / v) L
Elution was performed with 0.02 M Tris-HCl buffer (pH 7.5) containing ubrol PX, and 2.0 ml fractions were collected. For each fraction obtained by elution, the ability of thrombin to accelerate protein C activation was measured by the method described above. At the same time, the absorbance (A ₂₈₀ ) of each fraction at a wavelength of 280 nm was measured using a spectrophotometer UV-240 manufactured by Shimadzu (Japan).

Fractions capable of activating protein C were collected and washed with 0.1 M NaCl, 0.5 mM CaCl ₂ , 0.
0.02M with 05% (v / v) Lubrol PX
It was dialyzed against Tris-HCl buffer (pH 7.5). The resulting dialysate was subjected to the second DIP-thrombin-agarose column chromatography. That is, dialysate 1.5
It is applied to a DIP-thrombin-agarose column having a size of cmφ × 10 cm, 0.4 M NaCl, 0.5 mM
After washing with 0.02 M Tris-HCl buffer (pH 7.5) containing CaCl ₂ and 0.1% (v / v) Lubrol PX, 0.4 M NaCl and 0.1 mM EDT were added.
A, 0.1% (v / v) Lubrol PX containing 0.
Wash with 02 M Tris-HCl buffer (pH 7.5), then 1 M NaCl, 0.5 mM EDTA, 0.1%
Elution was performed with 0.02 M Tris-HCl buffer (pH 7.5) containing (v / v) Lubrol PX.

Fractions capable of activating protein C were collected and further added with 0.1 M NaCl, 0.05% (v / v)
It was dialyzed against 0.02 M Tris-HCl buffer (pH 7.5) containing Lubrol PX. The obtained dialysate was subjected to the third DIP-thrombin-agarose column chromatography. The column size, washing conditions and elution conditions were exactly the same as the conditions of the second DIP-thrombin-agarose column chromatography. Fractions obtained by elution were collected in 2 ml portions. The fraction capable of activating protein C was collected and
M NaCl, 0.05% (v / v) Lubrol P
After dialysis with 0.02 M Tris-hydrochloric acid buffer solution (pH 7.5) containing X, it was subjected to the fourth DIP-thrombin-agarose column chromatography with a size of 0.9 cmφ × 8 cm. 0.35M NaCl, 0.5mM C
After washing with 0.02 M Tris-HCl buffer (pH 7.5) containing aCl ₂ and 0.1% (v / v) Lubrol PX, 1 M NaCl, 0.5 mM EDTA, 0.1%
Elution was carried out with 0.02 M Tris-HCl buffer (pH 7.5) containing (v / v) Lubrol PX. Fractions obtained by elution were collected in 1.9 ml increments.

FIG. 1 shows the elution pattern of this fourth DIP-thrombin-agarose column chromatography.
Shown in. Fraction numbers 48 to 56 were collected. From the absorbance of the fractions purified in this way, the molecular extinction coefficient of the obtained purified product does not become the molecular extinction coefficient of general protein 10.0 (E ^1% _{1 cm}
280 nm = 10.0) and the amount of this purified product was calculated based on this, and it was about 500 μg. The obtained purified fraction was added to a polyacrylamide gel with a concentration of 5 to 1
SDS-polyacrylamide gel electrophoresis using a 0% gradient was performed at a voltage of 50 V for 2 hours, and the band was observed by silver staining, and only a single band was confirmed.

In addition, about 10 μg of this purified protein was added to 20
50 mM Tris-HCl buffer containing 0 mM NaCl (p
ConA equilibrated with the same buffer after dialysis against H7.5)
Sepharose (Pharmacia, Catalog No. 17-
0440) column (resin amount about 1 ml) and washed thoroughly with the same buffer solution, this protein was adsorbed on ConA Sepharose and was not eluted in the wash solution.
Then 0.5 M of methyl-α-D-mannopyranoside (Methyl-α-D-mannnopyranos) was used.
ide) (manufactured by Sigma, USA, catalog number M-68)
82) containing the same buffer solution as above,
This protein eluted. Therefore, it was found that this protein is a so-called glycopeptide containing sugar.

(3): (Amino acid sequence analysis of glycopeptide that promotes protein C activation of thrombin) The amino acid sequence of this glycopeptide was analyzed as follows. 0.1% (v / v) of purified glycopeptide
16 with sodium lauryl sulfate (SDS) aqueous solution at room temperature
It is dialyzed for a time to prepare a sample for amino acid sequence analysis. Using an amino acid sequencing analyzer (Model 470A) manufactured by Applied Biosystems (USA),
Method of RM Hewick et al. [The Journal of Biological Chemistry (J. Biol. Chem.) 256, 7990]
Page, (1981)], and Edman analysis was sequentially performed from the N-terminal side.

Free phenylthiohydantoin
Amino acids were analyzed using a device for high performance liquid chromatography (SP8100) manufactured by Spectrophysics (USA) and a Solvax ODS column manufactured by DuPont (USA) to determine the amino acid sequence. As a result, part of the amino acid sequence was clarified, and it was found that the eleventh amino acid sequence from the N-terminus had the following amino acid sequence. Ala-Pro-Ala-Glu-Pro-Gln-P
ro-Gly-Gly-Ser-Gln

(4): (Preparation of DNA probe encoding N-terminal amino acid sequence) A DNA probe encoding the N-terminal amino acid sequence of a glycopeptide that promotes protein C activation by thrombin is prepared from the above-mentioned N-terminal amino acid sequence. , Nucleic Acid Res., Vol. 9, R4, taking into account the frequency of use of the bases of the base sequence encoding amino acids in human-derived genes.
3 (1981)], 5'CTGGGG AGCC as a nucleotide sequence encoding an amino acid sequence from the N-terminus.
G CCGGG CTGGG GCTCG GCGGG
The 33 mer of the GGC 3'is also used by Otsuka et al. [E. Ohtsuka, et.
l. ), The Journal of Biological Chemistry (J. Biol. Chem.) Volume 260, 26.
05, 1985], deoxyinosine (indicated by "1") is used instead of thymidylate as a nucleotide sequence encoding an amino acid sequence from the N-terminus. 2) 5'GCICC IGCIG AGCCI CAACC IGG3 '(3) 5'GCICC IGCIG AGCCI CACCC IGG3' (4) 5'GCICC IGCIG AACCI CAACC IGG3 'Four kinds of 23mers made by US Applied Biosystems p3e ps eds p.
Synthesized with a model 80A DNA synthesizer and purified according to the manufacturer's manual. F. Maniatis EF, et al, Molecular Clon.
ing), page 122, 1982) and labeled with T ₄ DNA kinase and γ- ³² P-ATP.

(5): (Protein C by thrombin
Glycopeptide Antibody Having an Action of Promoting Activation) The rabbit antibody against the glycopeptide having an action of promoting the protein C activation of thrombin has the action of promoting the protein C activation by thrombin purified as described above. Using a glycopeptide from a human lung,
Book [L. Hudson et al.
l. ), Practical Immunology (Practi)
cal Immunology, page 9 (1976)
Year), Blackwell Scientific Publications (Blackwell Scientific)
c Publications)].

This antibody is protein C by thrombin
It was confirmed that it reacts with a human lung-derived glycopeptide having an action of promoting activation as follows. That is, about 10 ng of the purified protein obtained by the method described in Reference Example 2- (2) is spotted on a nitrocellulose filter. After air-drying well, this antibody was reacted with a protein on a nitrocellulose filter as a primary antibody, and then biotinylated anti-rabbit IgG prepared by goat (Zymet Laboratories, USA, Catalog No. 62-1) was used.
840) as a secondary antibody, and then avidin / biotinylated horseradish-derived peroxidase (Amersham Japan, Japan, catalog number RPN.105).
When the color was developed by the method of acting 1), a blackish brown spot was given.

(6): (Collecting and culturing human umbilical cord endothelial cells) Human umbilical cord endothelial cells were dispase II (purified joint liquor,
The method of Mano et al. [Y.
no, et al. ), Experin Encia (Exp
erientia), Vol. 39, p. 1144, (19
1983)], and was collected and cultured from a vein obtained from a fresh human umbilical cord provided by a private hospital.

Reference Example 3 (Acquisition of Recombinant DNA) (1): (Preparation of poly (A) ⁺ RNA) From human endothelial cells, the method of Chargin et al. [JM]
Chirgwin, JM et a
l. ), Biochemistry (Biochemistr)
y), Vol. 18, p. 5294 (1979)], poly (A) ⁺ RNA was prepared.

(2): (Screening from human lung cDNA library) A cDNA library (manufactured by Clontech, USA) in which cDNA prepared from human lung poly (A) ⁺ RNA was incorporated into bacteriophage λgt ₁₁ was used. According to the manual, the one infected with E. coli Y1090 (manufactured by Clontech, USA) was transplanted onto an LB medium plate so that about 100,000 plaques per 15 cm diameter plate were obtained. After culturing at 42 ° C for 3.5 hours, 10 mM IPTG (isopropy) was previously prepared.
l-β-D-thiogalactopyranosi
Place a nitrocellulose filter (BA85 membrane filter, Schleicher & Shell GmbH, Germany) soaked in de) and dried on the plate. 3
Incubate for 3.5 hours at 7 ° C
It is induced by TG and transferred to a nitrocellulose filter.

According to the manual, this nitrocellulose filter has an action of promoting the protein C activation of thrombin prepared in rabbits, Reference Example 2- (5).
The antibody against the glycopeptide obtained in 1. was reacted as a primary antibody, and then biotinylated anti-rabbit IgG (manufactured by Zymed Laboratories, Inc., US Catalog No. 62-1840) prepared with goat was reacted as a secondary antibody, and then avidin. A positive clone was isolated by color development with a biotinylated horseradish-derived peroxidase (Amersham Japan, Japan, catalog number RPN.1051). Recombinant c possessed by this positive clone
The cDNA fragment contained in DNA / λgt ₁₁ was designated as TM13.

(3): (Hybridization with DNA probe encoding N-terminal amino acid sequence) The DNA fragment TMI3 obtained in Reference Example 3- (2) is the N-terminal amino acid prepared in Reference Example 2- (4). Whether or not it hybridizes with a DNA probe encoding a sequence is described in an experimental document [Silhavy et al., Experiments with Gene Fusions (Experim).
ents With Gene Fusions), 1
Page 91, (1984) Cold Spring Harbor Laboratory (Cold Spring Harbo)
r Laboratory)]. DN
It was found that the A fragment TM13 did not hybridize with the DNA probe encoding any N-terminal amino acid sequence.

(4): (Nucleotide sequence of TM13) DNA contained in the clone obtained in Reference Example 3- (2)
The nucleotide sequence of the fragment TM13 was analyzed by the method of Sanger et al. (Sanger, F, et al.), Proceeding of National Academy of Sciences.
Science USA (Proc. Natl. Ac
ad. Sci. USA), 74, 5463 (197).
7 years). The results are shown in FIGS. 2 and 3.

[0062] (5) :( DNA fragment TM13 human lung cDNA library screening as a probe) restriction DNA fragment TM13 enzymes Kpn I and Pvu II
It was digested with to obtain a DNA fragment of about 440 base pairs, which was labeled with ³² P by the nick translation method. This DN
Plaque hybridization was carried out from a human lung cDNA library using the A fragment as a probe to screen positive clones. In other words, separated by polyacrylamide gel electrophoresis by digestion of DNA clones TM13 with Kpn I and Pvu II in a conventional manner, extracted to obtain a purified fragment of about 500ng of the purified approximately 440 bp. This DNA was labeled with nick translation kit (catalog number N.5000) manufactured by Amersham Japan (Japan) and with α- ³² P-dCTP according to the attached user manual.

This ³² P-labeled DNA fragment is used as a probe in an experiment manual [Maniatis et al.,
Molecular Clone (Molecular C
Longing, 320 pages, 1982, Cold Spring Harbor Laboratory (Cold Spr)
ing Harbor Laboratory)], and plaque hybridization of the human lung cDNA library was performed. A positive clone was isolated, and the recombinant contained in the clone was analyzed with various restriction enzymes. It was found that a likely DNA fragment of about 2,400 bp was incorporated. This DNA fragment was called TM137.

(6): (Nucleotide sequence of DNA fragment TM137) The nucleotide sequence of the DNA fragment TM137 obtained in (5) above was determined in the same manner as in Reference Example 3- (4). The results are shown in FIGS. From this result, DNA fragment TM
It was found that 137 does not contain the nucleotide sequence encoding the N-terminal amino acid sequence described in Reference Example 2- (3).

(7): (Primer extension) Among the base sequences of the DNA fragment obtained in Reference Example 3- (4), three kinds of synthetic DNAs are referred to based on the N-terminal side sequence of the DNA fragment TM13. It was prepared in the same manner as described in Example 2- (4) and named HTM131, HTM132, and HTM133. In designing the synthetic DNA, the base sequence on the side that hybridizes with mRNA prepared from human umbilical cord endothelial cells was used. The base sequences of the synthetic DNAs are as follows, and the DNs to which the synthetic DNAs correspond
The position of the A fragment TM13 is shown in FIG. 2, and the position of TM137 is shown in FIG.

HTM131: 5′GACGCAGAGGTAGCTAGTTT 3 ′ (20mer) HTM132: 5′AACATCTGGCACCTG 3 ′ (15mer) HTM133: 5′GACAGGCAGTCTGGTTGCAA 3 ′ (20mer)

Next, poly prepared from human umbilical cord endothelial cells obtained by the method described in Reference Example 3- (1) The HTM133 as a primer (A) ⁺ using a RNA, so-called primer extension (Primer E
xtension) method to perform DNA fragment TM13
A further 5'upstream part of 7 was synthesized. That is, about 1μ
About 27 ng / μ in 5 μl of g / μl of poly (A) ⁺ RNA
20 μl of HTM133 solution (1) was added, the mixture was heated at 65 ° C. for 20 minutes, and then cooled to room temperature over about 1 hour. After that, the cDNA synthesis system (Amersham Japan,
CDNA was synthesized according to its manual using catalog number RPN1256), Japan. However, cDNA
This was done using HTM133 instead of the oligo (dT) primer included in the synthesis system.

The synthesized cDNA was prepared in accordance with the experimental manual [Maniatis et al., Molecular Cloning, 241.
Page, 1982, Cold Spring Harbor Laboratory
Laboratory)], pBR322 (ATC) with C-tails at both ends and G-tails at both ends
C37017) and heated at 65 ° C for 5 minutes, then 57
After heating at ℃ for 2 hours, slowly returning to room temperature E. coli K12MC1061 (obtained from Beckman City of Hope Medical Institute, USA)
Was transformed. Specifically, E. coli K12MC1061
A colony of the strain was cultured in LB medium until the absorbance at 550 nm was 0.3. 50 ml of the culture
And collect 25 ml of 10 mM RbCl in 10 mM
3- (N-morpholino) propane-sulfonic acid (MO
PS) (pH 7.0) solution, then 50 mM
CaCl ₂ , 10 mM RbCl in 25 ml of 0.
It was resuspended in 1M MOPS (pH 6.5).

The obtained suspension was ice-cooled for 30 minutes, centrifuged, and the supernatant was removed, and 30 μl of DMSO and 50 mM were added.
It was suspended in a mixed solution of 2.0 ml of 0.1 M MOPS (pH 6.5) containing CaCl ₂ and 10 mM RbCl. 200 μl of the suspension was dispensed, and 10 μl of the above-mentioned plasmid DNA solution was added to each. The mixture was ice-cooled for 30 minutes, heat-shocked at 44 ° C for 60 seconds, and immediately heated to 37 ° C for 5 m.
1 LB medium was added. After culturing this solution at 37 ° C. for 1 hour, each solution was centrifuged and the supernatant was removed to obtain a cell pellet. LB medium was added to the cell pellet, stirred, and then made into a suspension. The suspension was spread on an LB agar plate containing 5 μg / ml of tetracycline and cultured at 37 ° C. overnight.

From the cDNA bank thus obtained, colony hybridization was carried out using HTM131 and HTM132 each having a 5 ′ end labeled with ³² P as a probe according to the method described in Reference Example 2- (4). It carried out by the method similar to (3). Colony
By screening about 70,000 transformants by hybridization, HTM131 and HTM132
6 clones were obtained which reacted with both probes. From these 6 clones, the experiment manual [Maniatis (M
aniatis) et al., Molecular cloning (M
Molecular Cloning), 366, 19
1982, Cold Spring Harbor Laboratory
plasmid DNA (this is referred to as "pTMP5") according to the following procedure, digested with various restriction enzymes, and analyzed by electrophoresis. The plasmid DNAs obtained from 6 clones were all the same, About 9
It was found to consist of a DNA fragment with a size of 00 bp and a vector. This DNA fragment was named TMP5.

(8): (Hybridization of DNA fragment TMP5 with a DNA probe encoding the N-terminal amino acid sequence) In the same manner as in Reference Example 3- (3), the DNA fragment TMP5 contains the N-terminal amino acid sequence. It was investigated whether or not it hybridized with a DNA probe encoding It was found that the DNA fragment TMP5 did not hybridize with any N-terminal DNA probe, that is, did not encode the N-terminal amino acid sequence portion.

(9): (Base sequence of DNA fragment TMP5) The base sequence of the DNA fragment TMP5 was determined in the same manner as in Reference Example 3- (4). The results are shown in FIGS.

(10): (Second primer extension) HTM134, HTM based on the nucleotide sequence of the DNA fragment TMP5 in the same manner as in Reference Example 3- (7).
135, HTM136 3 synthetic DNA of 20mer
To create. The positions in the DNA fragment TMP5 corresponding to these synthetic DNAs are shown in FIG. Reference Example 3- (7)
The primer extension was performed using HTM136 as a primer and HTM134 and HTM135 as probes in the same manner as described in 1. About 50,0
HTM134, and HTM1 from 00 transformants
One type of transformant that hybridized with 35 was obtained. The DNA fragment contained in the recombinant carried by this transformant was designated as TMP26.

(11): (Hybridization of DNA fragment TMP26 with a DNA probe encoding an N-terminal amino acid sequence) In the same manner as in Reference Example 3- (3), the DNA fragment TMP26 contains an N-terminal amino acid. It was examined whether or not it hybridizes with the DNA probe encoding the sequence. As a result, the DNA fragment TMP26 was a DNA encoding the 33-mer N-terminal amino acid sequence synthesized in Reference Example 2- (4).
Hybridized with a probe and a mixed probe of four 25-mer probes. That is, DNA fragment TM
It was found that P26 encodes the N-terminal amino acid sequence portion.

(12): (Base sequence of DNA fragment TMP26) The base sequence of the DNA fragment TMP26 was determined in the same manner as in Reference Example 3- (4). DNA fragment TMP
The nucleotide sequence of about 540 bases from the carboxyl end of 26 is shown in FIG.

(13): (DNA fragment TMP26, TM
Joining of P5 and TMP137) Four DNA fragments (TM13, TM137, TMP) obtained in Reference Example 3- (1) to (12) and having their nucleotide sequences determined
5 and TMP26) and their corresponding nucleotide sequences and a simple restriction map are shown in FIG. As shown in FIG. 11, when an open reading frame was assembled from the first ATG upstream from the nucleotide sequence encoding the N-terminal amino acid sequence contained in DNA fragment TMP26, DNA fragment T was formed.
It was found that it consists of 1,725 bp that passes through MP26 and TMP5 and continues to the middle of TM137. This each DN
DNA fragments TMP26, T to obtain a DNA fragment encoding an open reading frame spanning the A fragment.
MP5 and TM137 were spliced according to a conventional method as follows.

(13-1) (DNA fragments TM137 and T
MP5 splicing) First, D inserted in the Eco RI site of λgt ₁₁
The NA fragment TM137 was isolated and used in plasmid pUC18
(Pharmacia, Sweden, Catalog No. 27
-4949-01) was inserted into the Eco RI site to obtain plasmid pUC18TM137. Then plasmid p
UC18TM137 is a restriction enzyme Hinc II, Eco RI
Digested with 4% (v / v) polyacrylamide gel electrophoresis and separated by electrophoresis extraction device (Japan, Art Co.,
MAX-YIELD ^R ) and a D of about 2,300 bp
The NA fragment was recovered and purified by ethanol precipitation.

On the other hand, the TMP obtained in Reference Example 3- (7)
The plasmid p in which 5 was integrated into the plasmid pBR322
After TMP5 was completely digested with Dde I, the cut ends were
E. E. coli DNA polymerase (Klenow Po
a blunt end with about 100 bp DN
The A fragment was recovered, and this DNA fragment was Sma of pUC18.
It was inserted into the I site to obtain the plasmid pUC18TMP5. Next, this plasmid pUC18TMP5 was completely digested with restriction enzymes Bam HI and Hinc II to obtain about 600
to obtain a bp of Bam HI- Hinc II fragment.

As described above, the plasmid pUC18T
About 2,300 bp DNA fragment prepared from M137 and about 60 prepared from plasmid pUC18TMP5
Bam HI of 0 bp DNA fragment plasmid pUC18
And inserted into a vector prepared by digestion with Eco RI to obtain plasmid pUC18TMJ1. This step is shown in FIG. (13-2) (Splicing of DNA fragment TMJ1 and TMP26) Plasmid pUC18TMJ1 was digested with restriction enzymes Dde I, K
Complete digestion with pn I and Bam HI recovered fragments of approximately 950 bp and approximately 1,500 bp.

On the other hand, the DNA fragment TMP26 was inserted into the restriction enzyme Pst I site of the plasmid pUC13 (Pharmacia, Sweden, Catalog No. 27-4954-01) to obtain the plasmid pUC13TMP26.
After completely digesting this with Bbe I, the cleaved end was digested with T ₄ DN.
Blunt ends using A polymerase and then the restriction enzyme
It was completely digested with BglII to obtain a DNA fragment of about 170 bp. Separately, the plasmid pUC13TMP26 was added to B
about 280 bp DN by complete digestion with gl II and Dde I
A fragment was obtained. Next, about 170bp and about 280b
DNA fragments of p and about 950 bp were joined by using T ₄ DNA ligase, digested with restriction enzyme Kpn I, and then 5
It was purified and isolated by subjecting it to 1.3% low melting point agarose gel electrophoresis at 4 ° C. for 2 hours at a voltage of 0 V to obtain a DNA fragment of about 1,400 bp.

Separately, plasmid pUC18 was added to Sph
After complete digestion with I. E. After blunting the cleaved ends with E. coli DNA polymerase, it was completely digested with Bam HI to prepare a vector. The above-mentioned DNA fragments of about 1,400 bp and about 1,500 bp were transferred to this vector by T
₄ Inserted using DNA ligase to generate plasmid pUC
18TMJ2 was obtained. This step is shown in FIG.

Reference Example 4 (Screening of target gene from human chromosome) Screening of target gene from human chromosome library was carried out as follows. λ phage vector E
The human chromosome library contained in MBL-3 is available from Clontech Laboratories.
es, Inc. 922 Industrial Ave.
Palo Alto, CA 94303) (catalog number HL1006). Screening was performed in the same manner as in Reference Example 3- (5) using the DNA fragment TM13 obtained in Reference Example 3- (2) from this library as a probe. As a result, a chromosome containing about 20,000 bp insert One clone was obtained. This chromosome clone was completely digested with the restriction enzyme Bam HI to obtain 1.0%
Agarose gel electrophoresis was performed and the experiment manual [Maniatis et al., Molecular Cloning, page 382,
1982, Cold Spring Harbor Lab (Cold Spring Harbor Lab)
Southern blot hybridization was carried out with the same purobs according to

[0083] As a result, to obtain a band of strong positive for the DNA fragment of about 4,000bp isolated fragments thereof in accordance with a conventional method, it was subcloned into the Bam HI site of the plasmid pUC18. When the nucleotide sequence of this DNA of about 4,000 bp was determined, it was found to be completely identical with the nucleotide sequence of the DNA fragment inserted in the plasmid pUC18TMJ2 prepared in Reference Example 3- (13-2).

Example 1 (Preparation of plasmid pSV2TMJ2) Construction of plasmid pSV2TMJ2 Plasmid pSV2-dhfr (ATCC37146)
Was completely digested with HindIII and BglII to obtain a vector having an SV40 early transcription promoter and an SV40 transcription terminator. Next, Reference Example 2- (13-2)
The plasmid pUC18TMJ2 prepared in step 2 was added to Hind II
After partial digestion with I, complete digestion with Bam HI to about 2,9
A 00 bp DNA fragment was isolated. This DNA fragment is
It was called MJ2. The 2,900 bp DNA fragment and the vector prepared as described above were ligated together using T4 DNA ligase to obtain plasmid pSV2TMJ2.
The process of constructing the plasmid pSV2TMJ2 is shown in FIG. The obtained plasmid pSV2TMJ2 was deposited with the American Type Culture Collection (ATCC) under the deposit number 67 according to the provisions of the Budapest Treaty.
Deposited as No. 283.

Example 2 (of COS-1 cells with plasmid pSV2TMJ2)
Transformation) COS-1 cells (ATCC CRL165
0) in an incubator at 10% (v / v) fetal bovine blood
The best of Dulbecco with the addition of Kiyoshi (hereinafter abbreviated as "FCS")
Small essential medium (hereinafter abbreviated as "MEM") [US, Flow
Laboratories (Flow Laboratories)
Manufactured by Catalog No. 10-331) at 37 ° C for 5
% Until the logarithmic growth phase in the carbon dioxide incubator
Culture and add 0.1% trypsin and 0.02% EDTA.
Peel off the cells that have adhered and grown in the incubator from the incubator.
Hanks Balanced Salt Solution [Flow Laboratory, USA
(Flow Laboratories), Catalo
No. 17-101-22] about 1 x 10 ⁷Pieces / ml
Suspended to a concentration.

Plasmid pSV2T obtained in Example 1
MJ2 was suspended in 1 mM Tris-HCl buffer (pH 8.0) to a concentration of about 2 μg / μl. 5 μl of the resulting plasmid suspension containing about 10 μg of plasmid pSV2TMJ2 was placed in a 1.5 ml volume Eppendorf tube and then COS-1 obtained as described above in this tube.
200 μl of cell suspension of cells was added and left at 0 ° C. for 10 minutes. The suspension in the test tube was added to US D. E. P. SYST
The cells were transferred to a cuvette of a cell fusion device FPH1001 manufactured by EM, and an electric pulse was applied twice at a condition of 1.2 kV and 40 μsec. After that, the suspension was transferred again to the original Eppendorf type test tube and left at 0 ° C. for 5 minutes, and then 10% (v / v)
10 ml of Dulbecco's MEM with FCS was transferred to a 10 cm diameter tissue culture plate as follows. That is, Dulbecco's MEM containing a small amount of 10% (v / v) FCS was added to the suspension and the mixture was transferred to a tissue culture plate. The test tube was then washed several times with the rest of Dulbecco's MEM and the wash was added to the same plate. After that, the plate was placed at 37 ° C. in the presence of 5% CO ₂ for 24 hours.
Incubated for hours.

(Confirmation of the action of promoting protein C activation by thrombin) After completion of the culture, the medium of the plate was subjected to F
The medium was replaced with Dulbecco's MEM without CS and the cells were cultured for 48 hours. 5 μl of the culture supernatant was sampled, and the stimulatory effect of protein C activation was measured by the method described in Reference Example 1 using this as a sample. In addition, cells for one tissue culture plate having a diameter of 10 cm are transferred to a US coaster (Coaste).
r) Scraper (Cell Scrape)
r) (catalog number 3010), and collected by centrifugation at 800 rpm for 10 minutes. Using this pellet as a sample, the effect of promoting the activation of protein C was measured by the method described in Reference Example 1. As a control, plasmid pSV2-d
Culture supernatants and cell pellets of hfr-transformed COS-1 cells were used as samples.

The promoting action of protein C activation by thrombin of the peptide produced by the cells transformed with the plasmid pSV2TMJ2 was measured. as a result,
The cell pellet sample showed a strong protein C activation promoting action, and the amount of protein C produced was about 300 ng. On the other hand, the plasmid pS used as a control
This activity was not detected in cells transformed with V2-dhfr.

Example 3 (Transformation of CHO cells with plasmid pSV2TMJ2 and measurement of stimulatory effect of protein C activation by thrombin of peptides produced by the transformed cells) About 4 μg
Plasmid pSV-2-neo (ATCC 3715
0) and about 20 μg of the plasmid pSV2TMJ2 prepared in Example 1 were mixed and ethanol-precipitated. After air-drying the precipitate, it was dissolved in 450 μl of TE (pH 7.9, 1 mM Tris-HCl buffer, 0.1 mM EDTA), and
00 μl of 2 × HBS (50 mM HEPES, 280
mM NaCl, 1.5 mM Na2HPO ₄ , pH
7.12) was added. Then 50 μl of 2.5 M Ca
Cl ₂ was dropped and the mixture was left at room temperature for 10 minutes. On the other hand, 10%
Ham's F-1 containing (v / v) FCS and 1 v / v% penicillin-streptomycin (Catalog No. 16-700-49, manufactured by Flow Laboratory, USA).
CHO-KI strain (ATCCCCLD 61) in which about 5 × 10 ² cells per plate were seeded on a tissue culture plate having a diameter of 6 cm using 2 medium (Catalog No. 10-421-20, manufactured by Flow Laboratory, USA). Was cultured overnight, the medium was replaced with a fresh medium, and the cells were further cultured for 3 hours.

A plasmid DNA solution in which the above-mentioned CaCl ₂ was dropped was layered on this CHO-KI and cultured at 37 ° C. for about 8 hours. The cells were washed twice with 5 ml of PBS (-) (Catalog No. 28-103-05, manufactured by Flow Laboratory, USA), further washed with 5 ml of the above-mentioned medium, and then fresh medium was added for about 16 hours. It was further cultured. Cells attached to the plate were treated with 0.25% trypsin, 0.0
It was peeled off using a 2% EDTA solution, spread on four tissue culture plates with a diameter of 10 cm, and cultured. After 24 hours, the medium was replaced with a selective medium. The composition of the selective medium was Geneticin C-418 so that the medium was 400 μg / ml.
(Gibco, USA, Catalog No. 860-181
1) is added. The transformed cells were cloned by culturing for about 2 weeks while changing the medium every 3 to 4 days. The cell clones obtained by this operation were grown on a tissue culture plate with a diameter of 10 cm until they became confluent. FCS of the medium on the way
The culture was switched to a medium whose concentration was reduced from 10% to 1% and cultured.

The promoting action of protein C activation by the peptide produced by cells transformed with the plasmid pSV2TMJ2 by thrombin was measured. as a result,
The cell pellet sample exhibited a strong protein C activation promoting action. On the other hand, this activity was not detected in the cells transformed with only the plasmid pSV2-neo used as a control and the culture supernatant thereof.

Example 4 (Transformation of C ₁₂₇ I cells with plasmid pSV2TMJ2 and measurement of promoting action of protein C activation by thrombin of peptide produced by transformed cells) The plasmid pSV2TMJ2 prepared in Example 1 was used as Hind.
After complete digestion with III, the cut ends were made blunt using DNA polymerase and treated with T ₄ DNA ligase to obtain plasmid pSV2TMJ2-1 lacking the HindIII site of plasmid pSV2TMJ2. Then, this plasmid pSV2TMJ2-1 was added to PvuII.
And DN of about 4,100 bp after complete digestion with Bam HI
A fragment was obtained. This is the Hinc of the plasmid pUC18
It was inserted into a vector completely digested with II and Bam HI to obtain the plasmid pUCTMJ2-1.

On the other hand, the plasmid pBR322 (ATCC
37017) to the method of Cobas Rubias et al. [L. Covasrubias et a.
l), Gene, 13 , 25, (1981).
The plasmid pBR327 was constructed according to The resulting plasmid pBR327 was transformed with Bam HI and Hind.
A DNA fragment of about 2,960 bp obtained by digesting with III,
Plasmid pUCTMJ2-1 was added to Bam HI and Hin
The DNA fragment obtained by complete digestion with dIII was inserted to obtain the plasmid pBRTMJ2-1. This plasmid pBR
Complete digestion of TMJ2-1 with HindIII and plasmid pBPV-1 (9-1) (ATCC37111)
Was completely digested with Hind III to obtain T ₄ DNA.
Subsequent with ligase to obtain plasmid pdBPVTMJ2-1 for C ₁₂₇ cell expression. The above steps are shown in FIG.
~ Shown in FIG.

Next, according to the method described in Example 3 with pdBPVTMJ2-1, C ₁₂₇ I cells (ATCC CRL
1616) was transformed. 10% FCS and 1 (v / v)% penicillin-streptomycin (manufactured by Flow Laboratory, USA, Catalog No. 16-70)
0-49) and cultured in Dulbecco's MEM for about 3 weeks, 6 cells forming the foci were obtained.
The plate was grown to a confluence on a 0 cm tissue cell plate. Then, the medium was replaced with a medium containing no FCS and the cells were cultured. After culturing for 1 day in this medium, the pellet of the cultivated cells was scraped, and the stimulatory effect of protein C activation was measured by the method described in Reference Example 1, and strong activity was observed. On the other hand, this activity was not detected in cells transformed only with the plasmid pBPV-1 (9-1) used as a control.

Example 5 (Purification of Peptide Encoded by DNA of the Present Invention) CHO cells transformed with the plasmid pSV2TMJ2 and the plasmid pSV2-neo by the method described in Example 3 were used to prepare 25 tissue culture plates having a diameter of 10 cm. Was used for culturing. After culturing, the culture was centrifuged at 800 rpm for 10 minutes to collect the cells. In the obtained cell pellet,
0.5% (v / v) Triton X-100, 0.25M sucrose, 1 mM benzamidine hydrochloride, 0.5 mM CaCl ₂
0.02M Tris-HCl buffer (pH 7.5) containing 10
The cells were suspended in 0 ml and homogenized 5 times at 4 ° C. for 5 minutes using a Waring blender to obtain a cell extract. The obtained extract was centrifuged at 35,000 g and 10 ° C. for 60 minutes to collect the supernatant. From this supernatant, a purified product of the peptide encoded by the DNA of the present invention was obtained by the following procedure.

After the cell extract was adjusted to pH 7.5, D
Prepared by column chromatography of IP-thrombin-agarose. That is, NL Esmon et al. [The Journal of
Biological Chemistry (J. Biol. Che
m), 257, p. 859, (1982)], DIP-thrombin [diisopropylphosphorothrombin (diisopropoxylpho).
sporothrombin by the method of P. Cuatrecasas [The Journal of Biological Chemistry (J. Bi
ol. Chem, 245, 359, (1970.
(Year)] and was bound to bromocyanated agarose to prepare DIP-thrombin-agarose.

Next, DIP-thrombin-agarose was packed in a column having a size of 2.5 cmφ × 10 cm to give D
An IP-thrombin-agarose column was prepared and at room temperature, 0.1 M NaCl, 0.5 mM CaCl ₂ , 1 mM
Benzamidine hydrochloride, 0.5% (v / v) Lubrol
The column was equilibrated with 0.02 M Tris-HCl buffer (pH 7.5) containing PX (manufactured by Hanai Chemical Co., Ltd., Japan). Then, the above cell extract was applied to the column. Column is 0.3M NaCl, 0.5mM CaCl ₂ , 1mM
Benzamidine hydrochloride, 0.5% (v / v) Lubrol
0.02 M Tris-HCl buffer containing PX (pH 7.
After washing with 5), 1M NaCl, 0.1 mM ED
TA, 1 mM benzamidine hydrochloride 0.5% (v / v) L
0.02 M Tris-HCl buffer containing ubrolPX (p
Elution was performed with H7.5), and 2.0 ml fractions were collected.

For each fraction obtained by elution, the promoting action on protein C activation was measured by the method described above. At the same time, using a spectrophotometer UV-240 manufactured by Shimadzu (Japan), the wavelength of each fraction was 28.
Absorbance at 0nm the (A _{28 0)} was measured. Active fractions were collected, 0.1 M NaCl, 0.5 mM Ca
It was dialyzed against 0.02 M Tris-HCl buffer (pH 7.5) containing Cl ₂ and 0.5% (v / v) Lubrol PX (manufactured by Hanai Chemical Co., Ltd., Japan). Then, the obtained dialysate was subjected to the second DIP-thrombin-agarose column chromatography.

That is, the dialysate was passed through a DIP-thrombin-agarose column having a size of 1.5 cmφ × 10 cm, and 0.4 M NaCl, 0.5 mM CaCl ₂ ,
0.02 with 0.1% (v / v) Lubrol PX
After washing with M Tris-hydrochloric acid buffer solution (pH 7.5), the solution was further washed with 0.
4M NaCl, 0.1 mM EDTA, 0.1% (v
/ V) washed with 0.02M Tris buffer (pH 7.5) containing Lubrol PX, then 1M NaCl,
0.5 mM EDTA, 0.1% (v / v) Lubro
0.02 M Tris-HCl buffer containing PX (pH 7.
It eluted in 5). The active fraction was collected and the purified product was stored frozen at -80 ° C. The molecular extinction coefficient of this product does not match the molecular extinction coefficient of general proteins 10.0 (E ^1% _1cm・ 28
0 nm = 10.0), and the amount of the purified product was calculated based on this, and it was about 3 μg. In addition, when this purified product was subjected to SDS-polyacrylamide gel electrophoresis using a gradient of polyacrylamide concentration of 5 to 10% and the band was observed by silver staining, only a single band was confirmed.

Example 6 (Confirmation of action of promoting protein C activation by thrombin) The action of promoting the protein C activation of the peptide encoded by the purified DNA of the present invention was evaluated by the following method. That is, 0.1 M NaCl, 3.6 mM CaC
l ₂ , 0.0 containing 10 mg / ml bovine serum albumin
50 μg / ml in 2M Tris-HCl buffer (pH 7.5)
Protein C, 5 nM thrombin, and 5 nM of the purified DNA of the present invention encoded peptide.
The reaction was carried out at 7 ° C. 300 μg / ml antithrombin III (manufactured by Sigma, USA) and 5 mM ED were added to the reaction product.
The reaction was stopped by adding TA, and the amount of active protein C produced was measured by the method using the aforementioned synthetic substrate.

The results are shown in FIG. 17. The DN of the present invention is as follows.
When the peptide encoded by A was not added (B), the production of activated protein C was not observed (dotted line).
When the peptide encoded by the DNA of the present invention was added (A), the amount of activated protein C produced increased with the reaction time (solid line).

Example 7 (Confirmation of Anticoagulant Action) It was found that the peptide encoded by the DNA of the present invention inhibits the conversion of fibrinogen to fibrin by thrombin and substantially inhibits blood coagulation (Heinrich Amellung ( It was examined by measuring the blood coagulation time using a Coagulometer KC-10 manufactured by Germany. That is, 5 mM CaCl ₂ , 0.1 M
0.05M Tris-HCl buffer containing NaCl (pH
To 7.5), 3.0 μg of fibrinogen (manufactured by Sigma, USA, fraction I) was added, and 0 to 50 nM of the purified peptide of the present invention was added thereto, and then the total amount was 0.4 ml.
Was added to 10 nM thrombin to measure the coagulation time. The result is shown in FIG. It was confirmed that the blood coagulation time was extended as the amount of the purified peptide added increased compared to that of thrombin.

Example 8 The fact that the peptide encoded by the DNA of the present invention substantially inhibits the thrombin-aggregating action of thrombin was evaluated using a platelet aggregometer manufactured by SIENCO (USA). That is, 300,000 cells / μl of platelet liquid (Platelet Rich Plasma, P.
R. P. ) One unit of thrombin (about 0.4
Platelet agglutination did not occur when μg) was added, but platelet aggregation did not occur if the peptide encoded by the purified DNA of the present invention in an equimolar amount or more to the added thrombin was added before the addition of thrombin.

[0104]

[Application Examples] Application examples of the peptide of the present invention will be described below with reference to application examples, but the present invention is not limited to these application examples. Application Example 1 Purified peptide of the present invention 10 mg Purified gelatin 20 mg Mannitol 100 mg Sodium chloride 7.8 mg Sodium phosphate 15.4 mg The above components were dissolved in distilled water for injection 2 ml, put in a sterile vial, and at -35 ° C. Pre-freeze for 2 hours, dry at -35 ° C. under a vacuum of 0.075 Torr for 35 hours, then 3
Secondary dry for 5 hours at 0 ° C and vacuum degree 0.03 Torr,
An injection vial was manufactured. The obtained composition is dissolved in 500 ml of physiological saline or glucose injection immediately before administration and used for intravenous infusion.

Application Example 2 Purified Peptide of the Present Invention 2.5 mg Albumin 5 mg Mannitol 25 mg Sodium Chloride 1.95 mg Sodium Phosphate 3.85 mg For injection by substantially the same method as Application Example 1 with the above components A vial was manufactured.

[0106]

Industrial Applicability The peptide encoded by the DNA of the present invention, which has an action of promoting activation of protein C by thrombin, has an anticoagulant action, a platelet aggregation inhibitory action, a thrombolytic action, and a circulatory system with few side effects. It is an extremely useful substance as a therapeutic drug for diseases and the like. In addition to such pharmaceutical use, the present peptide can be used as a drug for preventing thrombus formation by binding to a medical artificial material such as an artificial blood vessel, an artificial organ or a catheter.

[Sequence list]

SEQ ID NO: 1 Sequence length: 557 Sequence type: Amino acid Sequence type: Protein sequence Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln A la Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro A la Asp Cys Asp 385 390 395 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser 465 470 475 480 Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His 485 490 495 Ser Gly Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu Val Val 500 505 510 Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala Ala 515 520 525 Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val Val 530 535 540 Leu Gln His Val Arg Thr Glu Arg Thr Pro Gln Arg Leu 545 550 555 SEQ ID NO: 2 Sequence length: 1671 Sequence type: Nucleotide sequence Sequence type: DNA sequence GCA CCC GCA GAG CCG CAG CCG GGT GGC AGC CAG TGC GTC GAG CAC GAC 48 TGC TTC GCG CTC TAC CCG GGC CCC GCG ACC TTC CTC AAT GCC AGT CAG 96 ATC TGC GAC GGA CTG CGG GGC CAC CTA ATG ACA GTG CGC TCC TCG GTG 144 CGC CGG CGC CTC TGG ATC GGC CTG CAG CTG CCA CCC GGC TGC GGC GAC 240 CCC AAG CGC CTC GGG CCC CTG CGC GGC TTC CAG TGG GTT ACG GGA GAC 288 AAC AAC ACC AGC TAT AGC AGG TGG GCA CGG CTC GAC CTC AAT GGG GCT 336 CCC CTC TGC GGC CCG TTG TGC GTC GCT GTC TCC GCT GCT GAG GCC ACT 384 GTG CCC AGC GAG CCG ATC GAG GAG CAG CAG TGC GAA GTG AAG GCC 432 GAT GGC TTC CTC TGC GAG TTC CAC TTC CCA GCC ACC TGC AGG CCA CTG 480 GCT GTG GAG CCC GGC GCC GCG GCT GCC GCC GTC TCG ATC ACC TAC GGC 528 ACC CCG CTC GCG GCC GGA GCG GAC TTC CAG GCG CTG CCG GTG GGC 576 AGC TCC GCC GCG GTG GCT CCC CTC GGC TTA CAG CTA ATG TGC ACC GCG 624 CCG CCC GGA GCG GTC CAG GGG CAC TGG GCC AGG GAG GCG CCG GGC GCT 672 TGG GTG TGC AGC GAG AAC GGC GGC TGC GAG CAC GCG TGC AAT GCG 720 ATC CCT GGG GCT CCC CGC TGC CAG TGC CCA GCC GGC GCC GCC CTG CAG 768 GCA GAC GGG CGC TCC TGC ACC GCA TCC GCG ACG CAG TCC TGC AAC GAC 816 CATC TGC GAGTTC TGC GTT CCC AAC CCC GAC CAG CCG GGC TCC TAC 864 TCG TGC ATG TGC GAG ACC GGC TAC CGG CTG GCG GCC GAC CAA CAC CGG 912 TGC GAG GAC GTG GAT GAC TGC ATA CTG GAG CCC AGT CCG TGT CCG CAG 960 CGC TGC AAC ACA CAG GGT GGC TTC GAG TGC CAC TGC TAC CCT AAC 1008 TAC GAC CTG GTG GAC GGC GAG TGT GTG GAG CCC GTG GAC CCG TGC TTC 1056 AGA GCC AAC TGC GAG TAC CAG TGC CAG CCC CTG AAC CAA ACT AGC TGC 1104 CTC GTC TGC GCC GAG GGC TTC GCG CCC ATT CCC CAC GAG CCG CAC 1152 AGG TGC CAG ATG TTT TGC AAC CAG ACT GCC TGT CCA GCC GAC TGC GAC 1200 CCC AAC ACC CAG GCT AGC TGT GAG TGC CCT GAA GGC TAC ATC CTG GAC 1248 GTC GGT TTC ATC TGC ACG GAC ATC GAC GAG TGC GAA AAC GGC GGC TTC 1296 TGC TCC GGG GTG TGC CAC AAC CTC CCC GGT ACC TTC GAG TGC ATC TGC 1344 GGG CCC GAC TCG GCC CTT GTC CGC CAC ATT GGC ACC GAC TGT GAC TCC GGC AAG GTG GAC GGT GGC GAC AGC GGC TCT GGC GAG CCC CCG CCC AGC 1440 CCG ACG CCC GGC TCC ACC TTG ACT CCT CCG GCC GTG GGG CTC GTG CAT 1488 TCG GGC TTG CTC ATA GGC ATC TCC ATC GCG AGC CTG TGC CTC TG GTG GTG 1536 GCG CTT TTG GCG CTC CTC TGC CAC CTG CGC AAG AAG CAG GGC GCC GCC 1584 AGG GCC AAG ATG GAG TAC AAG TGC GCG GCC CCT TCC AAG GAG GTA GTG 1632 CTG CAG CAC GTG CGG ACC GAG CGG ACG CCG CAGC CTC 1671

[Brief description of drawings]

FIG. 1 is a graph showing the results of subjecting a peptide having an action of promoting protein C activation by thrombin obtained by purifying human lung to the fourth DIP-thrombin-agarose column chromatography in Reference Example 2. Is.

FIG. 2 shows the nucleotide sequence of DNA fragment TM13 obtained in Reference Example 3- (2) of the present invention.

FIG. 3 shows the nucleotide sequence of DNA fragment TM13 following FIG.

FIG. 4 shows the nucleotide sequence of DNA fragment TM137 obtained in Reference Example 3- (5) of the present invention.

FIG. 5 shows the nucleotide sequence of DNA fragment TM137 following FIG.

FIG. 6 shows the nucleotide sequence of DNA fragment TM137 following FIG.

FIG. 7 shows the nucleotide sequence of DNA fragment TM137 following FIG.

FIG. 8 shows the nucleotide sequence of DNA fragment TMP5 obtained in Reference Example 3- (7) of the present invention.

FIG. 9 shows the nucleotide sequence of DNA fragment TMP5 following FIG.

FIG. 10: DN obtained in Reference Example 3- (10) of the present invention
1 shows the base sequence of A fragment TMP26.

FIG. 11: DNA fragments TMP13, TM137, TMP
5 and TMP26 and Reference Examples 3- (13-1) and 3-
DNA fragments TMJ1 and TMJ2 obtained in (13-2)
3 shows the correspondence between each restriction enzyme map and the nucleotide sequences of these DNA fragments, and shows that the overlapping portions of the DNA fragments in the vertical direction have a common nucleotide sequence. In the figure, the portion including the hatched portion and the hatched portion in the restriction enzyme map of the DNA fragment TMJ2 is a possible open reading frame, and the nucleotide sequence encoding the peptide of the present invention is present in the hatched portion. .

FIG. 12 is a flow chart showing the construction of plasmid pUC18TMJ1 containing TMJ1 and TMP5 bound thereto.

FIG. 13 is a flow chart showing the construction of plasmid pUC18TMJ2 containing TMJ1 and TMP26 bound thereto.

FIG. 14: TMJ2 was used as an expression vector for animal cell host
Plasmid pSV2TMJ by inserting MJ2
It is a flowchart which shows the construction of 2.

FIG. 15 is a flow chart showing the construction of the plasmid pdBPVTMJ2-1 which is the replicable recombinant DNA of the present invention.

16 is a flow chart showing the construction of plasmid pdBPVTMJ2-1 which is a replicable recombinant DNA following FIG.

FIG. 17 is a graph showing the relationship between the amount of activated protein C produced by the reaction of protein C and thrombin in the presence and absence of the peptide encoded by the purified DNA of the present invention and the reaction time.

FIG. 18: Coagulation time of blood added with a peptide encoded by the purified DNA of the present invention and purified DNA of the present invention
3 is a graph showing the relationship with the added amount of the peptide encoded by.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 5/10 C12P 21/02 C 9282-4B // (C12P 21/02 C12R 1:91) 7729 -4B C12N 5/00 B

Claims (8)

[Claims] Claims 1. Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Ser Gln Cys Val Glu His Asp Cys Phe Ala Leu Tyr Gy Pro Pro Ala Thru Gure Pro Gly Pro Ala Thr Aur Thru Aur Thru Phe Alu Thru Aur Thru Phe Alu Thru Alu His Leu Met Thr Val Arg Ser Ser Val Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp sn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro Val Gly Ser Ser Ala Ala al Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr rg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro la Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His er Gly Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu Val Val Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys Lys Gln Gly Ala Ala Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala Pro Ser Lys Glu Val Val Leu Gln A DNA having a amino acid sequence of HisValArgThrGluArgThrProGlnArgLeu and having an action of promoting activation of protein C by thrombin or a homologous variant thereof. Wherein the following formula (II): GCACCCGCAG AGCCGCAGCC GGGTGGCAGC CAGTGCGTCG AGCACGACTG CTTCGCGCTC TACCCGGGCC CCGCGACCTT CCTCAATGCC AGTCAGATCT GCGACGGACT GCGGGGCCAC CTAATGACAG TGCGCTCCTC GGTGGCTGCC GATGTCATTT CCTTGCTACT GAACGGCGAC GGCGGCGTTG GCCGCCGGCG CCTCTGGATC GGCCTGCAGC TGCCACCCGG CTGCGGCGAC CCCAAGCGCC TCGGGCCCCT GCGCGGCTTC CAGTGGGTTA CGGGAGACAA CAACACCAGC TATAGCAGGT GGCACGGCT CGACCTCAAT GGGGCTCCCC TCTGCGGCCC GTTGTGCGTC GCTGTCTCCG CTGCTGAGGC CACTGTGCCC AGCGAGCCGA TCTGGGAGGA GCAGCAGTGC GAAGTGAAGG CCGATGGCTT CCTCTGCGAG TTCCACTTCC CAGCCACCTG CAGGCCACTG GCTGTGGAGC CCGGCGCCGC GGCTGCCGCC GTCTCGATCA CCTACGGCAC CCCGTTCGCG GCCCGCGGAG CGGACTTCCA GGCGCTGCCG GTGGGCAGCT CCGCCGCGGT GGCTCCCCTC GGCTTACAGC TAATGTGCAC CGCGCCGCCC GGA CGGTCC AGGGGCACTG GGCCAGGGAG GCGCCGGGCG CTTGGGACTG CAGCGTGGAG AACGGCGGCT GCGAGCACGC GTGCAATGCG ATCCCTGGGG CTCCCCGCTG CCAGTGCCCA GCCGGCGCCG CCCTGCAGGC AGACGGGCGC TCCTGCACCG CATCCGCGAC GCAGTCCTGC AACGACCTCT GCGAGCACTT CTGCGTTCCC AACCCCGACC AGCCGGGCTC CTACTCGTGC ATGTGCGAGA CCGGCTACCG GCTGGCGGCC GACCAACACC GGTGCGAGGA CGTGGATGAC TGCATACTGG AGCCCAGTCC GTGTCC GCAG CGCTGTGTCA ACACACAGGG TGGCTTCGAG TGCCACTGCT ACCCTAACTA CGACCTGGTG GACGGCGAGT GTGTGGAGCC CGTGGACCCG TGCTTCAGAG CCAACTGCGA GTACCAGTGC CAGCCCCTGA ACCAAACTAG CTACCTCTGC GTCTGCGCCG AGGGCTTCGC GCCCATTCCC CACGAGCCGC ACAGGTGCCA GATGTTTTGC AACCAGACTG CCTGTCCAGC CGACTGCGAC CCCAACACCC AGGCTAGCTG TGAGTGCCCT GAAGGCTACA TCCTGGACGA CGGTTTCATC TGCACGGACA TCGACGAG G CGAAAACGGC GGCTTCTGCT CCGGGGTGTG CCACAACCTC CCCGGTACCT TCGAGTGCAT CTGCGGGCCC GACTCGGCCC TTGTCCGCCA CATTGGCACC GACTGTGACT CCGGCAAGGT GGACGGTGGC GACAGCGGCT CTGGCGAGCC CCCGCCCAGC CCGACGCCCG GCTCCACCTT GACTCCTCCG GCCGTGGGGC TCGTGCATTC GGGCTTGCTC ATAGGCATCT CCATCGCGAG CCTGTGCCTG GTGGTGGCGC TTTTGGCGCT CCTCTGCCAC CTGCGCAAGA AGCAGGGCGC CGCCAGGGCC AAGATGGAGT A CAAGTGGCGC GGCCCCTTTCC AAGGAGGTAG TGCTGCAGCA CGTGCGGACC GAGCGGACGC CGCAGAGACT C at least one base is substituted based on the degeneracy of the genetic code or D according to claim 1.
NA. 3. The DNA according to claim 1 or 2.
DNA complementary to. 4. A replicable recombinant DNA containing the DNA according to claim 1, 2 or 3 and a replicable expression vector. 5. The replicable recombinant D according to claim 4.
A microorganism or cell transformed with NA. 6. At least an amino acid sequence of formula (I)
A method for producing a peptide having the amino acid sequence of 1) having the action of promoting activation of protein C by thrombin, or a homologous variant thereof, comprising: (a) containing a base sequence encoding the peptide or a homologous variant thereof. DN
A is ligated to a replicable expression vector to obtain a replicable recombinant DNA containing the DNA and the replicable expression vector, and (b) a microorganism or cell with the replicable recombinant DNA. To form a transformant, (c) the transformant is selected from the parent cells of the microorganism or cells, (d) the transformant is cultured, and the transformant is transformed with the DNA. Is produced to produce the peptide or a homologous variant thereof, and (e) the peptide or a homologous variant thereof is isolated from a cultured transformant. . 7. An effective amount of a peptide having an action of promoting activation of protein C by thrombin produced by the production method according to claim 6, or a homologous variant thereof, and at least one drug. A pharmaceutical composition, characterized in that it contains possible carriers, diluents or excipients and is free of other human-derived peptides. 8. The pharmaceutical composition according to claim 7, which is a pharmaceutical composition for cardiovascular disease.