WO1994029448A1

WO1994029448A1 - Dna and protein coded for thereby

Info

Publication number: WO1994029448A1
Application number: PCT/JP1994/000946
Authority: WO
Inventors: Atsushi Nii; Yasukazu Nagase; Seiichi Mizushima
Original assignee: Nawata, Hajime
Priority date: 1993-06-11
Filing date: 1994-06-10
Publication date: 1994-12-22
Also published as: JPH07132095A

Abstract

A novel DNA represented by SEQ ID NO. 3 or 4 in the Sequence Listing. A novel protein represented by the amino acid sequence of SEQ ID NO. 1 or 2 and coded for by this DNA can be prepared by means of a transformant obtained by using this DNA. This protein promotes PGI2 production and serves as medicines efficacious for diseases such as hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral embolism, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral infarction, hyperlipemia, diabetes, cardiac failure, angina pectoris, ischemic heart disease, congestive heart disease, choroidal circulatory disturbance, bronchial disease, gastric ulcer and eclampsia of pregnancy on the basis of the platelet aggregation inhibitor activity, smooth muscle relaxant activity and gastric secretion inhibitor activity of PGI2.

Description

DNA and its encoded proteins

[Technical field]

The present invention acts on vascular endothelial cells, prostaglandin 1 ₂ from the cells (another name; prostacyclin (P Rosta cy clin), referred to as PG I ₂ below) proteinaceous physiologically active substance having a production stimulating activity (Hereinafter referred to as PG I 2 production stimulating factor), a method for producing the PG I ₂ production stimulating factor using the DNA, and the PG I 2 production stimulating factor produced and recognizing the PG I ₂ production stimulating factor Antibodies and uses thereof.

[Background technology]

Prostaglandins (pr 0 stag 1 andin, PG) were released in 1930 by Kurtzrock et al. [Proc. Soc. Exp. Biol. Med. 28, p. 268, 1930] and was reported as a uterine muscle contractile active substance present in semen. Since then, basic research on prostaglandins has developed rapidly. Prostaglandin is a collective term for biologically active substances based on prostanoic acid in human and animal tissues and organs, and a series of chemical reactions called arachidonic acid cascades starting from arachidonic acid in cells. Produces several kinds of prostaglandins having different chemical structures. They are attracting attention as important active substances involved in maintaining the homeostasis of the endocrine system, nervous system, immune system and other organisms, and are widely used in various mechanisms such as inflammatory response, thrombus formation, peripheral circulation, arteriosclerosis, and aging. [Clinical Science, 17, 958, 1981]. In addition, prostaglandin plays an important role as a vasoactive substance for vascular smooth muscle [Pathophysiology, Vol. 2, p. 792, 1983], and thromboxane is an active prostanoid derived from platelets and vascular wall. A ₂ (TXA ₂ ) [Proceding of the National Academy of Sien Sov the United States of America (Proc. Natl.

Ac ad. Sci. USA). 72, 2994, 1975] and PG I

2 [Nature, 263, 663, 1976] has received attention for its clinical significance in the physiology and pathology of platelets and vasculature. Among these prostaglandins, PG I ₂ exhibits potent platelet aggregation inhibitory action and vasorelaxant action, and acts antagonistically with platelet-derived TXA ₂ which has the opposite action, resulting in homeostasis in vivo. [Pretty Gian Nanoleoff] Pharmaco-Koji (Br. J. Pharmac.), 76, 3 pages, 1982.] Normal vascular endothelial cells are physically and chemically When the stimulus is applied, it mainly produces PG I ₂ and activates a mechanism that not only suppresses platelet activation but also regulates vascular wall tone (Tonus) to maintain local circulation homeostasis. The development of vascular disorders such as thrombosis and arteriosclerosis involves an imbalance in the production of TXA ₂ and PG I 2, especially a decrease in the production of PG I ₂ [Pretish Journal of Pharmacology— (Br J. Pha rma c.) , 76, 3, 1982] Mac Inyer et al., 1978 [Nature, 271, 549, 1978] reported that PG I 2 Remudge et al. Have reported that the presence of a factor that stimulates the production of this factor is a heat-labile macromolecule. Have been reported to be lower compared to healthy individuals [Lancet, 2, 871 (1978)], thrombotic thrombocytopenic purpura [Lanc et], 2, 748, 1979], Sickle cell anemia [Brit. J. Haema to l., 48, 545, 1981], sudden Myocardial infarction [Coronary, Vol. 2, p. 49, 1985], thrombotic and atherosclerotic diseases such as diabetes It has been clarified that the decrease in medium level is closely related to the etiology of the disease, and especially regarding the onset and progression of diabetic vasculopathy, increased production of platelet-derived TXA ₂ [Thrombosis Research (T hr omb) Res.), 19, 211, 1980, Journal of Laboratory and Clinical Medicine (J. Lab. Clin. Med.), 97, 87, 1981], and vascular origin It has been reported that reduced PG I ₂ production in platelets causes an increase in platelet aggregation in diabetic patients and experimental diabetic animals [Lanc et: 1, 325, 1979, Lancet] , Volume 2, 1365 pages, 1979, The Two Wings Land Journal of Medicine (N. Enl. J. Med.) 300, 366, 1979 and Life Science (Life Sc i.), 23, 351, 1978]. Moreover, the PG I ₂ production decreases, potential reduction of PG I 2 Stimulating activity present in the blood of above is partly has been suggested [Metabolism (Me tab 01 i sm). 38 , pp. 837, 1989, Ha emo stasis. Volume 16, 447, 1986 and Diabetis Research and Clinical Practice (Dia b. Res. Clin. Prac.) 3, 243, 1987]. As described above, PG I ₂ has a platelet aggregation inhibitory action, but also has a smooth muscle relaxing action on blood vessels and bronchi, a gastric acid secretion inhibitory action, and the like. While developing these physiological effects based on Hazuki PG I ₂ itself as a medical drug is considered, half-life of the PG I ₂ is extremely short as 5 minutes in water at 37 ° C neutral, chemically non It is a stable substance and has not been put to practical use. On the other hand, attempts have been made to develop chemically stable PGI ₂ analogs as pharmaceuticals such as anticoagulants and vasodilators while maintaining the natural PGI 2 -like action.

However, for living organisms, it is more desirable that PG I ₂ be chemically unstable and have a short life. It is the natural function of PG Ia that it is locally produced and acts while preventing unnecessary inhibition of platelet aggregation.When a large amount of chemically stable PG I 2 analog is given, PG I Responsiveness to PGI 2 may be reduced, and it may not respond to PGI 2 in an emergency. In fact, pretreatment with prostaglandins (PGEi) and stable PG I ₂ analogs has shown that in some cells, PG I ₂ does not result in an increase in cAMP, which should occur naturally. Prostaglandins (Prostagl and ins.). 19, 2, 2980.

Therefore, if the above-mentioned effects of PGI 2 are expected, rather than using chemically stable analogs, the required concentration of PGI ₂ will be produced at the required site when needed. Is considered to be more preferable for the living body. In vivo metabolism of PG I 2 is controlled by two factors, one of which is a stabilizing factor of PG I ₂ , which was recently identified as apolipoprotein A-1 (Apo A-1) [ The ja —Naluob Clinical Investigation (J. Clin. Invest.), 82, 803, 1988]. The other is a PG I ₂ production stimulating factor (Prosta cy clin P roduc ti on S t imu lati ng Fa cto r. PSF). Above PGI ₂ Already in blood as the Sansei剌intense activity (P rosta cyc lin S t imu lati ng Ac tivity, PSA) but it has been reported to be present, PG I ₂ Stimulating its active body Factors increase PG I 2 production from vascular endothelial cells and increase the PG I 2 concentration in blood to exert platelet aggregation inhibitory action, smooth muscle relaxing action, gastric acid secretion inhibitory action, and the like. The PG I ₂ production stimulating factor having such an effect is hemolytic uremic syndrome group, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia It can be used for the treatment of diabetes, heart failure, angina pectoris, ischemic heart disease, depressive heart disease, choroidal circulatory disorder, bronchial disease, gastric ulcer, pregnancy and eclampsia. It is expected that the local expression of the I 2 production stimulating factor will increase and decrease, and the concentration in the blood and urine will change.If such a change in the concentration of the PG I ₂ production stimulating factor can be detected, it will be possible to measure it. It is thought that the above-mentioned diseases can be diagnosed.

The present inventors have conducted intensive studies on the PG I 2 production stimulating activity present in the blood and in the culture supernatant of a human-derived cultured cell line, and found that the culture supernatant of normal human diploid fibroblasts After confirming that a substance having PG I 2 production stimulating activity was present at a high concentration, we succeeded in isolating and purifying PG I ₂ production stimulating factor from this culture supernatant, and determined a part of the amino acid sequence. [PCTZJ P 93/00294]. However, in order to obtain the proteinaceous physiologically active substances such as PG I ₂ Stimulating Factor in large quantities, that the make isolation and identification of genes, to establish a production method using recombinant techniques Was desired.

[Disclosure of the Invention]

An object of the present invention is to provide a gene encoding a PG I 2 production stimulating factor which stimulates vascular endothelial cells to promote PG I 2 production in view of the above situation, ₍₂₎ To provide a method for mass production of a production stimulating factor. In addition, Another object of Tomo is to provide a medical pharmaceutical composition for the aforementioned diseases, based on the action of PG I ₂ Stimulating Factor. It is still another object to provide a specific antibody against the PGI ₂ production stimulating factor and a method for diagnosing the above-mentioned diseases using the antibody.

The present inventors have conducted intensive research to achieve the above-mentioned object, and as a result,

A portion of the amino acid sequence of PG I 2 Stimulating Factor as a cue, to create a cDNA library of normal human diploid fibroblasts to obtain the cDNA of PG I 2 Stimulating Factor, then, PG I ₂ Stimulating The nucleotide sequence of the factor was determined, and the amino acid sequence deduced from the sequence was determined.

That is, the present invention provides a novel DNA containing a part or all of a base sequence encoding an amino acid sequence represented by the following formula [1] or [2] (SEQ ID NO: 1 or 2 in the sequence listing). I do.

Equation [1]

Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly

-25 -20 -15

Leu Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp

-10 -5 1

Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro

5 10 15

Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys

20 25 30

Pro Met Cys Ala Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Gly

35 40 45

Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu Cys Val Lys Ser

50 55 60

Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala Ala Gly Gly Pro

65 70 75

Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg Tyr Pro Val Cys

80 85 90 Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys Gin Leu Arg Ala

95 100 105

Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys Ala lie Thr Gin

110 115 120

Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser lie Val Thr Pro

125 130 135

Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin Val Tyr Leu Ser

140 145 150

Cys Glu Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys

155 160 165

Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro

170 175 180

Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly Gly Pro Glu

185 190 195

Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

200 205 210

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Ser Gin Gly

215 220 225

Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His

230 235 240

Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

245 250 255 256

Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly -25 -20 -15

Leu Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp -10 -5 1

Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro 5 10 15

Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys

20 25 30

Pro Met Cys Ala Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Gly

35 40 45

Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu Cys Val Lys Ser

50 55 60

Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala Ala Gly Gly Pro

65 70 75

Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg Tyr Pro Val Cys

80 85 90

Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys Gin Leu Arg Ala

95 100 105

Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys Ala lie Thr Gin 110 115 120

Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser lie Val Thr Pro 125 130 135

Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin Val Tyr Leu Ser 140 145 150

Cys Glu Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys 155 160 165

Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro 170 175 180

Gly Asp Arg Asp Asn Leu Alalie Gin Thr Arg Gly Gly Pro Glu 185 190 195

Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys 200 205 210

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Phe Gin Gly

215 220 225 Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His 230 235 240

Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

245 250 255 256 Further, the present invention provides a novel DNA containing a part or all of the nucleotide sequence represented by the following formulas [3] to [4] (SEQ ID NO: 3 or 4 in the sequence listing).

Expression [3]

GCCGCTGCCA CCGCACCCCG CCATGGAGCG GCCGTCGCTG CGCGCCCTGC TCCTCGGCGC 60 CGCTGGGCTG CTGCTCCTGC TCCTGCCCCT CTCCTCTTCC TCCTCTTCGG ACACCTGCGG 120 CCCCTGCGAG CCGGCCTCCT GCCCGCCCCT GCCCCCGCTG GGCTGCCTGC TGGGCGAGAC 180 CCGCGACGCG TGCGGCTGCT GCCCTATGTG CGCCCGCGGC GAGGGCGAGC CGTGCGGGGG 240 TGGCGGCGCC GGCAGGGGGT ACTGCGCGCC GGGCATGGAG TGCGTGAAGA GCCGCAAGAG 300 GCGGAAGGGT AAAGCCGGGG CAGCAGCCGG CGGTCCGGGT GTAAGCGGCG TGTGCGTGTG 360 CAAGAGCCGC TACCCGGTGT GCGGCAGCGA CGGCACCACC TACCCGAGCG GCTGCCAGCT 420 GCGCGCCGCC AGCCAGAGGG CCGAGAGCCG CGGGGAGAAG GCCATCACCC AGGTCAGCAA 480 GGGCACCTGC GAGCAAGGTC CTTCCATAGT GACGCCCCCC AAGGACATCT GGAATGTCAC 540 TGGTGCCCAG GTGTACTTGA GCTGTGAGGT CATCGGAATC CCGACACCTG TCCTCATCTG 600 GAACAAGGTA AAAAGGGGTC ACTATGGAGT TCAAAGGACA GAACTCCTGC CTGGTGACCG 660 GGACAACCTG GCCATTCAGA CCCGGGGTGG CCCAGAAAAG CATGAAGTAA CTGGCTGGGT 720 GCTGGTATCT CCTCTAAGTA AGGAAGATGC TGGAGAATAT GAGTGCCATG CATCCAATTC 780 CCAAGGACAG GCTTCAGCAT CAGCAAAAAT TACAGTGGTT GATGCCTTAC ATGAAATACG 840 AGTGAAAAAA GGTGAAGGTG CCGAGCTATA AACCTCCAGA ATATTATTAG TCTGCATGGT 900 TAAAAGTAGT CATGGATAAC TACATTACCT GTTCTTGCCT AATAAGTTTC TTTTAATCCA 960 ATCCACTAAC ACTTTAGTTA TATTCACTGG TTTTACACAG AGAAATACAA AATAAAGATC 1020 ACACATCAAG ACTATCATAAATCAAAAGAAAAAATCAAAAGAA GCCGCTGCCA CCGCACCCCG CCATGGAGCG GCCGTCGCTG CGCGCCCTGC TCCTCGGCGC 60 CGCTGGGCTG CTGCTCCTGC TCCTGCCCCT CTCCTCTTCC TCCTCTTCGG ACACCTGCGG 120 CCCCTGCGAG CCGGCCTCCT GCCCGCCCCT GCCCCCGCTG GGCTGCCTGC TGGGCGAGC

CCGCGACGCG TGCGGCTGCT GCCCTATGTG CGCCCGCGGC GAGGGCGAGC CGTGCGGGGG 240

TGGCGGCGCC GGCAGGGGGT ACTGCGCGCC GGGCATGGAG TGCGTGAAGA GCCGCAAGAG 300

GCGGAAGGGT AAAGCCGGGG CAGCAGCCGG CGGTCCGGGT GTAAGCGGCG TGTGCGTGTG 360

CAAGAGCCGC TACCCGGTGT GCGGCAGCGA CGGCACCACC TACCCGAGCG GCTGCCAGCT 420

GCGCGCCGCC AGCCAGAGGG CCGAGAGCCG CGGGGAGAAG GCCATCACCC AGGTCAGCAA 480

GGGCACCTGC GAGCAAGGTC CTTCCATAGT GACGCCCCCC AAGGACATCT GGAATGTCAC 540

TGGTGCCCAG GTGTACTTGA GCTGTGAGGT CATCGGAATC CCGACACCTG TCCTCATCTG 600

GAACAAGGTA AAAAGGGGTC ACTATGGAGT TCAAAGGACA GAACTCCTGC CTGGTGACCG 660

GGACAACCTG GCCATTCAGA CCCGGGGTGG CCCAGAAAAG CATGAAGTAA CTGGCTGGGT 720

GCTGGTATCT CCTCTAAGTA AGGAAGATGC TGGAGAATAT GAGTGCCATG CATCCAATTT 780

CCAAGGACAG GCTTCAGCAT CAGCAAAAAT TACAGTGGTT GATGCCTTAC ATGAAATACC 840

AGTGAAAAAA GGTGAAGGTG CCGAGCTATA AACCTCCAGA ATATTATTAG TCTGCATGGT 900

TAAAAGTAGT CATGGATAAC TACATTACCT GTTCTTGCCT AATAAGTTTC TTTTAATCCA 960

ATCCACTAAC ACTTTAGTTA TATTCACTGG TTTTACACAG AGAAATACAA AATAAAGATC 1020

ACACATCAAG ACTATCTACA AAAATTTATT ATATATTTAC AGAAGAAAAG CATGCATATC 1080

ATTAAACAAA TAAAATACTT TTTATCACAA AAAAAAAAAA AAAA 1124 Further, a DNA having a base sequence complementary to DNA encoding the novel amino acid sequence represented by the formula [1] or [2], and a formula [3] or [4] Provided is a DNA having a nucleotide sequence complementary to the novel DNA represented.

Furthermore, the present invention relates to a vector obtained by introducing a DNA encoding the amino acid sequence represented by the formula [1] or [2] or a DNA represented by the formula [3] or [4], and A transformant transformed with the vector is provided. Then, to provide a PGI ₂ production-stimulating factor obtainable by the process as well as the manipulation to produce PGI 2 production-stimulating factor by using the transformant.

The amino acid sequence of the PGI ₂ production stimulating factor of the present invention is represented by the formula [1] or [2]. The protein contains a part or all of the amino acid sequence to be obtained. It preferably has the amino acid sequence represented by the following formula [5] or [6] (SEQ ID NO: 5 or 6 in the sequence listing).

Expression [5]

Ser Ser Ser Asp Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro 1 5 10 15

Pro Leu Pro Pro Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala

20 25 30

Cys Gly Cys Cys Pro Met Cys Ala Arg Gly Glu Gly Glu Glu Pro Cys

35 40 45

Gly Gly Gly Gly Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu

50 55 60

Cys Val Lys Ser Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala

65 70 75

Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg

80 85 90

Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys

95 100 105

Gin Leu Arg Ala Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys

110 115 120

Ala lie Thr Gin Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser

125 130 135 lie Val Thr Pro Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin

140 145 150

Val Tyr Leu Ser Cys Glu Val lie Gly lie Pro Thr Pro Val Leu

155 160 165 lie Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr

170 175 180

Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg 185 190 195

Gly Gly Pro Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser

200 205 210

Pro Leu Ser Lys Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser

215 220 225

Asn Ser Gin Gly Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val

230 235 240

Asp Ala Leu His Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu

245 250 255

Leu

256

Phe Ser Ser Asp Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro

1 5 10 15

Pro Leu Pro Pro Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala

20 25 30

Cys Gly Cys Cys Pro Met Cys Ala Arg Gly Glu Gly Glu Glu Pro Cys

'' 35 40 45

Gly Gly Gly Gly Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu

50 55 60

Cys Val Lys Ser Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala

65 70 75

Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg

80 85 90 Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys

95 100 105 Gin Leu Arg Ala Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys

110 115 120 Ala lie Thr Gin Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser

125 130 135 lie Val Thr Pro Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin

140 145 150

Val Tyr Leu Ser Cys Glu Val lie Gly lie Pro Thr Pro Val Leu

155 160 165

He Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr

170 175 180

Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg

185 190 195

Gly Gly Pro Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser

200 205 210

Pro Leu Ser Lys Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser

215 220 225

Asn Phe Gin Gly Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val

230 235 240

Asp Ala Leu His Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu

245 250 255

Leu

256

This protein is characterized by its ability to stimulate vascular endothelial cells to promote PGI 2 production.

Furthermore, the present invention provides an antibody obtained by using a part or all of the amino acid sequence of the PG I ₂ production stimulating factor of the present invention as an antigen, and a method for immunologically measuring the PG I 2 production stimulating factor using the antibody.

The present invention also provides a pharmaceutical composition for preventing or treating the following diseases, which comprises the above-mentioned PG I 2 production stimulating factor as an active ingredient.

Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart failure, angina, ischemic heart disease, depression Hematologic heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnancy eclampsia. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram illustrating the COS expression vector pM953.

FIG. 2 is a schematic diagram illustrating the CHO expression vector pM954.

FIG. 3 is a drawing showing the results of gel electrophoresis, showing the results of SDS-PAGE of the purified sample of pM953 ZCOS cell culture supernatant.

Figure 4 is a graph showing the elution pattern by DEAE-5 PW anion exchange chromatography.

FIG. 5 is a graph showing an elution pattern obtained by heparin-1 5PW affinity chromatography.

FIG. 6 is a graph showing an elution pattern obtained by protein one-pack gel filtration column chromatography.

FIG. 7 is a schematic diagram showing the experimental results by SDS-PAGE. Hereinafter, the present invention will be described in detail.

That is, the present invention is a protein containing a part or all of an amino acid sequence represented by the following formula [1] or [2] and a DNA containing a part or the whole of a nucleotide sequence encoding the protein.

Equation [1] Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly

-25 -20 -15

Leu Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp

-10 -5 1

Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro

5 10 15

Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys

20 25 30

Pro Met Cys Ala Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Gly

35 40 45

Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu Cys Val Lys Ser

50 55 60

Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala Ala Gly Gly Pro

65 70 75

Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg Tyr Pro Val Cys

80 85 90

Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys Gin Leu Arg Ala

95 100 105

Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys Ala lie Thr Gin 110 115 120

Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser lie Val Thr Pro 125 130 135

Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin Val Tyr Leu Ser 140 145 150

Cys Glu Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys 155 160 165

Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro 170 175 180

Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly Gly Pro Glu 185 190 195

Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

200 205 210

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Ser Gin Gly

215 220 225

Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His

230 235 240

Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

245 250 255 256

Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly

-25 -20 -15

Leu Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp

-10 -5 1

Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro

5 10 15

Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys

20 25 30

Pro Met Cys Ala Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Gly

35 40 45

Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu Cys Val Lys Ser

50 55 60

Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala Ala Gly Gly Pro

65 70 75

Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg Tyr Pro Val Cys

80 85 90

Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys Gin Leu Arg Ala

95 100 105 Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys Ala lie Thr Gin

110 115 120

Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser lie Val Thr Pro

125 130 135

Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin Val Tyr Leu Ser

140 145 150

Cys Glu Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys

155 160 165

Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro

170 175 180

Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly Gly Pro Glu

185 190 195

Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

200 205 210

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Phe Gin Gly

215 220 225

Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His

230 235 240

Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

245 250 255 256 DNA containing a part or all of the base sequence represented by the following formula [3] or [4].

Expression [3]

GCCGCTGCCA CCGCACCCCG CCATGGAGCG GCCGTCGCTG CGCGCCCTGC TCCTCGGCGC 60

CGCTGGGCTG CTGCTCCTGC TCCTGCCCCT CTCCTCTTCC TCCTCTTCGG ACACCTGCGG 120

CCCCTGCGAG CCGGCCTCCT GCCCGCCCCT GCCCCCGCTG GGCTGCCTGC TGGGCGAGAC 180

CCGCGACGCG TGCGGCTGCT GCCCTATGTG CGCCCGCGGC GAGGGCGAGC CGTGCGGGGG 240

TGGCGGCGCC GGCAGGGGGT ACTGCGCGCC GGGCATGGAG TGCGTGAAGA GCCGCAAGAG 300 GCTGGTATCT CCTCTAAGC CCCTA AGGAAGGCGGGAAAT GGGCATGATAATTTAT TATAT

GGACAACCTG GCCATTCA CCGGGGA CCCGGGGTGG CCCAGAAAAG CATGAAGTAATGGTT

GACAGGAATA AAAAGGGGCCGGTC ACTTGGAGT TCAGGC GCTCCTGC CTGGTAAAAAAAA

TGGGCCTCAG GTGTACCCGTTGA GCGTGGGT CCGGAATC CCGCACCTG TCCTATTTAATA

GGGCCACTGC GAGC GGCACAAGGTC CCCATGT GACGCCCCCC AAGGACATCTGAATTTTA

o

GCGCGCCGCC AGCCCCAGAGGG CCGGGCCG CGGGGAGG GCCCCCC AGGTAGAAAAAAATA

CAGAGCACGCCGC TACCCGGCGC CCCCCCCCGCG GCGCATTGT GCGGAGAGGAA TACGAT

GCGGAAGGGT AAAGCCCC ¾GGCGGGCGTGTGGGGG CAGAGCCGG CGGTCGGGTAACG TT

C TGGGGCGCC GCGGGCAGGCGGCGGG GCGCAAAGGGT ACTGCGCGCC GGGATGA TGTAAA

CCGCGACGCGGGG TGCGGCCCGCC GGGCGAGC CGTGCGGTGCT GCCCTATGTG CGCGGAG

CCCCCTGGAG CCCGCGGGCGAGACGGCCTCC GCCCGCCCC GCCCCCGCTG GGCTGCT TTT

CGCTGGGCG CCCCGCGGTTGCTCCGCCCGCCCC CCCCTTCCCCTCTTCGG AATT TTTTT T

GCCGCGCGCCTCA CCGCCCCC CCGCCCTGC TCCTCGGACCG CCATGGAGCG GCCGTGTGG

ATTAAACAAA TAAAATACTT TTTATCACAA AAAAAAAAAA AAAA CC AAATCAGCG CATGCATATCA ATATCTAC AAATTATATTTAC AGAAGAAAAAATTATT A C ATCACTCGAA ACTTGCACAA AATAAAATCTATTA TATTACTGG TTTT AGAAAT

TAAAAGT CCCAGTATGGTC TTTTAATAATAAC TACATTACC GTCTTGCCT AATAAGTTTT

n ^ O CO D OO-I-^ 3

o C CD oo t σ¾ <=> CO ts3 OO CsD OO

CD CD CD CD C AGTGAAAAAA GGTGGGCTGCATGGTAAGTG CCGAGCCCTCCAG ATATTATTA TTATA AAAD CD CD

CCAGGAACAG GCGATCCTTCAGC CGCCG GGCCTTAC ATAAAATAAAAAAT TAAGTGTTAT

GCTGGTATC CTCTCGCCG CATCCAATTCTAAGTA AGGAAGTGCGGAGAATAT GAGTATA T

GGACACACTG GCC¾ CGGCGGGATTCAG CCCGGGG CCCGAG CGAAAATTTATGGAAAAAT

GAACAGGATA AAGGCCGC CTGGTGACCGAAGGTC ACTAGGGCAAAGGACA GAACTTTAT T

TG¾GCCCAG GGCCCGCCTCATCTGTTATTGA GCTGTGGG CTCGGAAC CCGACAT TATAT

GGGCACCGC G GGGCCTAGCAAGGC CCCCGCCCCCCGGACATCTAATTATTTATAGT GA AA

GCGCGCCGCCGCCCCGGTCAGCAA AGAGG CCGAGAGCCG CGGGGAGAG GCCATA AA

CAAGGCACGC TACCCGCGGCG GCTGCCAGCGTGT GCGGCAGCG CGGCACCACC TACCATA CCAAGGACAG GCTTCAGCAT CAGCAAAAAT TACAGTGGTT GATGCCTTAC ATGAAATACC 840 AGTGAAAAAA GGTGAAGGTG CCGAGCTATA AACCTCCAGA ATATTATTAG TCTGCATGGT 900 TAAAAGTAGT CATGGATAAC TACATTACCT GTTCTTGCCT AATAAGTTTC TTTTAATCCA 960 ATCCACTAAC ACTTTAGTTA TATTCACTGG TTTTACACAG AGAAATACAA AATAAAGATC 1020 ACACATCAAG ACTATCTACA AAAATTTATT ATATATTTAC AGAAGAAAAG CATGCATATC 1080 ATTAAACAAA TAAAATACTT TTTATCACAA AAAAAAAAAA PG I 2 is a protein of AAAA 1124 present invention In order to obtain the production stimulating factor cDNA, a general genetic engineering technique is used. That is, the amino acid sequence of the PG I 2 production stimulating factor of the present invention is clarified, a DNA probe is prepared based on the amino acid sequence, and an appropriate cDNA library, preferably from mRNA obtained from normal human diploid fibroblasts, is prepared. The cDNA of the PG I 2 production stimulating factor of the present invention can be obtained by screening the prepared cDNA library. Also, purified PG I

2 A part of the amino acid sequence determined by using a production stimulating factor or by amino acid sequence analysis or gene sequence analysis is synthesized as a synthetic peptide, which is used to immunize egrets and mice to obtain antibodies. Thereafter, the cDNA library can be screened using this antibody.

Preferably, cDNA encoding the PG I 2 production stimulating factor of the present invention can be obtained by the following method. Prepare total RNA from normal human diploid fibroblasts and prepare poly (A) + RNA (mRNA). For example, preparation of total RNA is possible by a general guanidine thiosinate method, an AGPC method or a hot phenol method. MRNA can be prepared by subjecting total RNA to affinity chromatography using oligo (dT) cellulose, poly-U-sepharose, or the like by a column method or a batch method. Next, a single-stranded cDNA is synthesized using reverse transcriptase, and then converted into a double-stranded cDNA using DNA polymerase. Acid Research (Nucl. Ac ids Res.), 1

8, 5322, 1990] etc. to transform E. coli. Alternatively, a phage vector for cloning: introduced into Igt10, λgt11, etc. Mouth packaging and infect E. coli. Thus, a cDNA library is prepared. cDNA can be synthesized according to the method of Gubler and Hoffman [Gene, 25, 263, 1983]. A commercially available cDNA synthesis kit (manufactured by Amersham, Boehringer, Invitrodin) may be used. The above-mentioned cDNA can be introduced into a plasmid vector or a phage vector by adding a linker or the like. The linker used is not particularly limited, but when using plasmid pEF-BOS, a BstXI linker is preferred. Addition of a linker and introduction of cDNA into plasmid and phage vector can also be performed using a commercially available ligation kit (Takara Shuzo). The Escherichia coli to be transformed is not particularly limited as long as it is a commonly used strain, but HB101, DH5 and MC1061 / P3 are preferred. When λgt10 is used as a phage vector, NM514 is particularly preferred. Plasmid DNA into which cDNA has been introduced can be introduced into Escherichia coli by the electroporation method or the calcium chloride method. In vitro packaging can also be performed using a commercially available in vitro packaging kit (Stratagene, Amersham).

CDNA encoding the PGI ₂ production stimulating factor of the present invention can be isolated by a combination of general cDNA screening methods. For example, a method of preparing a DNA probe based on the obtained partial amino acid sequence [PCTZJ P93Z00294] and directly screening a cDNA library, or preparing a PCR primer and selecting a clone having a DNA fragment amplified by the PCR method And so on. When using a library capable of expressing cDNA, for example, a library prepared using a λgt11 phage vector, a target clone can be selected using the antibody obtained by the above-described method. When using a library prepared using plasmid vector pEF-BOS, etc., with C S cells as the host, select the desired clone using the PGI ₂ production stimulating activity detected in the culture supernatant as an index. be able to.

The nucleotide sequence of cDNA contained in the obtained target clone can be determined by the dideoxy termination method. A preferred example of the determined DNA sequence is the following formula [3] Or [4].

Expression [3]

GCCGCTGCCA CCGCACCCCG CCATGGAGCG GCCGTCGCTG CGCGCCCTGC TCCTCGGCGC 60 CGCTGGGCTG CTGCTCCTGC TCCTGCCCCT CTCCTCTTCC TCCTCTTCGG ACACCTGCGG 120 CCCCTGCGAG CCGGCCTCCT GCCCGCCCCT GCCCCCGCTG GGCTGCCTGC TGGGCGAGC

CCGCGACGCG TGCGGCTGCT GCCCTATGTG CGCCCGCGGC GAGGGCGAGC CGTGCGGGGG 240

TGGCGGCGCC GGCAGGGGGT ACTGCGCGCC GGGCATGGAG TGCGTGAAGA GCCGCAAGAG 300

GCGGAAGGGT AAAGCCGGGG CAGCAGCCGG CGGTCCGGGT GTAAGCGGCG TGTGCGTGTG 360

CAAGAGCCGC TACCCGGTGT GCGGCAGCGA CGGCACCACC TACCCGAGCG GCTGCCAGCT 420

GCGCGCCGCC AGCCAGAGGG CCGAGAGCCG CGGGGAGAAG GCCATCACCC AGGTCAGCAA 480

GGGCACCTGC GAGCAAGGTC CTTCCATAGT GACGCCCCCC AAGGACATCT GGAATGTCAC 540

TGGTGCCCAG GTGTACTTGA GCTGTGAGGT CATCGGAATC CCGACACCTG TCCTCATCTG 600

GAACAAGGTA AAAAGGGGTC ACTATGGAGT TCAAAGGACA GAACTCCTGC CTGGTGACCG 660

GGACAACCTG GCCATTCAGA CCCGGGGTGG CCCAGAAAAG CATGAAGTAA CTGGCTGGGT 720

GCTGGTATCT CCTCTAAGTA AGGAAGATGC TGGAGAATAT GAGTGCCATG CATCCAATTC 780

CCAAGGACAG GCTTCAGCAT CAGCAAAAAT TACAGTGGTT GATGCCTTAC ATGAAATACC 840

AGTGAAAAAA GGTGAAGGTG CCGAGCTATA AACCTCCAGA ATATTATTAG TCTGCATGGT 900

TAAAAGTAGT CATGGATAAC TACATTACCT GTTCTTGCCT AATAAGTTTC TTTTAATCCA 960

ATCCACTAAC ACTTTAGTTA TATTCACTGG TTTTACACAG AGAAATACAA AATAAAGATC 1020

ACACATCAAG ACTATCTACA AAAATTTATT ATATATTTAC AGAAGAAAAG CATGCATATC 1080

ATTAAACAAA TAAAATACTT TTTATCACAA AAAAAAAAAA AAAA 1124

Expression [4]

GCCGCTGCCA CCGCACCCCG CCATGGAGCG GCCGTCGCTG CGCGCCCTGC TCCTCGGCGC 60

CGCTGGGCTG CTGCTCCTGC TCCTGCCCCT CTCCTCTTCC TCCTCTTCGG ACACCTGCGG 120

CCCCTGCGAG CCGGCCTCCT GCCCGCCCCT GCCCCCGCTG GGCTGCCTGC TGGGCGAGAC 180

CCGCGACGCG TGCGGCTGCT GCCCTATGTG CGCCCGCGGC GAGGGCGAGC CGTGCGGGGG 240

TGGCGGCGCC GGCAGGGGGT ACTGCGCGCC GGGCATGGAG TGCGTGAAGA GCCGCAAGAG 300

GCGGAAGGGT AAAGCCGGGG CAGCAGCCGG CGGTCCGGGT GTAAGCGGCG TGTGCGTGTG 360 CAAGAGCCGC TACCCGGTGT GCGGCAGCGA CGGCACCACC TACCCGAGCG GCTGCCAGCT 420

GCGCGCCGCC AGCCAGAGGG CCGAGAGCCG CGGGGAGAAG GCCATCACCC AGGTCAGCAA 480

GGGCACCTGC GAGCAAGGTC CTTCCATAGT GACGCCCCCC AAGGACATCT GGAATGTCAC 540

TGGTGCCCAG GTGTACTTGA GCTGTGAGGT CATCGGAATC CCGACACCTG TCCTCATCTG 600

GAACAAGGTA AAAAGGGGTC ACTATGGAGT TCAAAGGACA GAACTCCTGC CTGGTGACCG 660

GGACAACCTG GCCATTCAGA CCCGGGGTGG CCCAGAAAAG CATGAAGTAA CTGGCTGGGT 720

GCTGGTATCT CCTCTAAGTA AGGAAGATGC TGGAGAATAT GAGTGCCATG CATCCAATTT 780

CCAAGGACAG GCTTCAGCAT CAGCAAAAAT TACAGTGGTT GATGCCTTAC ATGAAATACC 840

AGTGAAAAAA GGTGAAGGTG CCGAGCTATA AACCTCCAGA ATATTATTAG TCTGCATGGT 900

TAAAAGTAGT CATGGATAAC TACATTACCT GTTCTTGCCT AATAAGTTTC TTTTAATCCA 960

ATCCACTAAC ACTTTAGTTA TATTCACTGG TTTTACACAG AGAAATACAA AATAAAGATC 1020

ACACATCAAG ACTATCTACA AAAATTTATT ATATATTTAC AGAAGAAAAG CATGCATATC 1080

ATTAAACAAA TAAAATACTT TTTATCACAA AAAAAAAAAA AAAA 1124 The DNA sequence of the present invention encodes the PG I 2 production stimulating factor of the present invention, and is a part of the DNA sequence represented by the formula [3] or [4]. Or all. Further, it may include a part of the DNA sequence represented by the formula [3] or [4].

The DNA sequence of the present invention may be synthesized using RNA as type III or may be synthesized organically. Moreover, you may obtain by amplifying by a PCR method.

In general, in the field of genetic recombination, since the genetic code is degenerate, at least one base defined by the DNA sequence of the gene can be replaced with another base. In this case, the amino acid sequence of the protein produced from the gene does not change. Therefore, the DNA sequence of the present invention also includes a partially modified base sequence due to the degeneracy of the genetic code. In this case, the amino acid sequence encoding the DNA sequence obtained by the substitution is the amino acid sequence represented by the formula [1] or [2]. In the present invention, a DNA sequence complementary to DNA encoding the amino acid sequence represented by the formula [1] or [2] and a DNA sequence complementary to the DNA sequence of the formula [3] or [4]

It is also possible to provide an NA sequence, in which case the complementary DNA sequence It may be complementary to the entire DNA sequence or may be complementary to a part thereof. Further, it may include a complementary DNA sequence as a part. The DNA of the present invention may be combined with a DNA complementary thereto to form a double-stranded DNA, or may be single-stranded DNAs complementary to each other.

The present invention provides an amino acid sequence derived from the DNA sequence represented by the formula [3] or [4], and a novel protein represented by the formula [1] or [2].

Equation [1]

Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly

-25 -20 -15

Leu Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp

-10 -5 1

Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro

5 10 15

Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys

20 25 30

Pro Met Cys Ala Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Gly

35 40 45

Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu Cys Val Lys Ser

50 55 60

Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala Ala Gly Gly Pro

65 70 75

Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg Tyr Pro Val Cys

80 85 90

Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys Gin Leu Arg Ala

95 100 105

Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys Ala lie Thr Gin

110 115 120

Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser lie Val Thr Pro

125 130 135 Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin Val Tyr Leu Ser

140 145 150

Cys Glu Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys

155 160 165

Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro

170 175 180

Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly Gly Pro Glu

185 190 195

Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

200 205 210

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Ser Gin Gly

215 220 225

Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His

230 235 240

Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

245 250 255 256

Expression [2]

Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly

-25 -20 -15

Leu Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp

-10 -5 1

Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro

5 10 15

Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys

20 25 30

Pro Met Cys Ala Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Gly

35 40 45

Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu Cys Val Lys Ser

50 55 60

Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala Ala Gly Gly Pro

65 70 75

Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg Tyr Pro Val Cys

80 85 90

Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys Gin Leu Arg Ala

95 100 105

Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys Ala lie Thr Gin 110 115 120

Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser lie Val Thr Pro 125 130 135

Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin Val Tyr Leu Ser 140 145 150

Cys Glu Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys 155 160 165

Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro

170 175 180 Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly Gly Pro Glu

185 190 195

Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

200 205 210

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Phe Gin Gly

215 220 225

Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His

230 235 240

Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

245 250 255 256 The protein of the present invention may be a part of the formula [1] or [2], or may be the whole. It may also include a part of the amino acid sequence represented by the formula [1] or [2]. In particular, it may include a part of the amino acid sequence represented by the formula [5] or [6], which represents the mature protein obtained by removing the signal peptide from the amino acid sequence represented by the formula [1] or [2]. It is good, or it may be all.

Equation [5]

Ser Ser Ser Asp Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro 1 5 10 15

Pro Leu Pro Pro Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala

20 25 30

Cys Gly Cys Cys Pro Met Cys Ala Arg Gly Glu Gly Glu Glu Pro Cys

35 40 45

Gly Gly Gly Gly Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu

50 55 60

Cys Val Lys Ser Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala

65 70 75

Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg 80 85 90

Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys

95 100 105

Gin Leu Arg Ala Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys

110 115 120

Ala lie Thr Gin Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser

125 130 135 lie Val Thr Pro Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin

140 145 150

Val Tyr Leu Ser Cys Glu Val He Gly He Pro Thr Pro Val Leu

155 160 .165 lie Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr

170 175 180

Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg

185 190 195

Gly Gly Pro Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser

200 205 210

Pro Leu Ser Lys Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser

215 220 225

Asn Ser Gin Gly Gin Ala Ser Ala Ser Ala Lys He Thr Val Val

230 235 240

Asp Ala Leu His Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu

245 250 255

Leu

256

Phe Ser Ser Asp Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro

1 5 10 15 Pro Leu Pro Pro Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala 20 25 30

Cys Gly Cys Cys Pro Met Cys Ala Arg Gly Glu Gly Glu Glu Pro Cys

35 40 45

Gly Gly Gly Gly Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu

50 55 60

Cys Val Lys Ser Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala

65 70 75

Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg

80 85 90

Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys

95 100 105

Gin Leu Arg Ala Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys

110 115 120

Ala lie Thr Gin Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser

125 130 135 lie Val Thr Pro Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin

140 145 150

Val Tyr Leu Ser Cys Glu Val lie Gly lie Pro Thr Pro Val Leu

155 160 165 lie Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr

170 175 180

Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg

185 190 195

Gly Gly Pro Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser

200 205 210

Pro Leu Ser Lys Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser

215 220 225

Asn Ser Gin Gly Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val 230 235 240

Asp Ala Leu His Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu

245 250 255

Leu

256 The protein of the present invention is characterized by having a PG I ₂ production stimulating activity, and the activity can be measured by the following method.

In other words, vascular endothelial cells collected from the thoracic aorta intima by the detachment method were subcultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and cultured until they reached a saturated cell density. After adding and incubating for 60 minutes, it is a stable metabolite of PGI _{2 in} the supernatant.

6—Keto—PGF

(Hereinafter referred to as 6-keto PGF 1α), the amount of PGI ₂ produced can be determined. For this measurement, a commercially available 6-keto PGF1α measurement kit may be used.

In general, polypeptides can mutate a portion of the amino acid sequence or the structure of the polypeptide by natural or artificial mutation without altering its intrinsic function. Thus, PG I ₂ production stimulators of the present invention may also contain an array of structures corresponding to homologous mutants of all Amino acid sequence described above.

Escherichia coli [DH5 (pM953) and DH5 (pM954)] transformed with a plasmid (pM953, pM954) containing the DNA of SEQ ID NO: 3 in the sequence listing encoding the PGI 2 production stimulating factor of the present invention are: [Deposit No .: FEEM P-13672 (Deposit: June 3, 1993)], [Deposit No .: FERM P-13673 (Deposit date: June 3, 1993) [FEM BP-4684] and [FEEM BP-4685], respectively, on May 31, 1994.

In order to obtain the PG I 2 production stimulating factor of the present invention by genetic engineering, it is generally used. A host-vector system can be used. For example, when Escherichia coli is used as a host, an expression vector containing Lac UV5, a tryptophan promoter, an IPL promoter, or the like can be used as a promoter. Alternatively, it can be obtained as a fusion protein with S-galactosidase using λ gt11 which is one of the sperm vectors. Furthermore, cultured mammalian cells can be used as a host, and monkey-derived COS cells, Chinese hamster-derived CHO cells, mouse-derived 3T3 cells, and human-derived fibroblasts are suitable. When these are used as hosts, expression vectors containing the SV40 early promoter, the adenovirus major late promoter, the / 5-actin promoter, the polypeptide chain growth factor promoter and the like can be used.

When such a host-vector system is used, the PGPI of the present invention can be obtained by culturing each host under conditions that allow it to grow suitably, and by giving conditions that allow each promoter to function. 2 Production stimulating factor can be obtained. The expression of the PG I ₂ production stimulating factor can be confirmed by the enzyme immunoassay (EIA) described below. The PGI ₂ production stimulating factor of the present invention obtained as described above can be purified by the following method. That is, the culture solution containing the PG I 2 production stimulating factor of the present invention is adsorbed to affinity mouth chromatography, for example, to a column or the like to which an antibody against heparin (HEPARIN) -5PW or PG I 2 production stimulating factor is bound. It can be isolated by elution with a linear concentration gradient method of sodium chloride.

Antibodies that recognize the PG I 2 production stimulating factor of the present invention can be prepared by immunizing a suitable animal with a suitable antigen. The antigen may be a protein represented by the formula [5] or [6] or a peptide fragment thereof. The animal used for immunization is preferably a mouse, a heron, a goat, a sheep, a poma, and the like. Furthermore, a mouse can be used to obtain a monoclonal antibody. Uses of the antibody include affinity chromatography, screening of cDNA libraries, immunological diagnostic methods, and the like. Further, in the immunological diagnosis method, an appropriate method can be selected from an immunoblot method, a radioimmunoassay method, an enzyme immunoassay method, and a fluorescence or luminescence measurement method. The present invention further provides a PGI 2 production stimulator of the present invention, which is effective for the prevention and treatment of the various diseases described below, since PGI ₂ has a platelet aggregation inhibitory action, a smooth muscle relaxing action, a gastric acid secretion inhibitory action, and the like. It is possible to provide a pharmaceutical composition containing the factor as at least one active ingredient.

Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart failure, angina, ischemic heart disease, depression Hematologic heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnancy eclampsia.

The dosage of the PGI 2 production stimulating factor of the present invention varies depending on the sex, age, body weight, type of disease, disease state and dosage form of the patient, but the effective dosage is 111 2 to 2111 8 per day. 2, preferably in the range of 110 118 to 200 8/1 ^ 8.

The dosage form of the pharmaceutical composition of the present invention may be any dosage form capable of supplying an effective amount to the lesion of the disease, such as tablets, powders, powders, capsules, ointments, and sprays. Alternatively, injections and the like can be mentioned. In addition, the pharmaceutical composition of the present invention may contain a commonly used pharmaceutical mixture such as a excipient, a stabilizing agent, or a solubilizing agent, as long as the pharmacological properties are not impaired. Good. Specific examples of the excipient include Ringer's solution, phosphate buffer, human serum albumin, hydrolyzed gelatin, sucrose, dextran, polyethylene glycol, and the like, which are appropriately selected and used depending on the dosage form.

Hereinafter, the present invention will be described more specifically with reference to Examples, but these are only examples of the embodiments of the present invention, and the present invention is not limited thereto. Unless otherwise noted, the scientific terms, abbreviations, and the like in the examples were in accordance with those generally used in the technical field. For basic operations related to gene manipulation, refer to Mania Teis et al., "Molecular Cloning Laboratory Manual" Cold Spring Harbor Laboratory, 1989, Masanori Muramatsu, "Lab Manual Genetic Engineering" Maruzen 1988, etc. Was carried out. Furthermore, the basic operations related to protein purification were carried out with reference to “Biological Chemistry Experiment Course, Vol. 1, Protein I Separation, Purification, and Properties”, edited by the Biochemical Society of Japan, Tokyo Chemistry, 1990. The usage of various devices and reagents was in accordance with the attached instruction manual. (Example 1) Preparation of cDNA library

Step 1 Preparation of normal human diploid fibroblasts

Normal human tetraploid fibroblasts were prepared in 4.8 × 10 ⁴ cells ZmL in Dulbecco's modified Eagle's medium containing 15% fetal serum. 3 L of this cell suspension was inoculated in a rotary culture vessel, and cultured at 37 ° C under 5% carbon dioxide and 95% air. After the cell implantation 3, the culture was replaced with 3 L of Dulbecco's modified Eagle's medium containing fresh 15% fetal calf serum, and the culture was continued for 2 days. Then, the medium was removed, Ca ^2+, the cells were washed with Mg ²⁺ free Dulbecco's monophosphate saline solution was added Hue Norure' de-free Dulbecco's modified Eagle's medium 3 L, 37 The culture was continued for 24 hours at ° C. Then, the medium was removed, Ca ^2+, Mg ²⁺ free physiological phosphate buffer (PBS (-)) washing the cells with, 8 X 1 0 ⁹ pieces from seven rotating culture vessels Cells were obtained.

Step 2 Preparation of total RNA

From normal human diploid fibroblasts, AGPC (Acid Guanidinium Thioio) from Chomczynski, P. et al. [Analytical Biochemistry (Anal. Biochem.) 162, 156, 1987] was used. RNA was prepared according to the cyanate-Phenol-Chloroform method. That is, about 1 × 10 ⁹ cells obtained in step 1 were added to 6 OmL of a 4 M guanidine thiocynate solution (25 mM sodium citrate (pH 7.0), 0.5% sarkosyl and 0.1 M 2-mercapto (Containing ethanol) and shaken vigorously. In addition, add 110 volumes of 2M sodium acetate (pH 4.0), 6 OmL of water-saturated phenol, and 15 volumes of a mixture of formaldehyde and isoamyl alcohol (49: 1) in a sequential order, and mix vigorously for another 10 seconds. After shaking, it was left on ice for 15 minutes. After centrifugation at 7000 xg for 20 minutes at 4 ° C, the aqueous layer was transferred to a new tube. An equal amount of 100% isopropanol as the obtained aqueous layer was added, and the mixture was left overnight at 120 ° C. 4 After centrifugation at 7,000 for 30 minutes, pellet obtained

5 OmL of a 4M guanidine thiocyanate solution (25 mM sodium citrate

(pH 7.0), containing 0.5% sarkosyl and 0.1 M 2-mercaptoethanol), and about 5 OmL of isopropanol was added. 1 o'clock at 20 ° C After standing for a while, the mixture was centrifuged at 4 ° C and about 7,000 xg for 30 minutes. The pellet was dissolved in about 30 mL of 0.5 mM EDTA, and ethanol was added thereto in the presence of sodium chloride to precipitate RNA, thereby obtaining about 9 mg of total RNA.

Step 3 Preparation of poly (A) + RNA

Poly (A) + RNA was separated from total RNA using an oligo (dT) cellulose column. Specifically, 50 Omg of oligo (dT) cellulose resin was suspended in 20 OmM tris-hydrochloric acid buffer solution (containing pH 7.5.2 OmM EDTA, 400 mM sodium chloride and 0.1% SDS), and the volume was about 2 mL. Was prepared. Dissolve the total RNA obtained in step 2 in 3.5 mL of 0.5 mM EDTA, heat treat it at 65 ° C for 5 minutes, quench, and equilibrate an equal volume of 40 OmM Tris-HCl buffer solution (pH 7.5, pH 7.5 4 OmM EDTA. 800 mM sodium chloride and 0, 2% SDS) were added. Apply this solution to an oligo (dT) cellulose column equilibrated with the above 20 OmM Tris-HCl buffer solution (pH 7.5, containing 20 mM EDTA, 40 OmM sodium chloride and 0.1% SDS), and adsorb RNA I let it. The column was washed with 1 OmL of 10 OmM Tris-HCl buffer solution (pH 7.5, 1 OmM EDTA. 20 OmM sodium chloride and 0.1% SDS). Next, poly (A) + RNA was eluted with 1 OmL of sterilized distilled water, and ethanol was added thereto in the presence of sodium chloride to precipitate poly (A) ⁺ RNA according to a conventional method. About 270 g of poly (A) ⁺ RNA (mRNA) was obtained.

Step 4 Synthesis of double-stranded cDNA

Double-stranded cDNA using a cDNA synthesis system kit (Amersham) constructed based on an improved version of the method of Gubler and Hoffman [Gene, 25, 263, 1983]. Was synthesized.

A buffer for 5X single-stranded cDNA synthesis reaction attached to the cDNA synthesis system kit, sodium pyrophosphate solution, human placental ribonuclease inhibitor, mixed solution of deoxynucleoside triphosphate, 925KB Q [ ³² P] dCTP solution and oligo (dT) primer were mixed in order. To this solution, the 1 O ^ g poly (A) + RNA solution obtained in step 3 was added and gently mixed, and then 200 U of reverse transcriptase was added. The reaction solution was 100 L. Mix gently and stir at 42 ° C for 40 minutes. It was cupped.

Buffer for double-stranded cDNA synthesis reaction attached to the above-mentioned cDNA synthesis system kit, 8 U E. coli ribonuclease 11, 230 U E. coli DNA polymerase I, 3.7 MBq [ ³² P] d CTP The solutions were added sequentially, adjusted to a total volume of 500 / L with sterile water, and gently mixed. The tube was reacted at 12 ° C for 60 minutes and then at 22 ° C for 60 minutes, and then incubated at 70 ° C for 10 minutes.

The reaction solution was returned to an ice bath, 20 U of T4 DNA polymerase was added, and the mixture was gently mixed, followed by reaction at 37 ° C for 10 minutes. The reaction was quenched by adding 20 / L 0.25M EDTA (pH 8.0) to the reaction solution, extracted with funor chloroform, and precipitated with ethanol in the presence of ammonium acetate. A single-stranded cDNA was obtained.

Process 5 Addition of Bs tXI linker

BstXI linker manufactured by Invitrodin was added to the double-stranded cDNA. The method was carried out using a DNA ligation kit (Takara Shuzo) according to the attached manual.

That is, 15 L of a solution obtained by adding about 500 ng of BstXI linker to about 500 ng of the double-stranded cDNA synthesized in Step 4 (100 mM Tris-HCl buffer (pH 7.6), 5 mM magnesium chloride, 300 mM (Containing sodium chloride). Next, 15 L of an enzyme solution (DNA Ligation Kit Solution B, hereinafter referred to as Solution B) was added, mixed well, and reacted at 10 ° C for 3 hours. Heat treatment was performed at 70 ° C for 10 minutes, and ethanol precipitation was performed in the presence of ammonium acetate. Next, electrophoresis was performed on a low-melting-point agarose gel, fractions of 500 bp or more were collected, heat-treated at 65 ° C to dissolve the gel, and then extracted with phenol Z-cloth form.After that, ethanol was added in the presence of sodium chloride. Settled. Finally, about 100 ng of linker-added cDNA was recovered.

Step 6 Introduction of cDNA vector ^ ^

The cDNA was introduced into a plasmid vector using a DNA ligation kit (Takara Shuzo).

That is, Plasmid p EF—BOS [Nucleic Acid Research (Nucl. Acids Res.), 18, 5322, 1990], and the large fragment was recovered by digestion with the restriction enzyme BstXI. A DNA solution containing 100 ng of the vector prepared in this manner and about 20 ng of the linker-added double-stranded cDNA obtained in step 5 was added to 4.5 μL of a 36 zL reaction buffer (DNA buffer). (A solution kit A) was added and mixed well. Next, 4.5 μL of the enzyme solution (solution B) was added, mixed well, and allowed to react at 16 ° C. overnight. The plasmid was precipitated with ethanol in the presence of ammonium acetate and dissolved in 20 zL of TE solution.

Step 7 Preparation of cDNA library

DNA was introduced into E. coli using the electroporation method. Elect opening Poreshiyon method, de Wa one et al. [Nucleic Ashi' de research (Nu c l. Ac ids Re s.), L 6 , pp. 6127, 1988] off the real h according to the method of ₀

E. coli MC1061ZP3 was inoculated from a single colony into 1 OmL of L-broth and cultured overnight. One OmL of the overnight culture was inoculated into 1 L of L-broth and cultured at 37 ° C until OD βοοηπ, = about 0.5. After the culture solution was ice-cooled, the cells were collected by centrifugation at about 7000 X g for 15 minutes at 4 ° C. After removing the supernatant by decantation, the suspension was resuspended in 1 L of ice-cold sterilized water. After centrifugation at about 7000 X g at 4 ° C for 15 minutes, the supernatant was removed, and the cells were suspended in 50 OmL of ice-cold sterilized water, centrifuged at about 7000 xg for 15 minutes at 4 ° C, and the supernatant was removed. The suspension was resuspended in 2 OmL of ice-cold 10% glycerol, centrifuged at about 3500 xg for 15 minutes at 4 ° C, and the supernatant was removed. The cells were resuspended in 2 mL of ice-cold 10% glycerol and dispensed in 40 to 50 portions into 1.5 mL tubes. A 10% glycerol solution containing Escherichia coli was rapidly frozen and frozen in ethanol containing dry ice, and stored frozen at about 170 ° C.

The solution in the 12 cryopreservation tubes was thawed on ice, and the DNA solution 1 prepared in step 6 was added per tube. After transferring the DNA-E. Coli mixture to a cuvette (distance between electrodes: 0.1 cm, manufactured by Bio-Rad), the conditions of electroporation (Gene Pulser, Bi0-Rad, Capacitance: 25

, Voltage: 1.8 kV, pulse controller: 200Ω), a pulse was applied once, and 1 mL of SOC medium was added to the cuvette to suspend E. coli. Suspension The solution was transferred to a culture tube and cultured for 1 hour. Then, an appropriate amount was spread on an LB plate containing 50 μg ZmL of ampicillin, and cultured at 37 ° C. for 1 hour. By the above operation, about 600,000 clones of the transformant were obtained.

(Example 2) Acquisition of cDNA encoding PG I 2 production stimulating factor

Step 1 Preparation of primer

Among the partial amino acid sequences determined in Reference Example 2, the DNA sequence encoding the N-terminal and C-terminal amino acid sequences of the 20 amino acid sequence represented by Formula [7] (SEQ ID NO: 8 in the sequence listing) was converted to a 394 DNAZRNA synthesizer. (Applied Biosystems) using the phosphoramidite method. That is, a DNA mixture consisting of 20 bases represented by the following formula [8] (SEQ ID NO: 9 in the sequence listing) as a DNA encoding a part of the N-terminal amino acid, and a DNA encoding a part of the amino acid at the C-terminal As a complementary strand sequence of DNA, a mixture of 20 bases of DNA represented by the following formula [9] (SEQ ID NO: 10 in the sequence listing) was prepared.

Formula [7] lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys Lys Gly

1 5 10 15

Glu Gly Ala Glu Leu

20

Formula [8] 5'-ATA ACA GTA GTA GAC GCA CT-3 '

C G G G T G T T C C C C T T T T

Formula [9] 5, -AA CTC AGC ACC CTC ACC CTT-3 '

GTGGTGTCCCTTT Using the above DNA mixture as a primer, the poly (A) + RNA prepared in step 3 of Example 1 was subjected to an RT-PCR method using the 铸 -form. That is, Super S cript ™ Preamp lification

Single-stranded cDNA was synthesized using System (manufactured by Lifetech Oriental) according to the attached manual. Add oligo (dT), 10X synthesis buffer, dNTP mixture, DTT, and 6000 U reverse transcriptase to 3 g of poly (A) + RNA to make a total volume of 600 L, 42 ° C for 2 hours, then 90 ° C For 5 minutes. After quenching on ice, 60 U (30 L) of RNaseH was added, and the mixture was incubated at 37 ° C for 20 minutes.

Add 1 Lg sense primer, 1 g antisense primer, 8 〃L 1 OxPCR buffer (1 O OmM Tris-HCl buffer (pH 8.3), 5 O OmM potassium chloride, 15 mM magnesium chloride, 0.01% (WZV) gelatin) and 2.5 U of Ampli Taq DNA. Polymerase were added to make the total amount 100. DNA Th e rma l Cy cler TM is set to (Perkin Elmer one Cetus Instruments Inc. _{(P er ki η E l me} r C etus I nstr ume nts)), 1 minute at 94, at 55 ° C The reaction for 2 minutes and 3 minutes at 72 ° C was repeated 30 times. Next, the reaction product was electrophoresed on a 10% polyacrylamide gel, and it was confirmed that a band appeared at around 59 bp. Next, the obtained amplification product DNA fragment of about 59 bp was extracted according to a conventional method, the ends were blunted using a conventional method using T4 DNA polymerase, and cloned into a small site of plasmid PUC118. The nucleotide sequence of the DNA fragment was determined according to the sequencing method described later. The sequence is shown by the following formula [10] (SEQ ID NO: 11 in the sequence listing).

Formula [10] 5'-ATTACGGTGG TTGATGCGTT ACATGAAATA CCAGTGAAAA

10 20 30 40

AAGGCGAAGG CGCCGAATT-3 '

50 59 A 20-base DNA fragment having a base sequence from 11 to 30 of the base sequence shown in Formula [10] (Formula [11] (SEQ ID NO: 12 in the sequence listing) and a base sequence from 31 to 50) A DNA fragment of 20 bases that is complementary to It was synthesized in the same manner as described above, and was used as a primer for screening by the PCR method.

Formula [11] 5 '-TTGATGCGTTACATGAAATA-3'

Formula [12] 5, -CCTTCGCCTTTTTTCACTGG-3 'Step 2 Screening of cDNA library of normal human diploid fibroblasts by PCR method

The transformant obtained in Example 1 was divided so as to contain about 14,000 clones per pool, and 46 pools were prepared.Each of them was inoculated into 3 mL of LB medium containing 50 μg ZmL of ampicillin, and inoculated at 37 ° C. With shaking overnight. Plasmid DNA was extracted according to the method described in Molecular Cloning Laboratory Manual (Cold Spring Harbor Laboratory 1989). That is, 1.5 mL of the culture solution was centrifuged at 5000 rpm for 3 minutes in a micro high-speed centrifuge to remove the supernatant, and the precipitate was concentrated in 100 L of a suspension solution (5 OmM glucose, 10 mM EDTA, The cells were suspended in a 25 mM Tris-HCl buffer solution (pH 8.0) and left at room temperature for 5 minutes. Add 200 zL alkaline solution (containing 0.2N sodium hydroxide and 1% SDS) in ice, mix gently, and leave on ice for 5 minutes. Then add 150 L of ice-cold potassium acetate solution. Mix well and leave on ice for 5 minutes. After centrifugation at 12000 rpm for 5 minutes at 4 ° C in a micro high-speed centrifuge, transfer the supernatant to another tube, add 400 μL phenol-chloroform solution, mix well, and transfer to a micro high-speed centrifuge. And centrifuged at 12000 rpm for 5 minutes. Two volumes (about 800 / L) of ethanol were added to the aqueous layer, mixed, and allowed to stand at room temperature for 2 minutes. After centrifugation at 12000 rpm for 5 minutes, the supernatant was completely removed, and the precipitate was washed with 70% ethanol, dried, and dissolved in a 50 / L TE solution containing 20 zg / mL RNaseA. 2.5 〃 of the obtained plasmid DNA solution was treated with the restriction enzyme & I at 37 ° C for 1 hour and cut.

Next, the reaction solution containing the above DNA solution was prepared using Gene Amp PCR Reent Kit (manufactured by Perkin Elmer Cetus Instruments, Inc.). / L (1 OmM Tris-HCl buffer solution (pH 8.3), 50 mM potassium chloride, 1.5 mM magnesium chloride, 200〃M dNTP each, l OngZmL sense primer, 10 ngZmL antisense primer and 0.025 U / / L The sample was prepared in a Gene Amp ™ PCR System 9600 (Perkin Elmer Cetus Instruments, Inc.), and then set in Gene Gene ™ PCR System 9600 (Perkin Elmer Cetus Instruments, Inc.). The reaction was repeated 25 times for 30 seconds at 55 ° C, 30 seconds at 55 ° C and 1 minute at 72 ° C. Next, the reaction product was electrophoresed on a 10% polyacrylamide gel, and a band around 40 bp was detected as positive. Escherichia coli DH5 was transformed with the plasmid DNA of one positive pool, and the obtained transformants were divided so as to contain about 1800 clones per pool, and 15 pools were prepared. A was prepared, and a band in which a band was detected around 40 bp by the PCR method was determined to be positive. Thereafter, this operation was repeated, and two positive clones were identified by a total of six screenings.

(Example 3) Determination of base sequence

The nucleotide sequence was determined according to the method described in Lab Manual Genetic Engineering (Masami Muramatsu, edited by Maruzen 1988). That is, the plasmid DNA of the positive clone obtained in Example 2 was digested with the restriction enzyme XbaI, and the excised DNA fragment was mixed with pUC119 digested with the restriction enzyme XbaI, and the ligation was performed as described above. Ligation was performed using a kit. Plasmids obtained by this operation were named pM950 and pM951. After transforming Escherichia coli JMl09 with pM950 and pM951, single-stranded DNA was prepared according to a conventional method. Using a DNA sequencer (373A, manufactured by Applied Biosystems), the nucleotide sequence of each single-stranded DNA prepared in accordance with the attached manual was determined.

As a result, the nucleotide sequences encoding the PGI 2 production stimulating factor of the present invention contained in plasmids PM950 and PM951, respectively, are shown in SEQ ID NOs: 7 and 14 in the sequence listing. The amino acid sequence deduced from the DNA sequence is shown in SEQ ID NO: 15 or 16 in the sequence listing.

(Example 4) Preparation of sphage cDNA library

The sphage cDNA library was prepared using a cDNA cloning system; Igt10 (manufactured by Amersham) according to the attached manual.

Step 1 Add EcoRI—BamHI—Kpnl—NcoI Adapter

A double-stranded cDNA was obtained in the same manner as in Step 4 of Example 1 using a random primer instead of the oligo (dT) primer. Double-stranded cDNA synthesized here 1.

Dissolve 2 g in 27 L of sterile pure water, add L / K buffer, 5 L Eco

RI—BamHI—Kpnl—Ncol adapter and 4 L of T4DNA ligase were added. Mix gently and allow to react at 15 ° C. EcoRI—Bam

An HI-Kpnl-Ncol adapter was added to the synthesized double-stranded cDNA. After the reaction, gel filtration was performed using an NI P517 column, and fractions of 500 bp or more were collected. Next, 350 μL of cDNA, 40 L of LZK buffer and 10 αL of T4 polynucleotide kinase were added to this fraction, and reacted at 37 ° C. for 2 hours. EcoRI-BamHI-Knl-NcoI adapter added with ethanol The cDNA was precipitated, and finally about 150 ng of phosphorylated adapter-added cDNA was obtained.

Step 2 Introduction of cDNA vector ^ ^

cDNA cloning system; 20 ng of double-stranded cDNA was added to 1 〃g of λΐ10 digested with Ec0RI attached to Igt10 (manufactured by Amersham), and 1 〃L of LZK buffer and 1 〃L of After adding pure water and 1 / L T4 DNA ligase, the mixture was gently mixed. The total amount of the reaction solution was 10 L, and the mixture was allowed to react at 15 ° C. This was carried out for two reactions.

Process 3 inv i tr o packaging

The recombinant phage DNA obtained in step 2 was packaged using an in vitro packaging kit (Stratagene) according to the attached manual. That is, 5 L each of the recombinant phage DNA solution prepared in Step 2 was added to 25 packaging extracts, and incubated at 22 ° C for 2 hours to obtain a phage particle mixed solution.

Step 4 Preparation of cDNA library

The phage particle mixture obtained in Step 3 was diluted to 5 × 10 ⁴ pfu / 200 / zL with SM buffer. 5 OmL of E. coli NM514 culture cultured in L medium containing _0.4 % maltose until OD _600nra = 0.5 was centrifuged at 900 xg at 4 ° C. The obtained precipitate was suspended in 15 mL of a 1 OmM magnesium sulfate solution. Mix 200 L of this NM5 14 suspension with 200 L of the phage dilution described above, incubate at 37 ° C for 15 minutes, mix with 1 OmL of soft agarose (0.7% agarose ZL medium), and mix It was overlaid on the L plate of diameter. This was incubated at 37 ° C for 10 hours. That is, 50,000 clones per plate, consisting of 600,000 clones in a total of 12 plates; a phage I cDNA library was obtained.

(Example 5) Screening of sphage cDNA library

Step 1 Preparation of labeled probe

Labeled probes were prepared using a random primer DNA labeling kit (Takara Shuzo) according to the attached manual. pM951 was treated with restriction enzymes Pvull and SmaI, and about 100 ng of the obtained 265 bp DNA was recovered and dissolved in 20 μL of TE. 8 L of this solution and 2 L of random primer were mixed, incubated at 95 ° C for 3 minutes, and cooled with ice for 5 minutes. Add 2.5 zL of 10X reaction buffer, 2. dNTP mixture, 1. 85 MBQ [ ³² P] dCTP solution, 4 μL of sterile pure water and 1 / L of K 1 en The ow fragment was added and incubated at 37 ° C for 2 hours. Thereafter, the mixture was incubated at 65 ° C. for 5 minutes, and the reaction solution was gel-filtered through a nick column G-50 (manufactured by Pharmacia) to fractionate, and the radioactivity of each fraction was measured. Step 2 Screening of sphage cDNA library

Screening of the λ phage cDNA library was performed by a plaque hybridization method according to a conventional method.

A Hy bond N filter (manufactured by Amersham) was superimposed on one of the λ phage cDNA libraries obtained in Example 4 to prepare a replica filter. The resulting replica filter was subjected to alkaline denaturation treatment with 0.5 M sodium hydroxide containing 1.5 M sodium chloride for 5 minutes, and then treated with 1.5 M sodium chloride-containing 0.5 M tris-chloride buffer (pH 7.0). The mixture was neutralized twice for 2 minutes, washed with 2XSSC, and air-dried. This was irradiated with 0.72 J of UV using a UV irradiator (UV stratulin ker ^™ 2400, manufactured by Stratagene) to fix the DNA.

Twelve replica filters prepared by this method are immersed in 5 OmL of a prehybridization solution (6xSSPE, 5xDenhardt's, 50% formamide, 0.1% SDS, 250 ^ gZmL salmon sperm DNA) at 42 ° C. The incubation was continued. The filter was then combined with 5 OmL of hybridization solution (6xSSPE, lxDenhardt's, 50% formamide, 0.1% SDS, 10% dextran sulfate, 100-111 1 ^ salmon sperm). 5 X 10 ⁷ cpm labeled probe obtained in step 1)

The mixture was incubated at 42 ° C. After hybridization, 2XSSC

/ 0. Washed three times with 1% SDS at room temperature for 20 minutes. Then 0.lxSSC

/ 0. Washed twice with 1% SDS at 44 ° C for 30 minutes. In addition, 0.lxS

The membrane was washed once with SC at room temperature for 10 minutes, and air-dried. At Autoradiography Radioactivity was detected, and 10 positive clones were detected. In order to isolate these 10 positive clones, secondary screening was performed in the same manner as in this step, and 8 positive clones were isolated.

(Example 6) Determination of base sequence

Of the eight positive phage clones obtained in step 2 of Example 5, the DNA of the clone having the longest cDNA was digested with the restriction enzyme BamHI. The excised cDNA fragment was mixed with pUC118 digested with the restriction enzyme BamHI and ligated using the above-described ligation kit. The nucleotide sequence of the obtained plasmid was determined in the same manner as in Example 3.

Shows the DNA sequence encoding the PG I ₂ production stimulators of the present invention in SEQ ID NO: 3 and 4 in Sequence Listing, The amino acid sequence deduced from the DNA sequence in SEQ ID NO: 1 and 2 in Sequence Listing.

The plasmid containing DNA shown in SEQ ID NO: 3 in the sequence listing was named pM952. (Example 7) Expression in COS cells

Step 1 Construction of expression vector

Expression vector ρEF—BOS [Nucleic Acid Research (Nucl. Acids Res.), 18, 5322, 1990] After digesting 2 g with BstXI, large fragments were recovered. One-half of the DNA and l と g of each of the synthetic DNAs of the following formulas [13] and [14] (SEQ ID NOS: 17 and 18 in the sequence listing) were ligated using a DNA ligation kit (manufactured by Takara Shuzo), and the expression vector pEF was obtained. I got BOS—KS.

Formula [13] 5'-CTGGTCGACGGATCCCGGGTACCAGCACA-3 '

Formula [14] 3′-ACACGACCAGCTGCCTAGGGCCCATGGTC-5 Then, after digesting pM952 with the restriction enzyme NcoI, a DNA fragment of about 1. 1 kbp is recovered, and the obtained DNA fragment is subjected to DNA branching kit ( (Takara Shuzo Co., Ltd.). After digestion of the expression vector pEF-BOS-KS with the restriction enzyme SmaI, a large DNA fragment was recovered. DNA ligation with blunted DNA fragments Ligation was performed using a kit (Takara Shuzo Co., Ltd.) to obtain the expression vector pM953 (Fig. 1).

Step 2 Introduction of expression vector pM953 into COS cells

The expression vector was introduced into COS cells by the method of Sompoyrac et al. [Proceding of the National Academy of Sciences of the United States of America. d. Sci. USA). 78, 7575, 1981].

Prepare 3 x 10 ⁵ COS cells in 2 mL of DME medium containing 10% fetal bovine serum, seed in a 3.5 cm petri dish, and in the presence of 5% carbon dioxide at 37 ° C for 20 to 24 hours Cultured. The medium was removed using an aspirator, and the cells were washed three times with .DME medium.

Next, a DNA-DEAE dextran mixture was prepared. That is, 10 TE solutions containing 0.5 g pM953, 20 mg ZmL DEAE dextran solution, 35 aL 1 M Tris-HCl solution (pH 7.4) and 648 / zL DME medium were mixed. Next, a mixture of DNA-DEAE dextran was gently overlaid on the COS cells, and cultured at 37 ° C for 4 hours in the presence of 5% carbon dioxide. After gently removing the DNA-DEAE dextran mixture with a Pittman, the cells were washed with DME medium, and DME medium was newly added. After culturing at 37 ° C for 96 hours in the presence of 5% carbon dioxide, measurement was performed by the EIA method described in Example 11, and PG I

2 The expression of production stimulating factor was confirmed.

(Example 8) Expression in CHO cells

Step 1 Construction of expression vector

After digesting the expression vector PM953 constructed in Step 1 of Example 7 with restriction enzymes XbaI and NotI, a DNA fragment of about 1.2 Kb was recovered. dh f r (di hy d r fo l a t e r e d u c t a se) having an expression unit, and capable of controlling the expression of the foreign gene by the EF promoter, at a foreign gene insertion site of an expression vector,

This fragment was ligated using a DNA ligation kit (Takara Shuzo). Then, the desired expression vector pM954 was obtained (FIG. 2).

Step 2 Introduction of expression vector pM954 into CHO cells

The electroporation method was used to introduce the expression vector into CHO cells. That is, using a Gene Pulser (B i 0- Rad Co.), in cuvette preparative (electrode distance 0. 4 cm), CHO cells 4. shoe containing 0 x 10 ⁶ and p M 954 20〃G The phosphate buffer containing sucrose was subjected to an electric pulse with a voltage of 0.40 kVZm and a capacitance of 25 F. The resulting pM954ZCHO culture supernatant was measured by EIA method shown in real施例11 was sure the expression of PG I ₂ Stimulating Factor. For the expressed strain, gene amplification was performed using methotrexate (manufactured by Nippon Redley Co., Ltd.), and a high expression strain was selected.

(Example 9) Purification of PG I 2 production stimulating factor

Process 1

An ultrafiltration apparatus in which 600 mL of the culture supernatant of pM953ZCOS cells prepared according to step 2 of Example 7 was set with an ultrafiltration membrane YM10 (fraction molecular weight: 10 kDa)

(Diaflow, manufactured by Grace Japan Co., Ltd.). When the solution was concentrated to 20 mL, it was centrifuged and dialyzed against 2 OmM Tris-HCl buffer (pH 7.5). Thereafter, the mixture was filtered through a 0.22 m filter (Mirex GV, manufactured by Nippon Millipore).

Process 2

The culture supernatant of the PM953 ZC OS cells prepared according to step 1 is

OmM Tris monohydrochloride-Heparin -5PW (manufactured by Tosoh Corporation) equilibrated with 0.1 M sodium chloride buffer (pH 7.5) In the eluate to which sodium chloride was added based on pH 7.5), elution was performed with a linear gradient of 0.1 to 1.0 M sodium chloride, and the elution profile was measured at an absorption wavelength of 280 nm. The concentration of the PG I 2 production stimulating factor was measured for each eluted fraction by the EIA method described in Example 11, and eluted when the concentration of sodium chloride was 0.3 to 0.6 M.

Fractions containing PG I 2 production stimulating factors were pooled. Process 3

The eluted fraction obtained in step 2 was dialyzed against 50 mM Tris-HCl buffer (pH 7.5) and applied to an antibody column previously equilibrated with the same buffer. Antibodies column is purified by antiserum base one force one bond A Bx column PG I ₂ production stimulators produced in Example 10 (di We one 'made tea Baie one force one company), formyl one Serurofa (Manufactured by Seikagaku Corporation). After adsorbing the protein in the eluted fraction, the adsorbed protein was eluted with 4.5 M magnesium chloride Z5 OmM Tris-HCl buffer (pH 6.4). Analysis of the adsorbed protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions confirmed that the PG I ₂ production stimulating factor was almost uniformly purified ( (Figure 3).

(Example 10) Method for producing antibody

A total of 13 amino acids with a cysteine added to the N-terminal of the C-terminal 12 amino acids of the formula [7] were synthesized using an automatic peptide synthesizer (43 OA type Applied Biosystems). An antigen for immunization was prepared by binding to the carrier protein KLH (keyhole lysate mosocyanin) via the N-terminal cysteine of the synthetic peptide.

The animals used were New Zealand White female egrets.

The immunization was carried out according to the method of MV Sofroniew et al. [Fresenius Zeissrift Fuyer Analytic Chemie. 290, 163, 1978]. That is, the antigen for immunization and complete Freund's adjuvant (FCA, manufactured by Difco Laboratories) or incomplete Freund's adjuvant (FIA, manufactured by Difco Laboratories) were sufficiently mixed and emulsified at 1: 1 (vZv). This was repeatedly administered to 20 or more sites in the back skin of the egret and immunized. After immunization five times every two weeks, the whole blood of the animal was collected to obtain antiserum. The obtained antiserum was stored at 140 ° C until use. (Example 11) Enzyme immunoassay (EIA)

As a reaction system, a competitive inhibition system in which the reaction between the antigen immobilized on the plate and the labeled antibody was quantified by blocking the reaction with the antigen in the sample was used. That is, the culture supernatant of the transformant diluted 10-fold is added to a 96-well culture dish at 100 L / well, and reacted at 4 ° C for 1 minute or at 56 ° C for 30 minutes, and then washed to fix the antigen. Phased. To this, 0.5% BSA / PBS was added in 150 LZ holes at a time, and reacted at 25 ° C for 1.5 hours or more, washed, and blocked. Next, anti-PG I 2 -producing stimulating factor antibody 110; / L, which had been labeled with 4 gZmL of horseradish peroxidase (HRPO) in advance, and 110 L of the sample were mixed, and pre-incubation was performed at 25 ° C. for 1 hour. This solution was added in 100 zz LZ holes, and reacted at 25 ° C. for 2 hours. Further 3 mg / mL of Orutofuwe two Renjiamin (OP.D) / 0. 027% H 2 0 2 Nomatsukiru base in buffer (pH 5. 0) to (MCB) was added in 100 z LZ holes and reacted for 10 minutes The color was developed, and 2N sulfuric acid was added in 100 LZ holes to stop the reaction. Color development was measured by absorbance at 490 nm.

(Example 12) Toxicity test

Using the PG I 2 production stimulating factor of the present invention obtained in Example 9, a toxicity test in mice was performed. That is, the PGI ₂ production stimulating factor obtained in Example 9 was intravenously administered once a week for one week for 5 weeks each to male and female 6-week-old ICR mice. The general characteristics of each animal were observed throughout the administration period. In this study, no deaths were observed in any of the animals in the highest dose group, 1 Omg / kg group, and there was no change in general characteristics.

(Example 13) Preparation of solution formulation for injection

10 Omg of the PG I2 production stimulating factor of the present invention obtained in Example 9 was dissolved in 10 OmL of a physiological saline solution containing 1 OmgZmL of hydrolyzed gelatin, and a filter having a pore size of 0.22im (Mirex GV Millipore The solution was sterilized by filtration using _c ). Aseptically, 2 mL of this solution was dispensed into glass vials and sealed, to give a solution for injection. (Example 14) Preparation of freeze-dried preparation for injection

10 Pmg of the PG I2 production stimulating factor of the present invention obtained in Example 9 was dissolved in 100 mL of 1 OmM PBS (pH 7.4) containing 10 OmgZ mL of human serum albumin, and the pore size was 0.22. The solution was sterilized by filtration using a zm filter-1 (Mirex GV Millipore). This was aseptically dispensed into glass vials in 3 mL portions, freeze-dried and sealed to give a freeze-dried preparation for injection.

(Reference example 1)

A culture supernatant was prepared by the following method, and a PG I ₂ production stimulating factor was purified to obtain a PG I 2 production stimulating factor. The measurement of PG I ₂ production stimulating activity is performed by the method described later.

(1) Preparation of culture supernatant of normal human diploid fibroblasts

4. Normal Hitoni fold body fibroblasts at 15% © shea fetal serum-containing Dulbecco's modified Eagle's medium was prepared in 8Xl0 ⁴ cells ZML. 3 L of this cell suspension was inoculated in a rotary culture vessel, and cultured at 37 ° C under 5% carbon dioxide and 95% air. After the cell implantation 3, the culture was replaced with 3 L of Dulbecco's modified Eagle's medium containing fresh 15% fetal calf serum, and the culture was continued for 2 days. Next, the culture solution was removed, and the cells were washed using a Dulbecco's monophosphate-free saline solution containing no Ca ²⁺ or Mg ²⁺ , and then 3 L of Dulbecco's modified Eagle's medium containing no norred was added. The cells were cultured at 37 ° C for 2 days. After collecting the culture, the cells were filtered through a 2.5 / zm filter (CN cartridge, 30 inch, manufactured by Millipore) to remove cell debris. Next, a two-stage process using a hollow fiber module (Molsep fiber FS-106 kDa cut-off, manufactured by Daicel Chemical Industries) and an ultrafiltration cassette (Omega mini-set, manufactured by Fuji Filter Industries, Ltd.) with a cut-off molecular weight of 1 OkDa And concentrated. The concentrated solution was dialyzed against 2 OmM Tris-HCl buffer (pH 7.8) at 4 ° C for 1 ° C using a dialysis tube having a molecular weight cutoff of 3.5 kDa (manufactured by Spectrum). Dialyze against fresh buffer of the same composition for another 8 hours at 4 ° C. 1. Filter

(DF Assembly, manufactured by Nippon Pall Corporation), 0.22 m filter system Filtration was performed successively through a system (manufactured by Koningu Co., Ltd.).

(2) Purification of PG I 2 production stimulating factor

Process 1

DEAE-5PW (manufactured by Tosoichi) anion-exchange chromatography column in which the culture supernatant of normal human diploid fibroblasts prepared according to (1) was previously equilibrated with 20 mM Tris-HCl buffer (pH 7.8) And the components in the supernatant were adsorbed. 2 Adsorbed components are eluted with a linear gradient elution solution of 0 to 1.0 M sodium chloride prepared based on OmM Tris-HCl buffer (pH 7.8), and absorbance of each fraction is simultaneously measured at 280 nm. did. The PG I 2 production stimulating activity of each eluted fraction was measured according to the method described below, and the fractions having PG I 2 production stimulating activity eluted with the eluate having a sodium chloride concentration of 50 to 15 OmM were pooled. (Figure 4). .

Process 2

Heparin (HE PAR IN) was eluted from the active fraction obtained in step 1 with 1 OmM phosphate buffer (pH 7.4) and equilibrated with 1 OmM phosphate buffer (pH 7.4). — 5PW (manufactured by Tosoichi Co., Ltd.) The active ingredient is adsorbed on an affinity column, and 0 to 1.0 M of the eluate containing sodium chloride added based on 1 OmM phosphate buffer (pH 7.4) is used. Elution was performed with a linear concentration gradient of sodium chloride, and the elution profile was measured by absorbance at 280 nm. The PG I ₂ production stimulating activity of each eluted fraction was measured, and the fractions having the PG I 2 production stimulating activity eluted when the concentration of sodium chloride was 450 to 500 mM were pooled (FIG. 5).

Process 3

The active fraction obtained in step 2 was concentrated with Centricon-1 10 (Amicon). Two columns of PROTE IN-PAK 300 (Nippon Millipore Limited Waters Chromatography Division) pre-equilibrated with 1 OmM phosphate buffer (pH 7.4) were connected, and the concentrated fraction was subjected to gel filtration. The elution profile was simultaneously measured at an absorption wavelength of 280 nm. When the activity of stimulating PG I 2 production in each fraction after elution was measured, the activity was observed at a molecular weight of around 30 kDa (FIG. 6). The numbers in FIG. 6 represent the molecular weight (kDa), Vt represents the total volume of the filler, and Vo represents the elimination volume. Process 4

The active fraction obtained in step 3 was applied to an IGF-affinity column equilibrated with 1 OmM phosphate buffer (pH 7.4). As an IGF-affinity column, a pressure-resistant ram in which recombinant IGF-I was bound as a ligand to Affibrep 10 (manufactured by Nippon Bio-Rad Laboratories) was used in this experiment. After adsorbing the active components, the adsorbed components were eluted with 0.5M acetic acid. When the PGI ₂ production stimulating activity of the adsorbed fraction and the non-adsorbed fraction was measured, the PGI ₂ production stimulating activity was observed in the non-adsorbed fraction.

Process 5

Step 0.1% The active fractions obtained in 4 Torifuruoro acetate containing 10% Asetonito C ₄ reverse phase HPLC column equilibrated with Lil aqueous solution (manufactured by Waters Corporation) adsorbed to _c columns subjected to the components 0.1% Elution was carried out with a linear gradient eluent of 10 to 60% acetonitrile in a trifluoroacetic acid noacetonitrile solution. The PG I 2 production stimulating factor of the present invention eluted as a single peak. (Reference Example 2) Measurement of physical properties of PG I 2 production stimulating factor derived from normal human diploid fibroblasts

① Measurement of molecular weight

The molecular weight of the PG I 2 production stimulating factor of the present invention obtained in Step 5 of Reference Example 1 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. It was recognized as a single band at a molecular weight of about 33 kDa (Fig. 7). FIG. 7 is a schematic diagram of a photograph showing the results after SDS-PAGE electrophoresis. In FIG. 7, lines A and C are bands of a standard sample having a known molecular weight, and line B is a band of the PG I ₂ production stimulating factor of the present invention. The numbers indicate the molecular weight (kDa) of the standard sample.

②Analysis of amino acid sequence

The PGI ₂ production stimulating factor of the present invention obtained in Step 5 of Reference Example 1 was digested with trypsin to obtain a peptide fragment. Balancing these fragments at each 0.1% Torifuruoro aqueous acetic acid was C ₈ reversed-phase H PLC column (Separeshiyon And eluted with a linear gradient elution solution of 0.08% trifluoroacetic acid / acetonitrile of 0 to 100% acetonitrile. The amino acid sequence of each peptide fragment was determined by Edman degradation using an automated gas phase sequencer (Applied Biosystems 477A-120A).

Then, the PG I 2 production stimulators of the present invention reduced carboxymethylated and desalted by C ₈ reversed-phase HP LC column (manufactured by Nippon Waters Co., Ltd.). Subsequently, peptide fragments were obtained by digesting with endoproteinase Glu-C (protease V8). These fragments were treated in the same manner as described above, and the amino acid sequence of each peptide fragment was determined.

The obtained amino acid sequences are shown in the following formulas [7] and [15:] to [25] (SEQ ID NOS: 8 and 19 to 29 in the sequence listing).

Equation [7]

lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys Lys Gly

1 5 10 15

Glu Gly Ala Glu Leu

20

Equation [15]

Ser Xaa Xaa Asp Thr Xaa Gly Pro

1 5 8

Equation [16]

Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys Val Lys 1 5 10 15

Arg Gly His Tyr Gly Val Gin Arg Thr Glu

20 25

Expression [17]

Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly 1 5 10 15

Gly Pro Glu

Equation [18] Cys His Ala Ser Asn Ser Gin Gly Gin Ala Ser Ala Ser Ala Lys 1 5 10 15 lie Thr Val Val

19

Equation [19]

Ala He Thr Gin Val Ser Lys

1 5

Equation C20]

Gly His Tyr Gly Val Gin Arg

1 5

Equation [21]

Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr 1 5 10 15

Arg

16

Equation [22]

Gly Ala Glu Leu

14

Equation [23]

Lys Ala lie Thr Gin Val Ser Lys Gly Thr Xaa Glu

1 5 10 12

Equation [24]

Xaa Glu Pro Xaa Gly Gly Gly Gly Ala Gly Arg Gly Tyr Xaa Ala 1 5 10 15

Pro Gly

17

Equation [25]

Gin Gly Pro Ser lie Val Thr Pro Xaa Lys Asp lie

1 5 10 12 (Reference Example 3) Measurement of PG I 2 production stimulating activity

Vascular endothelial cells were collected from the thoracic aorta intima by a detachment method. The obtained vascular endothelial cells were then passaged at 37 ° C in 5% carbon dioxide-95% air in Dulbecco's modified Eagle medium containing 10% OUZmL benicillin and 100 g / mL streptomycin containing 10% fetal calf serum. Cultured. The medium was changed twice a week and passaged for 5-10 passages. The vascular endothelial cells were treated with 0.05% trypsin to obtain a cell suspension. Next, the vascular endothelial cells were transferred to a 24-well culture dish containing 1 mL of Dulbecco's modified Eagle's medium containing 10% fetal calf serum, and cultured to 5 × 10 ⁴ cells / well. Dulbecco's Modified Eagle Medium 50 containing 10% or less of various measurement samples was added, and incubated at 37 ° C for 60 minutes.

Extraction and purification of 6-keto PGF 1α from the culture supernatant was performed according to the method of Jaff et al. [The Journal of Clinical Investigation (J. Clin. In Vest.). 52, 398 Page, 1973]. 1.5 mL of 0.1N hydrochloric acid was added to 1 mL of the culture supernatant, and extracted twice with 5 mL of ethyl acetate. Ethyl acetate was evaporated under nitrogen gas, and the residue was redissolved in ethanol to obtain an Atsushi sample. This sample was stored at 120 ° C until used for Atsushi. Ethanol was evaporated at 40 ° C, and the residue was dissolved in 0.1 M phosphate buffer (pH 7.2, 1 M sodium chloride, containing 1% gelatin).

6- ^[3 H] Keto PGF 1 alpha and anti-6-Keto PGF New England Nuclear one (N ew Eng l and Nuc lear ) manufactured 6 Keto PGF1 alpha measuring radio I Takeno assay I using 1 alpha antibody Using a kit, the concentration of 6-keto-PGF1α was measured according to the attached manual. PG I ₂ production level is 10 ⁴ cells 6-keto produce per hour - was determined by PGF 1 alpha production quantity (p GZL 0 ⁴ during the time of cell Z).

[The invention's effect]

The present invention provides a novel DNA. This DNA can be used to express the novel protein it encodes by genetic engineering techniques.This new protein promotes the production of PG I _{2 and} the platelet aggregation of PG I ₂ Inhibiting action, smooth Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, glucoseuria It is an effective medicine for diseases such as heart disease, heart failure, angina pectoris, ischemic heart disease, depressive heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, and pregnancy eclampsia.

[Sequence list]

Sequence number

Array length 2 8 2

Sequence type Amino acid

Topology linear

Sequence type Peptide

Array

Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly

-25 -20 -15

Leu Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp

-10 -5 1

Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro

5 10 15

Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys

20 25 30

Pro Met Cys Ala Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Gly

35 40 45

Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu Cys Val Lys Ser

50 55 60

Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala Ala Gly Gly Pro

65 70 75

Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg Tyr Pro Val Cys

80 85 90

Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys Gin Leu Arg Ala 95 100 105

Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys Ala lie Thr Gin

110 115 120

Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser lie Val Thr Pro

125 130 135

Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin Val Tyr Leu Ser

140 145 150

Cys Glu Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys

155 160 165

Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro

170 175 180

Gly Asp Arg Asp Asn Leu Ala He Gin Thr Arg Gly Gly Pro Glu

185 190 195

Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

200 205 210

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Ser Gin Gly

215 220 225

Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His

230 235 240

Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

245 250 255 256

SEQ ID NO: 2

Array length 2 8 2

Sequence type Amino acid

Topology linear

Sequence type Peptide

Array

Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly

-25 -20 -15

Leu Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Asp

-10 -5 1

Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro

5 10 15

Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys

20 25 30

Pro Met Cys Ala Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Gly

35 40 45

Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu Cys Val Lys Ser

50 55 60

Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala Ala Gly Gly Pro

65 70 75

Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg Tyr Pro Val Cys

80 85 90

Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys Gin Leu Arg Ala

95 100 105

Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys Ala lie Thr Gin

110 115 120

Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser lie Val Thr Pro

125 130 135

Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin Val Tyr Leu Ser 140 145 150

Cys Glu Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys

155 160 165

Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro

170 175 180

Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly Gly Pro Glu

185 190 195

Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

200 205 210

Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Phe Gin Gly

215 220 225

Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His

230 235 240

Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

245 250 255 256 SEQ ID NO: 3

Array length: 1124

Sequence type: Nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: cDNA o mRNA

Origin:

Array features

Characteristic symbol: CDS

Location: 23. .868

How the features were determined: S

Symbol indicating special features: s i g ep d e

Location: 23..100 How the features were determined: s

Characteristic symbol: m a t p e p t i d e

Location: 10 1.. 8 6 8

How the features were determined: S

Array

CCGCGACGCG TGCGGCTGCT GCCCTATGTG CGCCCGCGGC GAGGGCGAGC CGTGCGGGGG 240

TGGCGGCGCC GGCAGGGGGT ACTGCGCGCC GGGCATGGAG TGCGTGAAGA GCCGCAAGAG 300

GCGGAAGGGT AAAGCCGGGG CAGCAGCCGG CGGTCCGGGT GTAAGCGGCG TGTGCGTGTG 360

CAAGAGCCGC TACCCGGTGT GCGGCAGCGA CGGCACCACC TACCCGAGCG GCTGCCAGCT 420

GCGCGCCGCC AGCCAGAGGG CCGAGAGCCG CGGGGAGAAG GCCATCACCC AGGTCAGCAA 480

GGGCACCTGC GAGCAAGGTC CTTCCATAGT GACGCCCCCC AAGGACATCT GGAATGTCAC 540

TGGTGCCCAG GTGTACTTGA GCTGTGAGGT CATCGGAATC CCGACACCTG TCCTCATCTG 600

GAACAAGGTA AAAAGGGGTC ACTATGGAGT TCAAAGGACA GAACTCCTGC CTGGTGACCG 660

GGACAACCTG GCCATTCAGA CCCGGGGTGG CCCAGAAAAG CATGAAGTAA CTGGCTGGGT 720

GCTGGTATCT CCTCTAAGTA AGGAAGATGC TGGAGAATAT GAGTGCCATG CATCCAATTC 780

CCAAGGACAG GCTTCAGCAT CAGCAAAAAT TACAGTGGTT GATGCCTTAC ATGAAATACC 840

AGTGAAAAAA GGTGAAGGTG CCGAGCTATA AACCTCCAGA ATATTATTAG TCTGCATGGT 900

TAAAAGTAGT CATGGATAAC TACATTACCT GTTCTTGCCT AATAAGTTTC TTTTAATCCA 960

ATCCACTAAC ACTTTAGTTA TATTCACTGG TTTTACACAG AGAAATACAA AATAAAGATC 1020

ACACATCAAG ACTATCTACA AAAATTTATT ATATATTTAC AGAAGAAAAG CATGCATATC 1080

ATTAAACAAA TAAAATACTT TTTATCACAA AAAAAAAAAAAAA 1124 SEQ ID NO: 4

Array length 1 1 2 4

Sequence type Nucleic acid

Number of chains Double strand Topology: linear

Sequence type: cDNA t o mRNA

Origin:

Array features

Characteristic symbol: CDS

Location: 23. .868

How the features were determined: S

Characteristic symbol: s i g ep t i de

Location: 23..100

How the features were determined: S

Characteristic symbol: ma t p ep t i de

Location: 101..868

How the features were determined: S

Array

GCCGCTGCCA CCGCACCCCG CCATGGAGCG GCCGTCGCTG CGCGCCCTGC TCCTCGGCGC 60 CGCTGGGCTG CTGCTCCTGC TCCTGCCCCT CTCCTCTTCC TCCTCTTCGG ACACCTGCGG 120 CCCCTGCGAG CCGGCCTCCT GCCCGCCCCT GCCCCCGCTG GGCTGCCTGC TGGGCGAGAC 180 CCGCGACGCG TGCGGCTGCT GCCCTATGTG CGCCCGCGGC GAGGGCGAGC CGTGCGGGGG 240 TGGCGGCGCC GGCAGGGGGT ACTGCGCGCC GGGCATGGAG TGCGTGAAGA GCCGCAAGAG 300 GCGGAAGGGT AAAGCCGGGG CAGCAGCCGG CGGTCCGGGT GTAAGCGGCG TGTGCGTGTG 360 CAAGAGCCGC TACCCGGTGT GCGGCAGCGA CGGCACCACC TACCCGAGCG GCTGCCAGCT 420 GCGCGCCGCC AGCCAGAGGG CCGAGAGCCG CGGGGAGAAG GCCATCACCC AGGTCAGCAA 480 GGGCACCTGC GAGCAAGGTC CTTCCATAGT GACGCCCCCC AAGGACATCT GGAATGTCAC 540 TGGTGCCCAG GTGTACTTGA GCTGTGAGGT CATCGGAATC CCGACACCTG TCCTCATCTG 600 GAACAAGGTA AAAAGGGGTC ACTATGGAGT TCAAAGGACA GAACTCCTGC CTGGTGACCG 660 GGACAACCTG GCCATTCAGA CCCGGGGTGG CCCAGAAAAG CATGAAGTAA CTGGCTGGGT 720 GCTGGTATCT CCTCTAAGTA AGGAAGATGC TGGAGAATAT GAGTGCCATG CATCCAATTT 780 CCAAGGACAG GCTTCAGCAT CAGCAAAAAT TACAGTGGTT GATGCCTTAC ATGAAATACC 840

AGTGAAAAAA GGTGAAGGTG CCGAGCTATA AACCTCCAGA ATATTATTAG TCTGCATGGT 900 TAAAAGTAGT CATGGATAAC TACATTACCT GTTCTTGCCT AATAAGTTTC TTTTAATCCA 960 ATCCACTAAC ACTTTAGTTA TATTCACTGG TTTTACACAG AGAAATACAA AATAAAGATC 1020 ACACATCAAG ACTATCTACA AAAATTTATT ATATATTTAC AGAAGAAAAG CATGCATATC AAA ATAAAAAAAAAA

Array length 2 5 6

Sequence type Amino acid

Topology linear

Sequence type Peptide

Array

Ser Ser Ser Asp Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro 1 5 10 15

Pro Leu Pro Pro Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala

20 25 30

Cys Gly Cys Cys Pro Met Cys Ala Arg Gly Glu Gly Glu Glu Pro Cys

35 40 45

Gly Gly Gly Gly Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu

50 55 60

Cys Val Lys Ser Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala

65 70 75

Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg

80 85 90

Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys

95 100 105

Gin Leu Arg Ala Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys

110 115 120

Ala lie Thr Gin Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser

125 130 135 lie Val Thr Pro Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin 140 145 150

Val Tyr Leu Ser Cys Glu Val lie Gly lie Pro Thr Pro Val Leu

155 160 165 lie Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr

170 175 180

Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg

185 190 .195

Gly Gly Pro Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser

200 205 210

Pro Leu Ser Lys Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser

215 220 225

Asn Ser Gin Gly Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val

230 235 240

Asp Ala Leu His Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu

245 250 255

Leu

256 SEQ ID NO: 6

Array length: 2 5 6

Sequence type: Amino acid

Topology: linear

Sequence type: peptide

Array

Phe Ser Ser Asp Thr Cys Gly Pro Cys Glu Pro Ala Ser Cys Pro

1 5 10 15

Pro Leu Pro Pro Leu Gly Cys Leu Leu Gly Glu Thr Arg Asp Ala

20 25 30 Cys Gly Cys Cys Pro Met Cys Ala Arg Gly Glu Gly Glu Glu Pro Cys

35 40 45 Gly Gly Gly Gly Ala Gly Arg Gly Tyr Cys Ala Pro Gly Met Glu

50 55 60

Cys Val Lys Ser Arg Lys Arg Arg Lys Gly Lys Ala Gly Ala Ala

65 70 75

Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val Cys Lys Ser Arg

80 85 90

Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly Cys

95 100 105

Gin Leu Arg Ala Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu Lys

110 115 120

Ala lie Thr Gin Val Ser Lys Gly Thr Cys Glu Gin Gly Pro Ser

125 130 135 lie Val Thr Pro Pro Lys Asp lie Trp Asn Val Thr Gly Ala Gin

140 145 150

Val Tyr Leu Ser Cys Glu Val lie Gly lie Pro Thr Pro Val Leu

155 160 165 lie Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gin Arg Thr

170 175 180

Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg

185 190 195

Gly Gly Pro Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser

200 205 210

Pro Leu Ser Lys Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser

215 220 225

Asn Phe Gin Gly Gin Ala Ser Ala Ser Ala Lys lie Thr Val Val

230 235 240

Asp Ala Leu His Glu lie Pro Val Lys Lys Gly Glu Gly Ala Glu 245 250 255

Leu

256

SEQ ID NO: 7

Array Length 5 5 7

Sequence type Nucleic acid

Number of chains Double strand

Topology linear

Sequence type c D N A t o mR N A

Array

GGTCATCGGA ATCCCGACAC CTGTCCTCAT CTGGAACAAG GTAAAAAGGG GTCACTATGG 60 AGTTCAAAGG ACAGAACTCC TGCCTGGTGA CCGGGACAAC CTGGCCATTC AGACCCGGGG 120 TGGCCCAGAA AAGCATGAAG TAACTGGCTG GGTGCTGGTA TCTCCTCTAA GTAAGGAAGA 180 TGCTGGAGAA TATGAGTGCC ATGCATCCAA TTTCCAAGGA CAGGCTTCAG CATCAGCAAA 240 AATTACAGTG GTTGATGCCT TACATGAAAT ACCAGTGAAA AAAGGTGAAG GTGCCGAGCT 300 ATAAACCTCC AGAATATTAT TAGTCTGCAT GGTTAAAAGT AGTCATGGAT AACTACATTA 360 CCTGTTCTTG CCTAATAAGT TTCTTTTAAT CCAATCCACT AACACTTTAG TTATATTCAC 420 TGGTTTTACA CAGAGAAATA CAAAATAAAG ATCACACATC AAGACTATCT ACAAAAATTT 480 ATTATATATT TACAGAAGAA AAGCATGCAT ATCATTAAAC AAATAAAATA CTTTTTATATCA 540 CAAAAAAAAA 557 SEQ ID NO: 8

Array length 2 0

Sequence type Amino acid

Topology linear

Sequence type Peptide

Array

lie Thr Val Val Asp Ala Leu His Glu lie Pro Val Lys Lys Gly 1 5 10 15 Glu Gly Ala Glu Leu

20 SEQ ID NO: 9

Array length: 20

Sequence type: Nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Synthetic DNA

Array

5 '-ATA ACA GTA GTA GAC GCA CT-3'

C G G G T G T T C C C C T T T T SEQ ID NO: 10

Array length: 20

Sequence type: Nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Synthetic DNA

Array

5 '-AA CTC AGC ACC CTC ACC CTT-3'

G G T G

C C C

TTT SEQ ID NO Array length: 59

Sequence type: Nucleic acid

Number of chains: double strand

Topology: linear

Sequence type: PCR product

Array

5 '-ATTACGGTGG TTGATGCGTT ACATGAAATA CCAGTGAAAA

10 20 30 40

AAGGCGAAGG CGCCGAATT-3 '

50 59 SEQ ID NO: 12

Array length: 20

Sequence type • Nucleic acid

Number of chains Single strand

Topology linear

Sequence type Synthetic DNA

Array

5'-TTGATGCGTTACATGAAATA-3 'SEQ ID NO: 13

Array length: 20

Sequence type: Nucleic acid

Number of chains

Topology .linear

Sequence type Synthetic DNA

Array

5 '-CCTTCGCCTTTTTTCACTGG-3' SEQ ID NO: 1 4

Array length 8 0 2

Sequence type Nucleic acid

Number of chains Double strand

Topology linear

Sequence type c D NA o mR N A

Array

GCAGCCGGCG GTCCGGGTGT AAGCGGCGTG TGCGTGTGCA AGAGCCGCTA CCCGGTGTGC 60 GGCAGCGACG GCACCACCTA CCCGAGCGGC TGCCAGCTGC GCGCCGCCAG CCAGAGGGCC 120 GAGAGCCGCG GGGAGAAGGC CATCACCCAG GTCAGCAAGG GCACCTGCGA GCAAGGTCCT 180 TCCATAGTGA CGCCCCCCAA GGACATCTGG AATGTCACTG GTGCCCAGGT GTACTTGAGC 240 TGTGAGGTCA TCGGAATCCC GACACCTGTC CTCATCTGGA ACAAGGTAAA AAGGGGTCAC 300 TATGGAGTTC AAAGGACAGA ACTCCTGCCT GGTGACCGGG ACAACCTGGC CATTCAGACC 360 CGGGGTGGCC CAGAAAAGCA TGAAGTAACT GGCTGGGTGC TGGTATCTCC TCTAAGTAAG 420 GAAGATGCTG GAGAATATGA GTGCCATGCA TCCAATTCCC AAGGACAGGC TTCAGCATCA 480 GCAAAAATTA CAGTGGTTGA TGCCTTACAT GAAATACCAG TGAAAAAAGG TGAAGGTGCC 540 GAGCTATAAA CCTCCAGAAT ATTATTAGTC TGCATGGTTA AAAGTAGTCA TGGATAACTA 600 CATTACCTGT TCTTGCCTAA TAAGTTTCTT TTAATCCAAT CCACTAACAC TTTAGTTATA 660 TTCACTGGTT TTACACAGAG AAATACAAAA TAAAGATCAC ACATCAAGAC TATCTACAAA 720 AATTTATTAT ATATTTACAG AAGAAAAGCA TGCATATCAT TAAACAAATA AAATACTTTT 780 TATCACAAAA AAAAAAAAAA AA 802

SEQ ID NO: 15

Array length 1 0 0

Sequence type Amino acid

Topology linear

Sequence type Peptide

Array

Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys Val Lys 1 5 10 15

Arg Gly His Tyr Gly Val Gin Arg Thr Glu Leu Leu Pro Gly Asp

20 25 30

Arg Asp Asn Leu Ala lie Gin Thr Arg Gly Gly Pro Glu Lys His

35 40 45

Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys Glu Asp

50 55 60

Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Phe Gin Gly Gin Ala

65 70 75

Ser Ala Ser Ala Lys lie Thr Val Val Asp Ala Leu His Glu lie

80 85. 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu

95 100 SEQ ID NO: 1 6

Array length: 1 8 2

Sequence type: Amino acid

Topology: linear

Sequence type: peptide

Array

Ala Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val Cys Lys Ser 1 5 10 15

Arg Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro Ser Gly

20 25 30

Cys Gin Leu Arg Ala Ala Ser Gin Arg Ala Glu Ser Arg Gly Glu

35 40 45

Lys Ala lie Thr Gin Val Ser Lys Gly Thr Cys Glu Gin Gly Pro

50 55 60

Ser lie Val Thr Pro Pro Lys Asp lie Trp Asn Val Thr Gly Ala 65 70 75

Gin Val Tyr Leu Ser Cys Glu Val lie Gly lie Pro Thr Pro Val

80 85 90

Leu lie Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gin Arg

95 100 105

Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr

110 115 120

Arg Gly Gly Pro Glu Lys His Glu Val Thr Gly Trp Val Leu Val

125 130 135

Ser Pro Leu Ser Lys Glu Asp Ala Gly Glu Tyr Glu Cys His Ala

140 145. 150

Ser Asn Ser Gin Gly Gin Ala Ser Ala Ser Ala Lys lie Thr Val

155 160 165

Val Asp Ala Leu His Glu lie Pro Val Lys Lys Gly Glu Gly Ala

170 175 180

Glu Leu SEQ ID NO: 1 7

Array length: 2 9

Sequence type .Nucleic acid

Number of chains: single strand

Topology linear

Sequence type Synthetic D N A

Array

5'-CTGGTCGACGGATCCCGGGTACCAGCACA-3'SEQ ID NO: 18

Array length 2 9

Sequence type Nucleic acid Number of chains: single strand

Topology: linear

Sequence type: synthetic DNA

Array

(5'-CTGGTACCCGGGATCCGTCGACCAGCACA-3 ')

SEQ ID NO: 1 9

Array length: 8

Sequence type: Amino acid

Topology: linear

Sequence type: peptide

Array

Ser Xaa Aaa Asp Thr Xaa Gly Pro

1 5 8 SEQ ID NO: 20

Array length: 2 5

Sequence type: Amino acid

Topology: linear

Sequence type: peptide

Array

Val lie Gly lie Pro Thr Pro Val Leu lie Trp Asn Lys Val Lys 1 5 10 15

Arg Gly His Tyr Gly Val Gin Arg Thr Glu

20 25 SEQ ID NO: 2 1

Array length: 1 8

Sequence type: Amino acid

Topology: linear

Sequence type: peptide

Array

Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly 1 5 10 15

Gly Pro Glu SEQ ID NO: 2 2

Array length: 1 9

Sequence type: amino acid

Topology: linear

Sequence type: peptide

Array

Cys His Ala Ser Asn Ser Gin Gly Gin Ala Ser Ala Ser Ala Lys 1 5 10 15 lie Thr Val Val

19 SEQ ID No. • 2 3

Array length 7

Sequence type Amino acid

Topology linear

Sequence type Peptide

Array

Ala lie Thr Gin Val Ser Lys

1 5 SEQ ID NO: 2 4

Array length 7

Sequence type .Amino acid

Topology linear

Sequence type Peptide

Array

Gly His Tyr Gly Val Gin Arg

1 5 SEQ ID NO: 25

Array length: 1 6

Sequence type: Amino acid

Topology: linear

Sequence type: peptide

Array

Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala lie Gin Thr

1 5 10 15

Arg SEQ ID NO: 26

Array length 4

Sequence type Amino acid

Topology

Sequence type Peptide

Array

Gly Ala Glu Leu

1 4 SEQ ID No. • 2 7

Array length '1 2

Sequence type Amino acid

Topology linear

Sequence type Peptide

Array

Lys Ala lie Thr Gin Val Ser Lys Gly Thr Xaa Glu

1 5 10 SEQ ID NO: 2 8

Array length: 1 7

Sequence type: Amino acid

Topology: linear

Sequence type: peptide

Array

Xaa Glu Pro Xaa Gly Gly Gly Gly Ala Gly Arg Gly Tyr Xaa Ala 1 5 10 _ 15

Pro Gly

SEQ ID NO: 2 9

Array length 1 2

Sequence type Amino acid

Topology linear

Sequence type Peptide

Array

Gin Gly Pro Ser lie Val Thr Pro Xaa Lys Asp lie

1 5 10

Claims

The scope of the claims

1. A DNA containing a part or all of a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 1 or 2 in the sequence listing.

2. A DNA containing a part or all of the nucleotide sequence of SEQ ID NO: 3 or 4 in the sequence listing.

3. A DNA having a nucleotide sequence complementary to the DNA according to claim 1 or 2.

4. A vector containing the DNA according to any one of claims 1 to 3.

5. A transformant containing the DNA according to any one of claims 1 to 3.

6. A transformant transformed with the vector according to claim 4.

7. The transformant according to claim 5, wherein the host cell is a COS cell.

8. The transformant according to claim 5, wherein the host cell is a CHO cell.

9. A protein containing a part or all of the amino acid sequence of SEQ ID NO: 1 or 2 in the sequence listing.

10. The protein according to claim 9, wherein the protein containing a part of the amino acid sequence is a mature protein.

11. The protein according to claim 9 or 10, which acts on vascular endothelial cells and has an activity of stimulating the production of prostaglandin I ₂ (PGI 2) from the cells.

1 2. The method for producing a protein according to any one of claims 9 to 11, comprising culturing the transformant according to any one of claims 5 to 8 L.

1 3. An antibody obtained by using a part or all of the protein according to any one of claims 9 to 11 as an antigen.

14. The method for immunologically measuring a protein according to any one of claims 9 to 11, wherein the antibody according to claim 13 is used.

15. A pharmaceutical composition for treating at least one of the following diseases, comprising the protein according to any one of claims 9 to 11 as an active ingredient.

Hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, brain Ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart failure, angina, ischemic heart disease, congestive heart disease, choroidal circulatory disorder, bronchial disease, gastric ulcer, pregnancy eclampsia.