JPH06100589A - Novel protein bioactive substance and its DNA - Google Patents

Abstract

(57) [Summary] [Purpose] PGI₂Platelet aggregation inhibitory effect, smooth muscle
Hemolytic uremic disease based on relaxing action, gastric acid secretion inhibitory action, etc.
Group, thrombotic thrombocytopenic purpura, peripheral artery occlusion, heart failure
Blood, cerebral ischemia, arteriosclerosis, cerebral obstruction, hyperlipidemia, diabetes, heart
Insufficiency, angina, ischemic heart disease, congestive heart disease, choroid
Circulatory disorders, bronchial diseases, gastric ulcers, pregnancy eclampsia, etc.
The provision of novel protein bioactive substances that can be applied as [Structure] Prostaglandin I₂(PGI₂) Production stimulation
Novel active protein bioactive substance, more specifically blood vessels
Acting on endothelial cells, PGI from vascular endothelial cells ₂Production of
, A novel protein-based bioactive substance having the activity of promoting
Method for producing the same and encoding the protein bioactive substance
DNA.

Description

Detailed Description of the Invention

[0001]

The present invention relates to prostaglandin I
₂ (aka; Prostacyclin
in), hereinafter referred to as PGI ₂ ) A novel protein physiologically active substance having a production stimulating activity, more specifically, a novel protein property having an activity of acting on vascular endothelial cells and promoting production of PGI ₂ from vascular endothelial cells The present invention relates to a physiologically active substance, a method for producing the same, and a DNA encoding the proteinaceous physiologically active substance.

[0002]

2. Description of the Related Art Prostaglandins
andin, PG) in 1930 by Kurzlok et al. [Proceeding of the Society for Experimental Biology and Medicine (Proc. Soc. Exp. Biol. Med.),
28, 268, 1930] was discovered and reported as a uterine muscle contractile active substance present in semen. After that, basic research on prostaglandins made rapid progress. Prostaglandins are a generic name for physiologically active substances that have a basic structure of prostanoic acid contained in human and animal tissues and organs, and have a chemical structure formed by a series of chemical reactions called arachidonic acid cascades starting from arachidonic acid in cells. Of several different prostaglandins are biosynthesized. They are attracting attention as important active substances involved in the maintenance of homeostasis of the endocrine system, nervous system, inflammation, immune system and other organisms, and are widely used in various mechanisms such as inflammation, thrombus formation, peripheral circulation, arteriosclerosis and aging. Involved in [clinical science, 1
7, p. 958, 1981]. Furthermore, prostaglandins play an important role as vasoactive substances for vascular smooth muscle [pathophysiology, 2
Vol. 792, p. 1983], thromboxane A ₂ (TXA), which is an active prostanoid derived from platelets and blood vessel walls.
₂ ) [Proceeding of the National Academy of Science of the United
States of America (Proc. Natl. Ac
ad. Sci. USA), 72, 2994, 197.
5 years] and PGI ₂ [Nature,
263, 663, 1976] has received attention for its clinical significance in the physiology and pathology of platelets and the vascular system.

Among these prostaglandins, the
Ke PGI₂Is a potent inhibitor of platelet aggregation and vasorelaxation
And platelet-derived TXA₂In vivo by acting antagonistically with
Involved in maintaining homeostasis in
Shu Journal of Pharmacology (Br.
J. Pharmac. ), 76, p. 3, 1982
Year]. Normal vascular endothelial cells are exposed to physical and chemical stimuli.
In general, PGI₂Generate and activate platelets
Not only suppress it but also regulate the tonus of the blood vessel wall locally
Mechanisms that maintain homeostasis of the circulation are working. Sanawa
TX is used for the development of vascular disorders such as thrombosis and arteriosclerosis.
A₂And PGI₂Imbalance in production of PGI ₂Production of
Life decline is involved [British Journal
Of Pharmacology (Br.J.Pharma)
c. ), 76, p. 3, 1982].
It 1978 Mac Inner et al. [Nature (N
Aure), 271, 549, 1978]
PGI in blood vessel wall₂The presence of factors that stimulate production
It is confirmed that this factor is a heat-labile polymer substance.
Is reported. Hemolytic uremic afterwards by Lemudge et al.
Since it was reported that this factor was reduced in the illness syndrome
[Lancet, Volume 2, pp. 871, 19
1978], thrombotic thrombocytopenic purpura [Lancet (L
ancet), Volume 2, 748, 1979], red sickle
Hemocytic anemia [British Journal of Hemat
Rosie (Br. J. Haematol.), 48, 5
45, 1981], acute myocardial infarction [Coronary (C
oronary), 2, p. 49, 1985], Diabetes.
Is a thrombotic / arteriosclerotic disease such as a disease
It has been revealed that it is closely related to the cause. When
In addition, small blood pressure as a factor of onset and progress of diabetic angiopathy.
Platelet-Derived TXA for Platelet Acceleration Mechanism₂Production of
Enhancement [Thrombosis Research (Thromb. Re
s. ), 19: 211, 1980, Journal
Of Laboratory And Clinical Medicine
(J. Lab. Clin. Med.), Vol. 97, 87.
P., 1981], and PGI derived from blood vessels.₂Decreased production
Has been reported on diabetic patients and experimental diabetic animals
[Lancet, Volume 1, 325, 19
1979, Lancet, Volume 2, 1365
Page, 1979, The New England Journal
Le of Medicine (N. Engl. J. Me
d. ), 300, 366, 1979 and Life
 Science (Life Sci.), Volume 23, 351
P. 1978]. Moreover, this decreased production is due to
Existing PGI₂Partly due to the decrease in production stimulating activity
Have been suggested [metabolism (Met
abolism), 38, 837, 1989, to
Hamostasis, Volume 16, 44
7 pages, 1986 and Diabetis Research
And Clinical Practice (Diab. Res.
Clin. Pract. ) 3, p. 243, 1987
Year].

As mentioned above, PGI₂Is a platelet aggregation inhibitor
However, other than this, other than flat such as blood vessels and bronchi
It has a smooth muscle relaxing action and a gastric acid secretion inhibiting action. these
PGI based on physiological action₂As a pharmaceutical product
It is possible that PGI₂Has a half-life of 37 ° C in neutral water
It is a substance that is extremely chemically unstable for 5 minutes, and it has reached practical use
Not On the other hand, natural PGI₂While maintaining a similar action,
Chemically stable PGI ₂Analogs are anticoagulants, blood vessels
Attempts were made to develop it as a medicine such as an extender.
ing. However, for living organisms, PGI₂Is unstable
In some cases, a shorter life may be desirable. You
That is, PGI that is quickly generated in an emergency
₂On the other hand, the living body must always be ready to react
Not a stable PGI₂By giving a large amount of analogs
The PGI of the cell₂Responsiveness to
In case PGI₂May not be able to respond to
It In fact, prostaglandin E₁(PGE₁) And stable
Na PGI₂Due to pretreatment of analogues, PGI₂Against
Cells in which the cAMP elevation reaction that should occur naturally has ceased
Examples of [Prostaglandins (Pro
stalandins. ), Vol. 19, page 2, 1980
Year].

Therefore, if the above-mentioned action of PGI ₂ is expected, PGI ₂ is produced at a required concentration at a required site at a required time, rather than using a chemically stable analog. It is considered to be more preferable for the living body. PGI ₂ metabolism in vivo is regulated by two factors, one of which is a PGI ₂ stabilizing factor, which was recently identified as apolipoprotein A-1 (ApoA-1) [The Journal of Clinical Investigation (J. Clin. Invests
t. ), 82, 803, 1988]. The other one is PGI ₂ production stimulating factor (Prostacyclin P
production StimulatingFact
or PSF). As mentioned above, there is already P in the blood.
GI ₂ production stimulating activity (Prostacyclin St
It has been reported that there is an activating activity (PSA), but its active body (PS
F) has not been identified and its identification is desired. PS
By stimulating PGI ₂ production from vascular endothelial cells and increasing the PGI ₂ concentration in blood, F can exert the platelet aggregation inhibitory action, smooth muscle relaxation action, gastric acid secretion inhibitory action and the like that PGI ₂ has. PS based on such action
F is hemolytic uremic syndrome, thrombotic thrombocytopenic purpura,
Peripheral artery occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, cerebral occlusion, hyperlipidemia, diabetes, heart failure, angina, ischemic heart disease, congestive heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, pregnancy It has the potential to become a drug such as eclampsia.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to purify and isolate a novel substance that stimulates PGI ₂ production from vascular endothelial cells and provide the novel protein bioactive substance. Moreover, this invention is providing the manufacturing method. Further, the present invention provides a gene encoding the physiologically active proteinaceous substance, and provides a genetically engineered method for mass-producing the physiologically active proteinaceous substance.

[0007]

[Means for Solving the Problems] In order to achieve the above-mentioned objects, the present inventors have conducted extensive studies on PGI ₂ production stimulating activity present in blood and culture supernatant of human cell lines, PG in the culture supernatant of human diploid fibroblasts
It was confirmed that a substance having an I ₂ production stimulating activity was present at a high concentration. Then, a novel protein physiologically active substance having a PGI ₂ production stimulating activity was isolated and purified from this culture supernatant, and a part of the amino acid sequence was successfully determined. Furthermore, the present inventors obtained the cDNA of the protein physiologically active substance from the cDNA library of normal human diploid fibroblasts by using the amino acid sequence as a clue, and mass-produced the protein physiologically active substance by genetic engineering. Opened the way to.

That is, the present invention provides a novel proteinaceous substance having at least one amino acid sequence represented by any of the following formulas [1] to [11] (SEQ ID NOS: 1 to 11 in the sequence listing). Provide a physiologically active substance.

Formula [1] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu 20 25 Formula [2] Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr Arg Gly 1 5 10 15 Gly Pro Glu

Formula [3] Cys His Ala Ser Asn Ser Gln Gly Gln Ala Ser Ala Ser Ala Lys 1 5 10 15 Ile Thr Val Val 19 Formula [4] Ile Thr Val Val Asp Ala Leu His Glu Ile Pro Val Lys Lys Gly 1 5 10 15 Glu Gly Ala Glu Leu 20

Formula [7] Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr 1 5 10 15 Arg Formula [9] Lys Ala Ile Thr Gln Val Ser Lys Gly Thr Cys Glu 1 5 10

Formula [10] Xaa Glu Pro Xaa Gly Gly Gly Gly Ala Gly Arg Gly Tyr Gln Ala 1 5 10 15 Pro Gly Formula [11] Gln Gly Pro Ser Ile Val Thr Pro Xaa Lys Asp Ile 1 5 10

The present invention further provides a novel protein bioactive substance containing an amino acid sequence represented by the following formula [12] or [13] (SEQ ID NO: 12 or 13 in the sequence listing).

Formula [12] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu Leu Pro Gly Asp 20 25 30 Arg Asp Asn Leu Ala Ile Gln 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 Gln Gly Gln Ala 65 70 75 Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile 80 85 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu 95 100

Formula [13] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu Leu Pro Gly Asp 20 25 30 Arg Asp Asn Leu Ala Ile Gln 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 Ser Gln Gly Gln Ala 65 70 75 Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile 80 85 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu 95 100

The novel physiologically active proteinaceous substance is suitable when the molecular weight thereof is 30 to 36 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions.
It is more preferable when it is characterized by having prostaglandin I ₂ (PGI ₂ ) production stimulating activity. Also,
The present invention provides a DNA having a nucleotide sequence encoding the protein physiologically active substance of the present invention. Furthermore, the following formula [1
4] or [15] (SEQ ID NO: 14 or 1 in the sequence listing)
A DNA having the nucleotide sequence represented by 5) is provided.

[0017] Formula [14] 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 CTTTTTATCA 540 CAAAAAAAAA

[0018] Formula [15] GGTCATCGGA ATCCCGACAC CTGTCCTCAT CTGGAACAAG GTAAAAAGGG GTCACTATGG 60 AGTTCAAAGG ACAGAACTCC TGCCTGGTGA CCGGGACAAC CTGGCCATTC AGACCCGGGG 120 TGGCCCAGAA AAGCATGAAG TAACTGGCTG GGTGCTGGTA TCTCCTCTAA GTAAGGAAGA 180 TGCTGGAGAA TATGAGTGCC ATGCATCCAA TTCCCAAGGA 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 CTTTTTATCA 540 CAAAAAAAAA

The present invention also provides a production method characterized in that a raw material containing the above novel protein physiologically active substance is purified using at least one of the following steps (a) to (c): provide. (A) Ion exchange chromatography (b) Affinity chromatography (c) Gel filtration chromatography

The present invention will be described in detail below. The protein physiologically active substance of the present invention is a novel protein having at least one amino acid sequence represented by any of the following formulas [1] to [11] (SEQ ID NOS: 1 to 11 in the sequence listing). It is a sex bioactive substance. Formula [1] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu 20 25 Formula [2] Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr Arg Gly 1 5 10 15 Gly Pro Glu

Formula [3] Cys His Ala Ser Asn Ser Gln Gly Gln Ala Ser Ala Ser Ala Lys 1 5 10 15 Ile Thr Val Val 19 Formula [4] Ile Thr Val Val Asp Ala Leu His Glu Ile Pro Val Lys Lys Gly 1 5 10 15 Glu Gly Ala Glu Leu 20

Formula [7] Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr 1 5 10 15 Arg Formula [9] Lys Ala Ile Thr Gln Val Ser Lys Gly Thr Cys Glu 1 5 10 Formula [10] Xaa Glu Pro Xaa Gly Gly Gly Gly Ala Gly Arg Gly Tyr Gln Ala 1 5 10 15 Pro Gly Formula [11] Gln Gly Pro Ser Ile Val Thr Pro Xaa Lys Asp Ile 1 5 10

The portion of the amino acid sequence of any of the above formulas [1] to [11] (SEQ ID NOS: 1 to 11 in the sequence listing) may be any portion of the proteinaceous physiologically active substance of the present invention. The amino acid sequence portions of the above formulas [1] to [11] (SEQ ID NOS: 1 to 11 in the sequence listing) may be adjacent to each other, or any amino acid sequence may be included between them.

Furthermore, the proteinaceous physiologically active substance of the present invention is
The following formula [12] or [13] (SEQ ID NO: 12 in the sequence listing)
Alternatively, it is a novel protein physiologically active substance containing the amino acid sequence represented by 13).

Formula [12] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu Leu Pro Gly Asp 20 25 30 Arg Asp Asn Leu Ala Ile Gln 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 Gln Gly Gln Ala 65 70 75 Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile 80 85 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu 95 100

Formula [13] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu Leu Pro Gly Asp 20 25 30 Arg Asp Asn Leu Ala Ile Gln 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 Ser Gln Gly Gln Ala 65 70 75 Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile 80 85 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu 95 100

The novel protein physiologically active substance is suitable when the molecular weight is 30 to 36 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. Also,
The novel protein bioactive substance of the present invention is suitable when it has prostaglandin I ₂ (PGI ₂ ) production stimulating activity. The method for measuring this activity will be described later.

Next, a method for producing the novel protein bioactive substance of the present invention will be described. The novel protein physiologically active substance of the present invention may be of any origin, but is preferably of human origin, more preferably of normal human diploid fibroblast, and Those produced in the culture supernatant of diploid fibroblasts are preferred. The novel protein bioactive substance of the present invention can be obtained by, for example, culturing normal human diploid fibroblasts in an appropriate growth medium and collecting a culture solution having a PGI ₂ production stimulating activity. It can be obtained by purifying Qing. For the culture of normal human diploid fibroblasts, a commonly used medium may be used, but Dulbecco's modified Eagle medium, RPMI1640 medium, Eagle's minimum essential medium or ham medium is particularly preferable. Further, serum may or may not be added, but the substance of the present invention is purified from the culture supernatant,
It is preferable to use a serum-free medium because it is isolated.
The culture supernatant after completion of the culture is preferably concentrated by a method such as a filter method or an ultrafiltration method.

The raw material containing the novel protein physiologically active substance of the present invention obtained as described above is purified by using at least one of the following steps, and the following steps (a), (b) and (c): Each of) may be performed according to a known protocol. (A) Ion exchange chromatography (b) Affinity chromatography (c) Gel filtration chromatography The order may be arbitrary and may be repeated if necessary. Further, operations such as concentration and dialysis may be added before or after one step and the other steps, and further, if necessary, for example, chromatofocusing, adsorption chromatography, hydrophobic chromatography, reverse phase. It may be combined with other chromatography such as chromatography, thin layer chromatography and partition chromatography, and other protein purification methods such as isoelectric focusing, polyacrylamide electrophoresis and salt folding.

After the steps (a) to (c), the collected fraction may be subjected to reverse phase high performance liquid chromatography for the purpose of isolation and purification, confirmation of purity and the like. Each of the steps (a), (b) and (c) may be low pressure liquid chromatography or high pressure liquid chromatography (high performance liquid chromatography, HPLC). As for the elution method in each chromatography, appropriate elution conditions and eluate may be selected in consideration of each chromatographic carrier, the target substance, the physical properties of the contained contaminants, and the like. The elution may be linear gradient elution or stepwise gradient elution.

Using the above steps (a) to (c),
Or the novel protein of the present invention from the culture supernatant by the following method.
The sexually bioactive substance can be isolated. Double normal humans
Anion exchange chromatograph of the culture supernatant of somatic fibroblasts
E.g. DEAE-5PW (manufactured by Tosoh Corporation), Mon
oQ (Pharmacia) or Q-Sepharose color
Sodium chloride (made by Pharmacia)
Elute by the linear concentration gradient method of. Each fraction eluted
PGI₂The production stimulating activity is measured, and the active fraction is
obtain. PGI₂The production stimulating activity will be described later.
It Furthermore, this fraction is subjected to affinity chromatography.
-For example, heparin-5PW (manufactured by Tosoh Corporation)
And elute with a linear concentration gradient method of sodium chloride. P
GI₂The production stimulating activity was similarly measured, and the active fractions were collected.
It This fraction is then subjected to gel filtration chromatography, eg
Ba Protein-Pack (Japan Millipore Limited)
Data Chromatography Division), Sefakuri
Le S-100HR (Pharmacia) or TSK
Gel G3000SW _XLUsing a column (manufactured by Tosoh Corporation), etc.
Unfold and PGI₂Biological activity by measuring production stimulating activity
Fractions can be obtained. Furthermore, this fraction is affinite
Chromatography, eg insulin-like growth factors
Child (Insuline-like growth fa
cor, IGF) -I or II as a ligand
IGF-affinity column or wheat germ
WGA-Affinite bound with agglutinin as a ligand
Column, PGI₂Production stimulating activity
It is possible to isolate and purify a novel protein bioactive substance exhibiting

The desired fraction of the physiologically active proteinaceous substance of the present invention in each chromatography can be obtained by measuring the PGI ₂ production stimulating activity of each eluted fraction and collecting the active fractions. . The PGI ₂ production stimulating activity can be measured by the following method. That is, vascular endothelial cells collected from the bovine thoracic aortic intima by a detachment method were subcultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, and cultured until a saturated cell density was reached. A stable metabolite of PGI _{2 in} the supernatant after a minute of incubation,

[Chemical 1] The PGI ₂ production amount can be determined by measuring (hereinafter, referred to as 6-keto-PGF1α). A commercially available 6-keto-PGF1α measurement kit may be used for this measurement.

The following method can be used to determine the amino acid sequence of the novel protein physiologically active substance of the present invention having a purified PGI ₂ production stimulating activity. For example, when an automatic gas phase sequencer is used, the substance of the present invention can be directly analyzed by the sequencer, or
Peptide fragments obtained by enzymatic cleavage of trypsin, endoproteinase Glu-C (protease V8), lysyl endopeptidase and the like may be analyzed, and the amino acid sequence of each can be determined. The novel protein bioactive substance of the present invention is a novel substance characterized by at least one amino acid sequence of SEQ ID NOs: 1 to 11 in the sequence listing. Further, the novel protein bioactive substance of the present invention is a novel substance characterized by the amino acid sequence of SEQ ID NO: 12 or 13 in the sequence listing. The novel protein bioactive substance production method of the present invention stimulates PGI ₂ production from vascular endothelial cells and is a novel protein that can be used for the treatment of the above-mentioned various diseases caused by decreased PGI ₂ production. A sexually physiologically active substance can be obtained.

In order to use the novel proteinaceous physiologically active substance of the present invention for pharmaceutical use, it is necessary to develop a method capable of producing it in a large amount and with high purity. Examples of the method include a method using a genetic engineering method other than the method obtained from the normal human diploid fibroblasts described above. As a general genetic engineering method, the amino acid sequence of the protein physiologically active substance of the present invention is clarified, a DNA probe is prepared based on the amino acid sequence, and an appropriate cDNA library, preferably normal human diploid fibroblast, is prepared. Prepare mRNA from cells
By screening the cDNA library obtained by synthesizing A, the cDNA of the novel protein physiologically active substance of the present invention can be obtained. In addition, using the purified protein physiologically active substance, or synthesizing a part of the amino acid sequence determined by amino acid sequence analysis or gene analysis as a synthetic peptide, and using this to immunize rabbits, mice, etc. Once obtained, this antibody is used to
It is also possible to screen the A library. Preferably, the cDNA encoding the novel protein bioactive substance of the present invention can be obtained by the following method. Total RNA is prepared from normal human diploid fibroblasts and poly (A) ⁺ RNA (mRNA) is prepared. For example, the preparation of total RNA can be carried out by the general guanidine thiocyanate method, hot phenol method or AGPC method. In addition, 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. Then, double-stranded cDNA is synthesized using reverse transcriptase, and various plasmid vectors such as plasmid pUC119 and plasmid pEF-BOS are synthesized.
[Nucleic Acid Research (Nucl. Ac
ids Res. ), 18, p. 5322, 1990
Year] etc., or a phage vector λ for cloning
A cDNA library is prepared by introducing E. coli into gt10, λgt11 or the like and transforming E. coli or in vitro packaging. cDNA synthesis is
Gubler and Hoffman's Method [Gene, 25
Vol. 263, 1983] and the like. Commercially available cDNA synthesis kit (Amersham, Boehringer, Invitrogen)
May be used. Introduction of cDNA into the above-mentioned plasmid vector or phage vector is a synthetic cDNA
It can be carried out by adding a linker or the like. The linker used is not particularly limited, but when using the plasmid pEF-BOS, the BstXI linker is preferable. The addition of a linker and the introduction of cDNA into a plasmid or a phage vector can also be performed using a commercially available ligation kit (Takara Shuzo). Escherichia coli to be transformed is not particularly limited as long as it is a commonly used strain, but HB101, DH5 and MC
1061 / P3 is preferred. The method for introducing the plasmid DNA into which Escherichia coli has been introduced can be carried out by the electroporation method, the calcium chloride method or the like. In vitro packaging can also be performed using a commercially available in vitro packaging kit (Stratagene, Amersham).

The cDNA encoding the novel protein bioactive substance of the present invention can be selected by combining general cDNA screening methods. For example, a probe is prepared based on the obtained partial amino acid sequence to directly screen a cDNA library or a phage library, or a PCR primer is prepared to prepare a PC.
A method of selecting a clone having a DNA fragment amplified by the R method can be considered. When a library capable of expressing cDNA, for example, a library prepared by using the λgt11 phage vector is used, clones can be selected using the antibody obtained by the above method. Among them, the screening method using the PCR method is preferable because a large number of clones can be processed at one time. The nucleotide sequence of the cDNA contained in the obtained target clone can be determined by the dideoxy termination method. The determined DNA sequence is represented by the following formula [14] or [15] (SEQ ID NO: 14 or 15 in the sequence listing).

[0036] Formula [14] 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 CTTTTTATCA 540 CAAAAAAAAA

[0037] Formula [15] GGTCATCGGA ATCCCGACAC CTGTCCTCAT CTGGAACAAG GTAAAAAGGG GTCACTATGG 60 AGTTCAAAGG ACAGAACTCC TGCCTGGTGA CCGGGACAAC CTGGCCATTC AGACCCGGGG 120 TGGCCCAGAA AAGCATGAAG TAACTGGCTG GGTGCTGGTA TCTCCTCTAA GTAAGGAAGA 180 TGCTGGAGAA TATGAGTGCC ATGCATCCAA TTCCCAAGGA 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 CTTTTTATCA 540 CAAAAAAAAA

The DNA sequence of the present invention encodes the proteinaceous physiologically active substance of the present invention and has the formula [14] or [1].
5] (SEQ ID NO: 14 or 15 in Sequence Listing)
It may be a part or all of the NA sequence.
In addition, the formula [14] or [15] (SEQ ID NO: 1 in the sequence listing)
4 or 15) may be included in part. The DNA sequence of the present invention may be synthesized using RNA as a template or may be synthesized organically. Alternatively, it may be obtained by amplification by the PCR method. In general,
In the field of gene recombination, according to the degeneracy of the genetic code, at least one base of the DNA sequence of the gene can be replaced with another base without changing the amino acid sequence of the protein produced from the gene. Therefore, the DNA sequence of the present invention also contains a nucleotide sequence changed by substitution based on the degeneracy of the genetic code. In this case, the amino acid sequence deduced from the DNA sequence obtained by the substitution is represented by the formula [12] or [13] (SEQ ID NO: 12 in the sequence listing).
Alternatively, it matches the amino acid sequence shown in 13).

In the present invention, the formula [14] or [15] is used.
It is also possible to provide a DNA sequence complementary to the DNA sequence of (SEQ ID NO: 14 or 15 of the sequence listing), in which case the complementary DNA sequence has the formula [14] or [15].
It may be complementary to the entire DNA sequence (SEQ ID NO: 14 or 15 in the sequence listing) or may be complementary to a part thereof. Further, it may partially include a complementary DNA sequence. Also, the formula [14] or [15]
The DNA of (SEQ ID NO: 14 or 15 of the sequence listing) and the DNA complementary thereto are bound to each other in a complementary manner to form a double-stranded DN.
A may be formed, or complementary single-stranded DNA may be alone. Formula [14] or [15] (SEQ ID NO: 1 in the sequence listing)
The amino acid sequence derived from the DNA sequence represented by 4 or 15) is represented by the formula [12] or [13] (SEQ ID NO: 12 or 13 in the sequence listing).

Formula [12] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu Leu Pro Gly Asp 20 25 30 Arg Asp Asn Leu Ala Ile Gln 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 Gln Gly Gln Ala 65 70 75 Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile 80 85 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu 95 100

Formula [13] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu Leu Pro Gly Asp 20 25 30 Arg Asp Asn Leu Ala Ile Gln 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 Ser Gln Gly Gln Ala 65 70 75 Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile 80 85 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu 95 100

The novel protein physiologically active substance of the present invention may contain a part of the amino acid sequence represented by the formula [12] or [13] (SEQ ID NO: 12 or 13 in the sequence listing). However, it may include all. It is possible to mutate a part of the structure of DNA and the structure of the polypeptide deduced therefrom by natural mutation or by artificial mutation without changing its original function. Therefore, the novel protein physiologically active substance of the present invention can also contain a sequence having a structure corresponding to a homologous variant of all the amino acid sequences described above.

Escherichia coli DH5 (pM95 transformed with the plasmid pM950 containing the DNA of SEQ ID NO: 14 in the sequence listing, which encodes the novel protein bioactive substance of the present invention.
0) has been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology [Accession No .: Microindustrial Research Institute No. 13172 (FERM P-13172, deposit date: September 21, 1992)].

In order to obtain the novel protein physiologically active substance of the present invention by genetic engineering, a commonly used host-vector system can be used. For example, when Escherichia coli is used as a host, LacUV5 is used as a promoter,
An expression vector containing a tryptophan promoter, a λP _L promoter or the like can be used. It can also be obtained as a fusion protein with β-galactosidase by using λgt11 which is a λ phage type vector.
Furthermore, mammalian cultured cells can be used as a host, and monkey-derived COS cells, Chinese hamster-derived CHO cells, mouse-derived 3T3 cells, human-derived fibroblasts, etc. are preferable. When these are used as hosts, the SV40 early promoter,
An expression vector containing an adenovirus major late promoter, a β-actin promoter, a polypeptide chain elongation factor promoter, or the like can be used. When these host-vector systems are used, the novel protein physiology of the present invention can be obtained by culturing under conditions in which each host can grow properly and by providing conditions under which each promoter functions. An active substance can be obtained.

The novel protein bioactive substance of the present invention is P
GI ₂ has an inhibitory effect on platelet aggregation, a smooth muscle relaxing effect,
It is possible to provide a pharmaceutical composition containing the protein physiologically active substance of the present invention as at least one active ingredient against the above-mentioned various diseases based on the gastric acid secretion inhibitory action and the like. The pharmaceutical form of the pharmaceutical composition of the present invention may be any as long as it can supply an effective amount to the lesion of the disease, and examples thereof include tablets, powders, powders, capsules, injections and the like. Further, the pharmaceutical composition of the present invention, as long as it does not impair its pharmacological properties, is a excipient that is a commonly used pharmaceutical mixture,
It may contain a stabilizer or a solubilizing agent.
Specific examples include Ringer's solution, phosphate buffer, human serum albumin, hydrolyzed gelatin, sucrose, dextran, polyethylene glycol, etc., which are appropriately selected and used depending on the drug form.

[0046]

EXAMPLES The present invention will be described more specifically below with reference to Examples, which is one example of the embodiments of the present invention.
The present invention is not limited to this. The scientific terms, abbreviations, etc. in the examples are according to those generally used in the relevant technical field, unless otherwise specified. The basic operations related to protein purification were carried out with reference to "Chemistry Chemistry Experiment Course, Vol. 1, Protein I Separation, Purification, Properties" edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin, 1990. Furthermore, the basic manipulations related to gene manipulation were carried out with reference to Maniatis et al., “Molecular Cloning Laboratory Manual,” Cold Spring Harbor Laboratory, 1989, and Masami Muramatsu, “Lab Manual Genetic Engineering,” Maruzen 1988. The usage of various devices and reagents was in accordance with the attached instruction manual.

(Example 1) Purification of protein-based physiologically active substance A culture supernatant was prepared and purified by the following method to obtain a novel protein-based physiologically active substance of the present invention. The PGI ₂ production stimulating activity was measured by the following method. The vascular endothelial cells were collected from the bovine thoracic aortic intima by a detachment method. The obtained vascular endothelial cells were then added to 100 U containing 10% fetal bovine serum.
/ ML penicillin and 100 μg / mL streptomycin in Dulbecco's modified Eagle medium was subcultured at 37 ° C under 5% carbon dioxide-95% air. The medium was changed twice a week, and after passage 5-10, the vascular endothelial cells were replaced with 0.0
It was treated with 5% trypsin. Then, the vascular endothelial cells were transferred to a 24-well culture dish containing 1 mL of Dulbecco's modified Eagle medium containing 10% fetal bovine serum, and cultured up to 5 × 10 ⁴ cells / well. After adding 500 μL of Dulbecco's modified Eagle medium containing 10% or less of various samples, the mixture was incubated at 37 ° C. for 60 minutes. 6-keto-PGF from culture supernatant
The extraction and purification of 1α was performed by the method of Jaffe et al. [The Journal of Clinical Investigation (J. C.
lin. Invest. ), 52, 398, 197
3 years] was improved. 1.5 mL of 0.1N hydrochloric acid was added to 1 mL of the culture supernatant, and the mixture was extracted twice with 5 mL ethyl acetate. Ethyl acetate was evaporated under nitrogen gas and redissolved in ethanol. Stored at -20 ° C until used in assay. Ethanol was evaporated at 40 ° C. and dissolved in 0.1 M phosphate buffer (pH 7.2, containing 1 M sodium chloride and 1% gelatin). New England Nuclea using 6- [ ³ H] keto PGF1α and anti-6-keto-PGF1α antibody.
r) 6-keto-PGF1α measurement radioimmunoassay kit manufactured by r) and 6-keto-P according to the attached manual
The concentration of GF1α was measured. The PGI ₂ production was determined by the 6-keto-PGF1α production per 10 ⁴ cells (pg / 10 ⁴ cells / hour) for 1 hour.

(1) Culture of normal human diploid fibroblasts
Preparation of supernatant Normal human diploid fibroblasts were diluted with 15% fetal bovine serum
4.8 × 10 in Lubeco modified Eagle medium^FourCells / m
Prepared to L. Inoculate 3 L of this cell suspension into a rotary culture vessel.
Incubate at 37 ° C in 5% carbon dioxide-95% air.
did. 3 days after the cell implantation, the culture solution was replaced with fresh 15% bovine
Change to Dulbecco's modified Eagle medium 3L containing fetal serum
Then, the culture was continued for 2 days. Then remove the culture
Leave, Ca ²⁺, Mg²⁺Dulbecco's phosphate-free saline
After washing the cells with the solution, phenol red-free
Of Dulbecco's modified Eagle medium (3 L),
Cultured for 2 days. 2.5 μm fill after collecting the culture solution
Tar (CN cartridge 30 inches, Millipore)
And filtered to remove cell debris. Then hollow fiber
Module (Molsep Fiber FS-10 6kD
a Cut-off, manufactured by Daicel Chemical Industries, Ltd., molecular weight cutoff
10 kDa ultrafiltration cassette (Omega miniset, wealth
Concentration by a two-stage concentration operation by Shishi Filter Industry Co., Ltd.)
Contracted. This concentrated solution was dialyzed with a molecular weight cutoff of 3.5 kDa.
20 mM Tris using a tube (manufactured by Spectrum)
-Dialysis against hydrochloric acid buffer (pH 7.8) at 4 ° C overnight. same
After dialysis with buffer for 8 hours at 4 ℃, 1.2μm fill
Tar (Differentiation assembly, manufactured by Nippon Pole),
0.22μm filter system (made by Corning)
It was sequentially filtered.

(2) Purification of PGI ₂ production stimulating factor Step 1 The culture supernatant of normal human diploid fibroblasts prepared according to (1) was previously prepared in 20 mM Tris-HCl buffer (pH 7.
Adsorbed on DEAE-5PW (manufactured by Tosoh Corp.) anion exchange chromatography column equilibrated in 8), and 20 mM Tris-
0 to 1.0 prepared using hydrochloric acid buffer (pH 7.8)
Elute with a linear gradient method of sodium chloride of M, and
Absorbance was measured simultaneously at 80 nm. The eluted PGI ₂ production-stimulating activity of each fraction was measured according to the method described above, it was pooled fractions with the eluted PGI ₂ production stimulating activity at sodium chloride concentrations of 50 to 150 mM (Fig. 1). Step 2 The active fraction obtained in Step 1 was treated with 10 mM phosphate buffer (p
After dialysis with H7.4), a 10 mM phosphate buffer solution (pH 7.
4) equilibrated with Heparin-5PW
Adsorbed on an affinity column (manufactured by Tosoh Corp.) for 10
0 prepared using mM phosphate buffer (pH 7.4)
Elution was performed with a linear concentration gradient method of 1.0 M sodium chloride, and the absorbance was simultaneously measured at 280 nm. The PGI ₂ production stimulating activity of each eluted fraction was measured,
Fractions having a PGI ₂ production stimulating activity, which were eluted at a sodium chloride concentration of 450 to 500 mM, were pooled (FIG. 2).

Step 3 The active fraction obtained in Step 2 was concentrated with Centricon-10 (manufactured by Amicon), and then 10 mM phosphate buffer (p
H-7.4) equilibrated protein-pack (PROT
EIN-PAK) 300 (Japan Millipore Limited)
It was developed using two gel filtration columns (Waters Chromatography Division) connected to each other, and the absorbance was measured at 280 nm at the same time. PG of each fraction
When the I ₂ production stimulating activity was measured, the activity was 30
It was observed in the vicinity of kDa (Fig. 3). The numbers in FIG. 3 represent the molecular weight (kDa), Vt represents the total volume of the filler, and Vo represents the excluded volume. Step 4 The active fraction obtained in Step 3 was treated with 10 mM phosphate buffer (p
H7.4) was applied to an IGF-affinity column equilibrated. As the IGF-affinity column, a pressure resistant column in which recombinant IGF-I was bound as a ligand to Affiprep 10 (manufactured by Nippon Bio-Rad Laboratories) was prepared and used in this experiment. 0.5 after sample adsorption
Elute with M acetic acid. When the PGI ₂ production stimulating activity was measured, the PGI ₂ production stimulating activity was found in the non-adsorbed fraction.

Step 5 The active fraction obtained in Step 4 was applied to a C ₄ reverse-phase HPLC column (manufactured by Waters) equilibrated with a 10% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid, and then the eluate was adjusted to 0. Elution was performed with a linear concentration gradient method of 10 to 60% acetonitrile produced using a 1% trifluoroacetic acid / acetonitrile solution. As a result, the novel protein bioactive substance of the present invention was eluted as a single peak.

(3) Measurement of Physical Properties of PGI ₂ Production Stimulating Factor Derived from Normal Human Diploid Fibroblasts Measurement of Molecular Weight Molecular weight of novel protein physiologically active substance of the present invention obtained in step 5 of (2) Under non-reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS
-PAGE), the molecular weight was 30-36 kDa.
Was recognized as a single band (Fig. 4). Figure 4 SD
It is a schematic diagram of the reference photograph after S-PAGE migration. Figure 4
Lines A and C are bands of a standard sample having a known molecular weight, and line B is a band of the novel protein bioactive substance of the present invention. The numbers represent the molecular weight (kDa) of the standard sample. Analysis of amino acid sequence The novel protein bioactive substance of the present invention obtained in step 5 of (2) is digested with trypsin or after reductive carboxymethylation, and then subjected to a C ₈ reverse phase HPLC column (Nippon Waters Co., Ltd.). Desalting, and then endoproteinase G
Digested with lu-C (Protease V8) to fragment the peptide. Each of these samples was applied to a C ₈ reverse-phase HPLC column (manufactured by Separations Group) equilibrated with a 0.1% trifluoroacetic acid aqueous solution, and then 0.
Elution was performed by a linear concentration gradient method of 0 to 100% acetonitrile prepared using a 08% trifluoroacetic acid / acetonitrile solution. The amino acid sequence of each peptide fragment was determined by Edman degradation using an automated gas phase sequencer (Applied Biosystems 477A-120A). The obtained amino acid sequence is represented by the following formula [1]
[11] (SEQ ID NOS: 1 to 11 in the sequence listing).

Formula [1] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu 20 25 Formula [2] Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr Arg Gly 1 5 10 15 Gly Pro Glu

Formula [3] Cys His Ala Ser Asn Ser Gln Gly Gln Ala Ser Ala Ser Ala Lys 1 5 10 15 Ile Thr Val Val 19 Formula [4] Ile Thr Val Val Asp Ala Leu His Glu Ile Pro Val Lys Lys Gly 1 5 10 15 Glu Gly Ala Glu Leu 20

[0055] Formula [7] Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr 1 5 10 15 Arg

[0056] Formula [9] Lys Ala Ile Thr Gln Val Ser Lys Gly Thr Cys Glu 1 5 10 Formula [10] Xaa Glu Pro Xaa Gly Gly Gly Gly Ala Gly Arg Gly Tyr Gln Ala 1 5 10 15 Pro Gly Formula [11] Gln Gly Pro Ser Ile Val Thr Pro Xaa Lys Asp Ile 1 5 10

(Example 2) Preparation of cDNA library Step 1 Preparation of normal human diploid fibroblasts Normal human diploid fibroblasts were supplemented with 15% fetal calf serum-containing DA.
4.8x10 in Lubeco's modified Eagle medium^FourCells / m
Prepared to L. Inoculate 3 L of this cell suspension into a rotary culture vessel.
Incubate at 37 ° C in 5% carbon dioxide-95% air.
did. 3 days after the cell implantation, the culture solution was replaced with fresh 15% bovine
Change to Dulbecco's modified Eagle medium 3L containing fetal serum
Then, the culture was continued for 2 days. Then remove the culture
Leave, Ca ²⁺, Mg²⁺Dulbecco's phosphate-free saline
After washing the cells with the solution, phenol red-free
Of Dulbecco's modified Eagle medium (3 L),
It lasted 24 hours. Then, the culture solution is removed and Ca²⁺,
Mg²⁺Using a physiological phosphate buffer solution (PBS (-)) that does not contain
Wash cells with 8x10 from 7 rotating culture vessels⁹Individual
Cells were obtained.

Step 2 Preparation of Total RNA From normal human diploid fibroblasts,
czynski, P) et al. [Analytical Biochem., 162, 156].
Page, 1987] AGPC (Acid Guanid
inium Thiocyanate-Phenol-
RNA was prepared according to the Chloroform) method.
In other words, 60m for approximately 10 ⁹ cells obtained in step 1
L 4M guanidine thiocyanate solution (pH 7.0,
25 mM sodium citrate, 0.5% sarcosyl and 0.1 M 2-mercaptoethanol) was added,
I shook it strongly. In addition, 1/10 volume of 2 in cell lysate
M sodium acetate (pH 4.0), 60 mL of water-saturated phenol, and 1/5 volume of chloroform-isoamyl alcohol mixed solution (49: 1) were sequentially added and mixed, further shaken vigorously for 10 seconds, and then kept in ice for 15 minutes. I left it. 85 at 4 ° C
After centrifugation at 00 rpm for 20 minutes, the aqueous layer (upper part) was transferred to a new tube. The same amount of 100% isopropanol as that of the obtained aqueous layer was added, and the mixture was left overnight at -20 ° C. After centrifugation at 8500 rpm for 30 minutes at 4 ° C., the resulting pellet was added to about 50 mL of 4M guanidine thiocyanate solution (pH
7.0, containing 25 mM sodium citrate, 0.5% sarcosyl and 0.1 M 2-mercaptoethanol) and added about 50 mL of isopropanol. −
After leaving it at 20 ° C. for 1 hour, it was centrifuged at 4 ° C. and 8500 rpm for 30 minutes. Approximately 30 mL of 0.5 mM ED pellet
After dissolution in TA, ethanol precipitation was carried out in the presence of sodium chloride according to a conventional method, and the pellet was added to 3.5 mL of 0.5 mM.
Dissolved in EDTA. About 9 mg of total RNA was obtained.

Step 3 Preparation of poly (A) ⁺ RNA From the total RNA, an oligo (dT) cellulose column was used.
Poly (A) ⁺ RNA was isolated. That is, oligo (d
T) Cellulose resin 500 mg was added to 200 mM Tris-hydrochloric acid buffer solution (pH 7.5, 20 mM EDTA, 400).
The column was suspended in mM sodium chloride and 0.1% SDS) to prepare a column having a volume of about 2 mL. The total RNA (3.5 mL) obtained in step 2 was heat-treated at 65 ° C. for 5 minutes, then rapidly cooled, and the same amount of 400 mM Tris-hydrochloric acid buffer solution (pH 7.5, 40 mM EDT) as the total RNA solution.
A, containing 800 mM sodium chloride and 0.2% SDS). This solution was added to the above-mentioned 200 mM Tris-hydrochloric acid buffer solution (pH 7.5, 20 mM EDTA, 400
It was adsorbed on an oligo (dT) cellulose column equilibrated with mM sodium chloride and 0.1% SDS. 1
0 mL of 100 mM Tris-HCl buffer solution (pH 7.
5, containing 10 mM EDTA, 200 mM sodium chloride and 0.1% SDS). Then 10 mL
Poly (A) ⁺ RNA was eluted with the sterilized distilled water of 1. and ethanol precipitation was carried out in the presence of sodium chloride according to a conventional method. About 270 μg of poly (A) ⁺ RNA (mRNA) was obtained.

Step 4 Synthesis of Double-stranded cDNA Gubler and Hoffman's method [Gene, 25
Double-stranded cDNA was synthesized using a cDNA synthesis system kit (manufactured by Amersham), which was constructed based on the improved system of Vol. 263, 1983]. 5x1 chain cDNA attached to this cDNA synthesis system kit
A synthesis reaction buffer, sodium pyrophosphate solution,
A human placenta ribonuclease inhibitor, a deoxynucleoside triphosphate mixed solution, and an oligo (dT) primer were mixed in this order with 10 μg of the poly (A) ⁺ RNA solution obtained in step 3 and gently mixed, Reverse transcriptase was added. The reaction solution was 100 μL.
After gently mixing, the mixture was incubated at 42 ° C for 40 minutes, and the reaction tube was ice-cooled. Double-stranded cDNA synthesis reaction buffer, E. coli ribonuclease H, E. coli DNA polymerase I attached to the above-mentioned cDNA system synthesis kit
Were sequentially added, and the total volume was adjusted to 500 μL with sterilized water and gently mixed. The tube was reacted at 12 ° C. for 60 minutes, then at 22 ° C. for 60 minutes, and further incubated at 70 ° C. for 10 minutes. Put it back in the ice bath and get 20U of T4D
Add NA polymerase and mix gently, then at 37 ° C for 10
Let react for minutes. Add 20 μL of 0.25M ED to the reaction solution.
The reaction was stopped by adding TA (pH 8.0), followed by phenol / chloroform extraction and ethanol precipitation in the presence of ammonium acetate according to a conventional method to purify the double-stranded cDNA.

Step 5 Addition of BstXI linker A double strand c of BstXI linker manufactured by Invitrogen was added.
Added to DNA. The method was carried out using a DNA ligation kit (Takara Shuzo) according to the attached manual. That is, about 50 double-stranded cDNA synthesized in step 4
30 μL of a solution obtained by adding about 500 ng of BstXI linker to 0 ng (100 mM Tris-HCl buffer solution (pH
7.6) 5 mM magnesium chloride, containing 300 mM sodium chloride) was prepared. Next, 30 μL of enzyme solution (DNA Ligation Kit solution B, hereinafter referred to as solution B) was added and mixed well, and reacted at 10 ° C. for 3 hours. 7
After heat treatment at 0 ° C. for 10 minutes, ethanol precipitation was performed in the presence of ammonium acetate. Next, electrophoresis was carried out on a low-melting point agarose gel, and fractions of 500 bp or more were collected.
After the gel is dissolved by heat treatment at ℃, phenol /
Chloroform extraction and ethanol precipitation were performed in the presence of sodium chloride. Finally, about 100 ng of the linker-added cDNA was recovered.

Step 6 Introduction of cDNA into Vector Insertion of cDNA into a plasmid vector was performed using a DNA ligation kit (Takara Shuzo). That is, 100 ng of the plasmid pEF-BOS digested with the restriction enzyme BstXI [Nucleic Acids Res.], Vol. 18,
5322, 1990] and a DNA solution containing about 20 ng of the linker-added double-stranded cDNA obtained in Step 5.
36 μL of reaction buffer (DNA ligation kit A solution) was added to 5 μL and mixed well. Then, 4.
5 μL of enzyme solution (solution B) was added and mixed well, and the mixture was reacted at 16 ° C. for 30 minutes. After ethanol precipitation in the presence of ammonium acetate, the precipitate was dissolved in TE solution to obtain a ligated product of cDNA and plasmid vector. DNA for E. coli
Was introduced by electroporation. The electroporation method is performed by Dowar et al. [Nucl. Acids Res.],
16, p. 6127, 1988].

E. coli MC1061 / P3 was inoculated into 10 mL of L-broth from a single colony and cultured overnight. The overnight culture of 10mL was inoculated into L- broth 1L, and cultured at 37 ° C. until the A _{60 0} = about 0.5. The culture was ice-cooled and then centrifuged at 6000 rpm for 15 minutes at 4 ° C to collect the cells. After removing the supernatant by decantation, it was resuspended in 1 L of ice-cold sterile water. After centrifugation at 6000 rpm for 15 minutes at 4 ° C, the supernatant was removed and
Suspend in 00 mL of ice-cold sterile water and spin at 6000 rpm for 4
After centrifuging at ℃ for 15 minutes, the supernatant was removed. 20 mL ice cold 1
Resuspend in 0% glycerol, 4 ° C at 6000 rpm
After centrifuging at 15 minutes, the supernatant was removed. 2 mL ice-cooled 10%
The cells were resuspended in glycerol and dispensed in appropriate amounts into 1.5 mL tubes. After rapid cooling and freezing with dry ice ethanol, it was frozen and stored at around -70 ° C. After thawing 40 to 50 μL of cryopreserved E. coli on ice, 1 μL of the above-mentioned cDNA was added.
A DNA solution containing the ligation product of A and the vector was added. D
NA-E. Coli mixed solution was cuvette (distance between electrodes 0.1c
m) and then electroporation conditions (Gene Pulser, Bio-Rad, capacitance: 2)
5μF, voltage: 1.8kV, pulse controller: 2
00 Ω), pulse once, 1 mL SOC
The medium was added to the cuvette to suspend E. coli. After the suspension was transferred to a culture tube and cultured for 1 hour, an appropriate amount was spread on a 50 μg / mL ampicillin-containing LB plate and cultured at 37 ° C. overnight. About 600,000 clones of transformants were obtained by the above operation.

(Example 3) Acquisition of cDNA encoding protein physiologically active substance Step 1 Preparation of primer Among the partial amino acid sequences determined in Example 1, the formula [4]
The DNA sequence encoding the N-terminal and C-terminal amino acid sequences of the 20 amino acid sequence represented by (SEQ ID NO: 4 in the Sequence Listing) is subjected to the phosphoamidide method using a 394 type DNA / RNA synthesizer (manufactured by Applied Biosystems). Synthesized. That is, a DNA mixture consisting of 20 bases represented by the following formula [16] (SEQ ID NO: 16 in the sequence listing) was used as the DNA encoding the N-terminal 7 amino acids, and the complementary strand of the DNA encoding the C-terminal 7 amino acids was used. A mixture of DNA consisting of 20 bases represented by the following formula [17] (SEQ ID NO: 17 in the sequence listing) as a sequence was prepared.

[0065]

PCR was carried out using the above-mentioned DNA mixture as a primer and the double-stranded cDNA prepared in step 4 of Example 2 as a template.
The law was implemented. That is, Gene Amp PCR
Reagent Kit (Perkin Elmer Cetus Instruments (Perkin Elmer
Cetus Instruments))
20 μL of reaction solution containing NA solution (10 mM Tris-HCl buffer solution (pH 8.3), 50 mM potassium chloride,
1.5 mM magnesium chloride, 200 μM dNT each
P, 1 μM sense primer, 1 μM antisense primer and 0.025 U / μL AmpliTaq D
NA Polymerase) and prepare GeneAmp ^™ P
CR system 9600 (Perkin Elmer Cetus Instruments (Perkin Elmer
Cetus Instruments)),
The reaction at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 minute was repeated 25 times. Next, the reaction product was electrophoresed on a 10% polyacrylamide gel, and it was confirmed that a band appeared near 59 bp. Then, the obtained DNA fragment of the amplification product around 59 bp was extracted according to a conventional method to obtain T4 DNA.
The ends were blunted using a polymerase according to a conventional method, and cloned into the SmaI site of the plasmid pUC118. The nucleotide sequence of the DNA fragment was determined according to the sequencing method described below. The sequence is shown in the following formula [18] (SEQ ID NO: 18 in the sequence listing).

Formula [18] 5′-ATTACGGTGG TTGATGCGTT ACATGAAATA CCAGTGAAAA 10 20 30 40 AAGGCGAAGG CGCCGAATT-3 ′ 50 59

A 20-mer DNA fragment (formula [19] (SEQ ID NO: 19 of the sequence listing) having a base sequence of 11 to 30 of the base sequence represented by the formula [18] (SEQ ID NO: 18 of the sequence listing) 20m complementary to the base sequence from 31 to 50
er DNA fragment (formula [20] (SEQ ID NO: 2 in the sequence listing)
0) was synthesized by the same method as described above and used as a screening primer by the PCR method below.

Formula [19] 5′-TTGATGCGTTACATGAAATA-3 ′ Formula [20] 5′-CCTTCGCCTTTTTTCACTGG-3 ′

Step 2 Screening of cDNA Library of Normal Human Diploid Fibroblasts by PCR Method The transformant obtained in Example 2 was used for about 14 cells per pool.
000 clones were divided and 46 pools were prepared, each containing LB containing 50 μg / mL ampicillin.
The cells were inoculated into 3 mL of the medium and cultured at 37 ° C. with shaking overnight. Extraction of the plasmid DNA was carried out according to the method of Molecular Cloning Laboratory Manual (Cold Spring Harbor Laboratory 1989). That is, 1.5 mL of the culture solution at 5000 rpm
After centrifuging at 37 ° C for 3 minutes to remove the supernatant, the precipitate was mixed with 100 μL glucose-lysozyme solution (50 mM glucose, 10 m
M EDTA, 25 mM Tris-HCl buffer solution (pH
8.0) and 4 mg / mL lysozyme),
It was left at room temperature for 5 minutes. 200 μL of alkaline solution (containing 0.2N sodium hydroxide and 1% SDS) was added in ice, mixed gently, and allowed to stand in ice for 5 minutes. next,
150 μL potassium acetate solution (4 ° C.) was added, mixed well and placed on ice for 5 minutes. 4 ° C, 5 at 12000 rpm
After centrifuging for minutes, the supernatant was transferred to another tube, 400 μL phenol-chloroform solution was added, and the mixture was stirred well,
It was centrifuged at 12000 rpm for 5 minutes. 2 volumes (about 800 μL) of ethanol was added to the aqueous layer, mixed, and then left at room temperature for 2 minutes. After centrifugation at 12000 rpm for 5 minutes, the supernatant was completely removed, the precipitate was washed with 70% ethanol,
50 μL dried and containing 20 μg / mL RNase
Dissolved in TE solution. 2.5 μL of the obtained plasmid DNA solution was treated with a restriction enzyme XbaI at 37 ° C. for 1 hour to cut the plasmid and the DNA.

Next, Gene Amp PCR Rea
gen Kit (Perkin Elmer Cetus Instrument Co. (Perkin Elmer Ce
using Tus Instruments))
20 μL of reaction solution containing NA solution (10 mM Tris-HCl buffer solution (pH 8.3), 50 mM potassium chloride,
1.5 mM magnesium chloride, 200 μM dNT each
P, 1 μM sense primer, 1 μM antisense primer and 0.025 U / μL AmpliTaqDN
A Polymerase) and prepare Gene Amp ^™ P
CR system 9600 (PerkinElmer Citas Instruments (PerkinElmer C
etus Instruments)) and set 9
The reaction at 4 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 minute was repeated 25 times. Next, the reaction product was electrophoresed on a 10% polyacrylamide gel, and the one in which a band was detected near 40 bp was judged to be positive. Escherichia coli DH5 was transformed with one positive pool of plasmid DNA, the resulting transformants were divided so that each pool contained about 1800 clones, and 15 pools were prepared and DNA was prepared in the same manner as described above. It was prepared and judged to be positive when a band was detected around 40 bp by the PCR method. This operation was repeated thereafter, and positive clones were identified by a total of 6 screenings.

Example 4 Determination of Base Sequence The base sequence was determined according to the method described in Laboratory Manual Genetic Engineering (edited by Masamitsu Muramatsu, Maruzen 1988). That is, the cDNA of the positive clone obtained in Example 3 was digested with the restriction enzyme XbaI and excised D
The NA fragment was mixed with pUC119 digested with the restriction enzyme XbaI and ligated using the above-mentioned ligation kit. After transforming Escherichia coli JM109 with the ligation mixture, single-stranded DNA was prepared according to a conventional method. Prepare the prepared single-stranded DNA using a DNA sequencer (3
73A, manufactured by Applied Biosystems) was used to determine the nucleotide sequence according to the attached manual. The results are shown in SEQ ID NO: 14 or 15 of the sequence listing. Also the DN
The amino acid sequence deduced from the A sequence is shown in SEQ ID NO: 12 or 13 of the sequence listing.

[0073]

INDUSTRIAL APPLICABILITY The novel protein physiologically active substance of the present invention has an action of stimulating PGI ₂ production from vascular endothelial cells. Therefore, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, peripheral arterial occlusion, cardiac ischemia, cerebral ischemia, arteriosclerosis, based on the platelet aggregation inhibitory action, smooth muscle relaxation action, gastric acid secretion inhibitory action and the like of PGI ₂ . Brain obstruction, hyperlipidemia, diabetes,
It can be expected to be applied as a medical use for heart failure, angina, ischemic heart disease, congestive heart disease, choroidal circulation disorder, bronchial disease, gastric ulcer, eclampsia of pregnancy and the like. It is also possible to obtain a large amount of the novel protein physiologically active substance of the present invention by genetic engineering using the DNA encoding the protein physiologically active substance of the present invention.

[Sequence list]

SEQ ID NO: 1 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu 20 25

SEQ ID NO: 2 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr Arg Gly 1 5 10 15 Gly Pro Glu

SEQ ID NO: 3 Sequence length: 19 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Cys His Ala Ser Asn Ser Gln Gly Gln Ala Ser Ala Ser Ala Lys 1 5 10 15 Ile Thr Val Val 19

SEQ ID NO: 4 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ile Thr Val Val Asp Ala Leu His Glu Ile Pro Val Lys Lys Gly 1 5 10 15 Glu Gly Ala Glu Leu 20

SEQ ID NO: 5 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 6 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 7 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr 1 5 10 15 Arg

SEQ ID NO: 8 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 9 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Lys Ala Ile Thr Gln Val Ser Lys Gly Thr Cys Glu 1 5 10

SEQ ID NO: 10 Sequence length: 17 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Xaa Glu Pro Xaa Gly Gly Gly Gly Ala Gly Arg Gly Tyr Gln Ala 1 5 10 15 Pro Gly

SEQ ID NO: 11 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Gly Pro Ser Ile Val Thr Pro Xaa Lys Asp Ile 1 5 10

SEQ ID NO: 12 Sequence length: 100 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu Leu Pro Gly Asp 20 25 30 Arg Asp Asn Leu Ala Ile Gln 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 Gln Gly Gln Ala 65 70 75 Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile 80 85 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu 95 100

SEQ ID NO: 13 Sequence length: 100 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu Leu Pro Gly Asp 20 25 30 Arg Asp Asn Leu Ala Ile Gln 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 Ser Gln Gly Gln Ala 65 70 75 Ser Ala Ser Ala Lys Ile Thr Val Val Asp Ala Leu His Glu Ile 80 85 90 Pro Val Lys Lys Gly Glu Gly Ala Glu Leu 95 100

SEQ ID NO: 14 Sequence length: 557 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA sequence GGTCATCGGA ATCCCGACAC CTGTCCTCAT CTGGAACAAG GTAAAAAGGG GTCACTATGG 60 AGTTCAAAGG ACAGAACTCC CCTGTCTGGTGA 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 CTTTTTATCA 540 CAAAAAAAAA AAAAAAA 557

SEQ ID NO: 15 Sequence length: 557 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA sequence GGTCATCGGA ATCCCGACAC CTGTCCTCAT CTGGAACAAG GTAAAAAGGG GTCACTATGG 60 AGTTCAAAGG ACAGAACTCC CCTGTCTGGTGA AGACCCGGGG 120 TGGCCCAGAA AAGCATGAAG TAACTGGCTG GGTGCTGGTA TCTCCTCTAA GTAAGGAAGA 180 TGCTGGAGAA TATGAGTGCC ATGCATCCAA TTCCCAAGGA 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 CTTTTTATCA 540 CAAAAAAAAA AAAAAAA 557

SEQ ID NO: 16 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA

SEQ ID NO: 17 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA

SEQ ID NO: 18 Sequence length: 59 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: PCR product Sequence 5'-ATTACGGTGG TTGATGCGTT ACATGAAATA CCAGTGAAAA 10 20 30 40 AAGGCGAAGG CGCCGAATT -3 '50 59

SEQ ID NO: 19 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence 5'-TTGATGCGTTACATGAAATA-3 '

SEQ ID NO: 20 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence 5'-CCTTCGCCTTTTTTCACTGG-3 '

[Brief description of drawings]

FIG. 1 is a graph showing an elution pattern by DEAE-5PW anion exchange chromatography.

FIG. 2 is a graph showing an elution pattern by heparin-5PW affinity chromatography.

FIG. 3 is a graph showing an elution pattern by protein-pack gel filtration column chromatography.

FIG. 4 is a schematic diagram showing an experimental result by SDS-PAGE.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C07K 7/08 7537-4H 7/10 7537-4H 13/00 8517-4H C12N 15/12 C12P 21 / 02 C 8214-4B // A61K 37/02 ABX 8314-4C ACB 8314-4C ACD 8314-4C ACL 8314-4C ADP 8314-4C C07K 99:00 8318-4H (72) Inventor Atsushi Tokyo Metropolitan Shinjuku 1-7 Yotsuya, Tokyo Mochida Pharmaceutical Co., Ltd. (72) Inventor, Anzu Nagase 1-7 Yotsuya, Shinjuku, Tokyo Tokyo Mochida Pharmaceutical Co., Ltd.

Claims (9)

[Claims] 1. A novel protein physiologically active substance having at least one amino acid sequence represented by any of the following formulas [1] to [11] (SEQ ID NOS: 1 to 11 in the sequence listing). Formula [1] Val Ile Gly Ile Pro Thr Pro Val Leu Ile Trp Asn Lys Val Lys 1 5 10 15 Arg Gly His Tyr Gly Val Gln Arg Thr Glu 20 25 Formula [2] Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr Arg Gly 1 5 10 15 Gly Pro Glu Formula [3] Cys His Ala Ser Asn Ser Gln Gly Gln Ala Ser Ala Ser Ala Lys 1 5 10 15 Ile Thr Val Val 19 Formula [4] Ile Thr Val Val Asp Ala Leu His Glu Ile Pro Val Lys Lys Gly 1 5 10 15 Glu Gly Ala Glu Leu 20 Formula [7] Thr Glu Leu Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr 1 5 10 15 Arg Formula [9] Lys Ala Ile Thr Gln Val Ser Lys Gly Thr Cys Glu 1 5 10 Formula [10] Xaa Glu Pro Xaa Gly Gly Gly Gly Ala Gly Arg Gly Tyr Gln Ala 1 5 10 15 Pro Gly Formula [11] Gln Gly Pro Ser Ile Val Thr Pro Xaa Lys Asp Ile 1 5 10 2. A novel protein bioactive substance containing part or all of the amino acid sequence of SEQ ID NO: 12 in the sequence listing. 3. A novel protein bioactive substance containing part or all of the amino acid sequence set forth in SEQ ID NO: 13 of the Sequence Listing. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PA) under non-reducing conditions.
The novel proteinaceous physiologically active substance according to any one of claims 1 to 3, which has a molecular weight of 30 to 36 kDa in GE). 5. A prostaglandin I ₂ (PGI ₂ ) production stimulating activity.
The novel protein bioactive substance according to any one of 1. 6. D having a base sequence encoding the novel protein bioactive substance according to any one of claims 1 to 5.
NA. 7. The DN according to claim 6, wherein the DNA is a part or the whole of the nucleotide sequence of SEQ ID NO: 14 in the sequence listing.
A. 8. The DN according to claim 6, wherein the DNA is a part or all of the nucleotide sequence of SEQ ID NO: 15 in the sequence listing.
A. 9. A raw material containing the novel protein physiologically active substance according to any one of claims 1 to 5, which is prepared from the following (a) to (a):
6. The method for producing a novel physiologically active proteinaceous substance according to any one of claims 1 to 5, wherein the method is purified by using at least one of the steps (c). (A) Ion exchange chromatography (b) Affinity chromatography (c) Gel filtration chromatography