WO1994029448A1 - Adn et proteine codee par celui-ci - Google Patents

Adn et proteine codee par celui-ci Download PDF

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WO1994029448A1
WO1994029448A1 PCT/JP1994/000946 JP9400946W WO9429448A1 WO 1994029448 A1 WO1994029448 A1 WO 1994029448A1 JP 9400946 W JP9400946 W JP 9400946W WO 9429448 A1 WO9429448 A1 WO 9429448A1
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gly
ala
pro
ser
glu
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PCT/JP1994/000946
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English (en)
Japanese (ja)
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Atsushi Nii
Yasukazu Nagase
Seiichi Mizushima
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Nawata, Hajime
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Publication of WO1994029448A1 publication Critical patent/WO1994029448A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • PG I 2 production stimulating factor proteinaceous physiologically active substance having a production stimulating activity
  • 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.
  • 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.
  • TXA 2 TXA 2
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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).
  • 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.
  • 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).
  • This protein is characterized by its ability to stimulate vascular endothelial cells to promote PGI 2 production.
  • 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.
  • 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.
  • 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.
  • Gly Asp Arg Asp Asn Leu Ala lie Gin Thr Arg Gly Gly Pro Glu 185 190 195
  • GCCGCGCGCCTCA CCGCCCCC CCGCCCTGC TCCTCGGACCG CCATGGAGCG GCCGTGTGG
  • AAAA 1124 is a protein of AAAA 1124 present invention.
  • a general genetic engineering technique is used.
  • 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
  • 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.
  • cDNA encoding the PG I 2 production stimulating factor of the present invention can be obtained by the following method.
  • 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.
  • 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
  • a phage vector for cloning introduced into Igt10, ⁇ gt11, etc. Mouth packaging and infect E. coli.
  • 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 ⁇ gt10 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.
  • a library capable of expressing cDNA for example, a library prepared using a ⁇ gt11 phage vector
  • a target clone can be selected using the antibody obtained by the above-described method.
  • 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].
  • GCCGCTGCCA CCGCACCCCG CCATGGAGCG GCCGTCGCTG CGCGCCCTGC TCCTCGGCGC 60 CGCTGGGCTG CTGCTCCTGC TCCTGCCCCT CTCCTCTTCC TCCTCTTCGG ACACCTGCGG 120 CCCCTGCGAG CCGGCCTCCT GCCCGCCCCT GCCCCCGCTG GGCTGCCTGC TGGGCGAGC
  • 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.
  • the DNA sequence of the present invention also includes a partially modified base sequence due to the degeneracy of the genetic code.
  • the amino acid sequence encoding the DNA sequence obtained by the substitution is the amino acid sequence represented by the formula [1] or [2].
  • 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].
  • 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.
  • 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.
  • 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 1 ⁇ the amount of PGI 2 produced can be determined.
  • a commercially available 6-keto PGF1 ⁇ measurement kit may be used.
  • 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.
  • 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.
  • a host-vector system can be used.
  • Escherichia coli 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.
  • it can be obtained as a fusion protein with S-galactosidase using ⁇ gt11 which is one of the sperm vectors.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PBS (-) physiological phosphate buffer
  • 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.
  • OmM tris-hydrochloric acid buffer solution containing pH 7.5.2 OmM EDTA, 400 mM sodium chloride and 0.1% SDS
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the cDNA was introduced into a plasmid vector using a DNA ligation kit (Takara Shuzo).
  • Plasmid p EF—BOS Nucleic Acid Research (Nucl. Acids Res.), 18, 5322, 1990]
  • 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 ⁇ L of a 36 zL reaction buffer (DNA buffer).
  • a solution kit A was added and mixed well.
  • 4.5 ⁇ L 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.
  • 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.
  • 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.
  • 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
  • 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
  • a mixture of 20 bases of DNA represented by the following formula [9] SEQ ID NO: 10 in the sequence listing was prepared.
  • the poly (A) + RNA prepared in step 3 of Example 1 was subjected to an RT-PCR method using the ⁇ -form. That is, Super S cript TM 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.
  • System manufactured by Lifetech Oriental
  • DNA Th e rma l Cy cler TM is set to (Perkin Elmer one Cetus Instruments Inc. (P er ki ⁇ 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.
  • the reaction product was electrophoresed on a 10% polyacrylamide gel, and it was confirmed that a band appeared at around 59 bp.
  • 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).
  • 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.
  • Example 1 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 ⁇ g 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).
  • 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.
  • 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
  • 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 TM PCR System 9600 (Perkin Elmer Cetus Instruments, Inc.), and then set in Gene Gene TM 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.
  • 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.
  • 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.
  • 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.
  • amino acid sequence deduced from the DNA sequence is shown in SEQ ID NO: 15 or 16 in the sequence listing.
  • 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.
  • 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 ⁇ L of cDNA, 40 L of LZK buffer and 10 ⁇ L 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.
  • cDNA cloning system 20 ng of double-stranded cDNA was added to 1 ⁇ g of ⁇ 10 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.
  • 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.
  • 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.
  • the phage particle mixture obtained in Step 3 was diluted to 5 ⁇ 10 4 pfu / 200 / zL with SM buffer.
  • the obtained precipitate was suspended in 15 mL of a 1 OmM magnesium sulfate solution.
  • 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 ⁇ L 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.
  • Step 2 Screening of sphage cDNA library
  • Screening of the ⁇ 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 ⁇ 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 TM 2400, manufactured by Stratagene) to fix the DNA.
  • UV irradiator UV stratulin ker TM 2400, manufactured by Stratagene
  • 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.
  • 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.
  • Expression vector ⁇ EF 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 ⁇ 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.
  • 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].
  • 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
  • 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,
  • 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 ⁇ 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.
  • methotrexate manufactured by Nippon Redley Co., Ltd.
  • 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)
  • 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.
  • 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.
  • 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).
  • 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).
  • 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)
  • 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.
  • 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.
  • 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 9 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 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
  • Example 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.
  • 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.
  • 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.
  • 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). .
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • lines A and C are bands of a standard sample having a known molecular weight
  • 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.
  • 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).
  • 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.
  • Lys Ala lie Thr Gin Val Ser Lys Gly Thr Xaa Glu
  • 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.
  • 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.
  • 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-PGF1 ⁇ 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 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.
  • GCCGCTGCCA CCGCACCCCG CCATGGAGCG GCCGTCGCTG CGCGCCCTGC TCCTCGGCGC 60 CGCTGGGCTG CTGCTCCTGC TCCTGCCCCT CTCCTCTTCC TCCTCTTCGG ACACCTGCGG 120 CCCCTGCGAG CCGGCCTCCT GCCCGCCCCT GCCCCCGCTG GGCTGCCTGC TGGGCGAGC
  • Lys Ala lie Thr Gin Val Ser Lys Gly Thr Cys Glu Gin Gly Pro

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Abstract

Nouvel ADN représenté par le numéro d'identification de séquence 3 ou 4 dans la liste des séquences. Une nouvelle protéine représentée par la séquence d'aminoacides ayant le numéro d'identification 1 ou 2 et codée par ledit ADN peut être préparée à l'aide d'un transformant obtenu au moyen dudit ADN. Cette protéine stimule la production de PGI2 et constitue un agent thérapeutique efficace, utile pour lutter contre des affections telles que le syndrome hémolytique et urémique, le purpura thrombocytopénique thrombotique, l'embolie périphérique, l'ischémie cardiaque, l'ischémie céréabrale, l'artériosclérose, l'infarctus cérébral, l'hyperlipémie, le diabète, l'insuffisance cardiaque, l'angine de poitrine, la cardiopathie ischémique, la cardiopathie congestive, les troubles circulatoires choroïdiens, les maladies bronchiques, l'ulcère gastrique et l'éclampsie, l'efficacité de ladite protéine étant fondée sur l'activité inhibitrice de l'agrégation plaquettaire, l'activité spasmolytique de la musculature lisse, et l'activité inhibitrice de la sécrétion gastrique de la PGI2.
PCT/JP1994/000946 1993-06-11 1994-06-10 Adn et proteine codee par celui-ci WO1994029448A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000035473A3 (fr) * 1998-12-18 2000-11-09 Scios Inc Methodes de detection et d'utilisation de genes exprimes de façon differentielle dans des maladies
WO2001073022A1 (fr) * 2000-03-29 2001-10-04 Kyowa Hakko Kogyo Co., Ltd. Gene associe a la glomerulonephrite proliferative
WO2002081515A1 (fr) * 2001-04-03 2002-10-17 Kyowa Hakko Kogyo Co., Ltd. Proteine de liaison de facteur de croissance de type insuline
US6709855B1 (en) 1998-12-18 2004-03-23 Scios, Inc. Methods for detection and use of differentially expressed genes in disease states
EP1524524A1 (fr) * 1998-12-18 2005-04-20 Scios Inc. Méthodes de détection et d'utilisation de OSF-2 exprimés de façon différentielle pour diagnose l'hypertrophie cardiaque
US7109030B2 (en) 2000-03-31 2006-09-19 Nuvelo, Inc. Methods of therapy and diagnosis using insulin-like growth factor binding protein-like polypeptides and polynucleotides
EP2498095A2 (fr) 2007-01-25 2012-09-12 Roche Diagniostics GmbH Utilisation de IGFBP-7 dans l'estimation d'une insuffisance cardiaque

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6045533A (ja) * 1983-08-11 1985-03-12 Kureha Chem Ind Co Ltd プロスタグランジン調節剤

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6045533A (ja) * 1983-08-11 1985-03-12 Kureha Chem Ind Co Ltd プロスタグランジン調節剤

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000035473A3 (fr) * 1998-12-18 2000-11-09 Scios Inc Methodes de detection et d'utilisation de genes exprimes de façon differentielle dans des maladies
US6709855B1 (en) 1998-12-18 2004-03-23 Scios, Inc. Methods for detection and use of differentially expressed genes in disease states
EP1524524A1 (fr) * 1998-12-18 2005-04-20 Scios Inc. Méthodes de détection et d'utilisation de OSF-2 exprimés de façon différentielle pour diagnose l'hypertrophie cardiaque
WO2001073022A1 (fr) * 2000-03-29 2001-10-04 Kyowa Hakko Kogyo Co., Ltd. Gene associe a la glomerulonephrite proliferative
EP1275717A1 (fr) * 2000-03-29 2003-01-15 Kyowa Hakko Kogyo Co., Ltd. Gene associe a la glomerulonephrite proliferative
EP1275717A4 (fr) * 2000-03-29 2004-10-06 Kyowa Hakko Kogyo Kk Gene associe a la glomerulonephrite proliferative
US7109030B2 (en) 2000-03-31 2006-09-19 Nuvelo, Inc. Methods of therapy and diagnosis using insulin-like growth factor binding protein-like polypeptides and polynucleotides
WO2002081515A1 (fr) * 2001-04-03 2002-10-17 Kyowa Hakko Kogyo Co., Ltd. Proteine de liaison de facteur de croissance de type insuline
EP2498095A2 (fr) 2007-01-25 2012-09-12 Roche Diagniostics GmbH Utilisation de IGFBP-7 dans l'estimation d'une insuffisance cardiaque
US10488422B2 (en) 2007-01-25 2019-11-26 Roche Diagnostics Operations, Inc. Use of IGFBP-7 in the assessment of heart failure
US10996229B2 (en) 2007-01-25 2021-05-04 Roche Diagnostics Operations, Inc. Use of IGFBP-7 in the assessment of heart failure

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