WO1998042744A1 - Proteine inhibant la mort cellulaire - Google Patents

Proteine inhibant la mort cellulaire Download PDF

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Publication number
WO1998042744A1
WO1998042744A1 PCT/JP1998/001319 JP9801319W WO9842744A1 WO 1998042744 A1 WO1998042744 A1 WO 1998042744A1 JP 9801319 W JP9801319 W JP 9801319W WO 9842744 A1 WO9842744 A1 WO 9842744A1
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Prior art keywords
protein
cell death
ipia
antibody
dna
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PCT/JP1998/001319
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English (en)
Japanese (ja)
Inventor
Hideo Nakamura
Hideyuki Saya
Nobuo Hanai
Akiko Furuya
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Kyowa Hakko Kogyo Co., Ltd.
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Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Priority to AU65174/98A priority Critical patent/AU6517498A/en
Publication of WO1998042744A1 publication Critical patent/WO1998042744A1/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
    • C07K14/4747—Apoptosis related proteins
    • 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 relates to a protein that suppresses cell death, DNA encoding the protein, a recombinant vector containing the DNA, and a transformant containing the recombinant vector. Further, the present invention provides a method for detecting the protein using the DNA encoding the protein, and a cell death containing the protein, the DNA encoding the protein or a recombinant vector containing the DNA as an active ingredient. A method for screening a substance that inhibits the cell death inhibitory protein by using an inhibitor and the protein, DNA, recombinant vector, and transformant, and a substance that promotes cell death by using the method. About the method. Furthermore, the present invention relates to an antibody against the cell death inhibitory protein, a cell producing the antibody, and a method for detecting a cell death inhibitory protein using the antibody. Background technology
  • the p53 gene which is a tumor suppressor gene
  • the p53 gene is a tumor suppressor gene in which mutations are most frequently found in human cancer [Holstein (M. Hoilstein) et al., Science, 253, 49, (1991)]. Mutations in the regulation of apoptosis by the tumor suppressor gene product p53 are thought to be particularly involved in cancer development, proliferation, and malignancy.
  • the mutation of the p53 gene product As a result, the autonomous cell death mechanism inherent to the cell does not work, resulting in 1) cells with excessive damage to the gene survive cell death, become cancer cells, and increase the frequency of malignant cells, 2) hypoxia Cells that have been exposed to a poor environment due to abnormal proliferation of cells at the center of a cancer in a state survive cell death and survive.3) Induction of cell death to cancer cells by anticancer drugs and the like is unlikely to occur. Is considered to be involved in the development, growth, and malignancy of cancer in that it becomes resistant to anticancer drugs. [Linda J. Ko et al .: Jeans & Development (Genes & Dev.)] , 10. 1054 (1996)].
  • the factor that suppresses apoptosis caused by p53 is considered to be associated with various diseases, in particular, canceration, malignancy of cancer, and resistance to anticancer drugs. Therefore, it is expected that diseases such as cancer can be treated by inhibiting the cell death suppressing activity or suppressing the gene expression. Furthermore, factors that regulate p53-induced apoptosis are not only involved in the regulation of p53-dependent cell death, but also involved in the regulation of total cell death, including apoptosis in which p53 is not involved. Probably.
  • cancer By suppressing or promoting the function expression of such cell death inhibitory factors, cancer, autoimmune disease, viral infection, acquired immunodeficiency syndrome (AIDS), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmented It is expected to be able to treat diseases involving abnormal cell death such as retinopathy, cerebellar degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, liver disease due to alcohol, and periodontal disease.
  • diseases involving abnormal cell death such as retinopathy, cerebellar degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, liver disease due to alcohol, and periodontal disease.
  • antisense RNA / antisense DNA technology To suppress the expression of genes that suppress this cell death, especially cell death by p53, antisense RNA / antisense DNA technology [Tokuhisa: Biosa S. 322 (1992), Murakami: Chemistry, 4 ⁇ , 681 (1991)] or Triple helix technology [Chubb and Hogan: Trens Trends in Biotechnology, 10, 132 (1992)] is useful.
  • antisense RNA / antisense DNA technology Yamahisa: Biosa S. 322 (1992), Murakami: Chemistry, 4 ⁇ , 681 (1991)] or Triple helix technology [Chubb and Hogan: Trens Trends in Biotechnology, 10, 132 (1992)] is useful.
  • information on the gene or the base sequence of the gene is required. Therefore, cloning of the desired gene and its base sequence information are required. It is important to analyze
  • AIDS acquired immunodeficiency syndrome
  • Alzheimer's disease Parkinson's disease
  • amyotrophic lateral cords It is expected to be able to treat diseases caused by enhanced cell death, such as sclerosis, pigmented retinopathy, cerebellar degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, liver disease due to alcohol, and periodontal disease.
  • diseases caused by enhanced cell death such as sclerosis, pigmented retinopathy, cerebellar degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, liver disease due to alcohol, and periodontal disease.
  • a so-called gene therapy method [Shimada: Experimental Medicine, II, 303 (1994)] is useful.
  • the gene or the nucleotide sequence information of the gene is required. Therefore, the desired gene is cloned, and the nucleotide sequence information is analyzed. It is important to do so.
  • an antibody that specifically reacts with a protein that suppresses cell death, particularly cell death caused by P53, is useful in analyzing the pathology and treating various diseases described above involving the protein, and examining the function of the protein. It is.
  • An object of the present invention is to provide a protein that suppresses apoptosis, in particular, cell death caused by overexpression of p53 (hereinafter abbreviated as ⁇ - ⁇ ), a DNA containing the ⁇ - ⁇ , and a set containing the DN ⁇ . It is another object of the present invention to provide a recombinant vector and a transformant containing the vector. Further, the IPIA- ⁇ , a DNA encoding the protein, a cell death inhibitor containing a recombinant vector containing the DNA as an active ingredient, and a Northern hybrididal using the DNA encoding the IPIA- ⁇ .
  • the present invention relates to a protein ⁇ - ⁇ comprising the amino acid sequence represented by SEQ ID NO: 2, a DNA encoding the IPIA- ⁇ , a recombinant vector containing the DNA, a transformant containing the recombinant vector, and The antibody specifically reacting with the IPIA-Q;
  • the IPIA- ⁇ of the present invention includes a protein comprising the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence in which one or several amino acids have been deleted, replaced or added in the amino acid sequence of SEQ ID NO: 2. And a protein having an activity of suppressing cell death.
  • any cell death is included in the present invention as long as the cell autonomously loses its function.
  • the IPIA- ⁇ of the present invention suppresses cell death based on the tumor suppressor gene product ⁇ 53. Preferably suppressed.
  • IPIA- ⁇ of the present invention has SEQ ID NO: 2
  • the N-terminal methionine residue of the amino acid sequence is also cleaved by processing.
  • Examples of the DNA encoding the IPIA- ⁇ of the present invention include: (a) a DNA containing the nucleotide sequence shown in SEQ ID NO: 1, (b) —a plurality of genetic codes exist for one amino acid, or human DNA that contains a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 1 due to spontaneous mutations that occur in each animal, including DNA, and (c) the DNA defined in (a) and (b) DNA homologous to DNA introduced with mutations such as substitution mutations, deletion mutations, and insertion mutations within a range that does not lose the inhibitory activity, for example, the DNA defined in (a) or (b) is encoded by ⁇ - ⁇ Includes DNA having properties.
  • the DNA having this homology means a DNA that hybridizes with a DNA containing the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein having an activity of suppressing cell death.
  • the experimental method of hybridization is described in Molecular Cloning.
  • the IPIA- ⁇ of the present invention includes all IPIA- ⁇ encoded by the DNAs defined in (a), (b) and (c) above.
  • a cDNA library is constructed by incorporating, into an expression cloning vector, a cDNA synthesized as type III using mRNA extracted from a human-derived cell line or tissue. After introducing this cDNA library into animal cells, the cells are infected with a virus incorporating p53, thereby forcibly overexpressing p53 and inducing cell death. Resistant to cell death induced by p53 overexpression as described above After isolation of the surviving cells, a cDNA encoding the desired IPIA- ⁇ is obtained from the cells.
  • the human-derived cell line or tissue used as the basis for preparing the cDNA library used in the above method may be any cell line or tissue expressing the IPIA- ⁇ of the present invention. be able to.
  • HeLa S3 cells ATCC CCL2.2
  • the like are used.
  • Any vector can be used as a vector incorporating cDNA synthesized as type III using mRNA extracted from these cells as long as the vector can incorporate and express the cDNA.
  • pDR2 (Clontech, Catalog No. 6167-1) is preferably used when a cell line that highly expresses the EBNA-1 gene is used as a host cell because high expression of the integrated gene is expected. .
  • a commercially available cDNA library prepared by introducing cDNA obtained from a cell line or tissue expressing IPIA ⁇ into an appropriate expression vector may be used.
  • Clontech Human HeLa 5'-stretch cDNA catalog number HL1152y or the like is used.
  • Any animal cell into which the above cDNA library is introduced can be used as long as it can introduce and express the cDNA library.
  • cells in which cell death is likely to occur due to the p53 gene for example, Saos-2 cells (ATCC HTB-85) are preferably used.
  • any virus can be used as long as it can express p53 and induce cell death.
  • adenovirus vectors are preferred.
  • Ad- ⁇ 53 an adenovirus vector incorporating the p53 gene [Toshiyo shi Fujiwara et al .: Cancer Res., 54. 2287 (1994)]
  • p53 By adjusting the number of particles, almost all cells can be infected and p53 can be highly expressed, so that it is preferably used.
  • Animal cells into which the cDNA library has been introduced are infected with a virus incorporating the p53 gene, cell death is induced by overexpressing p53, and then surviving cells are isolated.
  • Known methods from the cells thus obtained for example, The alkaline method (ADR2 & pDR2 cloning and expression system, library, protocol, handbook & pDR2 Cloning and Expression System Library Protocol Handbook; Clontech, catalog number PT10 11-1)
  • a plasmid containing DNA encoding ⁇ or a DNA fragment containing the cDNA is recovered.
  • Examples of the plasmid containing DNA encoding the protein of the present invention include PDR2 / IPIA- ⁇ .
  • Escherichia coli DH5a / pDR2 / IPIA- ⁇ which is Escherichia coli containing PDR2 / IPIA- ⁇ , has been deposited on March 19, 1997 with the National Institute of Bioscience and Human Technology as FERM II-5879.
  • the DNAs defined in (b) and (c) above are based on the DNIA encoding IPIA- ⁇ obtained by the above-mentioned production method. It can be produced using well-known recombinant DNA technology [molecular cloning]. The DNA encoding IPIA- ⁇ of the present invention can also be produced by using a chemical synthesis method.
  • the host cell to be used any cells such as prokaryotic cells, animal cells, yeasts, molds, and insect cells can be used as long as they have been used in recombinant DNA technology.
  • prokaryotic cells include Escherichia coli K12 strain, ⁇ 522 strain (D.Hanahan J. Mol.
  • DH5 ⁇ strain Molecular 'cloning A.10
  • examples include CHO cells (ATCC CCL-61), which are Chinese hamster cells, and COS 7 cells (ATCC CRL-1651), which are African green monkey cells.
  • any vector can be used as long as it can incorporate the DNA encoding the IPIA-a and can produce the IPIA-a.
  • the IPIA-a of the present invention can be produced as a fusion protein with another protein.
  • Vectors used for expressing this fusion protein include the IPIA- Any vector can be used as long as it can incorporate a DNA encoding the same and can express the fusion protein.
  • An example is PGEX-2TH [Maruta, Experimental Medicine, 1082 (1991)].
  • Examples of a plasmid that expresses the IPIA- ⁇ protein of the present invention as a fusion protein with glutathione-1 S-transferase (hereinafter abbreviated as GST) include pGST / IPIA- ⁇ .
  • Escherichia coli ⁇ 522 / pGST / IPIA-a containing pGST / ⁇ - ⁇ has been deposited as FERM BP-5880 on March 19, 1997 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology.
  • the IPIA- ⁇ protein of the present invention can be purified using a commonly known protein purification method. Further, the protein can be efficiently purified by affinity chromatography using an antibody against the protein itself. When the protein is expressed as a protein fused with another protein, the protein can be purified by affinity chromatography using a substance having an affinity for the fused protein.
  • IPIA- ⁇ of the present invention is produced as a fusion protein with GST and affinity chromatography using glutathione as a ligand [Smith (DBSmitli) et al., Gene, 67, 31, (1988) ].
  • the method described in Molecular Cloning can be used.
  • any method known to date can be used.
  • the Lipoff-Exion method Philip L. Felgner et al .: Proceeding of the National Academy of Sciences (Proc. Natl. Acad. Sci.), USA, _84 ⁇ 7413 (1987)].
  • the DNA encoding the IPIA- ⁇ of the present invention can be used for the treatment of diseases such as cancer, autoimmune diseases, and viral infections using antisense RNAZDNA technology or gene therapy technology. It can also be used for the detection of those diseases using the istan hybridization method or the PCR method.
  • antisense RNA ZDNA technology [Tokuhisa: Bioscience and Industry, 322-326 (1992), Murakami: Chemistry, 46 »681- 684 (1991), Miller r): Biotechnology, 358-362 (1992), Cohen: Trends in Biotechnology, 10, 87-91 (1992), Agrawal ): Trends in Biotechnology, 10, 152-158 (1992)] or Triple Helix Technology (Chubb and Hogan): Trends in Biotechnology (Trends in Biotechnology) Biotechnology), 10, 132-136 (1992)] can suppress the expression of the IPIA- ⁇ activity.
  • an oligonucleotide is designed and prepared based on a partial nucleotide sequence of DNA encoding ⁇ - ⁇ of the present invention, preferably a nucleotide sequence of 10 to 50 nucleotides in a translation initiation region, and By administering a preparation containing an oligonucleotide as an active ingredient into a living body, the production of ⁇ - ⁇ can be suppressed.
  • the nucleotide sequence of the synthetic oligonucleotide may be a nucleotide sequence that completely matches a part of the nucleotide sequence of the antisense strand of DNA encoding ⁇ - ⁇ of the present invention, or suppresses the expression of ⁇ - ⁇ activity. Those modified within the range not losing the activity to be used can be used.
  • the base sequence of a synthetic oligonucleotide is designed based on the base sequence information of both the sense and antisense strands.
  • the production of IPIA- ⁇ of the present invention can be detected by the Northern hybridization method or the PCR method.
  • a synthetic oligonucleotide based on DN DN encoding ⁇ - ⁇ of the present invention or a base sequence thereof is used. Prepare nucleotides.
  • the Northern hybridization method and the PCR method are carried out according to known methods (Molecular Cloning, and Innis et al .: PCR Protocols, Academic Press, 1990).
  • the cell death in the living body in particular, the ⁇ 53 Suppresses cell death based on function, acquired immunodeficiency syndrome group (AIDS), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmented retinopathy, cerebellar degeneration, aplastic anemia, myocardial infarction, Stroke, reperfusion injury, alcohol Diseases caused by enhanced cell death, such as liver disease and periodontal disease.
  • AIDS acquired immunodeficiency syndrome group
  • Alzheimer's disease Parkinson's disease
  • amyotrophic lateral sclerosis pigmented retinopathy
  • cerebellar degeneration aplastic anemia
  • myocardial infarction myocardial infarction
  • Stroke reperfusion injury
  • alcohol Diseases caused by enhanced cell death such as liver disease and periodontal disease.
  • a DNA encoding the full-length or a part of the IPIA- ⁇ of the present invention is converted into a vector derived from adenovirus or murine leukemia virus [Miyake et al., Proceeding of the National Academy of Sciences ( Proc. Natl. Acad. Sci.), USA, 93, 1320 (1996), NAEglitis et al., Bioteclmidiies, 6 ⁇ 608, (1988), ADMiller et al., Bio. Technique (Bioteclmidues), 7_, 980 (1989)], and the IPIA- ⁇ is produced by in vivo administration or by in vitro administration of a gene-transfected cell in vivo. Cell death can be suppressed.
  • adenovirus or murine leukemia virus [Miyake et al., Proceeding of the National Academy of Sciences ( Proc. Natl. Acad. Sci.), USA, 93, 1320 (1996), NAEglitis et al., Bio
  • a substance that inhibits IPIA-a is screened using the DNA encoding ⁇ - ⁇ or the protein of the present invention, a recombinant vector containing the DNA, and a transformant containing the vector.
  • the construction of the screening method is performed by a known method, for example, the method of Hayakawa et al. [Journal of anti-biotics
  • compositions containing a substance that inhibits IPIA- ⁇ or a substance that promotes cell death as an active ingredient promotes cell death in the living body, especially cell death based on the function of ⁇ 53, when administered in vivo. It can treat diseases caused by reduced cell death, such as cancer, autoimmune diseases, and viral infections.
  • the medicament containing the IPIA- ⁇ production inhibitor, the cell death inhibitor or the substance that inhibits IPIA- ⁇ or the substance that promotes cell death of the present invention as an active ingredient can be used as it is or in various pharmaceutical forms. can do.
  • the pharmaceutical composition of the present invention comprises, as an active ingredient, an effective amount of the IPIA- ⁇ of the present invention, DNA encoding the protein, a recombinant vector containing the DNA, a transformant containing the vector or IPIA.
  • any useful pharmacologically acceptable Carrier can be used.
  • Oral liquid preparations such as suspensions and syrups include water, sugars such as sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, P -Can be manufactured using preservatives such as hydroxybenzoic acid esters and flavors such as stove beef flavor and peppermint.
  • the solution of the injection can be prepared using a carrier comprising distilled water, salt solution, glucose solution or a mixture of salt water and glucose solution. At this time, it is prepared as a solution, suspension or dispersion using an appropriate solubilizing agent and suspending agent according to a conventional method.
  • the medicament comprising the IPIA- ⁇ production inhibitor, the cell death inhibitor or the substance that inhibits ⁇ - ⁇ or the substance that promotes cell death of the present invention as an active ingredient is orally or in the above pharmaceutical form. It can be administered parenterally as an injection.
  • the antibody specifically reacting with ⁇ - ⁇ of the present invention can be used to examine the expression level of a protein that suppresses cell death, in particular, cell death by ⁇ 53, to detect cell death such as cancer, autoimmune disease, and virus infection.
  • a protein that suppresses cell death in particular, cell death by ⁇ 53
  • cell death such as cancer, autoimmune disease, and virus infection.
  • ⁇ IDS acquired immunodeficiency syndrome
  • Alzheimer's disease Parkinson's disease
  • amyotrophic sclerosis amyotrophic sclerosis
  • pigmented retinopathy adenotrophic sclerosis
  • cerebellar degeneration aplastic anemia
  • myocardial infarction stroke
  • diseases caused by enhanced cell death such as reperfusion injury, liver disease due to alcohol, and periodontal disease.
  • the function of the IPIA- ⁇ of the present invention can be suppressed in the living body, and cell death in the living body, in particular, cell death based on the function of ⁇ 53 can be suppressed.
  • Pharmaceuticals containing the antibody of the present invention inhibit the IPIA-Q of the present invention except that the antibody of the present invention is used instead of the substance of the present invention that inhibits IPIA- ⁇ or the substance that promotes cell death. It is manufactured and administered using the same method as for a drug containing a substance or a substance that promotes cell death.
  • the antibody that specifically reacts with IPIA- ⁇ of the present invention may be a polyclonal antibody or a monoclonal antibody.
  • the antibody of the present invention can be produced by a method generally used for producing an antibody.
  • a hybridoma is prepared by using a partial peptide of IPIA- as an immunogen, a monoclonal antibody-producing hybridoma strain that specifically reacts with the peptide is established, and the hybridoma is placed in a medium. It can be obtained by culturing or administering to an animal to cause ascites cancer, and then removing the culture supernatant or ascites.
  • anti-IPIA-monoclonal antibody examples include anti-IPIA- ⁇ monoclonal antibody K2102 produced by hybridoma cell line KM2102. Can be.
  • This anti-IP IA- ⁇ monoclonal antibody KM2102 was deposited on March 17, 1999 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology as FERM II-6296.
  • IPIA- ⁇ or a partial peptide of IPIA- ⁇ is used as an antigen.
  • the IPIA- ⁇ partial peptide can be produced by a usual peptide synthesis method based on the amino acid sequence of IPIA- ⁇ shown in SEQ ID NO: 2. It can also be obtained by decomposing IPIA- ⁇ chemically or enzymatically.
  • the IPIA- ⁇ partial peptide As the IPIA- ⁇ partial peptide, a protein partial sequence of about 5 to 30 residues is selected. In order to obtain an antibody recognizing the protein in a state having an undenatured natural structure, it is necessary to select a partial sequence existing on the surface of the protein in a three-dimensional structure as an antigen peptide. is there. For the portion existing on the protein surface due to its three-dimensional structure, use a commercially available protein sequence analysis software such as Genety X Mac to predict a highly hydrophilic partial sequence. That can be inferred. That is, generally, a portion having low hydrophilicity is often present inside the protein in a three-dimensional structure, and a portion having high hydrophilicity is often present on the protein surface. In addition, the N-terminus and C-terminus of a protein are often present on the protein surface. However, the partial peptide thus selected does not always serve as an antigen for establishing an intended antibody.
  • a cysteine is added to the terminal of the IPIA- ⁇ partial peptide in order to crosslink with the protein.
  • the ⁇ -terminal of the peptide is acetylated and the C-terminal is amidated as necessary.
  • ⁇ - ⁇ partial peptide can be synthesized by a general liquid phase, solid phase peptide synthesis method, a method of appropriately combining them, or a method analogous thereto.
  • an IPIA- ⁇ partial peptide can be synthesized using an automatic peptide synthesizer.
  • Peptide synthesis using a peptide synthesizer is performed by a peptide synthesizer manufactured by Shimadzu Corporation and manufactured by Applied Biosystems, Inc., USA.
  • ACT ChemTech Inc., USA, hereafter abbreviated as ACT
  • ACT peptide synthesizer
  • Protected amino acids and carrier resin required for peptide synthesis are available from ABI, Shimadzu, Kokusan Chemical, Nova Biochem, Watanabe Chemical, ACT, or Peptide Research Institute. And so on.
  • protected amino acids, protected organic acids, and protected organic amines required for peptide synthesis follow known synthesis methods.
  • the human IPIA- ⁇ partial peptide used as an antigen can be used to enhance immunogenicity, such as keyhole lysate hemocyanin (hereinafter abbreviated as KLH), ⁇ ⁇ serum albumin (hereinafter abbreviated as BSA), etc.
  • KLH keyhole lysate hemocyanin
  • BSA ⁇ ⁇ serum albumin
  • MBS cross-linking agent
  • a mouse or rat aged 3 to 20 weeks is immunized with a conjugate of human IPIA- ⁇ partial peptide with a carrier protein or human IPIA- ⁇ , and antibody-producing cells are obtained from the animal's spleen, lymph node, and peripheral blood.
  • Collect. Immunization can be performed subcutaneously, intravenously, or intraperitoneally in an animal using an appropriate adjuvant (eg, Freund's complete adjuvant), or aluminum hydroxide gel and pertussis vaccine. ] Together with the administration of the antigen.
  • the administration of the antigen is performed 5 to 10 times every 1 to 2 weeks after the first administration. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reactivity of the serum with the antigen is examined by enzyme immunoassay [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988].
  • a mouse or rat whose serum shows a sufficient antibody titer against the peptide used for immunization is used as a source of the antibody-producing cells.
  • spleens are excised from the immunized mice or rats 3 to 7 days after the final administration of the antigenic substance, and splenocytes are collected. Slice the spleen in a serum-free basal medium (hereinafter referred to as “washing medium”). After centrifugation (1200 rpm, 5 minutes), discard the supernatant, and treat with Tris ammonium chloride buffer (PH7.65) for 1 to 2 minutes to remove red blood cells and wash. Wash three times with the culture medium to provide as splenocytes for fusion.
  • washing medium a serum-free basal medium
  • myeloma cells cell lines obtained from mice are used.
  • 8-azaguanine-resistant mouse derived from BALB / c
  • P3-U1 myeloma cell line P3-X63Ag8-U1 [Current 'Topics in Microbiology and Immunology 1 (Cu rr en t To picsin M icrobiolo gy and I mmu no 1 ogy— 1), Eu rope an J.
  • These cell lines include 8-Azaguanin medium [RPMI one 1 6 4 0 medium glutamine (1.
  • PBS polyethylene glycol-1,100
  • the suspension 9 6-well culture plate dispensed at 1 00 H 1 / well min, in 5% C0 2 incubator primary, in at 37? Incubate for ⁇ 14 days.
  • the human ⁇ - ⁇ partial peptide used as an antigen is bound using a protein different from that used for immunization as a carrier protein and using a cross-linking agent such as glutaraldehyde ⁇ MBS.
  • an antibody labeled with biotin, an enzyme, a chemiluminescent substance or a radioactive compound as a second antibody is used.
  • the well to be selected is selected as an anti-IPIA- ⁇ monoclonal antibody.
  • mice 8- to 10-week-old mice treated with pristane (0.5 ml of 2,6,10,14-tetramethylpenedecane (Pristane) administered intraperitoneally and bred for 3 to 10 days) or into nude mice, intraperitoneal injection of 2 X 1 0 7 ⁇ 5 X 1 0 ⁇ cell mice resulting anti IPIA- alpha monoclonal antibody-producing High Priestess dormer cells (3). In 10 to 21 days, the hybridoma becomes ascites cancer.
  • pristane 0.5 ml of 2,6,10,14-tetramethylpenedecane (Pristane) administered intraperitoneally and bred for 3 to 10 days
  • mice Ascites was collected from the mice, centrifuged (3,000 rpm, 5 minutes) to remove solids, and then salted out with 40-50% saturated ammonium sulfate, and subjected to force prillic acid precipitation, DEAE-Sepharose. After passing through a column, protein A-column or gel filtration column, the IgG or IgM fraction is collected and used as a purified monoclonal antibody.
  • the antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. Quantification of protein amount is calculated by the Lowry method or from the absorbance at 280 nm.
  • the anti-IPIA- ⁇ monoclonal antibody selected in (4) specifically reacts with human IPIA- ⁇ is examined by Western blotting. After fractionating human IPIA- ⁇ by SDS-PAGE, plot it on a nitrocellulose membrane or PVDF membrane. After blocking with BSA solution, purify or purify the anti-IPIA- ⁇ monoclonal antibody or react with 1 to 10 gZm1 at room temperature for 2 hours or at 4 overnight. After washing well with PBS or Tween-PBS, add 1 to 5 Og / ml of anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody labeled with piotin, enzyme, chemiluminescent substance or radioactive compound as the second antibody.
  • Western blotting was performed to determine whether the anti-IPIA- ⁇ monoclonal antibody selected in (4) reacted specifically with human IPIA- ⁇ protein expressed in cells. You. That is, the total protein is extracted from the cells producing ⁇ - ⁇ , fractionated by SDS-PAGE, and then blotted on a nitrocellulose membrane or PVDF membrane. After blocking with BSA or skim milk solution, the culture supernatant of the anti-IPIA- ⁇ monoclonal antibody or 1 to 10 gZml of the purified antibody is reacted at room temperature for 2 hours, at 37 for 1 hour or at 4 for 1 hour.
  • the anti-IPIA- ⁇ monoclonal antibody selected in (4) specifically reacts with human IPIA- ⁇ present in human cells is examined by Western blotting.
  • an anti-mouse immunoglobulin antibody or anti-rat immunoglobulin antibody 1-50 / 1111 labeled with a second antibody such as piotin, an enzyme, a chemiluminescent substance, or a radioactive compound is used at room temperature. 1 to 2 hours or 37 to 30 to 60 minutes. After washing well, perform a reaction according to the labeling substance of the second antibody, and confirm that the anti-IPIA- ⁇ monoclonal antibody specifically reacts with the band corresponding to the molecular weight of human IPIA- ⁇ .
  • the IPIA- ⁇ of the present invention present in cells or tissues of healthy subjects and subjects is immunologically detected or quantified, and the amount is compared between healthy subjects and subjects, and the expression level is increased. Or whether it is decreasing,
  • diseases caused by reduced cell death such as cancer, autoimmune diseases, and viral infections, acquired immunodeficiency syndrome (AIDS), Alzheimer's disease, Parkinson's disease, amyotrophic chondrosclerosis, retinitis pigmentosa, and cerebellum
  • AIDS acquired immunodeficiency syndrome
  • Alzheimer's disease Parkinson's disease
  • amyotrophic chondrosclerosis retinitis pigmentosa
  • cerebellum It is possible to detect whether or not a disease caused by enhanced cell death such as degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, liver disease due to alcohol, periodontal disease, etc. is detected.
  • the antibody of the present invention may be used for diseases caused by reduced cell death such as cancer, autoimmune disease, and viral infection, as well as acquired immunodeficiency syndrome (AIDS), Alzheimer's disease, Parkinson's disease, and amyotrophic rule sclerosis.
  • diseases caused by enhanced cell death such as sclerosis, pigmentary retinopathy, cerebellar degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, liver disease due to alcohol, and periodontal disease Can be.
  • the antibody of the present invention can also be used as a reagent for studying the function of IPIA- ⁇ .
  • FIG. 1 is a diagram showing the structure of plasmid PDR2 / IPIA- ⁇ .
  • FIG. 2 is a diagram showing expression of ⁇ - ⁇ RNA in human tissues.
  • the left lane shows the tissues of heart, brain, placenta, lung, liver, skeletal muscle, kidney, and knee, respectively. Arrows indicate IPIA- ⁇ mRNA expression.
  • FIG. 3 shows the expression of IPIA- ⁇ mRNA in human tissues.
  • the left lane shows the spleen, thymus, prostate, testis, ovary, small intestine, colon, and leukocyte tissues, respectively. Arrows indicate IPIA- ⁇ mRNA expression.
  • FIG. 4 is a view showing the expression of IPIA- ⁇ mRNA in a human cell line. From the left lane, the cell lines HL60, HeLa S3, K562, Molt 4, Raji, SW480, A549, and G361 are shown, respectively. Arrows indicate the expression of IPIA- ⁇ mRNA.
  • FIG. 5 is a diagram showing the structure of plasmid pGST / IPIA- ⁇ .
  • FIG. 6 is a diagram showing production of a GST --- ⁇ fusion protein using Escherichia cojj NM522 / pGST / IPIA- ⁇ .
  • the left lane shows the electrophoresis pattern before isopropyl 1 / 3-thiogalactoside (hereinafter abbreviated as IPTG) induction, and the right lane shows the electrophoresis pattern after IPTG induction.
  • IPTG isopropyl 1 / 3-thiogalactoside
  • FIG. 7 is a view showing an electrophoresis pattern of the purified GST-I ⁇ I ⁇ - ⁇ fusion protein.
  • Left lane and center lane are purified protein fractions, right lane is GST Are respectively shown.
  • the right arrow indicates GST-IPIA- ⁇ fusion protein, and the left arrow indicates GST.
  • Fig. 8 shows the binding reactivity of the serum of mice immunized with the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 to GST-IPIA- ⁇ fusion protein (upper) and GST protein (lower) using enzyme immunoassay. Is shown.
  • IPIA- ⁇ Ml-3 is a mouse immunized with a peptide having the amino acid sequence shown in SEQ ID NO: 7
  • IPIA- ⁇ 2 ⁇ 1-3 is a peptide having the amino acid sequence shown in SEQ ID NO: 8.
  • NMS shows the normal mouse serum.
  • FIG. 9 shows the binding reactivity of anti-IPIA- ⁇ monoclonal antibodies M2102, KM210K KM2103 and KM2104 to GST-IPIA- ⁇ fusion protein using enzyme immunoassay.
  • FIG. 10 shows the reactivity of the anti-IPIA- ⁇ monoclonal antibodies KM2102, KM210K KM2103 and KM2104 with the GST-IPIA- ⁇ fusion protein in Western blotting.
  • FIG. 11 shows the reactivity of the anti-IPIA- ⁇ monoclonal antibodies KM2102, KM2101 and KM2103 with IPIA- ⁇ / myc expressed in C0S7 cells during estamplotting.
  • FIG. 12 shows the reactivity of anti-IPIA- ⁇ monoclonal antibody KM2102 with IPIA- ⁇ in cells in Western blotting.
  • the adenovirus Ad-p53 [Fujiwara (Toshiyoshi Fuj iwara) et al .: Cancer Res., 54 »2287 (1994)] was used and the method of Fujiwara et al. [Fujiwara (Toshiyosh i Fuj iwara) The cells were infected according to Cancer Res., 54. 2287 (1994)]. From 2 ⁇ 10 7 cell lines, one cell line that survived for 7 days after Ad-p53 infection was isolated. ADR2 & pDR2 Cloning and Expression System Library Protocol Handbook; ADR2 & PDR2 Cloning and Expression System Library Protocol Handbook; Clontech, Cat. No.
  • RNA + RNA (mRNA) and various human cell lines human promyelocytic leukemia cell HL60 (ATCC CCL-240), human cervical cancer cell HeLa S3 (ATCC CCL-2.2), human chronic myeloid leukemia cell K562 (ATCC CCL-243), human lymphoblastic leukemia cells Molt 4 (ATCC CRL-1582), human patient kit lymphoma cells Raji (ATCC CCL-86), human colon cancer cells SW480 (ATCC CCL-228), human lung cancer cells A549 ( ATCC C
  • Oligonucleotide S-3 having the nucleotide sequence of SEQ ID NO: 3 and oligonucleotide AS-6 having the nucleotide sequence of SEQ ID NO: 4 were synthesized using a DNA synthesizer, and dissolved at a concentration of 20 pmol / Atl. Using both oligonucleotides as primers, PCR was performed using DNA of GENEBRIDGE 4 RADIATION HYBRID SCREENING PANEL (manufactured by Research Genetics) as type III. The PCR procedure is as follows: 1 l of DNA on the screening panel, 11 dNTP mixture, S-3 primer, AS-6 primer
  • a 10-fold concentration of buffer for PCR, distilled water 91 and Tad polymerase 0.5 (i ⁇ (total 15 U)) are placed in a test tube, and mineral oil 151 is overlaid on top. And left for 5 minutes at 94. Then, the reaction was repeated 35 times at 60 for 30 seconds, followed by 72 at 30 seconds, and 95 at 30 seconds, and then at 60 at 30 seconds. Finally, the PCR was completed by elongating the fragment by reacting for 10 minutes at 72. Thereafter, 5 l ⁇ ⁇ of the PCR product was subjected to electrophoresis using a 2.0 agarose gel, and Hudson (Hadson TJ) was used.
  • PDR2 / IPIA- ⁇ plasmid DNA was converted to type III, and PCR was performed using a synthetic DNA described in SEQ ID NO: 5 synthesized by a DNA synthesizer and a synthetic DNA described in SEQ ID NO: 6 as primers. That is, using rTth poly erase Kit manufactured by Perkin Elmer, a reaction solution was prepared according to the attached method, and left at 95 for 5 minutes. Thereafter, 35 cycles of 1 minute at 95 :, 1 minute at 62, and 1 minute at 72 were performed, and finally, reaction was performed at 72 at 10 minutes to amplify the DNA containing the IPIA-cDNA coding region.
  • the obtained DNA was digested with Hindlll and BamHI, and the fragment was recovered and prepared by an electrolysis method (Elec elution method; Molecular Cloning, p. 6.28).
  • pGEX-2TH prepared by the same method [Maruta, Experimental Medicine
  • ECL Western-Blot detect ion system Anatham mouse horseradish peroxidase; manufactured by Amersham, Inc., log number NA-9310
  • FIG. 6 shows the results. As shown in FIG. 6, when induced by IPTG, the expression of GST-IPIA- ⁇ fusion protein was specifically observed.
  • FIG. 7 shows the electrophoresis pattern of each fraction of the purified fusion protein.
  • peptides represented by the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 were synthesized as follows.
  • the abbreviations for the amino acids and their protecting groups used are those recommended by the IUPAC-IUB Joint Commission on Biochemical Nomenclature regarding biochemical nomenclature. ⁇ Biochemistry (European Journal o f Biochemistry), 138.9 (1984)].
  • the main abbreviations are as follows.
  • Trt Trityl
  • Fioc-Thr (t-Bu) -OH ⁇ -9-fluorenylmethyloxycarbonyl -0-t-butyl-L-threonine
  • Fmoc-Tyr (t-Bu) -OH ⁇ -9-fluorenylmethyloxycarbonyl-0-t-butyl -L-tyrosine
  • Fmoc-Arg (Pmc) -OH N ⁇ -9-Fluorenylmethyloxycarbonyl-N s -2,2,5,7,8-pentanomethylchroman-6-sulfonyl-L-arginine
  • step (a) a condensation reaction is carried out using Fmoc-VaOH 0H. After the washing step (b), and then the deprotection steps (c) and (d), H-Val-Ala- Cys (Trt) was synthesized on the support.
  • step (a) Fmoc-His (Trt) -0H, Fmoc-Arg (Pmc) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pmc) -0H, Fmoc-Phe-OH, Fmoc-Val -OH, Fmoc-Arg (Pmc) -OH, Fmoc-Thr (t-Bu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Leu-0 H, Fmoc -T r (t-Bu) -0H, Fmoc-Arg (Pmc)-0H, Fmoc-Trp-OH, Fmoc-Leu-OH, and (a) to (d) were repeated, followed by methanol Then, the resultant was washed with butyl ether in that order, and dried under reduced pressure for 12 hours
  • the product was purified by HPLC using a reversed-phase column (manufactured by Shiseido, CAPCELL PAK C18 30 column ID X25). Elution was performed by linear concentration gradient method in which a 903 ⁇ 4acetonitrile aqueous solution containing TFA 0.0.1 was added to a 0.13 ⁇ 4 TFA aqueous solution, and the fraction containing the peptide represented by the amino acid sequence represented by SEQ ID NO: 7 was detected at 220 nm. Obtained. This fraction was freeze-dried to obtain 6.0 mg of a peptide represented by the amino acid sequence represented by SEQ ID NO: 7.
  • KLH was dissolved in PBS to adjust to 1 Omg / ml, 1/10 volume of 25 mg / ml MBS (manufactured by Nacalai Tesque) was added dropwise, and the mixture was stirred and reacted for 30 minutes.
  • 2.5 mg of KLH-MB obtained by removing free MBS using a gel filtration column such as a Sephadex G-25 column equilibrated with PBS in advance in 0.1 M sodium phosphate buffer
  • the mixture was mixed with 1 mg of the peptide dissolved in the solution (pH 7.0), and the mixture was stirred and reacted at room temperature for 3 hours. After the reaction, a substance dialyzed against PBS-0.5 M sodium chloride was used as an immunogen. '
  • Example 2 50 ml of a culture solution of E. coli N522 / pGST / IPIA-a into which the expression plasmid pGST / IPI ⁇ - ⁇ containing the cDNA encoding ⁇ - ⁇ obtained in Example 2 was introduced, and 450 ml of LB medium (Containing 100 ⁇ g / ml of ampicillin) and cultured with shaking at 25.
  • LB medium Constaining 100 ⁇ g / ml of ampicillin
  • the cells are collected by centrifugation, suspended in 100 ml of a buffer solution [5 OmM Tris-HCl (pH 7.5), 25% sucrose], and 5 ml of 10% nonidat P—40 and 0% .5 ml of 1 M magnesium chloride was added. After disrupting the cells by sonication, the cells were centrifuged at 10,000 Xg for 15 minutes. Centrifuged supernatant is used for glucan thione-agarose beads
  • the protein was eluted with 5 mM glucan thione and 5 OmM Tris-HCl (pH 9.6). The eluted fractions were collected and dialysed [2 OmM Tris-HCl (pH 7.5), 2 mM magnesium chloride, ImM dithiothreate) And the resulting protein was dialyzed. The protein concentration of the solution was measured by the BCA method (manufactured by PI ERCE). By this method, 5 mg of GST-IPIA- ⁇ fusion protein was obtained from 50 Oml of E. coli culture.
  • peptide one was prepared by the method shown in KLH conjugate 1 0 0 g aluminum gel 2mg and pertussis vaccine (Chiba Serum Laboratory Ltd.) IX 1 0 9 fine with 5 Shureimesu mice (Ba 1 b / c), and two weeks later, 100 conjugates were administered once a week for a total of four times. Blood was collected from the fundus venous plexus, and its serum antibody titer was examined by the enzyme immunoassay shown below. From mice showing a sufficient antibody titer to the GST-IPIA- ⁇ fusion protein obtained in (2), final Three days after the immunization, the spleen was removed (Fig. 8).
  • the spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical), loosened with forceps, centrifuged (1200 rpm, 5 minutes), the supernatant is discarded, and Tris-ammonium chloride buffer (pH 7 The cells were treated for 1-2 minutes to remove red blood cells, washed three times with MEM medium, and used for cell fusion.
  • MEM medium manufactured by Nissui Pharmaceutical
  • the human IPIA- ⁇ partial peptide shown in SEQ ID NO: 7 and SEQ ID NO: 8 was crosslinked with thyroglobulin (hereinafter abbreviated as THY) by the following method. That is, 1 mg of the above peptide was dissolved in 0.1 M ammonium acetate buffer, and 5 mg of THY dissolved in the same buffer was added to make 1 ml. Under stirring, 0.02M glutaraldehyde 5401 was added dropwise, and the mixture was stirred and reacted at room temperature for 5 hours. After the reaction, one dialyzed against PBS was used as an antigen. The GST-IPIA- ⁇ fusion protein was also used as an antigen. GST protein was used as a control antigen.
  • THY thyroglobulin
  • Azaguanin-resistant mouse myeloma cell line P 3 was cultured an U 1 in the normal medium to ensure 2 X 1 0 7 or more cells during cells fused, it was used as a parent strain for cell fusion.
  • mice spleen cells obtained in (3) and the myeloma cells obtained in (5) were mixed at a ratio of 10: 1, and centrifuged (1,200 rpm, for 5 minutes). The supernatant is discarded, and the precipitated cell group is thoroughly loosened. Then, with stirring, at 37, 2 g of polyethylene glycol 1,1000 (PEG-1,000), 2 ml of MEM medium and di methyl sulfoxide 0. 7m l mixture to 0. 2 ⁇ 1 m 1 Z 1 0 8 mouse splenocytes were added, after MEM medium is added several times. 1 to 2m 1. 1 to every 2 minutes, MEM medium was added The total volume was adjusted to 50 ml. After centrifugation (900 rpm, 5 minutes), the supernatant was discarded, the cells were loosened gently, and the cells were gently suspended in 100 ml of HAT medium by aspiration and aspiration with a female pipette.
  • PEG-11000 polyethylene glycol 1,1000
  • the suspension was dispensed by 1 0 0 1Z hole min to 9 for 6-well culture plates, in 5% C0 2 incubator one and cultured 0-1 4 days at 37.
  • the culture supernatant is examined by the enzyme immunoassay described in (4), a well that specifically reacts with the IPIA- ⁇ partial peptide is selected, the medium is replaced with a normal medium, and cloning is repeated twice.
  • Hybridoma strains KM2101, KM2102, KM2103 and KM2104 producing ⁇ - ⁇ monoclonal antibodies were obtained.
  • FIG. 9 shows the reactivity of the obtained monoclonal antibodies KM2101, KM2102, KM2103, and KM2104 with the GST- ⁇ fusion protein and the GST protein.
  • the antibody class was determined to be IgG1 by enzyme immunoassay using a subclass typing kit.
  • the obtained plasmid ⁇ - ⁇ / myc is purified by a conventional method, and ⁇ - ⁇ / myc in 5 ⁇ 10 6 C0S7 cells is allowed to act together with lipofectamine (GibcoBRL, catalog number 18324-012) 12 n ⁇ . , In RPMI medium containing fetal bovine serum 103 ⁇ 4, 37:!, They were cultured for 48 hours under 53 ⁇ 4C0 2.
  • chilled cell lysis buffer [0.5 Triton X, 0.05 SDS, 150 mM sodium chloride, 1 M pepstatinA, 100 ⁇ , leupeptin, 19 tg / ml 4- (2-aminoe thyl) -benzenesulfonyl fluoride 1 ml was added and kept for 4-5 minutes to lyse the cells.
  • the buffer containing the cells was centrifuged at 12,000 ⁇ g for 15 minutes to obtain a supernatant containing the protein.
  • the protein content of this supernatant was measured using the BCA method (manufactured by PIERCE), and proteins were separated by SDS-PAGE using samples prepared to have the same protein content (500) for each lane.
  • the separated protein was transferred to a Hybond, nitrocellulose membrane (manufactured by Amershani) using the electro-plotting method. After blocking with 10% skim milk (Snow Brand Milk Products), anti-IPIA- ⁇ monoclonal antibodies KM2102, KM2101 or KM2103 (2 / zg / ml) were reacted as primary antibodies at 37 for 1 hour. After washing well with Tween-PBS, horseradish peroxidase conjugate mouse-IgG (manufactured by Amersham) was further reacted as a secondary antibody at 37 for 40 minutes at 37, followed by thorough washing with Tween-PBS. .
  • chilled cell lysis buffer (0.5% Triton X, 0.05% SDS, 15 OmM sodium chloride, 1 M pepstatinA, 100 zM leupeptin, 19 ig / ml 4- (2 -aminoe thyl) -benzenesu Ifonyl fluoride] 11111 was added, and the mixture was
  • the lysate was centrifuged at 1200 xg for 15 minutes to obtain a protein-containing supernatant fraction.
  • the protein content of the supernatant was measured by the BCA method (PIE RCE).
  • Samples containing 0.5 mg of protein per lane were fractionated by SDS-PAGE. Elect the separated protein sample It was transferred to a Hybond membrane (Amersham) using the toro-blotting method.
  • anti-IPIA- ⁇ monoclonal antibody K ⁇ 210 concentration 2 g / ml was reacted as a primary antibody at 37 for 1 hour.
  • the present invention relates to acquired immunodeficiency syndrome (AIDS), Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmented retinopathy, cerebellar degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, Useful for the detection and treatment of diseases caused by enhanced cell death such as liver disease and periodontal disease caused by alcohol, and diseases caused by reduced cell death such as cancer, autoimmune disease, and viral infection. .
  • AIDS acquired immunodeficiency syndrome
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid Number of chains: double strand
  • Sequence type nucleic acid
  • Xaa represents N-acetyl-L-leucine
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

L'invention concerne une protéine IPIA-α ayant la fonction d'inhiber la mort cellulaire, un ADN codant cette protéine, des vecteurs recombinés contenant cet ADN, des transformants contenant ces vecteurs ainsi qu'un anticorps réagissant spécifiquement avec la protéine précitée. Cette protéine IPIA-α est utile dans la détection ou dans le traitement de maladies provoquées par une dégradation de la mort cellulaire telles que le cancer, des maladies autoimmunes et des infections virales, ainsi que des maladies provoquées par une mort cellulaire accélérée telles que le syndrome de l'immunodéficience acquise SIDA, la maladie d'Alzheimer, la maladie de Parkinson, la sclérose latérale amyotrophique, la rétinite pigmentaire, la dénaturation cérébelleuse, l'anémie aplasique, l'infarctus du myocarde, l'apoplexie, les troubles de reperfusion, les épatopathies alcooliques ainsi que les parodontolyses.
PCT/JP1998/001319 1997-03-25 1998-03-25 Proteine inhibant la mort cellulaire WO1998042744A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0892127A (ja) * 1994-09-28 1996-04-09 Ono Pharmaceut Co Ltd アポトーシス関与疾患の予防および／または治療剤、アポトーシス調節物質のスクリーニング方法、およびアポトーシス関与疾患の診断方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0892127A (ja) * 1994-09-28 1996-04-09 Ono Pharmaceut Co Ltd アポトーシス関与疾患の予防および／または治療剤、アポトーシス調節物質のスクリーニング方法、およびアポトーシス関与疾患の診断方法

Non-Patent Citations (3)

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Title
HAN T., ET AL.: "THE E1B 19K PROTEIN BLOCKS APOPTOSIS BY INTERACTING WITH AND INHIBITING THE P53-INDUCIBLE AND DEATH-PROMOTING BAX PROTEIN.", GENES AND DEVELOPMENT., COLD SPRING HARBOR LABORATORY PRESS, PLAINVIEW, NY., US, vol. 10., no. 04., 1 January 1996 (1996-01-01), US, pages 461 - 477., XP002910937, ISSN: 0890-9369 *
HAYAKAWA Y., ET AL.: "ANGUINOMYCINS C AND D, NEW ANTITUMOR ANTIBIOTICS WITH SELECTIVE CYTOTOXICITY AGAINST TRANSFORMED CELLS.", THE JOURNAL OF ANTIBIOTICS, NATURE PUBLISHING GROUP, GB, vol. 48., no. 09., 1 September 1995 (1995-09-01), GB, pages 954 - 961., XP002910938, ISSN: 0021-8820 *
JACKSON S., ET AL.: "HUMAN PAPILLOMAVIRUS E6 PROTEIN FROM BOTH CERVICAL AND CUTANEOUS TYPES INHIBIT P53-DEPENDENT AND P53-INDEPENDENT APOPTOSIS.", JOURNAL OF INVESTIGATIVE DERMATOLOGY, NATURE PUBLISHING GROUP, US, vol. 119., no. 04., 1 April 1998 (1998-04-01), US, pages 529., XP002910936, ISSN: 0022-202X *

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