[go: up one dir, main page]

WO1998039437A1 - Nouveaux adn associes a l'apoptose - Google Patents

Nouveaux adn associes a l'apoptose Download PDF

Info

Publication number
WO1998039437A1
WO1998039437A1 PCT/JP1998/000905 JP9800905W WO9839437A1 WO 1998039437 A1 WO1998039437 A1 WO 1998039437A1 JP 9800905 W JP9800905 W JP 9800905W WO 9839437 A1 WO9839437 A1 WO 9839437A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
apoptosis
seq
polypeptide
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1998/000905
Other languages
English (en)
Japanese (ja)
Inventor
Yoshiyuki Sakaki
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
KH Neochem Co Ltd
Original Assignee
Kyowa Hakko Kogyo Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Hakko Kogyo Co Ltd filed Critical Kyowa Hakko Kogyo Co Ltd
Priority to AU61201/98A priority Critical patent/AU6120198A/en
Publication of WO1998039437A1 publication Critical patent/WO1998039437A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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 DNA involved in apoptosis, a polypeptide encoded by the DNA, and an antibody recognizing the polypeptide.
  • Apoptosis refers to cell death in which cells that are no longer needed or have become abnormally self-destruct because the tissue retains its normal shape and function in the living body. For example, removal of unwanted cells during morphogenesis of developing tissues and organs and formation of neural networks, removal of old cells when cells in each tissue are replaced by new cells, elimination of immune cells that react to themselves, Apoptosis works to remove virus-infected cells.
  • Apoptotic cells are characterized by the accumulation of chromatin in the nucleus. The cells themselves contract and die, and are eliminated by phagocytic cells without inflammation. At this time, chromatin DNA is fragmented, and a ladder-like pattern is observed upon electrophoresis.
  • Apoptosis is cell death that is necessary for the body tissue to maintain its normal form and function, and is strictly controlled by various genes in the body. Therefore, it is considered that the abnormalities in the control have a great effect on the living body.
  • Nerve cells are strictly controlled by proliferation, elongation, and apoptosis from the developmental stage, and it is thought that the control forms a nerve network by nerve cells. Nerve cells cease to proliferate after differentiation, but survive for a long time. In recent years, it has been known that apoptosis is also involved in many pathological cell deaths in nerve cells. For example, neuronal necrosis due to cerebral ischemia In addition, death of a large number of nerve cells due to apoptosis has been observed along with necrosis [Journal of Cerebral Blood Flow and Metabolism, 16, 186 (1996), Stroke, 26, 1252 (1995)].
  • Alzheimer's disease [Experimental Neurology, 133-225 (1995)]
  • Parkinson's disease [Journal of Neurological Sciences, 137, 120 (1996)]
  • Huntington's chorea [Experimental Neurology, 133, 265 (1995), Neuro report, 6, 1053 (1995), Journal of Neuroscience, 1_5, 3775 (1995)]
  • Amyotrophic lateral sclerosis [Neuropathology and Applied Neurobiology, 21, 498 (1995)], etc., report that apoptosis occurs in nerve cells.
  • Amyloid ⁇ -peptide which accumulates in cells in Alzheimer's disease and is thought to be deeply involved in the pathogenesis, causes neuronal cell death, and cell death by this amyloid / peptide is also apoptosis [Pro Natl Acad. Sci. USA, 90, 7951 (1993)], and neuronal cell death due to human immunodeficiency virus (HIV) infection has also been reported to be due to apoptosis [American Journal of Pathology, 146, 1121 (1995), Acta Neuropathologies, 91, 169 (1996)].
  • HIV human immunodeficiency virus
  • bc 1 B cell lymphoma / leukemia-2 gene, science, 258, 302 (1992), Pro Natl. Acad. Sci. USA, 90, 4533 (1993)] has an inhibitory effect on apoptosis It is a protein gene that is thought to be involved in morphogenesis during ontogeny. This gene was found to be overexpressed in various cancer cells, and has become a target for anticancer drug development. Genta has shown that the use of the be1-2 antisense-oligoDNA can inhibit the growth of human colon cancer in animal studies. Dictionary, Nikkei Business Publications, 1995).
  • p53 [Proc. Natl. Acad. Sci. USA, 91, 7525 (1994)] arrests the cell cycle. In addition to controlling the cell cycle by this activity, it is thought to act to stop the cycle of cells damaged by radiation, drugs and the like, and to induce apoptosis. It is thought that if p53 becomes abnormal, the damaged cells will proliferate as they are, causing cancer. The mechanism of apoptosis induction of p53 is not yet known.
  • the present invention provides a novel DNA involved in apoptosis, a method for obtaining the DNA, a recombinant vector comprising the DNA and a vector, a transformant obtained by introducing the recombinant vector into a host, and a transformant.
  • a method for producing a novel polypeptide encoding the above-mentioned DNA, and the novel polypeptide, an antibody recognizing the polypeptide, the DNA, the polypeptide, a reagent for detecting apoptosis using the antibody, and a detection method The present invention relates to suppression of apoptosis using the above DNA, polypeptide, and antibody, and diagnosis, prevention, and treatment of a disease associated with apoptosis.
  • the DNA of the present invention is a DNA involved in apoptosis.
  • Gene monocyte chemoattractant protein-1 (MCP-1) gene, aldolase C gene, ⁇ -tubulin gene, represented by SEQ ID NOS: 1-8 And a DNA that hybridizes with the DNA under stringent conditions.
  • DNA that hybridizes under stringent conditions refers to a colony of a hybridized DNA using a DNA having a base sequence selected from the base sequences represented by SEQ ID NOS: 1 to 8 as a probe.
  • the SSC solution at a concentration of 0.1 to 2 times (the composition of the SSC solution at a concentration of 1 times) Can be identified by washing the filter with 150 mM sodium chloride and 15 mM sodium citrate) at 65 ° C.
  • the hybridizable DN is a DNA having at least 60% or more homology with the nucleotide sequence selected from the nucleotide sequences represented by SEQ ID NOS: 1 to 8, and preferably 80% or more. DNAs having homology, and more preferably DNA having homology of 95% or more can be mentioned.
  • the DNA of the present invention also includes oligonucleotides having a partial sequence of DNA involved in apoptosis and antisense oligonucleotides.
  • the oligonucleotide for example, the same sequence as the base sequence of consecutive 5 to 60 residues, preferably 10 to 40 residues in the base sequence selected from the base sequences represented by SEQ ID NOS: 1 to 8
  • an antisense-oligonucleotide for example, an antisense-oligonucleotide of the oligonucleotide.
  • the nucleotide include a nucleotide having a base sequence selected from the base sequences represented by SEQ ID NOS: 22 to 52, and the like.
  • polypeptide of the present invention examples include the polypeptide encoded by the above-described DNA of the present invention. Specifically, pi1, MCP-1 and aldolase C are exemplified. , "Tubulin, a polypeptide having an amino acid sequence selected from the amino acid sequence represented by SEQ ID NO: 9, or an amino acid of a polypeptide having an amino acid sequence selected from the amino acid sequence represented by SEQ ID NO: 9 Peptides having an amino acid sequence in which at least one amino acid in the sequence has been substituted, deleted or added, and having an activity relating to apoptosis can be mentioned.
  • Peptides having an amino acid sequence in which at least one amino acid in the amino acid sequence of the polypeptide has been substituted, deleted or added, and which has an activity involved in apoptosis are described in Molecular loning, A Laboratory Manual, Second Edition. , Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Pro Natl. Acad. Sci., USA, 79, 6409. (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Pro Natl. Acad. Sci USA, 82, 488 (1985), etc. Can be.
  • the antibodies of the present invention include antibodies that recognize the above-mentioned polypeptides.
  • BEST MODE FOR CARRYING OUT THE INVENTION
  • mRNA whose expression level fluctuates in the cells is identified by the differential display method, and c mRNA corresponding to the mRNA is identified.
  • any animal cell culturing method can be used.
  • neurons examples include human neuroblastoma cells, and preferably, SH—S Y 5 Y [Cancer Res., 33, 2643 (1973)].
  • a medium for culturing animal cells As a medium for culturing animal cells, a commonly used RPMI 164 medium, Eagle's MEM medium, or a medium obtained by adding fetal bovine serum or the like to such a medium can be used.
  • Culturing is carried out under conditions such as normal 5% C0 2 presence.
  • the culture temperature is preferably 35 to 37, and the culture time is usually 3 to 7 days.
  • the neuroblastoma cell line SH-SY5Y 100% fetal calf serum, 100 units 111 1 penicillin, 100 / gZml strebtomycin were used.
  • DMEM Dulbecco's modified Eagle's medium
  • the cultured neuroblastoma cells are differentiated into nerve cells.
  • a differentiation inducer such as retinoid is used for differentiation of cells.
  • SH-SY5Y cultured above As a method for differentiating the nerve cells, specifically, SH-SY5Y cultured above is used. . 1 to 5 X 1 0 4 pieces / footsteps planted cm 2 diameter 1 0 cm Petri dish for cell culture to a density 1 0-3 0 hours of culture in the same manner as described above, all-trans form retinoic acid [Sigma] can be added in an amount of 2 to 20 M, and further cultured for 5 to 10 days to differentiate the cells into nerve cells. -Inducing the differentiated cells to induce apoptosis using an apoptosis inducer.
  • a method for inducing apoptosis 0.1 to 10 M of colchicine (manufactured by Wako Pure Chemical Industries, Ltd.) is added to the differentiated cells, and the cells are cultured for 5 to 90 hours in the same manner as described above.
  • a method for inducing apoptosis can be exemplified.
  • apoptosis is induced in the cells means that DNA is extracted and purified from the cells by a conventional method, and the DNA is subjected to agarose gel electrophoresis, whereby fragmentation of the DNA occurs. It can be confirmed by observation.
  • RNA is extracted from each of these cells.
  • Methods for preparing total RNA from neural cells include the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)], the AGPC method [Experimental Medicine, 1937 (1991) 3]. And so on.
  • Fast-Track-mRNA 'isolation-kit [Fast T rack mRNA Isolation Kit; manufactured by Invitrogen), and Quick'Prep * mRNA / Purification Kit (Quick Prep mRNA Purification Kit; manufactured by Pharmacia).
  • MRNA can also be prepared directly from cells.
  • -CDNA is synthesized from each of the RNAs extracted from the nerve cells collected over time using an anchor primer in a conventional manner, and then the 5 'end of each cDNA is Perform PCR using the fluorescently labeled anchor primer and any primer to amplify cDNA.
  • An anchor primer is a primer obtained by adding an adenine, guanine, or cytosine oligonucleotide, excluding thymidine, at the 3 ′ end to an oligo dT sequence associated with the 3 ′ end poly A sequence of mRNA.
  • gT 15 MA (mixture of SEQ ID NO 1 0 ⁇ 1 2)
  • gT 15 MA ( mixture of SEQ ID NO: 1 3 ⁇ 1 5)
  • gT 15 MT ( mixture of SEQ ID NO: 1 6 ⁇ 1 8)
  • gT 15 MG , gT 15 MC (Mixtures of SEQ ID NOS: 19 to 21).
  • Fluorescent labeling can be performed by a conventional method using fluorescin / isothiocyanate (hereinafter abbreviated as FITC).
  • Optional primers are oligonucleotides that can amplify many types of cDNA sequences and obtain a large number of amplified cDNA fragments in a single reaction.
  • Operon 'Technologies Technologies 0 PA-1 to 20; OPB-1 to 20; OPC-1 to 20; OPD-1 to 20 and the like.
  • the optional primer preferably has a length of about 10 bases.
  • Each of the above-mentioned cDNAs amplified by the PCR is electrophoresed on a polyacrylamide gel, and the amount of fluorescence of the resulting band is measured using a fluoroimager for each.
  • the cDNA is amplified by PCR or the like.
  • the amplified DNA fragment is used as it is or after digestion with an appropriate restriction enzyme or the like and integrated into a vector by a conventional method.
  • a commonly used nucleotide sequence analysis method for example, the dideoxy method of Sanger et al. [Pro Natl. Acad. Sci. USA , 74, 5463 (1977)] or 373 A.
  • the nucleotide sequence of the DNA is determined by analysis using a nucleotide sequence analyzer such as DNA Sequencer (manufactured by Perkin Elmer).
  • Examples of the vector incorporating the amplified DNA fragment include pBluescript KS (+) (manufactured by Stratagene), pDIRECT [Nucleic Acids Research, 18, 6069 (1990)], pCR-Script Amp SK (+) (Stratagene Strategies, 5, 6264 (1992)], pT7Blue [manufactured by Novagen], pCR II [manufactured by Invitrodin, Biotechnology, 9, 657 (1991)]
  • nucleotide sequence determined in this way can be determined overnight by searching base sequence databases such as GenBank, EMBL and DDBJ using a homology search program such as biast. This can be confirmed by the fact that there is no nucleotide sequence showing obvious homology, which is considered to be identical to the nucleotide sequence in one copy.
  • Examples of the DNA having the novel base sequence obtained as described above include DNA having the sequence represented by SEQ ID NO: 3 or 4, and the like.
  • Escherichia coli DH5a / pG27T containing plasmid pG27TI having DNA
  • Plasmid pC46-5 having DNA having a sequence represented by SEQ ID NO: 3
  • Escherichia coli DH5 «/ pC46-5 or Escherichia coli DH5a / containing plasmid C55-3-1 containing DNA having the sequence represented by SEQ ID NO: 4 C 5 5—3—1 are FE RM BP—584 3, FE RM BP—5844, F ERM B P-584 5 and F ERM BP—584 6,
  • the DNA obtained by the above method is a partial DNA fragment of cDNA corresponding to mRNA whose expression varies due to apoptosis, the following (1), (2) or (3) ) Can be used to obtain the full-length cDNA.
  • the full-length cDNA can be obtained.
  • the following describes a method for preparing a cDNA library.
  • Methods for preparing DNA libraries include Molecular Cloning, 2nd Edition and Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereafter referred to as Current Protocols' In 'Molecular'). Or a commercially available kit, such as Superscript Plasmid System for cDNA Synthesis and And Plasmid Cloning [Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning; Gibco BRL) or Zap-1 cDNA, synthesis kit (ZAP-cDNA Synthesis Kit, Stratagene). In addition, there is a commercially available cDNA library itself, such as a human brain cDNA library from Clontech, which can be used.
  • Any phage vector, plasmid vector, or the like can be used as a closing vector for preparing a DNA library as long as it can replicate autonomously in the E. coli K12 strain.
  • ZAP Express [Stratagene, Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Rese arch, 17, 9494 (1989)], A zap II (Stratagene) GtlO, stll (DN A Cloning, A Practical Approach, 1, 49 (1985)), Lambda BlueMid (clo ExCell (Pharmacia), pT7T3 18U (Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)] and pUC18 [Gene, 33, 103 (1985) 3) Can be given.
  • any microorganism belonging to Escherichia coli can be used. Specifically, Escherichia cojj XLl-Blue MRF '[Stratagene, Strategies, 5, 81 (1992)], Escherichia coli C600 (Genetics, 39, 440 (1954)], Escherichia coli Y1088 [Science, 222, 778 (1983)], Escherichia coli Y1090 (Science, 222, 778 (1983)), Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966)] and Escherichia coli JM105 [Gene, 38, 275 (1985)].
  • the selection of cDNA clones from a single cDNA library includes colony hybridization using isotopic or fluorescently labeled probes, plaque hybridization hybridization (molecular), and cloning. 2nd edition).
  • the desired DNA can be obtained from the selected clone by a conventional method.
  • cDNA is synthesized from mRNA by the above-described method, and the cDNA is added to both ends of the cDNA, and PCR is performed with a primer based on the nucleotide sequence of the adapter and the nucleotide sequence of the amplified fragment.
  • RACE rapid amplification of cDNA ends
  • 3' RACE (Proc. Natl. Acad. Sci. USA, 85, 8998 (1988)) can obtain the desired DNA.
  • the nucleotide sequence of DNA obtained by these methods can be determined by the above-described nucleotide sequence determination method.
  • the novelty of the sequence can also be confirmed by the method described above.
  • Examples of the DNA having a novel nucleotide sequence confirmed by the method include DNA having the sequence represented by SEQ ID NO: 7 or 8.
  • the desired DNA can also be prepared by chemical synthesis using a DNA synthesizer.
  • the DNA synthesizer include a DNA synthesizer manufactured by Shimadzu Corporation using the Thoho phosphite method, and a DNA synthesizer m0de1 392 manufactured by Perkin Elmer Inc. using the phosphoramidite method. I can give it.
  • an oligonucleotide having a partial sequence of DNA involved in apoptosis and an antisense oligonucleotide can be prepared by a conventional method or a DNA synthesizer.
  • oligonucleotide or the antisense oligonucleotide for example, in a part of the nucleotide sequence of mRNA to be detected, a sense primer corresponding to a 5 ′ terminal nucleotide sequence and a nucleotide sequence at a 3 ′ terminal corresponding to a nucleotide sequence at a 5 ′ terminal side And the like.
  • the base corresponding to peracyl in mRNA is thymidine in the oligonucleotide primer.
  • the sense primer and the antisense primer are oligonucleotides whose melting temperature (Tm) and the number of bases thereof do not extremely change, and preferably have a base number of 10 to 40 bases.
  • An oligonucleotide having a partial sequence of DNA involved in apoptosis or an antisense oligonucleotide for example, a nucleotide having a base sequence selected from the base sequences represented by SEQ ID NOs: 22 to 52 Can be done.
  • derivatives of the nucleotide can also be used, and examples thereof include a methyl derivative of the nucleotide and a phosphotiotate derivative o.
  • a recombinant vector having an apoptosis-related DNA inserted downstream of a promoter of an appropriate expression vector is constructed, and the recombinant vector is introduced into a host cell to express the polypeptide of the present invention.
  • a transformant can be obtained.
  • any one that can express the gene of interest such as bacteria, yeast, animal cells, and insect cells, can be used.
  • those which can replicate autonomously in the host cell or can be integrated into the chromosome, and which contain a promoter at a position capable of transcribing apoptosis-related DNA are used.
  • apoptosis-related gene expression vectors can replicate autonomously in prokaryotes, as well as promoters, ribosome binding sequences, apoptosis-related genes, It is preferably composed of a transcription termination sequence.
  • a gene that controls the promoter may be included.
  • expression vectors include pBT ⁇ 2, pBTacl, and pBTac2 (all manufactured by Principle Mannheim), pKK233-2 (manufactured by Pharmacia), pGEX (manufactured by Pharmacia), and pSE280 (invitrogen).
  • PGEMEX-1 manufactured by Promega
  • pQE-8 manufactured by QIAGEN
  • pET-3 manufactured by Novazidin
  • pKYPIO Japanese Unexamined Patent Publication No. 58-110600
  • pKYP200 Agric. Biol. Chem., 48, 669 (1984)]
  • pLSAl [Aglic. Biol.
  • any promoter can be used as long as it can be expressed in host cells such as Escherichia coli.
  • promoters derived from Escherichia coli or phage such as t ⁇ promoter (Ptrp), iac promoter, PL promoter, PR promoter, PSE promoter, T7 promoter, SP01 promoter, SP02 promoter, penP promoter Etc.
  • Ptrp t ⁇ promoter
  • iac promoter iac promoter
  • PL promoter PL promoter
  • PR promoter PR promoter
  • PSE promoter T7 promoter
  • SP01 promoter SP02 promoter
  • penP promoter Etc penP promoter Etc.
  • the ribosomal binding sequence it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence and the initiation codon is adjusted to an appropriate distance (for example, 5 to 18 bases).
  • a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.
  • host cells include microorganisms belonging to the genus Escherichia, Corynebacterium, Brevipacterium, Bacillus, Microbacterium, Serratia, Pseudomonas, etc., such as Escherichia coli XLl-Blue, Escherichia coli XL2. -Blue, Escherichia col i DH1, Escherichia col ⁇ MC1000, Escherichia col i KY 3276, Escherichia col i W1485, Escher icnia col i JM109> Escherichia col H B101, Escherichia col i No.
  • Any method for introducing the recombinant DNA can be used as long as it is a method for introducing the DNA into the above host cells.
  • a method using calcium ions [Pr0 Natl. Acad. Sci. USA, 69 , 2110 (1972)]
  • the protoplast method Japanese Patent Application Laid-Open No. 63-2483942
  • the electroporation method [Nucleic Acids Research, 16, 6127 (1988)].
  • YEP13 ATCC37115
  • YEp24 ATCC37051
  • YCp50 ATCC37419
  • pHS19 pHS15 and the like
  • YEP13 ATCC37115
  • YEp24 ATCC37051
  • YCp50 ATCC37419
  • pHS19 pHS15 and the like
  • Any promoter can be used as long as it can be expressed in yeast strains.
  • Examples of the Hyodorou EQ vesicle include saccharomyces erev ⁇ _ae, Schizosaccharomvces Dombe, Kluyveromyces lactis Tr lchosporon potlulans ⁇ Schwann iomyces ailuvius and the like.
  • any method for introducing the recombinant DNA any method can be used as long as it introduces DNA into yeast.
  • electroporation method [Methods. Enzymol., 194, 182 (1990)], Suferoplus Natl. Acad. Sci. USA, 81, 4889 (1984)] and the lithium acetate method [J. Bacteriol., 153, 163 (1983)].
  • an expression vector such as pAGElO 7 (JP-A 3-22979), pAS3-3 (JP-A-2-227075), P CDM8 [Nature, 329, 840, (1987) ], (manufactured by Lee Nbitorojen Co.) pcDNAI / Amp, pREP4 (manufactured by Invitrogen) , PAGE103 [J. Biochem, 101, 1307 (1987)], pAGE210 and the like. Any promoter can be used as long as it can be expressed in animal cells.
  • the promoter of the immediate-early (IE) gene of cytomegalovirus (CMV), the early promoter of SV40 or the meta- Examples thereof include a promoter of a mouth protein, a retrovirus promoter, a heat shock promoter, and an SR "promoter.
  • An enhancer of the IE gene of human CMV may be used together with the promoter. .
  • Examples of the host cells include human cells such as Namalwa cells, monkey cells such as COS cells, Chinese hamster cells such as CH0 cells, and HBT5637 (JP-A-63-299). Can be given.
  • Any method for introducing the recombinant DNA can be used so long as it is a method for introducing the DNA into animal cells.
  • an electoral poration method [Cytotechnology, 3, 133 (1990)] ]
  • the calcium phosphate method Japanese Patent Laid-Open No. 2-227075
  • the ribofection method [Pro Natl. Acad. Sci. USA, 84, 7413 (1987)], and the like.
  • the recombinant gene transfer vector and Baculovirus were co-transfected into insect cells to obtain recombinant viruses in the culture supernatant of insect cells, and the recombinant viruses were further infected with insect cells to obtain the present invention.
  • the polypeptide can be expressed.
  • Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
  • Examples of the baculovirus include viruses that infect night insects such as Atographa, Californi, Nuclea, Polyhedrosis, and virus (Autographa cal l fornica nuclear polyhedros is virus).
  • S f 9 is an ovarian cell of Spodoptera f rugiperda
  • S f 2- 1 Bactetrachloride
  • High 5 manufactured by Invitrogen
  • Examples of a method for co-transferring the above-described recombinant gene introduction vector and the above baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a ribofusion method [Proc. Natl. Acad. Sc i. USA, 84, 7413 (1987)].
  • a method for expressing a gene in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular. Cloning, 2nd edition.
  • a sugar or a sugar chain-added polypeptide When expressed by yeast, animal cells or insect cells, a sugar or a sugar chain-added polypeptide can be obtained.
  • the polypeptide of the present invention is produced. it can.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium can be used as long as the medium can efficiently culture the transformants.
  • Any carbon source may be used as long as the organism can assimilate it, such as glucose, fructose, sucrose, molasses, starch or starch hydrolyzate containing these.
  • Carbohydrates such as pulverized products, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium or inorganic salts of organic acids, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, and corn starch. Plyka, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermentation cells, and digests thereof can be used.
  • Inorganic substances include potassium (II) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc. Can be used.
  • the culture is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the cultivation temperature is preferably 15 to 40 ° C, and the cultivation time is usually 16 to 96 hours.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture.
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • isopropyl-1,3-D-thiogalactobyranoside or the like is used when culturing a microorganism transformed with an expression vector using the promoter.
  • Monoacrylic acid or the like may be added to the medium.
  • Culture is usually p H 6-8, 1 to 7 days row also under such 30 ⁇ 40 ° C, 5% C 0 2 presence, during the culturing, if necessary kanamycin, the antibiotic penicillin such O Can be added to the medium
  • Examples of a medium for culturing the transformant obtained by using insect cells as a host include commonly used TNM-FH medium (Pharaingen), Sf-900II SFM medium (Gibco BRL), ExCell 400 ExCel 140 (both manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788 (1962)) and the like can be used.
  • Cultivation is usually carried out at pH 6-7, 25-30 ° C, etc. for 1-5 days.
  • an antibiotic such as gentamicin may be added to the medium during the culture.
  • an ordinary enzyme isolation and purification method may be used.
  • the polypeptide of the present invention when expressed in a dissolved state in a cell, after completion of the culture, the cell is recovered by centrifugation, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press. Then, the cells are disrupted using a mantongaulin homogenizer, a dynomill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method is used, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent.
  • an ordinary enzyme isolation and purification method is used, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent.
  • the cells When the polypeptide is expressed as an insoluble form in cells, the cells are similarly recovered, crushed, and centrifuged. After recovering the polypeptide by a method, the insoluble form of the polypeptide is solubilized with a polypeptide modifier. The lysate is diluted or dialyzed into a solution containing no polypeptide denaturing agent or diluted so that the concentration of the polypeptide denaturing agent does not denature the polypeptide. After the structure is formed, a purified sample can be obtained by the same isolation and purification method as described above.
  • the derivative of the polypeptide of the present invention or its modified sugar can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. Can be obtained.
  • the polypeptide expressed by the above method may be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tB0c method (t-butyloxycarbonyl method).
  • a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tB0c method (t-butyloxycarbonyl method).
  • Kuwawa Trading Advanced ChemTech, USA
  • Perkin-Elmer Japan Perkin-Elmer, USA
  • Pharmacia Biotech PharmaciaBiotech, Sweden
  • Aroka Protein Technologylnstrument ttM, USA
  • Kurabo US Synthecei, USA
  • It can also be synthesized using peptide synthesizers such as l-Vega, Japan Perceptive Limited (made by PerSeptive, USA) and Shimadzu Corporation.
  • Plasma is collected from the fundus venous plexus 3 to 7 days after each administration, and it is determined that the serum reacts with the antigen used for immunization by enzyme immunoassay [enzyme immunoassay (ELISA): published by Medical Shoin 1976 Year, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988].
  • enzyme immunoassay enzyme immunoassay (ELISA): published by Medical Shoin 1976 Year, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988].
  • Serum was obtained from a heron, goat, mouse, rat or hamster whose serum showed a sufficient antibody titer to the antigen used for immunization, and 40-50% saturated ammonium sulfate was obtained from the serum.
  • Purified antibodies can be obtained by standard methods such as salting out method using PEG, sedimentation method using DEP-Sepharose column, protein A-column, and chromatographic method using gel filtration column. .
  • the spleen cells of the animal immunized by the above method and the myeoma cells of the mouse are fused to produce a hybridoma, and the ability to culture the hybridoma is applied to the animal. Is transformed into ascites tumor, and the culture solution or ascites is collected to produce a monoclonal antibody against the polypeptide of the present invention.
  • the mRNA expression level depends on the amount of the hybridized probe according to the label of the probe.For example, the amount of radioactivity in the case of 32 P labeling, the amount of fluorescence in the case of fluorescent labeling It can be measured by measuring.
  • the amplified fragment can be measured by staining the amplified fragment with a DNA-specific fluorescent stain such as ethidium bromide ⁇ cyber green ⁇ and measuring the amount of fluorescence.
  • a DNA-specific fluorescent stain such as ethidium bromide ⁇ cyber green ⁇
  • RNA / DNA antisense 'oligonucleotide
  • RNA / DNA antisense 'oligonucleotide
  • polypeptide of the present invention can be obtained using the DNA described in 1. and the method described in 2.
  • an antibody can be produced by the method described in 3.
  • a polypeptide associated with apoptosis can be detected using the antibody described in 3.
  • Specific examples include the ELISA method using a microtiter plate, a fluorescent antibody method, and a detection method using an estanblot method.
  • the antibody described in 3. can be used for immunohistological staining of test nerve cells.
  • Apoptosis-related proteins derived from test nerve cells or normal nerve cells Quality is detected using the antibody described in 3., the amount of the protein in both cells is measured and compared, and a quantitative change is examined to detect apoptosis in the test nerve cells. be able to.
  • the antibody described above can be used for diseases involving suppression of apoptosis and apoptosis.
  • diseases involving suppression of apoptosis and apoptosis include, for example, diseases involved in the progressive decrease of nerve cells, such as Alzheimer's disease and Parkinson's disease.
  • the antibody can be used for diagnosis and treatment of apoptosis-related cancer.
  • Example 1 Differential display of neurons that induced apoptosis
  • Dulbecco's Modified Eagle Medium supplemented with human neuroblastoma cell line SH-SY5Y, supplemented with 10% fetal calf serum, 100 units / ml penicillin, and 100 / g / ml streptomycin.
  • DMEM manufactured by Nissui Seiyaku
  • hypotonic lysate [5 OmM Tris (hydroxymethyl) aminomethane hydrochloride (Tris—HC and pH 8.0), 1 OmM sodium diethylenetetraminetetraacetate (EDTA), 0.3% triton (T riton) X—100] was added and left in water for 30 minutes to lyse the cells.
  • the cell lysate was centrifuged at 27,000 ⁇ g for 20 minutes to separate into a chromatin fraction (precipitate) and a low molecular weight DNA fraction (supernatant).
  • the DNA was dissolved in TE buffer [1 OmM Tris-HC1 (pH 8.0), 1 mM EDTA], and subjected to 1.2% agarose gel electrophoresis.
  • RNAs were obtained. The specific method of acquisition was in accordance with the reagent protocol.
  • RNA was treated with ribonuclease-free deoxyribonuclease (Promega) to decompose and remove contaminating DNA.
  • RNA precipitation After extracting the RNA with phenol-chloroform, ethanol precipitation (precipitating and collecting RNA by adding 1/10 volume of 2M NaC to the aqueous layer and adding 2 volumes of ethanol). Purify and treat with 0.1% getyl pyrocarbonate (DE PC) Dissolved in distilled water. The solution can be stored at 180 ° C if necessary. .
  • RNA 2 5 g, 5 'end of Furuoresei N'i Sochioshiane preparative (hereinafter abbreviated as FITC) (a mixture of SEQ ID NO: 1 0-1 2) anchor primer g T 15 MA fluorescently labeled with, g T 15 MT (SEQ Mixture of numbers 13 to 15 ), gT15MG
  • cDNA preparation solution 80 ⁇ l of TE buffer was added to the reaction stop solution to obtain a cDNA preparation solution.
  • the preparation can be stored at 120 ° C. if necessary.
  • PCR device PHC-3 manufactured by Techne
  • heat at 94 ° C for 3 minutes and then heat at 95 ° C for 15 seconds, 40 ° C for 2 minutes, Twenty-five cycles were performed with a reaction step consisting of 1 minute at 2 ° C as one cycle, and finally PCR was performed at 72 ° C for 5 minutes.
  • One of the above four types of fluorescent-labeled anchor primers is used.
  • One of the optional primers is OPA-1-20, OPB-1-20, OPB manufactured by Operon's Technologies.
  • One of the 80 types of C—1 to 20 and OPD—1 to 20 is selected for use in the reaction, and by combining each of them, a total of 320 types of reactions are combined into one type of c. Performed for DNA.
  • the band fluorescence obtained on the gel after the electrophoresis was measured and output as an image. Based on the band measurement results obtained, the band whose fluorescence intensity fluctuated over time due to colchicine treatment (that is, the band corresponding to mRNA whose expression level fluctuated over time due to colchicine treatment) ) was picked up.
  • the total number of detected bands was about 200000, and some variation was observed in about 26.3, which is about 1.3% of the total.
  • Example 1- (3) the sample 561 used for polyacrylamide gel electrophoresis was similarly placed on a polyacrylamide gel (200 ⁇ 330 ⁇ 0.35 mm) containing 7 ⁇ urea, After electrophoresis at 1,500 V for 12 hours in TBE buffer, the band was amplified by measuring the fluorescence with Vistra Fluorlmager SI (manufactured by Molecular Dynamics). Was detected, and the image was output in full size.
  • Vistra Fluorlmager SI manufactured by Molecular Dynamics
  • the output image was combined with the gel, and a gel corresponding to the fluctuating band was cut out.
  • DNA was extracted from the gel by a conventional method, and the DNA was subjected to PCR in the same manner as in Example 1- (3) to amplify DNA.
  • the scale of the reaction was 501, and the combination of anchor primer and optional primer used for amplification of each band was used.
  • the amplified DNA was used for PCR fragment cloning vector pT7B1ueT—vector.
  • the PCR fragment was cut with Ec0RV and processed so that T was added to the 3 'end of both strands so that the PCR fragment could be directly cloned into the Ec0RV site (Novagen).
  • a DNA / ligation kit (Takara Shuzo).
  • transform Escherichia coli DH5a manufactured by Gibco BRL
  • Apply the transformant to LB agar medium containing 50 gZm1 of ampicillin, The cells were cultured at 37 ° C for 1 ⁇ .
  • Plasmid DNA was isolated from the grown transformant colonies by the alkaline SDS method (Molecular 'Cloning, 2nd edition).
  • the nucleotide sequence of the amplified DNA incorporated into the plasmid DNA was determined using SQ-5500 DNA Sequencer (manufactured by Hitachi, Ltd.) or 373 A. DNA Sequencer (manufactured by PerkinElmer). Were determined.
  • the specific reagents and methods for nucleotide sequence determination are as follows: SQ-5500 DNA sequencer, Thermosequenase.Premixed.Cycle-sequencing kit; Thermo Sequenase pre-mixed cycle sequencing kit; mersham, Inc.) and used a PRISM Dye primer cycle sequencing kit (PRISM Dye primer sequencing kit; No. 1 Kinelma) in the DNA sequencer. The test was performed according to the instructions attached to the kit.
  • Example 2 Detection of mRNA whose expression fluctuates due to apoptosis induction by RT-PCR
  • Example 1 In the Differential 'display of Example 1, for one type of mRNA whose expression level was thought to fluctuate due to apoptosis induction, the fluctuation was compared with a part of the DNA obtained in Example 1. Oligonucleotides and antisense oligonucleotides containing gene-specific 5 'sense primers and 3' Used as a primer, ⁇ 1?
  • RNA obtained in Example 1 was converted into a kit using a single-stranded cDNA synthesis kit, a superscript preamplification system-11 (manufactured by Gibco 81 ⁇ ). Was synthesized using the oligo dT primer attached to the kit. Specific reagents and methods followed the instructions attached to the kit.
  • the single-stranded cDNA 21A is then ligated with 50 Md NTP, a gene-specific 5 'sense primer, and a 3' antisense primer to 10
  • PCR buffer containing 1 unit of polymerase Gene Taq heat at 94 ° C for 3 minutes, and then start at 95 ° C for 30 seconds, 55 ° C for 1 minute, and 72 ° C for 1 minute.
  • This reaction step was performed 17 to 25 cycles as one cycle, and finally PCR was performed in which the reaction was performed at 72 for 5 minutes.
  • the nucleotide sequences of the gene-specific 5'-side sense primer and 3'-side antisense primer used were described in the column labeled RT-PCR in Table 2 using the SEQ ID NO in the sequence listing.
  • the primer was chemically synthesized by a conventional method based on the DNA obtained in Example 1 and involved in apoptosis.
  • the DNA amplified by the above PCR is subjected to 2% agarose gel electrophoresis, and then stained with 0.01% Cyber Green I (SYBR Green I; manufactured by Molecular Probes), and the amount of the amplified fragment is measured using a fluoroimager or UV. Detection was performed using a transilluminator, and the relative expression level of mRNA was determined.
  • Cyber Green I SYBR Green I; manufactured by Molecular Probes
  • the amplified fragment of C38-1-1 had a base sequence corresponding to positions 14464 to 1847 of the base sequence of SEQ ID NO: 7.
  • This base sequence is composed of a group of ESTs (expressed sequence tag; EST) which are considered to be the same gene as the cDNA including access number AA 0 1 9 4 3 4 in the base sequence database Gen Bank.
  • EST expressed sequence tag
  • the 3'-end and 5'-end nucleotide sequence information of many cDNA clones in the cDNA library was collected including duplications). No match was found in the nucleotide sequence data of the full-length cDNA in GenBank.
  • the amplified G27TI fragment had a nucleotide sequence corresponding to the nucleotide positions from 235 to 266 in the nucleotide sequence of SEQ ID NO: 8. This nucleotide sequence was consistent with the ESTs of access numbers N485593 and N40685 in the base sequence database GenBank (both are considered to be the same gene's cDNA). . There was no match in the nucleotide sequence data of the full-length cDNA in GenBank.
  • nucleotide sequence of the amplified fragment of C46-5 is shown in SEQ ID NO: 3
  • nucleotide sequence of the amplified fragment of C55-3-1 is shown in SEQ ID NO: 4. In both cases, no matching sequence was found in the base sequence database G EnBan k.
  • SEQ ID NO: 5 shows the nucleotide sequence of the C25-1-2 amplified fragment. This nucleotide sequence was consistent with a group of ESTs considered to be cDNAs of the same gene including access number W76643 in the nucleotide sequence database GEnBank. There was no match in the nucleotide sequence data of the full-length cDNA in GenBank.
  • the nucleotide sequence of the amplified fragment of G6 is shown in SEQ ID NO: 6. This nucleotide sequence was consistent with a group of ESTs considered to be cDNAs of the same gene, including accession number N51230 in the nucleotide sequence database GenBank.
  • the portion considered to be the 3 'end of G6 or EST (1933 to 42nd 2nd) is composed of the human Mab_21 gene in GenBank [Human Molecular Genetics, 5, 607 (1996). 5 'untranslated region of cDNA (7th to 23rd position). But this matches The portions are at the 3 'and 5' ends of the cDNA, and both cDNAs may not be derived from the same gene.
  • p i1 is a calcium-binding protein and binds to annexin II (annexinII) p36 to form a heterotetramer.
  • Annexin II complex is a protein that is phosphorylated by the protein kinase PKC in a calcium-dependent manner and is bound to the membrane, and is involved in secretion by exostosis of chromaffin cells in the adrenal gland. (Journal of Cell Biology, 114, 1135 (1191)) In relation to neurons, NGF was administered to cell line PC12 and differentiated into neurons.
  • MCP-1 is one of the chemokines, has an activity to induce chemotaxis to monocytes via receptors, and is thought to be involved in macrophage accumulation during inflammation .
  • MCP-1 is one of the chemokines, has an activity to induce chemotaxis to monocytes via receptors, and is thought to be involved in macrophage accumulation during inflammation .
  • the amplified fragment of C78-3-3 was obtained from human aldolase C gene [Nucieic Acids Res.,
  • Aldolase is an enzyme that degrades curtose 1,6-phosphate in the glycolysis system. Is a brain-specific type. No association with apoptosis has been reported.
  • Hepatic tubulin is a protein that forms tubulin together with tubulin and polymerizes to form microtubules. It has been reported that drugs such as colchicine that bind to tubulin and prevent microtubule formation induce apoptosis in cells [Cancer Letters, 23, 307 (1984), Experimental Cell Rese arch, 198, 367. (1992)] However, changes in the expression of ⁇ - tubulin associated with apoptosis have not been reported.
  • the nucleotide sequence of C38-1_1 was identical to a certain EST (Access No. AA019434) in GenBank, which was a cDNA clone (human). This is the base sequence at the 3 'side of the retinal cDNA clone 3 6 2 93 2), and the base sequence of the EST (accession number AA 018893) at the 5' side of the same clone can be obtained from the database.
  • EST acces No. AA019434
  • the 5 'specific sense primer shown in SEQ ID NO: 44 based on the base sequence of AA 018893 and the 3' specific antisense primer shown in SEQ ID NO: 45 based on the base sequence of C38-1-1-1 PCR was performed on SH-SY5Y cDNA using the sense primer in the same manner as in Example 2.
  • Plasmid DNA was prepared from the transformant in the same manner as in Example 1, and the nucleotide sequence of the cDNA was determined. The obtained nucleotide sequence was 157 It had a second base. From this nucleotide sequence and the nucleotide sequence of C38-1-1-1, the nucleotide sequence of cDNA of C38-1-1-1 shown in SEQ ID NO: 7 was obtained.
  • RACE shows G 27 TI shown in SEQ ID NOs: 48 and 49 A specific sense primer was used.
  • Plasmid DNA was isolated from each clone according to a known method (Molecular 'Cloning 2nd edition), and its nucleotide sequence was analyzed using 7-daza-1d GTP.
  • Lab station, thermosequenase, labeled primer, and cycle sequence Labstaion l'hermo Sequenase label led primer cycle sequenc ing kit with 7-deaza-dGTP; manufactured by Amersham Co., Ltd.] using the dideoxy-chain and yuichi-mination method [Prol. Natl. Acad. Sci., USA, 74, 5463 (1977)]. Decided more. The specific method followed the instructions attached to the kit.
  • G27TI-specific antisense primer shown in SEQ ID NO: 5-0 based on the base sequence near the 5 'end of the clone obtained by 5'-RACE
  • 5'-RACE was performed again in the same manner.
  • the cDNA sequence of G27TI cDNA shown in SEQ ID NO: 53 was obtained from the nucleotide sequence of each clone. However, this nucleotide sequence contained only a very short 0RF of 68 amino acids or less, and was considered to be a nucleotide sequence corresponding to a part on the 3 'side of full-length cDNA.
  • the power of comparing the homology with the base sequence database GenBank for this entire base sequence, and none of those showing homology were found in the database.
  • a novel DNA involved in apoptosis a method for obtaining the DNA, a recombinant vector comprising the DNA and a vector, a transformant obtained by introducing the recombinant vector into a host, and a transformant
  • the present invention can also provide a method for diagnosing, preventing, and treating a disease involving apoptosis and an apoptotic inhibitor using the DNA, polypeptide, or antibody described above.
  • Sequence type nucleic acid
  • Organism name human
  • Sequence type nucleic acid
  • Organism name human Cell line: neuroblastoma SH—SY5Y
  • Sequence type nucleic acid
  • Organism name human
  • Sequence type nucleic acid
  • Organism name human
  • Sequence type nucleic acid
  • Organism name human
  • Sequence type nucleic acid Number of chains: double strand
  • Sequence type nucleic acid
  • Organism name human
  • Lys Glu lie Ser Gly His Thr Ser Gly lie Lys Lys Ala Leu Trp Cys
  • Ser Glu Asp Lys Gin lie Leu Ser Ala Asp Asp Lys Thr Val Arg Leu
  • Sequence type nucleic acid
  • Organism name human
  • Tissue type brain
  • ATCACACGCC CACATACTGC AGTGTGGGAG TAGATTGGTA CGTACTGAGC ACTTAGAAAC 2520
  • Organism name human
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type other nucleic acids, synthetic DNA-sequence
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA Array
  • Sequence length 20-Sequence type: Nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type other nucleic acids, synthetic DNA-sequence
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA Array
  • Sequence length 20-Sequence type: Nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid.
  • Sequence type other nucleic acids, synthetic DNA Array
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Sequence type other nucleic acids, synthetic DNA
  • Sequence type nucleic acid
  • Organism name human

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention se rapporte à de nouveaux ADN associés à l'apoptose; à des procédés d'obtention de ces ADN; à des vecteurs de recombinaison constitués de ces ADN et de vecteurs; à des transformants que l'on obtient en transférant les vecteurs de recombinaison dans des hôtes; à des procédés de production de nouveaux polypeptides codés par les ADN ci-dessus utilisant lesdits transformants; à ces nouveaux polypeptides; à des réactifs et à des procédés de détection de l'apoptose au moyen des ADN décrits ci-dessus, de polypeptides et d'anticorps; à des médicaments conçus pour inhiber l'apoptose à l'aide de ces ADN, polypeptides et anticorps; et à des procédés permettant de diagnostiquer, prévenir ou traiter des maladies dans lesquelles l'apoptose joue un rôle à l'aide des ADN, polypeptides et anticorps décrits ci-dessus.
PCT/JP1998/000905 1997-03-05 1998-03-05 Nouveaux adn associes a l'apoptose Ceased WO1998039437A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU61201/98A AU6120198A (en) 1997-03-05 1998-03-05 Novel apoptosis-related dnas

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP5030297 1997-03-05
JP9/50302 1997-03-05

Publications (1)

Publication Number Publication Date
WO1998039437A1 true WO1998039437A1 (fr) 1998-09-11

Family

ID=12855111

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1998/000905 Ceased WO1998039437A1 (fr) 1997-03-05 1998-03-05 Nouveaux adn associes a l'apoptose

Country Status (2)

Country Link
AU (1) AU6120198A (fr)
WO (1) WO1998039437A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005526779A (ja) * 2002-03-26 2005-09-08 ベン−グリオン ユニバーシティー オブ ザ ネゲヴ 壊死の治療および予防のための組成物および方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICE, File 5: BIOSIS, Biosis Accession No. 99211679, NARANJO J.R. et al., "Differential Screening for NMDA-Induced Genes in Primary Neuronal Cultures"; & SOCIETY FOR NEUROSCIENCE ABSTRACTS, (1996), Vol. 22, Nos. 1 to 3, p. 709. *
IMAIZUMI K. et al., "Molecular Cloning of a Novel Polypeptide, DP5, Induced During Programmed Neuronal Death", J. BIOL. CHEM., (Jul. 1997), Vol. 272, No. 30, p. 18842-18848. *
SHIRVAN A. et al., "Induction of Mitosis-Related Genes During Dopamine-Triggered Apoptosis in Sympathetic Neurons", JOURNAL OF NEURAL TRANSMISSION SUPPLEMENT, (Feb. 1997), Vol. 50, p. 67-78. *
TERESA F.-A. et al., "CPP32, a Novel Human Apoptotic Protein with Homology to Caenorhabditis Elegans Cell Death Protein Ced-3 and Mammalian Interleukin-1beta-Converting Enzyme", J. BIOL. CHEM., (1994), Vol. 269, No. 49, p. 30761-30764. *
THOMAS P.D. et al., "cDNA Sequence of Human p11 Calpactin I Light Chain", GENOMICS, (1992), Vol. 13, No. 3, p. 866-868. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005526779A (ja) * 2002-03-26 2005-09-08 ベン−グリオン ユニバーシティー オブ ザ ネゲヴ 壊死の治療および予防のための組成物および方法

Also Published As

Publication number Publication date
AU6120198A (en) 1998-09-22

Similar Documents

Publication Publication Date Title
US7129338B1 (en) Secretory protein or membrane protein
JP2013063075A (ja) ＩｇＡ腎症関連遺伝子
WO1997042314A1 (fr) Acide nucleique codant pour l'heredo-ataxie cerebelleuse du type 2 et produits connexes
JP4112374B2 (ja) 血管新生マーカーとなるポリペプチドおよびそのｄｎａ
EA005921B1 (ru) Ген и белок, ассоциированные с шизофренией
US6025480A (en) Isolated nucleic acid molecules encoding P57KIP2
EP1038958A1 (fr) PROTEINE Nap1 HUMAINE
AU710551B2 (en) Nucleic acid encoding a nervous tissue sodium channel
US6673535B1 (en) Methods of detecting spinocerebellar Ataxia-2 nucleic acids
KR20030045083A (ko) β-카테닌 핵 국재화 단백질
JP2003521215A (ja) ８３個のヒト分泌タンパク質
WO1998039437A1 (fr) Nouveaux adn associes a l'apoptose
EP1180525B1 (fr) Proteine inhibitrice d'activation transcriptionnelle
US6790944B1 (en) DNA encoding IGA nephropathy indicating protein
WO2006006477A1 (fr) Polypeptide participant dans une maladie osseuse ou une maladie articulaire et adn de celui-ci
WO2000031132A1 (fr) Nouveau polypeptide
JPWO1999063085A1 (ja) ＩｇＡ腎症関連ＤＮＡ
JPWO2004005510A1 (ja) 新規Ｎｏｇｏ受容体様ポリペプチドおよびそのＤＮＡ
JPWO1998039437A1 (ja) 新規アポトーシス関連ｄｎａ
JP3824700B2 (ja) ヒトｓｉｍ遺伝子
JPWO2000040609A1 (ja) 新規蛋白質
JP2001352986A (ja) 新規ポリペプチド
JPWO2002064769A1 (ja) 新規疾患マーカー
JPWO2000031132A1 (ja) 新規ポリペプチド
CA2285605A1 (fr) Gene ii de fanconi

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BG BR CA CN CZ HU IL JP KR MX NO NZ PL RO SG SI SK UA US VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA