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WO2004076666A1 - Famille gasdermine - Google Patents

Famille gasdermine Download PDF

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Publication number
WO2004076666A1
WO2004076666A1 PCT/JP2003/002345 JP0302345W WO2004076666A1 WO 2004076666 A1 WO2004076666 A1 WO 2004076666A1 JP 0302345 W JP0302345 W JP 0302345W WO 2004076666 A1 WO2004076666 A1 WO 2004076666A1
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Prior art keywords
polypeptide
dna
gasdermin
differentiation
seq
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English (en)
Japanese (ja)
Inventor
Toshihiko Shiroishi
Masaru Tamura
Hiroki Sasaki
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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 the Gasdermin family and its use. Background art
  • Non-Patent Documents 1-2 are spontaneously mutated mouse found by Crew and Auerbach in 1939 and shows a curled beard and hair phenotype.
  • Recombinant- induced mutation - 3 (Rim3) and Re den is, their respective B10.BR (R228) and 1983 in Konjienikku system of B10.129, in a spontaneous mutant mice that were found in 1968, the representation
  • the types are epithelial hyperproliferation, hyperkeratosis, and hair loss (Non-Patent Documents 1, 3, and 4).
  • Rim3 and Re den is believed to be the allele of Re (Non-Patent Document 4).
  • These mutations are located on mouse chromosome 11 and in humans correspond to a region of chromosome 17 called 17ql2 amplicon that is closely associated with cancer.
  • Chromosomal instability has been well studied in human carcinogenesis. For example, deletion, amplification, and translocation of a part of a chromosome are phenomena that are remarkably observed in cancer cells. These genomic rearrangements are caused by DNA double-strand breaks ( ⁇ ), which are thought to lead to amplification of oncogenes and loss of tumor suppressor genes (non-patented patents). Reference 5).
  • gasdermin (later renamed mouse gasdermin A-1 (GasderminA-1 / GsdmA-l)) exists in a region homologous to human 17ql2 amplicon in mice (Non-Patent Document 8). ).
  • Non-Patent Document 1 Lyon MF, Rostan S. and Brown SDM Genetic Variants and Strains of the Laboratory Mouse. 3 rd edition (Oxford Univ. Press, 0 xford, 1996)
  • Non-Patent Document 2 Crew, F.A.E. and Auerbach, C. (1939) Rex: a dominant a utosomal monogenic coat texture character in the mouse.J. Genet. 38, 34 1-344.
  • Non-Patent Document 3 Snell, G.D., and Bunker, H.P. (1968) Mouse News Lett. 39.28.
  • Non-Patent Document 4 Sato, ⁇ ⁇ , Koide, T., Masuya, ⁇ ⁇ , Wakana, S., Sagai, T., Umezawa, A., Ishiguro, S., Tama, M., and Shiroishi, T (1998) A new mutation Rim3 resembling Re (den) is mapped close to retinoic acid receptor alpha (Rara) gene on mouse chromosome 11.Mamm Genome.; 9 (1), 20-25.
  • Non-Patent Document 5 Cottle, A., Pipiras, E., Toledo, F., But tin, G., and
  • Non-Patent Document 6 van de Vijver, M., van de Bersselaar, R., Devilee, P., Cornelisse, C., Peterse, J, and Nusse, R. (1987) .Amplification of the neu (c -erbB-2) oncogene in human mammary tumors is relatively frequent then is often accompanied by amplification of the linked c-erbA oncogene.Mol.Cell.Biol. 7, 2019-2023.
  • Non-Patent Document 7 Yokota, J., Yamamoto, T., Miyajima, N., Toyoshima, K., Nomura, N., Sakamoto, H., Yoshida, T., Terada, M., and Sugimura, T (19 88) .
  • Genetic alterations of the c-erbB-2 oncogene occur frequently in tu bular adenocarcinoma of the stomach and are often accompanied by amp 1 ifi cation of the v-erbA homologue.Oncogene 2, 283-287.
  • Non-Patent Document 8 Saeki, N., Kuwahara, Y., Sasaki, H., Satoh H., and Shi roishi, T. Gasdermin (Gsdi) localizing to mouse chromosome 11 is predom inantly expressed in upper gastrointestinal tract but significantly. S. ressed in human gastric cancer eel Is. Mammal an an Genome., 11 (9): 718-724, 2000. Disclosure of the Invention
  • the present invention has been made in view of such a situation, and an object of the present invention is to provide a Gasdamin family and a method of using the same.
  • the present inventors named the mouse gasdamin (later mousegasdermin A-GasderminA-1 / GsdmA-1). Modified), two novel gasdamine homologous genes were found next to the centromere in tandem. The full-length cDNAs of these two novel gasdermin homologous genes were isolated from a mouse skin and mouse stomach cDNA library by screening using mouse gasdamine cDNA as a probe. It was named Sgasdamine A-2 (GasderminA-2 / GsdmA-2) and mouse gasdamine A-3 (Gasdermin A-3 / GsdmA-3).
  • mouse gas dermin previously reported was mouse gas dermin A-1.
  • the present inventors have named mouse gasdermin Al, mouse gasdermin A_2, and mouse gasdermin A-3 as mouse gasdermin A cluster.
  • the MA fragment predicted to be the human gasdamine A-GasderminA-1 / GSDM A-1) gene was replaced with the Rim3 causative gene region and the human homologous region of human. Found on chromosome 17.
  • Oligonucleotide primers for PCR were set on the basis of the nucleotide sequence, and cDNA encoding the protein code region of human gasdermin A-1 was isolated by PCR.
  • the coding region of the human gasdermin A-1 cDNA protein consists of 1338 base pairs, and the protein predicted therefrom is composed of 445 amino acids, and has a homology of 70% or more with the mouse gasdermin A cluster one protein.
  • the mouse gasdermin A cluster gene is contained within the causative gene region of Rim3.
  • Rim3, phenotype Re den is a decision expected that it would be caused by any of mutation Gasudamin A class evening monogenic, Rim3, mouse gas Zehnder Min A cluster monogenic nucleotide sequence in Re den went.
  • the mutation in Rim3 changes the G (guanine) at position 1091 of mouse gasdermin A-3 to A (adenine), and the amino acid at position 342, A (alanine: a nonpolar amino acid), becomes T (threonine: Uncharged electrode amino acids).
  • 6 bases from 1279 to 1284 (GGAAGC) of mouse gasdamine A-3 are inserted at position 1285, and two amino acids EA to 407 to 408 (E: glutamic acid, A: Alanine).
  • the present inventors introduced the human gasdermin A-1 gene into a cancer cell line together with the Neo resistance gene in order to examine the cancer-suppressing activity of the human gasdermin A-1 gene.
  • the colony formation rate was examined (Colony format) 0 Human gasdermin A-1 gene and Neo resistance gene were simultaneously introduced into the MKN28 gastric cancer cell line and the TE10 esophageal cancer cell line, and G418 was used. As a result of selecting and comparing only the transfected cells, the cancer was dramatically increased compared to the control performed simultaneously with the vector alone or the control transfected with the negative chain (antisense) of the human gasdamine A-1 gene. Colony formation was suppressed. Therefore, it was shown that the human gasdamine A-1 gene has a tumor suppressor function.
  • Hitogasudamin A-1 gene was isolated as one of the family monogenic the gene responsible for epithelial morphogenesis abnormal mutant mice Rim3, Re de n. Rim3, the causative gene of Re den, mutation site of the mouse Gas Zehnder Min A- 3 gene is a region conserved in all between Gasudamin A cluster genes. Therefore, by introducing the same mutation as Re den in Hitogasudamin A-1 gene was performed experiments similar with MKN28, TE10 both cell lines. As a result, normal Hitogasudamin A - as compared to the 1 gene, Hitogasudamin A- 1 gene introduced mutations Re den type, significantly reduced the formation suppression effect was observed.
  • the present inventors considered the possibility that other gasdermin homologous genes exist, and, using a homology search program, searched the mouse EST database and the human genome database. While searching the mouse EST database and the human genome database, a number of families including the MLZE (Wat abe et., Al. Jpn J Cancer Res. 2001, 92 (2): 140-151) (named the Gasdermin family) ). Furthermore, the present inventors have isolated a novel human gasdermin B-1 (Gas derminB-1 / GSDMB-1) gene belonging to the gasdermin family. That is, the present invention provides the following [1:] to [28] regarding the gasdamine family and use thereof.
  • polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, or 10, wherein one or more amino acids have been substituted, deleted, inserted, and / or added.
  • a partial DNA of MA according to any one of (a) to (d), which is functionally equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, or 10 MA encoding a polypeptide.
  • [3] A vector into which the DNA of [1] has been inserted.
  • [4] A transformed cell carrying the DNA of [1] or the vector of [3].
  • [6] an oligonucleotide that hybridizes to the DNA of [1] and has a chain length of at least 15 nucleotides.
  • a disease caused by abnormal cell proliferation or differentiation which contains, as an active ingredient, DNA encoding a polypeptide belonging to the gasdamine family described in any of the following (a) to (e): Drugs for treatment or prevention.
  • polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, wherein one or more amino acids are substituted, deleted, inserted, and Z or added; : DNA encoding a polypeptide functionally equivalent to the polypeptide comprising the amino acid sequence of 2, 4, 6, or 8.
  • Cell proliferation comprising, as an active ingredient, a nucleotide or a nucleotide derivative for suppressing the expression of a polypeptide belonging to the gasdermin family described in any of the following (a) to (e): An agent for treating or preventing a disease caused by abnormal or abnormal differentiation.
  • SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38 Is a polypeptide consisting of an amino acid sequence in which a plurality of amino acids have been substituted, deleted, inserted, and added or deleted, and SEQ ID NOS: 10, 12, 14, 16, 18, 20, 22, 22, 24, 26, 28 39.
  • a polypeptide functionally equivalent to the polypeptide comprising the amino acid sequence according to 30, 32, 34, 36 or 38.
  • polypeptide a partial polypeptide of the polypeptide according to any of (a) to (d), which is SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, A polypeptide functionally equivalent to the polypeptide comprising the amino acid sequence of 30, 32, 34, 36 or 38.
  • the disease caused by abnormal cell proliferation or differentiation is cancer, non-neoplastic keratosis of the skin, ectopic epithelial mucosa of the gastrointestinal tract, or polyp; (7) or (8) The drug according to the above.
  • [10] A cell in which the expression of the DNA of [7] is artificially suppressed.
  • [11] A non-human animal in which the expression of the DNA according to [7] is artificially suppressed.
  • (b) measuring the proliferation or differentiation of the cell A method comprising determining that the test sample has an activity of suppressing abnormal cell growth or differentiation when the proliferation or differentiation is suppressed as compared to when the test sample is not contacted.
  • a method comprising determining that the test sample has an activity of suppressing abnormal cell growth or differentiation when the proliferation or differentiation is suppressed as compared to when the test sample is not contacted.
  • a method for evaluating whether or not a test sample has an activity of suppressing abnormal cell proliferation or differentiation comprising:
  • (c) a step of measuring the number of cells killed dependent on the introduction of the DNA according to (7), wherein the number of cells killed dependent on the introduction of the DNA is higher than when the test sample is not contacted.
  • a method for evaluating whether or not a test sample has activity to suppress abnormal cell proliferation or abnormal differentiation comprising:
  • test sample When the expression level of the reporter gene is not brought into contact with the test sample.
  • a method comprising determining that a test sample has an activity of suppressing abnormal cell growth or differentiation when the expression level of the reporter gene is reduced as compared to when the test sample is not contacted.
  • a method for screening a sample having an activity of inhibiting abnormal cell growth or differentiation comprising the following steps (a) and (b):
  • a method for screening a sample having an activity of inhibiting abnormal cell proliferation or differentiation comprising the following steps (a) to (e):
  • a method for detecting a disease caused by abnormal cell growth or differentiation which comprises a step of measuring the expression level of DNA encoding a polypeptide belonging to the gasdermin family.
  • a method for testing a disease caused by abnormal cell proliferation or differentiation comprising a step of detecting a mutation in a DNA region encoding a polypeptide belonging to the gasdamine family 1.
  • the disease caused by abnormal cell proliferation or differentiation is cancer, non-neoplastic keratosis of the skin, ectopic epithelial mucosa of the gastrointestinal tract, or polyp; [22] or Is the method described in [23].
  • a test agent for a disease caused by abnormal cell growth or differentiation comprising an oligonucleotide that hybridizes to a DNA region encoding a polypeptide belonging to the gasdamine family and has a chain length of at least 15 nucleotides.
  • test agent for a disease caused by abnormal cell proliferation or differentiation including an antibody that binds to a polypeptide belonging to Gasdermin Family.
  • polypeptide comprising the amino acid sequence of SEQ ID NO: 10 or 28 in which one or more amino acids have substitution, deletion, insertion, and Z or an added amino acid sequence, wherein SEQ ID NO: 10 Or a polypeptide functionally equivalent to a polypeptide comprising the amino acid sequence of 28.
  • the disease caused by abnormal cell proliferation or differentiation is cancer, non-neoplastic keratosis of the skin, ectopic epithelial mucosa of the gastrointestinal tract, or polyp, [25] to [ 27 7)
  • the test agent according to any one of the above.
  • the present inventors isolated a novel mouse gasdermin homologous gene and developed a mouse gasdermin A-2 (Gasdermin A-2 / GsdmA-2) and mouse gasdermin A-3 (GasderminA-3 / GsdmA-3). Furthermore, by comparing the mouse gasdermin family gene with the human genome draft sequence, the human gasdermin A-GasderminA-1 / GSDMA-1) gene was found, and the human gasdermin A-1 gene was isolated by the PCR method. A search for the presence of other Gasdermin homologous genes also surprisingly revealed the existence of the family (named the Gasdermin family). Furthermore, a new human gasdamine B-l (GasderminB-1 / GSDMB-1) belonging to the gasdamine family The gene was isolated.
  • Gasdamine Family 1 is classified into two subfamilies based on its characteristics.
  • the present inventors named gasdamine A cluster 1 and gasdamine A related gene family, respectively.
  • Gasdamine A cluster 1 includes human gasdamine A-1, mouse gasdamine A-2, mouse gasdamine A-3, mouse gasdamine A-1, and polyfunctional equivalents thereof.
  • Peptides. SEQ ID NOS: 1, 3, 5, and 7 show the nucleotide sequences of DNA encoding human gasdamine A_l, mouse gasdamine A-2, mouse gasdamine A-3, and mouse gasdamine A-1 respectively.
  • the amino acid sequence of the polypeptide encoded by the DM is shown in SEQ ID NOs: 2, 4, 6, and 8, respectively.
  • Gasdamine A class Yuichi has a function to suppress abnormal cell proliferation or differentiation. Therefore, DNA encoding the Gasdermin A cluster can be used as an agent for treating or preventing a disease caused by abnormal cell growth or differentiation.
  • the cells are not particularly limited, and include, for example, epithelial cells.
  • the diseases caused by abnormal cell proliferation or differentiation include cancer, non-neoplastic keratosis of the skin, ectopic epithelial mucosa of the gastrointestinal tract, and polyps.
  • Examples of cancer include, but are not limited to, stomach cancer, esophageal cancer, breast cancer, colon cancer, skin cancer, ovarian cancer, pelvic cancer, uterine cancer, kidney cancer, lung cancer, melanoma, and the like.
  • the non-neoplastic keratosis of the skin is not particularly limited.
  • ichthyosis vulgaris ichthyos is vulgar is
  • psoriatic psor i as is
  • ⁇ psoriasis ⁇ parapsor ias is
  • lichen planus l ichen planus
  • Gibert rose pityriasis pi tyri as is rosea Giberi
  • Examples of the ectopic epithelial mucosa of the gastrointestinal tract include, but are not limited to, the ectopic gastric mucosa of the esophagus and the ectopic gastric mucosa of the small intestine.
  • polyps include, but are not limited to, gastric hyperplastic polyps, Peutz-Jeghers syndrome, Cronkite-Canada syndrome group, and the like.
  • Gasdermin A-related gene families include Gasdamine B-1, PR02521 (AF119884), AK0 drawing 9, BC025682, AF258572, MLZE, Mlze, Gasdamine C-2, Gasdamine C-3, FLJ12150, Gasdamine One min Dl, A01693K HSA245876, AY008304, AK006248, and polypeptides functionally equivalent thereto.
  • Gasdamine Bl, PR0252KAF119884), AK000409, BC025682, AF258572, MLZE, Mlze, Gasdamine C-2, Gasdamine C-3, FLJ12150, Gasdamine D-1, AK01693K HSA245876, AY008304 and AK006248 are encoded by the DNAs as SEQ ID NOs: 9, 11, 13, 13, 15, 17, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 and 37, respectively.
  • the amino acid sequences of the polypeptides are shown in SEQ ID NOS: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38, respectively.
  • the gasdamine A-related gene family is expressed in cells with abnormal growth or differentiation.
  • the Gasdermin A-related gene family can be a diagnostic marker for diseases caused by abnormal cell proliferation or differentiation. Further, the gasdamine A-related gene family has a function of promoting abnormal cell proliferation or differentiation. Therefore, it can be a target for therapeutic drugs for diseases caused by abnormal cell proliferation or differentiation.
  • the Gasdermin family is a leucine-rich polypeptide.
  • the C-terminal is highly conserved, and in families other than Gasdermin B-1 / PR02521, the C-terminal Y (X) 3 A (X) 3 L (X) 3 Q (X) 3 L (X) 8 L 00 5 LV (X) 17 L (X) 3 LL (X) 5 E (X) 7 L (X) 4 GL (X) 6 P (X) 3 W (X) 9
  • ALY (X) 2 L (X ) 2 L (X) 2 L (SEQ ID NO: 39) is common.
  • Gasdermin family also includes (G / D / N) X3 (L / M) X2 (E / K) (V / L) X6 (V / L) X2 (
  • Milli is similar in structure as a whole, but one of its features is that it has a specific amino acid sequence in the center.
  • Gasdermin A cluster has a leucine zipper structure (yJSLSI ⁇ LGKKKEyiDLEEl (SEQ ID NO: 44)) in which L (leucine) is repeated every seven amino acids.
  • Gasdermin A cluster 1 is a unique group of polypeptides having no known motifs or domains other than the leucine zipper structure. The individual structural characteristics are that mouse gasdermin A-2 has a longer N-terminal amino acid than other gasdamine A, and mouse gasdermin A-3 has a longer C-terminal amino acid.
  • the unique sequences in the center include GEKSGEEKVIL IQASD (SEQ ID NO: 45) for human gasdermin A-1, LCVLQRQGSTVQMIS (SEQ ID NO: 46) for mouse gasdermin A-2, and mouse gasdermin A-3.
  • GEKPGEGKFILIQASD SEQ ID NO: 48
  • IRRVPCSAFISPTQMISEEPEEEKLI SEQ ID NO: 47.
  • Gasdermin B-1 (SEQ ID NO: 10) has a unique sequence (NIHFRGKTKSFPEEKDGASSCLGK (SEQ ID NO: 54)) in the center.
  • DNAs encoding human gasdermin A-1, mouse gasdermin A-2, mouse gasdermin A-3 and gasdamine B-1 of the gasdermin family were newly isolated in the present invention. .
  • the present invention relates to a DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, or 10, or a DNA comprising the coding region of the nucleotide sequence of SEQ ID NO: 1, 3, 5, or 9 I will provide a.
  • the present invention also provides a DNA encoding a polypeptide that is functionally equivalent to the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 10.
  • Such MAs include, for example, DNAs encoding variants, alleles, variants, homologs, etc., of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, or 10.
  • “functionally equivalent” means that the target polypeptide is a biological molecule equivalent to human gasdermin A-1, mouse gasdermin A-2, mouse gasdermin A-3, or gasdamine B-1. Functional (biological role, biological properties) and biochemical function (biochemical activity). Human gasdamine A-1, mouse gasdamine A-2, or mouse gasdamine T JP2003 / 002345
  • the biological functions of A-17-A-3 include its ability to suppress abnormal cell proliferation or differentiation, its ability to induce apoptosis, and its ability to suppress epithelial hyperproliferation, hyperkeratosis and hair loss.
  • the biochemical functions of human gasdamine A-1, mouse gasdamine A-2, and mouse gasdamine A-3 include the binding between gasdermin families, Modifications such as phosphorylation of clusters, protein transport to the nucleus, and the like.
  • the biological functions (biological role, biological properties) of gasdermin B-1 include its ability to be expressed in cells showing abnormal growth or abnormalities, and promote abnormal cell growth or abnormalities. Function.
  • DNA encoding human gasdermin A-1, mouse gasdermin A-2, mouse gasdermin A-3 or gasdermin B-1 is hybridized under stringent conditions to obtain human gasdermin A-.
  • DNA that encodes a polypeptide that is functionally equivalent to mouse gasdermin A-2, mouse gasdermin A-3 or gasdamine B-1 examples include, but are not limited to, homologues derived from human, monkey, mouse, rat, bush, and pest, human gasdamine A-1, mouse gasdermin A-2, Isolate DNA encoding a polypeptide functionally equivalent to mouse gasdermin A-3 or gasdermin B-1
  • the conditions for hybridization for the purpose can be appropriately selected by those skilled in the art.
  • the conditions for the hybridization include, for example, low stringent conditions.
  • the low stringency conditions are, for example, conditions of 42, 5xSS 0.1% SDS, preferably 50 ° C, 5xSSC, 0.1% SDS in washing after hybridization.
  • More preferable hybridization conditions include high stringency conditions.
  • Highly stringent conditions are, for example, conditions of 65 ° (:, 0.1xSSC and 0.1% SDS. Under these conditions, as the temperature is increased, DNA having higher homology can be efficiently produced. However, it is expected that a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of the hybridization, and those skilled in the art can appropriately select these factors. A similar stringency can be achieved.
  • DNA based on the sequence information of DNA (SEQ ID NO: 1, 3, 5, or 9) encoding human gasdamine A-1, mouse gasdamine A-2, mouse gasdamine A-3 or gasdermin B-1 Using a gene amplification method using synthesized primers, for example, polymerase chain reaction (PCR), human gasdamine Al, mouse gasdamine A-2, mouse gasdamine A-3 or gasdamine B-1 It is also possible to isolate DNA that encodes a functionally equivalent polypeptide.
  • PCR polymerase chain reaction
  • a polypeptide functionally equivalent to human gasdermin Al, mouse gasdermin A-2, mouse gasdermin A-3 or gasdermin B-1 encoded by DNA isolated by these hybridization techniques or gene amplification techniques Usually has high homology in amino acid sequence with human gasdermin A-1, mouse gasdermin A-2, mouse gasdermin A-3 or gasdermin B-1.
  • the polypeptide of the present invention includes human gasdamine A-1, mouse gasdamine A_2, mouse gasderin A-3 or gasdermin B-1, and is functionally equivalent to the amino acid of the polypeptide.
  • Polypeptides having high homology to the acid sequence are also included. High homology usually means at least 50% identity, preferably 75%, at the amino acid level.
  • the identity is usually at least 50% or more, preferably 75% or more. More preferably 85% or more identity, more preferably 95% or more identity.
  • It also comprises an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of human gasdamine Al, mouse gasdamine A_2, mouse gasdamine A-3, or gasdamine B-1; DM encoding a polypeptide equivalent to the above is also included in the MA of the present invention, which encodes a polypeptide having a function of suppressing abnormal cell growth or abnormal differentiation.
  • amino acid mutations can also occur in nature.
  • the number of mutated amino acids is usually within 30 amino acids, preferably within 15 amino acids, more preferably within 5 amino acids (eg, within 3 amino acids), and even more preferably within 2 amino acids.
  • the amino acid residue to be mutated is desirably mutated to another amino acid that preserves the properties of the amino acid side chain.
  • the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), and hydrophilic amino acids (R, D, N, E).
  • amino acids having aliphatic side chains G, A, V, L, I, P
  • amino acids having hydroxyl-containing side chains S, ⁇ , ⁇ )
  • Amino acids with sulfur atom-containing side chains C, M
  • amino acids with carboxylic acid and amide-containing side chains D, N, E, Q
  • amino acids with base-containing side chains R, H
  • amino acids having an aromatic-containing side chain H, F, Y, W
  • a method of introducing a mutation into a polypeptide is known.
  • those skilled in the art can use site-directed mutagenesis (Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol • 100, 468).
  • site-directed mutagenesis Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol • 100, 468).
  • -500 Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods.Enzymol. 154, 350-367, Kunkel'TA (1985) Proc Natl Acad Sci USA.
  • Polypeptides with multiple amino acid residues added to the amino acid sequence of human gasdermin A-1, mouse gasdermin A-2, mouse gasdermin A-3 or gasdermin B-1 Peptides include fusion polypeptides that include these polypeptides. Fusion port
  • a DNA encoding human gasdamine Al, mouse gasdamine A-2, mouse gasdamine A-3, or gasderin B-1, and other polypeptides may be encoded.
  • the DNAs to be ligated may be ligated so that their frames match, introduced into an expression vector, and expressed in a host.
  • the other polypeptide to be fused with the polypeptide of the present invention is not particularly limited.
  • peptides to be fused to the polypeptide of the present invention include, for example, FL AG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), and six Hiss ( Histidine) 6xHis, 10xHis, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7_tag, HSV-tag, E-tag, SV40T
  • peptides such as antigen fragments, lck tag, ⁇ -tubulin fragment, B-tag, and Protein C fragment can be used.
  • polypeptides to be fused with the polypeptide of the present invention include, for example, GST (daltathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, 3-galactosidase — And (maltose binding protein).
  • a fusion polypeptide can be prepared by fusing commercially available DNAs encoding these polypeptides with DNA encoding the polypeptide of the present invention, and expressing the fusion MA thus prepared. .
  • the DNA of the present invention may be in any form as long as it can encode the polypeptide of the present invention. That is, it does not matter whether it is cDNA synthesized from mRNA, genomic DNA, or chemically synthesized DNA. In addition, DNAs having any nucleotide sequence based on the degeneracy of the genetic code are included as long as they can encode the polypeptide of the present invention.
  • the DNA of the present invention can be prepared by a method known to those skilled in the art. For example, a cDNA library is prepared from cells expressing the polypeptide of the present invention, and Naturally-derived DNA can be prepared by performing hybridization using a part of the sequence of the DNA of the present invention (for example, SEQ ID NO: 1, 3, 5, or 9) as a probe.
  • the cDNA library may be prepared, for example, by the method described in the literature (Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)), or using a commercially available cDNA library. Is also good.
  • the sequence of the DNA of the present invention (for example, SEQ ID NO: 1, 3, 5, or 9) ) can be prepared by synthesizing an oligo DNA, performing a PCR reaction using this as a primer, and amplifying a cDNA encoding the polypeptide of the present invention.
  • genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.
  • mRNA is isolated from cells and tissues expressing the polypeptide of the present invention (eg, skin, esophagus, large intestine, breast, lung, thymus, oral squamous epithelium, etc.).
  • mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), the AGPC method (Chomczynski, P. and Sacchi, N., Anal. Prepare total RNA using Biochem. (1987) 162, 156-159) and purify niRNA from total RNA using mRNA Purification Kit (Amersham).
  • mRNA can be directly prepared using the QuickPrep mRNA Purification Kit (Amersham).
  • CDNA is synthesized from the obtained mRNA using reverse transcriptase.
  • cDNA can also be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like.
  • PCR polymerase chain reaction
  • the polymerase chain reaction may be performed according to a known method, for example, a method described in a literature (Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)), or a commercially available kit, For example, it can be performed by using TaKaRa PCR Amplification Kit (Yukara).
  • RNA can be converted to cDNA using TaKaRa One Step RNA PCR kit (Yukara Co., Ltd.) and PCR can be performed continuously.
  • the 5'-RACE method Frohman, MA et al., Proc. Natl. Acad. Sci.
  • 5'-Ampli FINDER RACE Kit manufactured by Clontech
  • PCR polymerase chain reaction
  • a target DNA fragment is prepared from the obtained PCR product, and ligated to vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector.
  • the base sequence of the target DNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.
  • a nucleotide sequence with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucleic Acids Research (1981)). ) 9, r43-74) 0
  • the DNA of the present invention can be modified by a commercially available kit or a known method. Examples of the modification include digestion with a restriction enzyme, insertion of a synthetic oligonucleotide or an appropriate DNA fragment, addition of a linker, insertion of an initiation codon (ATG) and Z or a termination codon (TAA, TGA, or TAG), and the like. Is mentioned.
  • the present invention provides a DNA which is a partial DNA of the above DNA and encodes a polypeptide having a function of suppressing abnormal cell growth or abnormal cell differentiation.
  • a polypeptide preferably has a function of suppressing abnormal cell proliferation or differentiation.
  • a C-terminal region of a polypeptide having -24- which includes amino acids 343 to 451 of mouse gas-dmin A-3 and amino acids 352 of mouse gas-damine A-2
  • the 460th amino acid and amino acids 333 to 441 of human gas A-1 are listed.
  • Such regions will, in mice, there are variations, such as mutant mice RIM-3, Re den, and highly homologous regions der Runode of between gas Zehnder Min family one to be described later, the cells It is considered to be a region having the function of suppressing abnormal growth or abnormal differentiation.
  • the present invention provides a polypeptide encoded by the DNA of the present invention.
  • the polypeptide of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chains, etc., depending on the cell or host producing the polypeptide described below, or the purification method. However, as long as the obtained polypeptide has a function equivalent to that of human gasdermin A-1, mouse gasdermin A_2, mouse gasdermin A-3 or gasdermin B-1, it is included in the present invention.
  • a prokaryotic cell for example, Escherichia coli
  • a methionine residue is added to the N-terminal of the amino acid sequence of the original polypeptide.
  • the polypeptide of the present invention also includes such a polypeptide.
  • the polypeptide of the present invention can be prepared as a recombinant polypeptide or a natural polypeptide by methods known to those skilled in the art.
  • a DNA encoding the polypeptide of the present invention is incorporated into an appropriate expression vector, and this is introduced into an appropriate host cell.
  • a transformant obtained is recovered, and an extract is obtained.
  • chromatography such as ion exchange, reverse phase, gel filtration, or the like, or by affinity chromatography on a force column, or further using these columns. It is possible to purify and prepare by combining several of these.
  • the polypeptide of the present invention is prepared by converting daltathione S-transferase protein When expressed in host cells (for example, animal cells or Escherichia coli) as a fusion polypeptide with the protein or as a recombinant polypeptide to which a plurality of histidines are added, the expressed recombinant polypeptide can be expressed on a glutathione column or a nickle. It can be purified using a gel column. After purification of the fusion polypeptide, if necessary, a region other than the target polypeptide in the fusion polypeptide can be cleaved with thrombin or Factor-I Xa and removed.
  • host cells for example, animal cells or Escherichia coli
  • the expressed recombinant polypeptide can be expressed on a glutathione column or a nickle. It can be purified using a gel column. After purification of the fusion polypeptide, if necessary, a region
  • polypeptide is a natural polypeptide
  • an antibody that binds to the polypeptide of the present invention described below against an extract of a tissue or a cell expressing the polypeptide of the present invention will be described later.
  • the protein can be isolated by the action of an affinity column to which the DNA is bound and purified.
  • the antibody may be a polyclonal or monoclonal antibody.
  • the present invention also provides a vector into which the DNA of the present invention has been introduced.
  • the vector of the present invention is useful for retaining the DNA of the present invention in a host cell or expressing the polypeptide of the present invention.
  • Escherichia coli when Escherichia coli is used as a host, the vector is amplified in Escherichia coli (E. coli, for example, JM109, DH5a, HB10K XLlBlue), etc.
  • Escherichia coli for example, JM109, DH5a, HB10K XLlBlue
  • E. coli selection gene for example, a drug resistance gene that can be distinguished by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)
  • the vector examples include an M13 vector, a pUC vector, pBR322, pBluescript, pCR-Script, and the like.
  • pGEM-T pDIRECT
  • pT7 pT7, etc. may be mentioned in addition to the above vectors.
  • Expression vectors include, for example, In the case of expression, in addition to having the above characteristics such that the vector is amplified in Escherichia coli, when the host is [Escherichia coli such as M109, DH5 «, thigh 01, XL1-Blue, etc. Promoters that can be efficiently expressed in E. coli, for example, IacZ Promo — Yuichi (Ward et al., Nature (1989) 341, 544-546; FASEB J.
  • Such vectors include pGEX-5X-1 (Amersham), QIAexpress system (Qiagen), pEGFP, or pET (in this case, the host is T7 RNA polymerase) Is preferred.
  • the vector as the signal sequence for may also be 9 polypeptide secretion to the signal sequence contains for polypeptide secretion, when produced in the Peripurazu beam of E. coli, pelB signal sequence (Lei, SP et al., J. Bacteriol. (1987) 169, 4379).
  • the introduction of the vector into the host cell can be performed using, for example, the calcium chloride method or the electoporation method.
  • a mammalian expression vector for example, pcDNA3 (manufactured by Invitrogen) or EGF-BOS (Nucleic Acids. Res.
  • insect cell-derived expression vectors eg, “Bac-to-BAC baculovairus expression syste mj (Invitrogen), pBacPAK8), plant-derived expression vectors (eg, pMHl, p-dish 2), expression vector derived from animal virus (for example, pHSV, pMV, pAdexLcw), expression vector derived from retrovirus (for example, pZIPneo), expression vector derived from yeast-(for example, pichia Expression Kitj (in vitro GenVI), NVIK SP-Q01) and Bacillus subtilis-derived expression vectors (eg, pPL608, pKTH50).
  • insect cell-derived expression vectors eg, “Bac-to-BAC baculovairus expression syste mj (Invitrogen), pBacPAK8), plant-derived expression vectors (eg, pMHl, p-dish 2), expression vector derived from animal virus (
  • promoters necessary for expression in cells such as the SV40 promoter (Muligan et al., Ature (1979) 277, 108), MMLV-LTR promoter, EF1a Mouth motor (izush ima et al., Nucleic Acids Res. (1990) 18, 5322) It is essential to have a CMV promoter, etc., and it is a gene to select for cell transformation. It is more preferable to have a drug (eg, a drug resistance gene that can be identified by a drug (neomycin, G418, etc.)). Vectors having such characteristics include, for example, MAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, p0P13, and the like.
  • a vector having the DHFR gene complementing the nucleic acid synthesis pathway-deficient CH0 cell is used.
  • pCHOI pCHOI
  • MTX methotrexate
  • a gene expressing the SV40 T antigen One method is to use a COS cell on the chromosome and transform it with a vector (such as pcD) having the SV40 origin of replication.
  • a vector such as pcD
  • the replication origin those derived from poliovirus, adenovirus, sipapirovirus (BPV) and the like can also be used.
  • expression vectors are used as selectable markers for amplification of gene copy number in the host cell system, such as aminoglycoside transferase (APH) gene, thymidine kinase (TI0 gene, Escherichia coli xanthinguanine phospholiposyltransferase (Ecogpt) gene). And a dihydrofolate reductase (dhfr) gene.
  • APH aminoglycoside transferase
  • TI0 thymidine kinase
  • Escherichia coli xanthinguanine phospholiposyltransferase Escherichia coli xanthinguanine phospholiposyltransferase
  • dhfr dihydrofolate reductase
  • the DNA of the present invention is incorporated into an appropriate vector and, for example, produced by the retrovirus method, ribosome method, cationic ribosome method, adenovirus method, or the like.
  • a method for introduction into the body This makes it possible to perform gene therapy for a disease caused by mutation of human gasdermin Al, mouse gasdermin A-2, mouse gasdermin A-3 or gasdermin B-1 of the present invention.
  • the vector used include, but are not limited to, an adenovirus vector (eg, pAdexlcw) and a retrovirus vector (eg, pZIPneo).
  • Book to vector General genetic manipulations such as ⁇ the DNA of the invention may be carried out ⁇ Tsu a conventional method (Molecular Cloning, 5.61-5.63) administration to 0 in vivo, be filed in ex / Facial method , In WTO method.
  • the present invention also provides a host cell into which the vector of the present invention has been introduced.
  • the host cell into which the vector of the present invention is introduced is not particularly limited.
  • Escherichia coli and various animal cells can be used.
  • the host cell of the present invention can be used, for example, as a production system for producing or expressing the polypeptide of the present invention.
  • Production systems for producing polypeptides include in 7 '/ ro and in vivo production systems. Examples of the in production system include a production system using eukaryotic cells and a production system using prokaryotic cells.
  • animal cells for example, animal cells, plant cells, and fungal cells can be used as hosts.
  • animal cells mammalian cells, for example, CHO (J. Exp .. Med. (1995) 108, 945), C0S, 3T3, Kazuma Mie, BHK (baby hamster kidney), HeLa, Vero, amphibian cells
  • African omega oocytes Valle, et al., Nature (1981) 291, 358-340
  • insect cells such as Si9, Sf21, and Tn5 are known.
  • CH0 cells in particular, DHFR-deficient CH0 cells such as dhfr-CHO (Proc. Natl.Acad.Sci.
  • the vector can be introduced into the host cells by, for example, the calcium phosphate method, the DEAE dextran method, the method using Cationic ribosome D0TAP (manufactured by Roche Diagnostics), the electoral poration method, or the lipofection method. It is possible to do.
  • Nicotiana tabacum As plant cells, for example, cells derived from Nicotiana tabacum (Nicotiana tabacum) are known as polypeptide production systems, which can be callus cultured.
  • Fungal cells include yeast, for example, the genus Saccharomyces, For example, Saccharomyces cerevisiae, filamentous fungi, for example, the genus Aspergillus, for example, Aspergillus niger (Aspergillus niger) are known.
  • bacteria cells When prokaryotic cells are used, there are production systems using bacterial cells.
  • bacterial cells include Escherichia coli (E. coli), for example: 1M109, DH5o !, HB101 and the like, and Bacillus subtilis.
  • the polypeptide can be obtained by transforming these cells with the desired DNA and culturing the transformed cells in vivo.
  • Culture can be performed according to a known method. For example, as a culture solution of animal cells, for example, DMEM, MEM, RPMI 1640, IMDM can be used. At that time, a serum replacement solution such as fetal calf serum (FCS) can be used together, or serum-free culture may be performed.
  • FCS fetal calf serum
  • the pH during the culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.
  • examples of a system for producing a polypeptide in / 'o include a production system using animals and a production system using plants.
  • the desired DNA is introduced into these animals or plants, and the polypeptide is produced in the animals or plants and collected.
  • the “host” in the present invention includes these animals and plants.
  • mice When using animals, there are production systems using mammals and insects. As mammals, goats, bushes, hidges, mice, and mice can be used (Vicki Glass, SPECTRUM Biotechnology Appli cats, 1993). When a mammal is used, a transgenic animal can be used.
  • the target DNA is prepared as a fusion gene with a gene encoding a polypeptide uniquely produced in milk, such as goat 3 casein.
  • the DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat.
  • the desired polypeptide can be obtained from milk produced by a transgenic goat born from the goat that has received the embryo or a progeny thereof.
  • Transgenica Hormones may be used in transgenic goats as appropriate to increase the amount of milk containing polypeptides produced from forage (Ebert, KM et al., Bio / Technology (1994) 12, 699-). 702).
  • silkworms can be used as insects, for example.
  • the desired polypeptide can be obtained from the body fluid of the silkworm by infecting the silkworm with a baculovirus into which the DNA encoding the polypeptide of interest has been introduced (Susumu,. Et al. , Nature (1985) 315, 592-594).
  • tobacco when using a plant, for example, tobacco can be used.
  • DNA encoding the desired polypeptide is introduced into a plant expression vector, for example, pMON530, and the vector is used to transform Agrobacterium tumefaciens into Agrobacterium tumefaciens.
  • a plant expression vector for example, pMON530
  • the bacteria are infected with tobacco, for example, Nicotiana tabacum, and the desired polypeptide can be obtained from the leaves of this tobacco (Julian K. -C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).
  • the polypeptide of the present invention thus obtained can be isolated from the inside or outside of the host cell (such as a medium) and purified as a substantially pure and homogeneous polypeptide.
  • the separation and purification of the polypeptide may be carried out by using the separation and purification methods used in ordinary polypeptide purification, and is not limited at all. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.
  • the polypeptide can be separated and purified by appropriate selection and combination.
  • chromatography examples include affinity mouth chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed D Five
  • chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPL FPLC.
  • the present invention also includes highly purified polypeptides using these purification methods.
  • the polypeptide can be arbitrarily modified or partially removed by reacting the polypeptide with a suitable protein modifying enzyme before or after purification.
  • a suitable protein modifying enzyme for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, dalcosidase and the like are used.
  • the present invention also provides an antibody that binds to human gasdamine A-1, mouse gasdamine A-2, mouse gasdamine A-3 or gasdermin B-1.
  • the type of the antibody is not particularly limited.
  • a monoclonal antibody and a polyclonal antibody can be used.
  • the antibody can be prepared by a method known to those skilled in the art.
  • a polyclonal antibody can be obtained, for example, as follows.
  • the peptide is immunized to small animals such as egrets to obtain serum.
  • ammonium sulfate precipitation, protein A, protein G columns, DEAE ion exchange chromatography, human gasdamine A-1, mouse gasdamine A-2, mouse gasdamine A-3, or gasdermin B-1 It is prepared by purifying a synthetic peptide using a coupled affinity column or the like.
  • a monoclonal antibody for example, a small animal such as a mouse is immunized with human gasdermin A-1, mouse gasdermin A-2, mouse gasdermin A-3 or gasdamine B-1 or a partial peptide thereof.
  • the spleen was excised from the mouse, crushed to separate cells, and the cells were fused with mouse myeloma cells using a reagent such as polyethylene glycol.
  • a clone producing an antibody that binds to human gasdamine Al, mouse gasdamine A-2, mouse gasdamine A-3 or gasdamine B-1 is selected from the resulting fused cells ( ⁇ ibridoma). I do.
  • the obtained hybridoma was transplanted into the abdominal cavity of a mouse, ascites was collected from the mouse, and the obtained monoclonal antibody was subjected to, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purifying human gasdermin A-1, mouse gasdermin A-2, mouse gasdermine A-3 or gasdermin B-1 or affinity peptide with synthetic peptide coupled affinity column. It is.
  • the present invention also provides oligonucleotides that hybridize to the DNA of the present invention and contain at least 15 nucleotides.
  • the oligonucleotide of the present invention can be used as a probe primer for detecting or amplifying a DNA encoding the polypeptide of the present invention, or as a probe primer for detecting the expression of the DNA. .
  • the oligonucleotide of the present invention can be used in the form of a DNA array substrate.
  • the present invention relates to a cell growth abnormality or DNA containing a DNA encoding a polypeptide belonging to the Gasdermin A cluster, or a DNA (including a partial DNA) encoding a polypeptide functionally equivalent thereto, as an active ingredient.
  • an agent for treating or preventing a disease caused by abnormal differentiation is provided.
  • the polypeptide encoded from the partial DNA is preferably a C-terminal region of a polypeptide belonging to the Gasdermin A cluster, and such a region is from the 343rd amino acid of mouse gasdermin A-3.
  • the present invention also relates to expression of a polypeptide belonging to the gasdermin A-related gene family or a polypeptide functionally equivalent thereto (including partial DNA).
  • an agent for treating or preventing a disease caused by abnormal cell proliferation or differentiation comprising a nucleotide or a nucleotide derivative for suppressing as an active ingredient.
  • the nucleotide or nucleotide derivative for suppressing the expression of a polypeptide belonging to Gasdamine A-related gene family 1 is not particularly limited, and examples thereof include an antisense oligonucleotide and a derivative thereof. .
  • Antisense oligonucleotides include, for example, antisense oligonucleotides that hybridize to any position in a DNA sequence encoding a polypeptide belonging to the gasdamine A-related gene family.
  • the antisense oligonucleotide is preferably an antisense oligonucleotide to at least 15 or more consecutive nucleotides in a DNA sequence encoding a polypeptide belonging to the Gasdermin A-related gene family. More preferably, it is an antisense oligonucleotide in which at least 15 or more consecutive nucleotides contain a translation initiation codon.
  • the antisense oligonucleotide derivatives and modifications thereof can be used.
  • the modified form include a modified lower alkyl phosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothioate modified form, a phosphoroamidated modified form, and the like.
  • Antisense oligonucleotides are not only those whose nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are all complementary sequences, but also that DNA or mRNA and oligonucleotides specifically hybridize to DNA sequences. Where possible, one or more nucleotide mismatches are included.
  • the antisense oligonucleotide derivative of the present invention acts on a polypeptide-producing cell belonging to Gasdermin A-related gene family 1 to transform the polypeptide.
  • a polypeptide-producing cell belonging to Gasdermin A-related gene family 1 By binding to DNA or mRNA that inhibits its transcription or translation, or by promoting the degradation of mRNA, and by suppressing the expression of the polypeptide, the action of the polypeptide is consequently effected. It has the effect of suppressing.
  • the DNA, the nucleotide or the nucleotide derivative can be formulated and administered by a known pharmaceutical method.
  • a known pharmaceutical method for example, tablets, capsules, elixirs, and microcapsules, which are sugar-coated as required, orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water / saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, It is possible to formulate a drug product by combining it as appropriate with preservatives, binders, etc., and mixing it in the unit dosage form generally required for drug practice.
  • the amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Additives that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • a suitable leavening agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, cocoa oil or cellulose are used.
  • the unit dosage form is a capsule, the above-mentioned materials can further contain a liquid carrier such as an oil or fat.
  • a sterile composition for injection can be formulated using a vehicle such as distilled water for injection according to normal pharmaceutical practice.
  • Aqueous injection solutions include, for example, saline, dextrose, and other adjuvants.
  • examples include isotonic solutions such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizers, such as alcohols, specifically, ethanol, polyalcohols, such as propylene glycol, and polyethylene. It may be used in combination with glycols, nonionic surfactants such as Polysorbate 80 (TM) or HCO-50.
  • oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. It may also be combined with a buffer, for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • a buffer for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • Administration to a patient can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. Can do better.
  • the dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the compound can be encoded by DNA
  • the DNA may be incorporated into a vector for gene therapy to perform gene therapy.
  • the dose and administration method vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
  • Antisense oligonucleotides and derivatives thereof are directly applied to the affected area of the patient. Or apply to the patient so that it can eventually reach the affected area, such as by intravenous administration.
  • an antisense encapsulating material that enhances durability and membrane permeability can be used.
  • ribosome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives thereof can be mentioned.
  • the dose of the DNA, nucleotide or nucleotide derivative can be appropriately adjusted depending on the condition of the patient, and a preferable amount can be used.
  • the present invention also relates to a polypeptide encoding the Gasdermin A cluster.
  • Cells including human-derived cells
  • non-human animals whose DNA expression is artificially suppressed.
  • the expression of a gene is artificially suppressed includes both complete suppression and partial suppression. It also includes cases where the expression of one of the gene pairs is suppressed.
  • a person skilled in the art can carry out the suppression by a generally known method. For example, a method using gene modification technology (including a conditional gene modification technology by introducing an enzyme that promotes recombination of a target gene site, for example, Cre in Cre_lox), a method using antisense DNA Or a method using RNAi technology.
  • non-human animal in the present invention means a vertebrate or an invertebrate that does not include a human.
  • Non-human animals capable of artificially suppressing gene expression using the gene modification technique include non-human mammals and insects. More preferably, non-human mammals (eg, mouse And rodents such as rats).
  • Gene-modified animals can be produced, for example, as follows. First, a genomic DNA fragment containing the target gene is cloned, and a homologous recombination vector for modifying the endogenous target gene is constructed based on this.
  • the homologous recombination vector includes a nucleic acid sequence in which at least a part of the target gene or its expression control region is deleted and mutated, a nucleic acid sequence in which a nucleotide or a polynucleotide is inserted into the target gene or its expression control region,
  • the deletion / mutation site and the insertion site are not limited as long as they include a nucleic acid sequence in which another gene is inserted into the target gene or its expression control region, but the nucleic acid sequence loses the activity of the target gene.
  • the inserted gene examples include a neomycin resistance gene, a thymidine kinase gene, a diphtheria toxin gene, and the like. A combination of these is also conceivable.
  • the basic skeleton of the homologous recombination vector is not particularly limited. For example, pKO Scrambler Series (Lexicon) or the like can be used.
  • Non-human cells capable of differentiating the constructed homologous recombination vector into individuals Gene-modified non-human animal cells are produced by introducing into a target cell (eg, embryonic stem cell (ES cell)) and homologously recombining with an endogenous target gene.
  • a target cell eg, embryonic stem cell (ES cell)
  • the cells can be obtained by a method of selecting cells with neomycin.
  • Introduction of the vector for homologous recombination into cells can be performed by a method known to those skilled in the art. Specifically, an elect-mouth poration method and the like can be exemplified.
  • chimeric embryos are prepared by injecting the cells into blastocysts and implanted into the uterus of a pseudopregnant animal. To obtain pups.
  • the external characteristics for example, coat color
  • the produced individual are determined by comparing the tissue derived from the genetically modified ES cell with the embryo. Blastocysts are selected so that they differ from the blastocyst-derived tissue.
  • whether or not the animal is a chimeric animal having a reproductive tissue derived from a genetically modified ES cell is generally determined by crossing the chimeric non-human animal with an appropriate strain of a homologous non-human animal.
  • the method is performed using the color of the offspring, but other methods include, for example, a method of detecting the presence or absence of the inserted gene by performing a PCR reaction using DNA extracted from the tail or ears of the chimeric non-human animal as a type III, or Southern hybridization. It is also possible to use the dimensioning method.
  • Whether or not the offspring obtained by crossing the chimeric animal with a homologous animal of an appropriate strain is a heterozygous genetically modified animal can be determined, for example, by PCR using DNA extracted from the animal cells as a ⁇ type. It can be determined by Southern hybridization. In addition, homozygous genetically modified animals can be produced by crossing between heterozygous genetically modified animals. The above determination method is used to determine whether the offspring obtained by mating are homozygous genetically modified animals.
  • the production of the genetically modified animal is not limited to the above method.
  • a genetically modified animal can be produced according to a technique for producing a somatic cell cloned animal.
  • Genetically modified animal cells can be produced using somatic cells other than ES cells (for example, skin cells) according to the same method as that for ES cells.
  • a genetically modified animal can be produced from the genetically modified animal cell by applying a somatic cell cloning technique.
  • the present invention also provides a cell (including a human-derived cell) or a non-human animal into which a DNA encoding a polypeptide belonging to the gasdamine A-related gene family has been introduced.
  • transgenic animals can be obtained by the method described in Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980). Specifically, DNA encoding a polypeptide belonging to the Gasdermin A-related gene family 1 is introduced into totipotent cells of an animal, and the cells are developed into an individual. A transgenic animal of interest can be produced by selecting, from the obtained individuals, those in which the transgene has been incorporated into somatic cells and germ cells. Totipotent cells into which the gene is introduced include fertilized eggs and early embryos, as well as cultured cells such as ES cells having multipotency. Those skilled in the art can appropriately modify the above method to produce transgenic animals in which the expression of a desired gene has been modified.
  • the above “DNA encoding a polypeptide belonging to the Gasdermin A-related gene family” is generally a recombinant gene construct (expression vector) linked to a promoter that can be expressed in cells of an animal into which the DNA is to be introduced. It is a target.
  • the recombinant gene construct of the present invention comprises, in a vector that can be cloned using an appropriate host, a DNA encoding a polypeptide belonging to the Gasdermin A-related gene family and a promoter upstream thereof. It can be constructed by cloning.
  • the promoter that can be used in the present invention is not particularly limited as long as it is a promoter that can be expressed in animal cells.
  • a promoter derived from a mammalian cell and viral promoters such as cytomegalovirus, retrovirus, poliovirus, adenovirus, and simian virus 40 (SV40).
  • viral promoters such as cytomegalovirus, retrovirus, poliovirus, adenovirus, and simian virus 40 (SV40).
  • SV40 simian virus 40
  • the above construct can be prepared according to the method of Muligan et al. (Nature (1990) 277, 108).
  • the vector that can be used in the present invention may be any vector that can induce the expression of the transgene in a wide range in the living body of an animal, and an expression vector known to those skilled in the art can be used.
  • the recombinant gene construct excised from the vector with an appropriate restriction enzyme is sufficiently purified and used for producing a transgenic animal.
  • a transgenic animal is produced by introducing the above-described construct into an unfertilized egg, a fertilized egg, a germ cell containing sperm and its progenitor cells, and the like.
  • the cells into which the construct is introduced are usually those at the stage of embryonic development in non-human mammal development, more specifically, at the stage of single cells or fertilized egg cells, usually at or before the 8-cell stage. You.
  • Known methods for introducing the above-described construct include, for example, the calcium phosphate method, the electoral poration method, the lipofection method, the agglomeration method, the microorifice injection method, the particle gun method, and the DEAE-dextran method.
  • a transgenic animal can be prepared by fusing the thus obtained transformed cells with the above-mentioned germinal cells.
  • a fertilized egg into which a construct can be introduced can be collected by mating a normal male mouse to a female mouse to which an ovulation-inducing agent has been administered.
  • the construct is generally introduced by microinjection into the male pronucleus.
  • the cells into which the construct has been introduced are cultured in vitro, and then the cells that are considered to have been successfully introduced are transplanted into the oviduct of the surrogate mother to produce a transgenic chimeric animal.
  • the surrogate mother is usually a female that has been bred to a vasectomized osseum and has been placed in a pseudopregnant state.
  • the transgenic chimeric animal is bred with a normal animal for the birth of an F1 animal after confirming that the DNA encoding the polypeptide belonging to the gasdermin A-related gene family has been incorporated.
  • multiple copies of foreign DNA introduced as a construct are integrated in series in the same part of the genome.
  • the higher the number of integrated copies the higher the gene expression, and a clearer phenotype can be expected.
  • the incorporation of a DNA encoding a polypeptide belonging to the Gasdermin A-related gene family into the somatic cell genome can be attributed to PCR using primers specific to the construct or Southern using a specific probe. It can be confirmed by blotting.
  • F1 animals born as a result of this mating those that have a foreign gene (DNA encoding a polypeptide belonging to the gasdermin A-related gene family) in their somatic cells (heterozygotes) must transmit the foreign gene to germ cells. It is a transgenic animal that can By selecting F1 animals that have foreign genes in somatic cells and using them as parents, homozygotes that are F2 animals can be obtained.
  • the transgenic non-human animal of the present invention is an animal of any generation of the above-described transgenic animal as long as the expression of the DNA encoding the polypeptide belonging to the gasderin A-related gene family is modified. There may be.
  • a transgenic animal that heterologously retains foreign DNA as long as it expresses a DNA encoding a polypeptide belonging to this exogenous gasdamine A-related gene family. It can be used as the transgenic non-human animal of the present invention.
  • the present invention also provides a cell line derived from the non-human animal.
  • a method for establishing such a cell line a known method can be used.
  • the method of primary culture of fetal cells (Shinsei Kagaku Kenkyusho, 18, 125-129 Tokyo Chemical Dojin, and mouse embryo operation manual, 262-264, modern Publication) can be used to establish cell lines.
  • the present invention provides a method for evaluating whether or not a test sample has an activity of suppressing abnormal cell proliferation or abnormal differentiation.
  • aspects of the evaluation method of the present invention will be exemplified, but the evaluation method of the present invention is not limited thereto.
  • a cell in which expression of a DNA encoding a polypeptide belonging to the gasdermin A cluster is artificially suppressed, or a gasdamine A-related gene family The test sample is brought into contact with cells into which DNA encoding a polypeptide belonging to the above has been introduced.
  • test sample in the method of the present invention is not particularly limited.
  • a single compound such as a natural compound, an organic compound, an inorganic compound, a polypeptide, and a peptide, and a compound library and a gene library are expressed.
  • Products, cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts can be mentioned.
  • the test sample can be appropriately labeled and used as necessary. Examples of the label include a radioactive label and a fluorescent label.
  • contact can be performed, for example, by adding a test sample to a cell culture solution.
  • test sample is a polypeptide
  • a vector containing DNA encoding the polypeptide can be introduced into the cells.
  • the proliferation or differentiation of the cells contacted with the test sample is then measured.
  • the measurement of cell proliferation or differentiation is not particularly limited.For example, cell number measurement by FACS, etc., colony formation method, soft agar method, differentiation marker staining and biochemical activity measurement, cell morphology observation, electron microscopy Observation of the fine cell structure and the like can be mentioned.
  • the test sample when the proliferation or differentiation is suppressed as compared to when the test sample is not contacted, the test sample has an activity of suppressing abnormal cell growth or abnormal differentiation. Is determined.
  • test sample is administered to a non-human animal into which the DNA encoding the polypeptide has been introduced.
  • Administration of the test sample can be performed, for example, orally, by injection, or the like, but is not limited thereto.
  • the test sample is a polypeptide
  • a viral vector having a gene encoding the polypeptide is constructed, and the infectivity is used to transfer the gene to the non-human higher animal of the present invention. It is also possible to introduce.
  • cell proliferation or differentiation in the non-human animal to which the test sample has been administered is then measured.
  • the measurement of cell proliferation or differentiation in animals is not particularly limited.For example, cell number measurement by FACS, etc., colony formation method, soft agar method, differentiation marker staining and biochemical activity measurement, cell morphology observation, Observation of a fine cell structure by an electron microscope and the like can be mentioned.
  • the test sample when the proliferation or differentiation is suppressed as compared to when the test sample is not contacted, it is determined that the test sample has an activity of suppressing abnormal cell growth or differentiation.
  • a third aspect of the evaluation method of the present invention is characterized by using the Colony format ion assay described in Examples. First, it provides a cancer cell into which DNA encoding a polypeptide belonging to the Gasdermin A cluster has been introduced.
  • the cancer cells the cancer cells described in Examples can be used, but are not limited thereto.
  • a test sample is brought into contact with the cancer cells.
  • the number of dead cells is determined depending on the introduction of DNA encoding the polypeptide belonging to the Gasdermin A cluster.
  • the method for measuring the number of dead cells can be the method described in Examples, but is not limited thereto.
  • the test sample has an activity of suppressing abnormal cell growth or abnormal differentiation. You.
  • a cell or a cell having a DNA in which a reporter gene is functionally bound downstream of a promoter region of a gene encoding a polypeptide belonging to the Gasdermin A cluster Provide the extract.
  • “functionally linked” means that the expression of the repo allele is induced by the binding of a transcription factor to the promoter region of the gene encoding a polypeptide belonging to Gasdermin A cluster 1.
  • the gene encoding a polypeptide belonging to Gasdermin A class evening As long as the expression of the fusion protein is induced by the binding of a transcription factor to the promoter region of the above, the term “functionally linked” is included in the meaning of “functionally linked”.
  • Cells having the above DNA can be produced, for example, by introducing a vector containing the above MA into the cells.
  • Vectors can be introduced into cells by a general method, for example, a micromouth injection method, a calcium phosphate coprecipitation method, a DEAE-dextran method, an electroporation method, a lipofection method, a liposome method, a ribofectamine (Invitrogen) ).
  • the cell extract containing the DNA include a cell extract contained in an in vitro transcription / translation system and a vector containing the DNA added thereto.
  • the in vitro transcription / translation system is not particularly limited, and a commercially available in vitro transcription / translation kit or the like can be used. .
  • the reporter gene is not particularly limited as long as its expression can be detected. Examples thereof include CAT gene, lacZ gene, luciferase gene, dalcuronidase gene (GUS), and CAT gene commonly used by those skilled in the art. GFP Genes and the like can be mentioned.
  • the reporter gene also includes a gene encoding a polypeptide belonging to the Gasdamine A cluster.
  • a test sample is then brought into contact with the cells or the cell extract.
  • the “contact” can be performed by adding a test sample to a cell culture solution or the cell extract.
  • the test sample is a polypeptide
  • the test can be performed, for example, by introducing a vector containing DNA encoding the polypeptide into cells, or by adding the vector to a cell extract. .
  • the expression level of the reporter gene in the cell or the cell extract is then measured.
  • the expression level of the reporter gene can be measured by a method known to those skilled in the art, depending on the type of the reporter gene used.
  • the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product.
  • the repo overnight gene is the lacZ gene, the coloration of the dye compound by the catalytic action of the gene expression product is detected, and when the repo overnight gene is the luciferase gene, the gene expression product is catalyzed.
  • the reporter By detecting the fluorescence of the fluorescent compound due to the action, and in the case of the i3-Darc mouth nidase gene (GUS), the luminescence of Glucuron (ICN) and 5-promote by the catalytic action of the gene expression product -4-Black mouth-3-Indolyl-) By detecting the color development of -Dalcuronide (X-Glue) and, if it is a GFP gene, by detecting the fluorescence of the GFP protein, the reporter The expression level of the gene can be measured.
  • GUS i3-Darc mouth nidase gene
  • the expression level of the gene can be measured by a method known to those skilled in the art.
  • the mRNA of the gene is extracted according to a standard method, and the transcription level of the gene is measured by performing a Northern hybridization method or an RT-PCR method using the mRNA as a type III. be able to.
  • the expression level of the gene can be measured using DNA array technology.
  • the fraction containing the polypeptide encoded from the gene is collected according to a standard method, and the expression of the polypeptide is detected by an electrophoresis method such as SDS-PAGE to measure the translation level of the gene. Can also be performed.
  • the level of translation of the gene can also be measured by detecting the expression of the polypeptide by carrying out a Western blotting method using an antibody against the polypeptide.
  • the antibody used for detecting the polypeptide is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.
  • the test sample when the expression level of the reporter gene is higher than when the test sample is not brought into contact, it is determined that the test sample has an activity of inhibiting abnormal cell growth or abnormal differentiation.
  • a cell having D in which a reporter gene is functionally bound downstream of the promoter region of a gene encoding a polypeptide belonging to the gasdermin A-related gene family or Provide cell extract Next, a test sample is brought into contact with the cells or the cell extract. Next, the expression level of the repo overnight gene in the cell or the cell extract is measured. In the fifth embodiment, when the expression level of the reporter gene is reduced as compared to when the test sample is not contacted, it is determined that the test sample has an activity of suppressing abnormal cell growth or abnormal differentiation. You.
  • the present invention provides a method for screening a sample having an activity of suppressing abnormal cell growth or differentiation.
  • the above-mentioned evaluation method is used to evaluate whether or not a plurality of test samples have an activity of suppressing abnormal cell proliferation or abnormal differentiation, A sample evaluated as having a suppressive activity on abnormal proliferation or abnormal differentiation is selected.
  • Gasdermin A class The test sample is contacted with a polypeptide belonging to the genus Acer or a gasderin A-related gene family. Next, the binding between the polypeptide and the test sample is detected.
  • the detection method is not particularly limited. The binding between the polypeptide and the test sample is detected, for example, by using a label attached to the test sample bound to the polypeptide (for example, a label capable of quantitative measurement such as a radiolabel or a fluorescent label). Can be. Further, a change in the activity of the polypeptide caused by the binding of the test sample to the polypeptide can be detected as an index.
  • test sample that binds to the polypeptide is then selected. Samples selected include those having activity to suppress abnormal cell proliferation or differentiation. In another embodiment, the test sample that binds to the polypeptide is then evaluated using the above-described evaluation method to determine whether it has an activity to suppress abnormal cell proliferation or abnormal differentiation, It is also possible to select a sample that has been evaluated as having an activity of suppressing abnormal differentiation.
  • the present invention provides a method for producing a pharmaceutical composition having an activity of suppressing abnormal cell proliferation or differentiation.
  • a sample that has been evaluated as having the activity of suppressing abnormal cell growth or abnormal differentiation by the above-described screening method is mixed with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants Agents, fluidity enhancers, flavoring agents, and the like, but are not limited thereto, and other conventional carriers can be used as appropriate.
  • the present invention also provides a method for testing a disease caused by abnormal cell proliferation or differentiation, which comprises a step of measuring the expression level of a DNA encoding a polypeptide belonging to the gasdamine family 1.
  • “DNA expression” includes not only mRNA expression but also polypeptide expression.
  • a sample of a subject is prepared.
  • the amount of RM encoding a polypeptide belonging to Gasdermin family 1 contained in the RNA sample is measured.
  • the measured amount of RNA is then compared to a control. Examples of such a method include a Northern blotting method, a DNA array method, and an RT-PCR method.
  • test method can be carried out as follows by measuring the expression level of a polypeptide belonging to the gasdamine family 1.
  • a polypeptide sample is prepared from a subject.
  • the amount of the polypeptide belonging to the gasdermine family 1 contained in the polypeptide sample is measured.
  • the measured amount of polypeptide belonging to the gas-damine family is then compared to a control.
  • examples of such methods include SDS polyacrylamide electrophoresis, Western blotting, dot blotting, immunoprecipitation, and enzyme-linked immunoassay using an antibody that binds to a polypeptide belonging to the Gasdermin family.
  • Assays (ELISA) and immunofluorescence can be exemplified. '
  • the present invention also provides a method for detecting a mutation in a DNA region encoding a polypeptide belonging to the gasdermin family, comprising the steps of: Provided is a method for testing a disease caused by the disease. If the test method has a mutation in a DNA region encoding a polypeptide belonging to the gasdermin family, the patient has already suffered from a disease caused by abnormal cell proliferation or differentiation, or Is determined to be possible.
  • the DNA region encoding a polypeptide belonging to Gasdermin Family 1 means a DNA encoding a polypeptide belonging to Gasdermin Family 1 and a region that affects the expression of the DNA.
  • the region affecting the expression of the DNA is not particularly limited, and examples thereof include a promoter region.
  • the mutation in the present invention is not limited in its type, number, site, etc., so long as it is a mutation that causes abnormal growth or differentiation of cells. Examples of the type of the mutation include deletion, substitution, insertion mutation, translocation, inversion, amplification, and the like.
  • test method including a step of detecting a mutation generated in a DNA region encoding a polypeptide belonging to Gasdamine family 1 will be described, but the method of the present invention is not limited to these methods. Absent.
  • DNA samples include, for example, chromosomal DNA or RNA extracted from the subject's blood, skin, oral mucosa, hair, bodily fluids, body wash, stool, sputum, tissues or cells collected or excised by surgery. Base.
  • MA containing a DNA region encoding a polypeptide belonging to the Gasdermin family is isolated.
  • the DNA region can be isolated by, for example, PCR using chromosomal DNA or RNA as a type II, using a primer that hybridizes to DNA containing the DNA region.
  • the base sequence of the isolated DNA is determined.
  • the nucleotide sequence of the isolated DNA can be determined by a method known to those skilled in the art.
  • a control refers to a DNA containing a DNA region encoding a polypeptide belonging to a normal (wild-type) gasdamine family 1.
  • the sequence of a DNA containing the DNA region of a healthy person is considered to be normal. Therefore, the term “compare with control” usually refers to a polypeptide belonging to the gasdamine family 1 of a healthy person. Comparison with the sequence of DNA containing the DNA region to be loaded.
  • Detection of a mutation in the present invention can also be performed by the following method.
  • a DNA sample is prepared from a subject.
  • the prepared DNA sample is cut with a restriction enzyme.
  • the DNA fragments are separated according to their size. The size of the detected DNA fragment is then compared to a control.
  • a DNA sample is prepared from a subject.
  • DNA containing a DNA region encoding a polypeptide belonging to the Gasdermin family 1 is amplified.
  • the amplified DNA is cut with a restriction enzyme.
  • the DNA fragment is separated according to its size. The size of the detected DNA fragment is then compared to a control.
  • Examples of such a method include a method using a restriction enzyme fragment length mutation (Restriction Fragment Length Polymorphism / RFLP) and a PCR-RFLP method.
  • a restriction enzyme fragment length mutation Restriction Fragment Length Polymorphism / RFLP
  • PCR-RFLP method Specifically, when there is a mutation in the recognition site of the restriction enzyme, or when there is a base insertion or deletion in the DNA fragment generated by the restriction enzyme treatment, the size of the fragment generated after the restriction enzyme treatment is different from that of the control. It changes in comparison. By amplifying the portion containing this mutation by PCR and treating it with each restriction enzyme, these mutations can be detected as a difference in the mobility of the band after electrophoresis.
  • the presence or absence of the mutation can be detected by treating the chromosomal DNA with these restriction enzymes, performing electrophoresis, and performing Southern plotting using the probe DNA of the present invention.
  • the restriction enzyme to be used can be appropriately selected according to each mutation.
  • RNA prepared from a subject can be converted into cDNA with a reverse transcriptase, which can be directly cut with a restriction enzyme and then subjected to Southern blotting.
  • this cDNA was used as a type II gas polymerase for PCR. It is also possible to amplify DNA containing a DNA region encoding a polypeptide belonging to family 1 and cut it with restriction enzymes before examining the difference in mobility.
  • a MA sample is first prepared from a subject.
  • DNA containing a DNA region encoding a polypeptide belonging to the Gasdermin family is amplified.
  • the amplified DNA is dissociated into single-stranded DNA.
  • the dissociated single-stranded DNA is separated on a non-denaturing gel. Compare the mobility of the separated single-stranded DNA on the gel with the control.
  • Examples of the method include, for example, a PCR-SSCP (Single-strand conformation polymorp ism, single-strand conformation mutation) method (Cloning and polymerase chain reaction- single-st rand conformation polymorphism analysis of anonymous Alu repeats on chro mosome 11. Genomics. 1992 Jan. 1; 12 (1): 139 146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphi sm analysis of polymerase chain reaction products.Oncogene. 1991 Aug 1; 6 (8): 1313- 1318., Multiple fluorescence-based PCR-SSCP analysis with post labeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.).
  • PCR-SSCP Single-strand conformation polymorp ism, single-strand conformation mutation
  • This method has advantages such as relatively simple operation and a small amount of test sample, and is particularly suitable for screening a large number of MA samples.
  • the principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When the dissociated DNA strands are electrophoresed in a polyacrylamide gel containing no denaturing agent, single-stranded DNAs of the same complementary length move to different positions depending on the structure of each higher-order structure. The substitution of a single base also changes the higher-order structure of the single-stranded DNA, indicating a different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, the presence of a mutation due to point mutation, deletion, insertion or the like in the DNA fragment can be detected.
  • the DNA containing the DNA region to be amplified is amplified by PCR or the like. As a range to be amplified, usually, a length of about 200 to 400 bp is preferable.
  • PCR can be performed by those skilled in the art by appropriately selecting reaction conditions and the like.
  • the amplified DNA product can be labeled by using a primer labeled with an isotope such as 32 P, a fluorescent dye, or biotin during PCR.
  • the amplified DNA product can be labeled by performing PCR by adding a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin to the PCR reaction solution.
  • labeling is also performed by adding a base base labeled with an isotope such as 32 P, a fluorescent dye, or biotin to the amplified DNA fragment using Klenow enzyme or the like. be able to.
  • the labeled DNA fragment thus obtained is denatured by heating or the like, and electrophoresed on a polyacrylamide gel containing no denaturing agent such as urea.
  • an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel, the conditions for separating DNA fragments can be improved.
  • electrophoresis conditions vary depending on the properties of each DNA fragment, but are usually performed at room temperature (20 to 25 ° C), and when the desired separation cannot be obtained, a temperature of 4 to 30 is a temperature that gives the optimal mobility.
  • the mobility of the DNA fragment is detected and analyzed by autoradiography using an X-ray film or a scanner that detects fluorescence. If a band with a difference in mobility is detected, this band can be directly excised from the gel, re-amplified by PCR, and directly sequenced to confirm the presence of the mutation. Even when labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium bromide silver silver staining.
  • a DNA sample is first prepared from a subject.
  • DNA containing a DNA region encoding a polypeptide belonging to the Gasdermin family is amplified.
  • the amplified DNA is separated on a gel with increasing concentrations of DNA denaturant.
  • the mobility of the separated DNA on the gel is then compared to a control.
  • denaturant gradient gel denaturant gradient gel electrophores is: DGGE method
  • the DGGE method is a method in which a mixture of DNA fragments is electrophoresed in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated based on differences in instability.
  • the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the movement speed is extremely slow. Therefore, the presence or absence of the mutation is detected by detecting the difference in the mobility. can do.
  • a DNA containing a DNA region encoding a polypeptide belonging to the Gasdermin family 1 prepared from a subject, and a substrate on which a nucleotide probe that hybridizes to the DNA is immobilized provide.
  • substrate means a plate-like material on which nucleotide probes can be immobilized.
  • nucleotides include ligated nucleotides and polynucleotides.
  • the substrate of the present invention is not particularly limited as long as the nucleotide probe can be immobilized thereon, but a substrate generally used in DNA array technology can be suitably used.
  • A-arrays are composed of thousands of nucleotides printed on a substrate at high density. Usually these DNAs are printed on the surface of a non-porous substrate.
  • the surface of the substrate is typically glass, but uses a permeable membrane, such as a nitrocellulose membrane be able to.
  • an array based on oligonucleotides developed by Affymetrix can be exemplified.
  • oligonucleotide array oligonucleotides are usually synthesized in situ.
  • photolithographic technology Affymetrix, Inc.
  • ink jet technology for immobilizing chemical substances
  • the nucleotide probe immobilized on the substrate is not particularly limited as long as it can detect a mutation in a DNA region encoding a polypeptide belonging to the gasdermin family. That is, the probe is, for example, a probe that hybridizes to a DNA containing a DNA region encoding a polypeptide belonging to the Gasdermin family. As long as specific hybridization is possible, the nucleotide probe need not be completely complementary to the DNA containing the DNA region.
  • the length of the nucleotide probe to be bound to the substrate is generally 10 to 100 bases, preferably 10 to 50 bases, and more preferably 15 to 25 bases when the oligonucleotide is immobilized. is there.
  • the DNA is then brought into contact with the substrate.
  • DNA is hybridized to the nucleotide probe.
  • the reaction solution and reaction conditions for the hybridization can vary depending on various factors such as the length of the nucleotide probe immobilized on the substrate, but can be generally performed by a method well known to those skilled in the art.
  • the intensity of hybridization between the DNA and the nucleotide probe immobilized on the substrate is detected.
  • This detection can be performed, for example, by reading the fluorescent signal with a scanner or the like.
  • DNA arrays DNA fixed on a slide glass is generally called a probe.
  • the labeled DNA in the solution is called the target. Therefore, the above nucleotide immobilized on the substrate is referred to herein as a nucleotide probe.
  • the detected hybridization intensity is further compared with a control.
  • Examples of such a method include a DNA array method (SNP gene mutation strategy, Kenichi Matsubara, Yoshiyuki Sakaki, Nakayama Shoten, P128-135, Nature Genetics (1999) 22: 164-167) and the like.
  • an Allele Specific 01 igonucleotide / ASO hybridization method can be used for the purpose of detecting only a mutation at a specific position.
  • an oligonucleotide containing a nucleotide sequence that is considered to have a mutation is prepared and hybridized with the DNA, the efficiency of octa-hybrid formation is reduced when the mutation is present. It can be detected by Southern blotting, a method utilizing the property of quenching by intercalating a special fluorescent reagent into the gap of the hybrid, or the like.
  • the MALDI-T0F / MS method SNP gene polymorphism strategy, Kenichi Matsubara • Yoshiyuki Sakaki, Nakayama Shoten, pl06-117, Trends Biotechnol (2000): 18: 77-84
  • TaqMan PCR method SNP genetic polymorphism strategy, Kenichi Matsubara and Yoshiyuki Sakaki, Nakayama Shoten, p94-105, Genet Anal.
  • the present invention provides a test agent for use in the test method of the present invention.
  • One embodiment is a test agent that hybridizes to a DNA region encoding a polypeptide belonging to the gasdamine family and contains an oligonucleotide having a chain length of at least 15 nucleotides.
  • the oligonucleotide includes a polynucleotide.
  • the oligonucleotide specifically hybridizes to DNA (normal DM or mutant DNA) containing a DNA region encoding a polypeptide belonging to the Gasdermin family.
  • “specifically hybridizes” means under ordinary hybridization conditions, preferably under stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor).
  • the oligonucleotide does not need to be completely complementary to DNA containing a DNA region encoding a polypeptide belonging to the gasdamine family.
  • Oligonucleotides that hybridize to DNA containing a DNA region encoding a polypeptide belonging to the Gasdermin family and have a chain length of at least 15 nucleotides are used as probes in the above-described test method of the present invention (the substrate on which the probes are immobilized). ) And primers.
  • the oligonucleotide is used as a primer, its length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp.
  • the primer is not particularly limited as long as it can amplify at least a part of a DNA region encoding a polypeptide belonging to the gasdamine family including a mutated portion.
  • the probe is not particularly limited as long as it specifically hybridizes to DNA containing a DNA region encoding a polypeptide belonging to the gasdamine family.
  • the probe may be a synthetic oligonucleotide and usually has a length of at least 15 bp or more.
  • the oligonucleotide of the present invention can be produced by, for example, a commercially available oligonucleotide synthesizer.
  • the probe can also be prepared as a double-stranded DNA fragment obtained by treatment with a restriction enzyme or the like.
  • Labeling can be performed by using T4 polynucleotide kinase to label the oligonucleotide by phosphorylation of the 5 'end with 32 P, or by using a DNA polymerase such as Klenow enzyme to form a random hexamer oligonucleotide.
  • a method of incorporating a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin using a nucleotide or the like as a primer can be exemplified.
  • test agent of the present invention is a test agent containing an antibody that binds to a polypeptide belonging to the gasdermin family.
  • the antibody is not particularly limited as long as it can be used for the test.
  • the antibody is labeled if necessary.
  • DNA that encodes human gasdamine B-1 or human gasdamine D-1 or an endogenous polypeptide functionally equivalent thereto is hybridized with at least 15%.
  • a test agent containing an oligonucleotide having a nucleotide chain length, or an antibody that binds to human gasdamine B-1 or human gasdamine D-1, or an endogenous polypeptide functionally equivalent thereto can be used as test agents for diseases caused by abnormal cell proliferation or differentiation (preferably cancer, more preferably gastric cancer, breast cancer, colon cancer, esophageal cancer, etc.).
  • test reagents of the present invention include, for example, sterilized water, physiological saline, vegetable oils, surfactants, lipids, solubilizing agents, buffers, and protein stabilizers ( BSA, gelatin, etc.), preservatives and the like may be mixed as necessary.
  • FIG. 1 is a photograph showing 5 months of age of dominant mutant mouse Rim-3 (Recombinant induced mutated ion 3) (a) and Re den (b) considered to be allele. These Mutant mice exhibit a phenotype of hair loss and hyperkeratinization of the epidermis, and opacity of the cornea (epithelialization). Rim-3 is a mutant mouse originally obtained in our laboratory at the National Institute of Genetics in the early 1980s.
  • FIG. 2 is a diagram showing mouse chromosome 11.
  • the Rim-3 causative gene was shown to be present between Grb7 and iMit, about 200 kbp.
  • FIG. 3 is a diagram showing the results of a more detailed examination of the region shown in FIG.
  • Two genes very similar to gasdamine (mouse gasdamine A-1) were shown to be present upstream of gasdamine (mouse gasdamine A-1). Arrows in the figure indicate the direction of transcription.
  • FIG. 4 shows human GSDMA-1 (human gasdermin A-1) and mouse GsdmA-1 (mouse gasdermin gasdamin A_l), mouse GsdmA-2 (mouse gasdermin A-2), mouse GsdmA-3 (mouse gasdera).
  • FIG. 4 is a view showing the homology of the amino acid sequence of min A_3). One in the amino acid sequence indicates a blank, and ⁇ indicates that it is the same amino acid as human gasdermin A-1. The lower part of mouse gasdermin A-3 is shown below.,:, * Represent amino acids with similar properties, amino acids with further similar properties, and amino acids that are conserved in all.
  • FIG. 5 is a diagram showing human chromosome 17 (a) and mouse chromosome 11 (b). The region of mouse chromosome 11 where mouse gasdermin A-1, A-2, A-3 is present is present on chromosome 17 in humans. It was also shown that there is only one gasdamine in humans. In addition, at the position where gasdamine A-2 and gasdamine A-3 are present in mice, a gene similar to (but distinctly different from) gasdamine B-1 in humans is located in humans. It was shown to be present.
  • FIG. 6 is a diagram showing mutation at the base level.
  • (A) is a diagram showing mutation of bases in Rira3. G at position 1091 has been replaced with A. The mutant sequence in Rim3 is shown in SEQ ID NO: 55.
  • (B) shows the insertion of 6 bases in Reden . Six bases at positions 1279-1284 are inserted at position 1285. Arrange the mutant sequence in Re den Column number: shown in 56
  • FIG. 7 is a diagram showing mutations at the amino acid level.
  • A is a diagram showing mutations of amino acids in Rim3. The change from alanine to threonine indicates that the nature of the amino acid changes to something completely different.
  • the mutant sequence in Rim3 is shown in SEQ ID NO: 57.
  • B is a diagram showing a 6 base ⁇ in Re den. ⁇ This does not cause a frame shift. It also shows a mutation in which two amino acids that do not generate a stop codon due to the insertion are inserted.
  • the mutation sequence in Reden is shown in SEQ ID NO: 58.
  • FIG. 8 is a photograph showing expression analysis of gasdamine A-1 and A-3 using the RT-PCR method.
  • Mouse gasdamine A-1 was expressed in epithelium, stomach and colon, and mouse gasdamine A-3 was expressed only in epithelium.
  • FIG. 9 is a photograph and a diagram showing expression analysis of mouse gasdamine A-1 and A-2 using RT-PCR in tissues cut out for each region of the stomach. It was shown that mouse gasdamine A-1 was expressed specifically in squamous epithelium, and mouse gasdamine A-2 was expressed specifically in glandular epithelium.
  • FIG. 10 is a photograph showing expression analysis of gasdamine A class Yuichi in the stomach during fetal life.
  • FIG. 11 is a photograph and a diagram showing the expression analysis of mouse gasdermin A cluster in mouse tissue using the in situ hybridization method. Esophageal expression is similar to that in the forestomach. Mouse gasdermin A-1 expression is found not in the stem cell region but in the cell group just before terminal differentiation, and mouse gasdamine A-2 expression is found in the fundic region and in the cervix of the glandular epithelium. In the pyloric region, it was shown to be expressed in the central part.
  • FIG. 12 is a photograph showing the results of double staining of a stem cell marker, PCNA (Proliferating Cell Nuclear Antigen) and mouse gasdamine A-2 in W. Mouse gasdermin A-2 is expressed in cells immediately after stem cell differentiation toward the cervix Was shown to begin.
  • PCNA Proliferating Cell Nuclear Antigen
  • FIG. 13 is a photograph showing the expression of mouse gasdamine A-1 and mouse gasdamine A-3 in epithelium in /.
  • Figure 14 shows the histological analysis of the ventral epithelium of Rim-3 and normal mice C57BL / 10 (strains with the same genetic background of Rim-3) at 30 days, 90 days, and 300 days after birth. It is a photograph. Rim-3 already shows abnormal hair follicles and epidermal hyperproliferation 30 days after birth. At 90 and 300 days, C57BL / 10 appears to have fewer hair follicles, but this is due to the hair cycle and is normal. On the other hand, in Rim-3, hair follicles were still observed on the 90th day, but on the 300th day, it was shown that the hair follicle itself had almost disappeared.
  • FIG. 15 is a photograph showing abnormal proliferation of ventral skin cells 30 days after birth of Rim3. ((A) to (d)). As a result of observing the tissue section of the ventral epithelium on the 30th day, hyperproliferation of epithelium and follicles was observed ((a), (b)). Cell proliferation was observed using BrdU (promo-doxy, lysine) incorporation. It was shown that BrdU-positive cells increased about 7 to 8 times as compared to control C57BL / 10 (B10) ( (c), (d)).
  • BrdU promo-doxy, lysine
  • FIG. 16 is a photograph showing the results of analysis of the abdominal skin of Rini3 30 days after birth using various cell layer cell marker antibodies.
  • A), (c) and (e) are tissue sections of the control B10, (b), (d) and (e) are Rim-3 tissue sections, and (a) and) are anti-keratin-14.
  • C) and (d) are photographs showing the results of immunohistochemical staining using an anti-keratin-10 antibody, and (e) and (f) are photographs showing the results of immunohistochemical staining using an anti-keratin-6 antibody. It was shown that abnormal epithelial cell differentiation was caused in the epithelium of Rim-3.
  • FIG. 17 is a diagram showing the 17ql 2a immediate 1 icon region of human chromosome 17. It shows which gene in this region is amplified and how much is involved in canceration.
  • White (normal) indicates that there is no change from the normal genome amount, polka dots (3x-7, diagonal lines (8x-15x), black (16x-26X), grids (> 27 for 3 to 7 times, respectively) It indicates that the amplification is more than 8 to 15 times, 16 to 26 times, and 27 times human gasdermin A-1 (GSDMA-1) geno
  • GSDMA-1 human gasdermin A-1
  • FIG. 18 is a photograph showing that the expression of human gasdermin A-1 in human tissues was detected by the Northern method.
  • A D Human gasdamine A-1 was shown to be expressed in the stomach.
  • 4B is a photograph showing that expression of human gasdermin A-1 in cancer cells was detected by the Northern method (B). It was shown that expression in the stomach and in the mammary gland was abolished irrespective of whether the genome was normal or amplified.
  • FIG. 19 is a diagram and a photograph showing the results of introducing human gasdamine A-1 into an esophageal cancer cell line and evaluating its cell growth inhibitory effect. It was clarified that the overexpression of human gasdermin A-1 (sense) significantly suppressed the proliferation as compared to the control (vector only, antisense). Further, by introducing a mutation of mutant mice Re den type Hitogasuda one Min A-1 (GSDMA- 1 Rex) , its ability to suppress proliferation was shown to be reduced.
  • FIG. 20 shows the results obtained by overexpressing human gasdamine A-1 in an esophageal cancer cell line (TE10) and a gastric cancer cell line (MKN28, MKN74) using a viral vector and assaying cell proliferation (FIG. 20).
  • TE10 esophageal cancer cell line
  • MKN28, MKN74 gastric cancer cell line
  • FIG. 20 shows the results obtained by overexpressing human gasdamine A-1 in an esophageal cancer cell line (TE10) and a gastric cancer cell line (MKN28, MKN74) using a viral vector and assaying cell proliferation (FIG. 20).
  • GSDMA-1 esophageal cancer cell line
  • FIG. 4B is a photograph and a diagram showing suppression of cancer colony formation using a plasmid vector (B).
  • AS expresses the reverse chain of control human gasdamine A-1; S expresses human gasdamine A-1; B-a, B-c, MKN28 cancer cell line; B-b, B -d is a graph showing the results using the TE10 and MKN74 cancer cell lines, respectively. It was shown to suppress the proliferation not only in the esophageal cancer cell line (TE10) but also in the gastric cancer cell lines (MKN28, MKN74).
  • FIG. 21 is a photograph showing induction of apoptosis using the TUNEL method. (A) shows no infection and (b) shows infection.
  • FIG. 22 is a photograph showing an expression analysis of Gasdermin B-1. Normal and cancer in the figure Each shows normal stomach tissue and stomach cancer tissue by surgical findings. Pre-cancerous refers to tissues that have abnormalities such as intestinal metaplasia and are considered to be in the pre-stage of cancer.
  • FIG. 23 is a diagram showing an amino acid sequence predicted from the base sequences of the Gasdermin A cluster and one of the Gasdermin A-related gene families.
  • FIG. 24 is a diagram showing an amino acid sequence predicted from the nucleotide sequence of the gasdamine A class and the gasdamine A-related gene family.
  • Figure 25, which is a continuation of Figure 26, shows the results of creating a phylogenetic tree of the amino acid sequences predicted from the base sequences of the Gasdamine A cluster 1 and the Gasdamine A related gene family using the ClustalW program.
  • FIG. The underlined lines indicate mice, and the underlined lines indicate human gene products.
  • FIG. 26 is a diagram showing a comparison of the highly conserved C-terminal sequences of the amino acid sequences predicted from the base sequences of the Gasdermin A cluster and the Gasdamine A related gene family 1.
  • One in the amino acid sequence represents a blank, and ⁇ represents the same amino acid as mouse gas dermine A-1.
  • ⁇ represents the same amino acid as mouse gas dermine A-1.
  • * Represent amino acids with similar properties, amino acids with similar properties, and amino acids conserved in all. Note that the value in 0 indicates the storage stability when gasdermin B-1 is excluded.
  • FIG. 27 is a photograph showing an ES cell-derived mouse in which the gasdermin A-2 gene has been deleted.
  • C57BL / 6J and C57BL / 10J are from Jackson Laboratory (Bar Harbor, ME., USA) Purchased.
  • Mutant mouse ⁇ Rim3 was independently systematized at the National Institute of Genetics, and mutant mouse .Re ⁇ was provided by Dr. Hiroyuki Sasaki of Kyushu University (now National Institute of Genetics). These mouse strains were bred and maintained at the National Institute of Genetics and the Research Center for Systematic Biology, and animals bred at the Research Center for Systematic Biology were used for experiments (Fig. 1).
  • human skin and human stomach total RNA those purchased from Invitrogen were used.
  • Adult mouse skin and stomach total RNA were purified using Isogen (Nippon gene).
  • Human skin, mouse skin and mouse stomach-specific cDNA libraries are available from Lambda ZAP II cDNA library construction kit (STRATAGENE) and Gigapack
  • Mouse gasdamine A-2 and mouse gasdamine A-3 were isolated from these cDNA libraries using the mouse gasdamine A-1 cDNA as a probe by a plaque hybridization method.
  • Human gas gene A-1 cDNA is firstly derived from Human draft Genome Sequence
  • the Min A-1 protein code region cDNA was isolated.
  • the nucleotide sequence was determined by reading both plus and minus strand directions using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and a Perkin-Elmer Model 3700 sequencer (Applied Biosystems).
  • mice were killed by cervical dislocation and fresh tissue was quickly removed. Tissue pieces were lightly washed with PBS (phosphate-buffered saline), and then fixed at 4 ° C. using 4% paraformaldehyde / PBS. The fixed tissue piece was replaced with a 30% sucrose solution, and then embedded in a 0.1 T compound. The sections were cut to a thickness of 25 xm using a cryosection microtome (Leica CM3050) and attached to a slide glass to prepare a preparation for in w '/ tf.
  • PBS phosphate-buffered saline
  • the preparation for in W / i / was dried at 42 ° C. for 1 hour, and then fixed with 4 paraformaldehyde / PBS. Then, wash twice with PBS for 5 minutes, then treat with Proteinase-K (Roche Applied Science) at a concentration of 50 g / ml, wash once with PBS for 5 minutes, and fix again with 4% paraformaldehyde / PBS Was done. After fixation, the cells were treated in distilled water for 5 minutes, and then placed in a 0.1 M triethanolamine (H8.0) solution stirred with a stirrer. Acetic anhydride was added thereto to a concentration of 0.25%.
  • H8.0 triethanolamine
  • the stirrer was stopped, and the mixture was allowed to stand for 10 minutes. Then, the cells were treated with PBS for 5 minutes and 0.85% NaCl for 5 minutes.
  • a hybridization solution 50% Formamide, 5X SSC, 1 mg / ml Yeast tRNA, 100 g / ml Heparin, IX Denhardt's
  • the probe for in was synthesized with cRNA using mouse gasdermin A-1, mouse gasdamine A-2, mouse gasdamine A-3 and human gasdamine A-1 as type III.
  • the probe was labeled by incorporating digoxigenine-labeled-UTP (Roche Applied Science).
  • the probe in order to improve the penetration into the tissue with 40 mM NaOCO 3/60 mM Na 2 C0 3 (H 10.2) solution, the time length of the pro part is 250 bases, 60 ° Incubation was performed at C to perform fragmentation.
  • the time (T) at which the length of the probe becomes 250 bases is calculated by dividing ⁇ (length of first cRNA, kb)-0.25 ⁇ by ⁇ 0.11 X (length of first cRNA, kb) x 0.25 ⁇ . I derived it.
  • Alkaline phosphatase recovery solution 337.
  • Alkaline phosphatase was visualized using Nitro blue tetrazolium, 175 g / ml, 5-bromo-4-chrolo-3-indoyl phosphate / Alkaline phosphatase buffer).
  • the preparation was fixed with a 4% formaldehyde solution, and then covered with a gabber glass and observed using an upright optical microscope (OLYMPUS BX51).
  • the deparaffinized sample is flushed with 2N hydrochloric acid for 20 minutes at 37 ° C, twice for 5 minutes at room temperature with 100 mM Tris-HCl (pH 7.5), and for 5 minutes with 0.05% Trypsin at 37. (Tap water) for 10 minutes and 0.3% H 2 O 2 / MtOH for 30 minutes at room temperature to suppress endogenous peroxidase activity.
  • the specimen after antibody staining was covered with a glass cover and observed using an upright optical microscope (OLYMPUS BX51).
  • Double staining using In 5 // ⁇ hybridization and PCNA-1 antibody Double staining using in situ hybridization of mouse gasdamine ⁇ -2 and PCNA antibody is usually performed first. 5 / Prepare tissue section for hybridization and detect mouse gasdermin A-2 gene expression by in situ hybridization method using mouse gasdermin A-2 cRNA probe did. Thereafter, the same tissue section was subjected to antibody staining using a primary antibody, an antibody recognizing PCNA, a proliferating cell marker (anti-proliferating cell nuclear antigen, diluted 500-fold, Santa Cruze). The procedure for antibody staining was the same as in the analysis of dividing cells in In W cells.
  • HSC39, HSC43, HSC44, HCS58, HSC59, HSC60, OCUM 2M, and KAT0III Eight poorly differentiated gastric cancer cell lines, HSC39, HSC43, HSC44, HCS58, HSC59, HSC60, OCUM 2M, and KAT0III, and four differentiated gastric cancer cell lines, HSC57, MKN7, MKN28, and MKN74, in 10% embryos Perforated serum, RPMI 1640 supplemented with 0.15% sodium bicarbonate, 2 mM L-glutamine, and benicillin streptomycin.
  • DAC 5-aza-2'-dexoxycytidine
  • TSA histone deacetylase inhibitor
  • Transformation takes 2 hours 24 hours after the transfection, 5 transfected cells lxlO are seeded on a 10 cm dish and placed in RPMI 1640 medium supplemented with 600 g / ml G418 (Invitrogen Corp., Carlsbad, CA). Maintained. After 12-14 days, surviving colonies were counted after May-Giemsa staining.
  • Rim3 has two independent strains: ⁇ 1> (C57BL / 10-RIM3 / + X MSM) F1 X C57BL / 10, ⁇ 2> (C57BL / 10-RIM3 / + X JF1) F1 X C57BL / 10
  • ⁇ 1> C57BL / 10-RIM3 / + X MSM
  • ⁇ 2> C57BL / 10-RIM3 / + X JF1
  • F1 X C57BL / 10 As a result of the large-scale linkage analysis used, it was confirmed that it was located within approximately 200 kbp (200,000 base pairs) between Grb7 and DllMit on mouse chromosome 11 (Sato et al., Mammalian Genome 9, 20). -25, 199 8).
  • the Rim3 'causal gene region was amplified by three BACs (227J19, 372A19, 321H6), and the BAC clone 321H6 was transformed into DllMi by determining the base sequence of the BAC clone and performing Southern hybridization. It was shown to contain tl4 and gasdermin (Saeki et al., Mammalian Genome 11, 718-724, 2000) ( Figure 2).
  • the present inventors aimed at elucidating the causative gene of Rim3 by analyzing the base sequence of BAC, the cDNA selection method, and the human genome draft sequence database (http: @www. Ncbi. Nlm). nih. gov /) was used for positional 'cloning and candidate gene' mapping.
  • two novel mouse gasdermin homologous genes existed in tandem next to the centromere side of gasdamine ( Figures 3 and 4).
  • the full-length cDNAs of these two new mouse gasdamine homologous genes were isolated from mouse skin and mouse stomach cDNA libraries by screening using the mouse gasdamine cDNA as a probe.
  • A-2 and Mouse Gasdermin A-3 was used for positional 'cloning and candidate gene' mapping.
  • mouse gasdermin A-1 The previously reported mouse gasdermin was named mouse gasdermin A-1.
  • mouse gasdamine A-1, mouse gasdamine A-2, and mouse gasdamine A-3 were named mouse gasdamine A cluster 1.
  • mouse gasdermin A cluster gene By comparing the mouse gasdermin A cluster gene with the human genome draft sequence, a DNA fragment predicted to be the human gasdermin A-1 gene was found on human chromosome 17 in the Rim3 causative gene region and the human homologous region.
  • Figure 5 Based on the nucleotide sequence, an oligonucleotide primer for PCR (polymerase chain reaction) was set up, and the human gas dermin protein-encoding cDNA was isolated by PCR. In addition, human stomach cDNA was used for type II.
  • the coding region of the human gasdermin cDNA protein consists of 1338 base pairs, and the protein predicted therefrom is composed of 445 amino acids, and has a homology of 70% or more with the mouse gasdermin A cluster 1 protein.
  • the nucleotide sequences of mouse gasdermin A-2, mouse gasdermin A-3 and human gasdermin A-1 are Acce, respectively.
  • ssion No. AB103372, No. AB103373, No. AB093591 were registered in the Japan DNA Database (DDBJ).
  • the mouse gasdamine A cluster gene is contained within the causative gene region of Rim3.
  • Rim3, phenotype Re den is a decision expected that it would be caused by any of the mutations Gasuda one Min A cluster genes, Rim3, Mausugasuda one Min A class evening monogenic nucleotide sequence in Re den I went.
  • Rim3, Re den both mutations in Mausugasuda one Min A- 3 gene revealed Fig. 6, 7).
  • mouse gasdermin A-1 is specifically expressed in skin, esophagus and stomach
  • mouse gasdermin A-2 is specifically expressed in stomach
  • mouse gasdermin A-3 is specifically expressed in skin.
  • Fig. 8 A more detailed examination of the expression in the stomach revealed that mouse gasdermin A-1 was expressed in the forestomach region of the stomach. It was revealed that mouse gasdamine A-2 was specifically expressed in the fundus and pylorus (glandular epithelium) of the stomach (Fig. 9).
  • Mouse gasdermin A-3 was not expressed in the stomach or esophagus, but was expressed only in the skin.
  • mouse gas dermin A cluster gene may be selectively regulated in specific epithelia.
  • Examination of the expression of the mouse gasdermin A class Yuichi gene during fetal life revealed that mouse gasdamine A-1 began to be expressed in the stomach from around embryonic day 12, and mouse gasdamine A-2 was expressed from around embryonic day 16 Starts (Fig. 10). This mode of expression is consistent with the formation of gastric squamous and glandular epithelium. In the skin, only mouse gasdermin A-1 was expressed around embryonic day 12, and mouse gasdermin A-3 was not expressed. The expression of mouse gasdermin A-3 was first confirmed in the skin at 2 days of age.
  • the in situ hybridization was performed using frozen sections of the skin and the upper gastrointestinal tract.
  • mouse gasdamine A-1 was expressed not in stem cells but in cells in the granular layer to the transitional layer (Fig. 11).
  • Mouse gasdamine A-2 was expressed in the apical region of the glandular epithelium in the fundus stomach and in the central region of the glandular epithelium in the pylorus (Fig. 11). It is known that the stem cells of the gastric gland epithelium exist in the central part of the glandular epithelium and differentiate independently toward the upper and lower ends (Fig. 12).
  • mice gasdermin A-2 was found in the epithelium as in the esophagus and forestomach squamous epithelium. From the results, it was clarified that it was expressed not only in the cells in the transition layer but also in the hair follicle. Taken together that causes gene for this result and Rim3, Re den is Gasuda one Min A- 3, mouse gas Zehnder Min A- 3 is It is expected to be expressed in hair follicles and to be associated with hair formation and the hair cycle.
  • PCNA Proliferating Cell Nuclear Antigen
  • human gasdamine A-1 In human tissues, the expression of the human gasdamine A-1 gene was determined by RT-PCR.As a result, it was found that human gasdamine A-1 was specifically expressed in the skin, esophagus, stomach, mammary gland, and lung. It became clear.
  • Rim3 hair loss and epidermal hyperproliferation in Re den has been reported by Sato et al. (Ma thigh al i an Genome 9, 20-25, 1998).
  • the present inventors further examined the abdominal skin of Rim3 30 days after birth by histological analysis. As a result, overgrowth of epithelium and overgrowth of the upper follicle region were observed (FIG. 14).
  • BrdU (5-Bromo-2'-deoxyuridine, 5-bromo-2'-deoxyduridine) is incorporated into DNA in place of thymidine (T) during DNA replication, and it specifically affects dividing cells (proliferating cells). Label. Therefore, when the proliferating cells of the Rim3 ventral skin at this time were examined using BrdU, BrdU-positive cells were observed 6 to 7 times as much as the control wild type (C57BL / 10J) (Fig. 1). 5 (c) (d)). In other words, in Rim3, it was expected that the cell growth control mechanism of epithelium and hair follicles would break down and abnormal cell growth would occur.
  • Rim3 30-day-old abdominal skin was examined immunohistologically using various cell layer cell marker antibodies (Fig. 15).
  • keratin 14 is specifically expressed in the basement membrane layer
  • keratin 10 is specifically expressed in the cell layer above the basement membrane layer.
  • keratin 14 was mainly expressed in the basement membrane layer, but its expression was extended to the cell layer above the basement membrane layer.
  • keratin 10 was expressed in whole epithelial cells and in hyperproliferative hair follicles. This indicated that the hair follicles were epithelialized in Rim3 (FIG. 16).
  • Human gasdermin A-1 is present on chromosome 17, (17ql 2), and this region It has been reported that the genomic region is amplified in some cancers, such as gastric cancer, breast cancer, and esophageal cancer. This region is called 17ql 2a immediately 1 i con (Fig. 17). Amplification of an oncogene and its overexpression are remarkable phenomena in human carcinogenesis. The present inventors have previously shown that the expression of the human gasdermin A-1 gene is lost in normal stomach tissues but is lost in gastric cancer tissues, gastric cancer cell lines, esophageal cancer cell lines, and ovarian healing cell lines. (Saeki et al., Mammal i an Genome 11, 718-724, 2000).
  • Example 4 Inhibitory effect of gasdermin A-1 gene on human ⁇ cell colonies
  • gasdamine A-1 gene was added to cancer cell lines together with Neo resistance gene. introduced, introducing examined the percentage of colony formation (Col ony format i on assay) 0 to MKN28 gastric cancer cell lines and TE10 esophageal cancer cell lines Gasuda Min a-1 gene and the Neo-resistant gene at the same time, the gene transfer G418
  • cancer colonies were dramatically increased as compared to the case where only the control was performed simultaneously or the minus strand (antisense) of gasdamine A-1 gene was introduced. Formation was suppressed (Fig. 19). Therefore, it was shown that the gasderin A-1 gene has a tumor suppressor function.
  • Hitogasudamin A-1 gene was isolated as one of the family monogenic the gene responsible for epithelial morphogenesis abnormal mutant mice Rim 3, Re den. Rim3, the causative gene of Re den, mutation site of the mouse Gas Zehnder Min A-3 gene is a region conserved in all between Gasudamin A class evening monogenic. Therefore, by introducing the same mutation as Re den in Hitogasuda one Min A- 1 gene was carried out by the same kind of experiments using MKN28, TE10 both cell lines. As a result, compared to normal Hitogasudamin A-1 gene, Hitogasudamin A- 1 gene introduced mutations Re den type, significantly reduced the formation suppressing effect was observed (Fig. 2 0).
  • Example 5 Induction of apoptosis (cell death) by transduction of gasdermin A-1 gene
  • the present inventors verified the induction of apoptosis by forcibly expressing the human gasdermin A-1 gene by introducing the human gasdermin A-1 gene into MKN28 gastric cancer cells using an adenovirus gene transfer system.
  • MKN28 for gastric cancer cells After transfection of the human gasdermin Al gene, examination using the T-ring EL (tunnel / tunnel) method revealed that apoptosis was dramatically induced 40 hours after the transfection (Fig. 21). Similar results were obtained by flow cytometry analysis using Annexin V-FITC and PI in the presence of the caspase inhibitor Z-VAD-FMK. From this result, it is considered that the cancer-suppressing effect of the gasdermin gene indicated by the colony formation assay is due to the death of cancer cells through the apoptotic pathway.
  • MLZE (Watabe et., Al. Jpn J Cancer Res. 2001, 92) was searching the mouse EST database and human genome database. The present inventors have found that there are a large number of DNA fragments considered to be gasdermin-related genes, including (2): 140-151). These genes were called gasdamine A-related genes. Interestingly, MLZE, one of the members of the gasdamine A-related gene, was originally isolated as a melanoma-causing gene and is selectively expressed in metastatic melanoma.
  • Human MLZE maps to chromosome 8 8q24.1-2, which contains the oncogene c-Myc and is often a metastatic melanoma, with genomic amplification observed.
  • Mouse Mlze reported by Wa tabe et al. At the same time as human MLZE, is, of course, a member of the gasdermin A-related gene.
  • the present inventors also reported that three types of gasdamine A-related gene members in the mouse, gasdamine C-2 (GsdmC-2: Accession No. AB103382) and gasdamine C_3 (GsdmC-3 : Accession No. AK033603) and Gasdermin Dl (GsdmD-1: Accession No.
  • Gasdamine D-1 Analysis of the base and amino acid sequences of Gasdamine D-1 revealed two human homologous genes, FU12150 and AK007710, at Gasdamine D-1. It is considered an alternative transcript from chromosome 8q24.3. Therefore, the gasdermin family human MLZE (GSDMC-1) and human FLJ12 150 / AK007710 (GSDMD-1) exists as a cluster very close to each other on chromosome 8q24, and probably these mouse homologous genes are also homologous regions of human chromosome 8 chromosome 8q24. It is thought that it exists as a cluster on chromosome 15. When the expression of gasdermin D-1 was examined by RT-PCR, no expression was observed in normal stomach and colon tissue, but expression was observed in stomach cancer and colon cancer tissue. This suggests that gasdamine D-1 can be used for cancer diagnosis.
  • gasdermin A-related gene family Another member of the gasdermin A-related gene family, which is located next to gasdamine A-1 on human chromosome 17 17ql2.
  • B-1 is considered to be fully utilized as a cancer diagnostic method.
  • samples that have started to be slightly expressed even in normal-looking tissues. This may be normal even though it looks normal, but it is likely that the cancer has already begun. From this, it is considered that by using Gasdamin B-1, it is possible to judge that although it is apparently normal, it has already entered the precancerous stage.
  • the alanine in which the amino acid conversion was caused by the Rii3 mutation is an amino acid conserved in the gasdermin A cluster and the entire gene family related to gasdamine A (Fig. 26).
  • the present inventors have found the Gasdermin family and its use.
  • abnormalities in epithelial cell differentiation in which the gasdermin family can be expressed and controlled such as cancer, non-neoplastic keratosis of the skin, ectopic epithelial mucosa of the gastrointestinal tract, or polyps And other diseases caused by abnormal cell proliferation or differentiation.

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Abstract

Au cours de la clarification de gènes pathogènes de Rim3 par clonage positionnel et mappage de gène candidat, de nouveaux gènes homologues Gasdermine ont été isolés et appelés Gasdermine de souris A-2 et Gasdermine de souris A-3. En outre, ces gènes sont comparés à des séquences de trait de génome humain, et c'est ainsi qu'a été découvert un gène Gasdermine humain (appelé Gasdermine humain A-1). Ce gène est isolé au moyen du procédé PCR. Le gène Gasdermine humain A-1 est transféré dans une lignée cellulaire cancéreuse et le rapport de formation de colonie de cellules est examiné. Ainsi on a découvert que ce gène possède un effet carcinostatique. Lors de l'examen de l'occurrence d'autres gènes homologues Gasdermine, on a découvert qu'il existait une famille Gasdermine.
PCT/JP2003/002345 2003-02-28 2003-02-28 Famille gasdermine Ceased WO2004076666A1 (fr)

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CN107312080A (zh) * 2017-07-23 2017-11-03 复旦大学 一种来源于gsdmd蛋白的抗菌肽及其应用
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Publication number Priority date Publication date Assignee Title
WO2006019153A1 (fr) * 2004-08-20 2006-02-23 Inter University Research Institution Corporation, Research Organization Of Information And Systems ANIMAL NON HUMAIN AVEC FONCTION INHIBÉE DU GÈNE GdsmA ET FONCTION FAVORISÉE OU INHIBÉE DU GÈNE ASSOCIÉ À LA CANCÉRISATION
JP2006061108A (ja) * 2004-08-27 2006-03-09 Research Organization Of Information & Systems ＧａｓｄｅｒｍｉｎＢを標的とした癌診断および創薬
WO2008084566A1 (fr) * 2007-01-11 2008-07-17 Research Organization Of Information And Systems Souris modèle, sa méthode d'élaboration et ses utilisations
JPWO2008084566A1 (ja) * 2007-01-11 2010-04-30 大学共同利用機関法人情報・システム研究機構 モデルマウス、その製造方法およびその用途
CN109415767A (zh) * 2016-06-29 2019-03-01 株式会社爱茉莉太平洋 用于确认是否暴露于细微灰尘中的生物标记物及利用其的确认方法
CN109415767B (zh) * 2016-06-29 2022-07-05 株式会社爱茉莉太平洋 用于确认是否暴露于细微灰尘中的生物标记物及利用其的确认方法
CN107312080A (zh) * 2017-07-23 2017-11-03 复旦大学 一种来源于gsdmd蛋白的抗菌肽及其应用
CN107312080B (zh) * 2017-07-23 2021-02-26 复旦大学 一种来源于gsdmd蛋白的抗菌肽及其应用
CN110917357A (zh) * 2019-12-16 2020-03-27 中南大学湘雅二医院 人gsdmb基因的用途及相关产品

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