Cytotoxic protein and utilization thereof

Technical field The present invention relates to M toxin (mucous layer devastating toxin), which is a novel cytotoxic protein produced by Helicobacter pylori, and its utilization. Background technology Many gastritis, gastric ulcers or gastric cancers have been attributed to Helicobacter pylori, but to date, obvious direct cytotoxic factors that cause gastric epithelial cell destruction and irreversible cell death, which are the beginnings of these diseases. Has not been identified. Factors that change the pH environment and immune response in the stomach, adhesion factors of Helicobacter pylori to gastric epithelial cells, and motility of the bacteria themselves have been pointed out as requirements for establishing the disease, but all of gastritis, gastric ulcer, and gastric cancer. Until now, it was unclear what kind of process the gastric mucosal destruction, which can be said to be the starting point of the disease, goes through, and what is the direct responsible factor. The only cytotoxic vacuolar toxin has been isolated, but it has weak cytotoxic activity and remains reversible cytotoxic activity, and is a lethal virulence factor that is pathogenic both in vivo and in vitro. It has not been recognized as a factor. As mentioned above, it is presumed by many researchers that Helicobacter pylori secretes a direct impairing factor to gastric mucosal cells in the gastric environment of the living body, and considering the importance of the disease, all of the genes Although the sequence was established in 1996, culture conditions that are still difficult to use with serum and unestablished isolation and purification conditions and evaluation systems have hampered the isolation and identification of putative toxins. Problems to be solved by the invention Therefore, we found the responsible protein that causes gastritis, gastric ulcer, gastric cancer, etc. associated with Helicobacter pylori infection, established a method for preparing a large amount of the toxin protein, and identified, diagnosed, and screened the new toxin M toxin. Establishing was one issue. In addition, using them, suppress the action of the toxin responsible for gastric mucosal cell damage, pave the way for the development of preventive / therapeutic agents for gastritis, gastric ulcer, gastric cancer, etc., and also begin the application method of the toxin. Was desired. Disclosure of invention As a result of intensive research to solve the above problems, the present inventor has found that irreversible cells are grown when Helicobacter pylori is grown under serum-free culture conditions, which is different from the conventional one and is closer to the gastric environment. We have identified a novel toxin that causes death. It is 1000 to 100,000 times more toxic per unit of the vacuoled toxin described above and causes irreversible cell death not only in gastric epithelial cells but also in a wide range of warm blood cells including immune system cells. I found that. The present inventor has completed the present invention as a result of further studies based on these findings. That is, the present invention is a cytotoxic protein characterized by (1) a protein having at least 70% identity with respect to the amino acid sequence represented by SEQ ID NO: 1, and (2) an amino acid represented by SEQ ID NO: 1. A partial peptide of the protein according to (1), which is characterized by having cytotoxic activity equal to that of the sequence, (3) the cytotoxic protein according to (1) 1 or (2) produced by helicobacter pyrori, (4) ( The above-mentioned (1) or (2), which comprises culturing a transformant transformed with a recombinant vector containing the DNA of SEQ ID NO: 2 encoding the cytotoxic protein according to 1) or (2). Cell-damaging protein, (5) The transformant has been deposited with the National Institute of Advanced Industrial Science and Technology (IPOD) under deposit number FERM BP-8218 (4) Cell-damaging protein, (6) (1) 1 Alternatively, an antitumor agent using the cytotoxic protein described in (2), a monoclonal antibody specific for the cytotoxic protein obtained by immunizing a mammal with the cytotoxic protein described in (7) (1) or (2), (8). ) Deposit number FERM The monoclonal antibody according to (7) produced using the hybridoma clone of BP-8222, (9) the monoclonal antibody according to (7) produced using the hybridoma clone of deposit number FERM BP-8223, (10) deposit. Number FERM Specific to the cytotoxic protein obtained by immunizing mammals with the monoclonal antibody according to (7) and the cytotoxic protein according to (11) (1) or (2) produced using the hybridoma clone of BP-8224. (13) A method and diagnostic method for detecting a cytotoxic protein according to (1) or (2) using the polyclonal antibody, the monoclonal antibody according to any one of (12), (7) to (11), or the polyclonal antibody. ) A prophylactic or therapeutic agent for gastric cancer, gastric inflammation, and gastric ulcer caused by the cytotoxic protein according to (1) or (2) using the monoclonal antibody or polyclonal antibody according to any one of (7) to (11). (14) Negative or negative using warm-blooded animal cells for compounds that promote or inhibit the activity of the protein according to (1) or (2), characterized by the use of the protein according to (1) or (2). A method of screening the compound by cell growth inhibitory activity, cytotoxic activity or cell death, as compared to a positive control group, comprising the protein according to (15) (1) or (2), (1) or (2) Obtained using a kit for screening a compound or a salt thereof that promotes or inhibits the activity of the described protein, the screening method described in (16) (14) or the screening kit described in (15), (1). Alternatively, a compound or a salt thereof that promotes or inhibits the activity of the protein according to (2), or a compound or a compound having an activity that inhibits the cytotoxic activity of warm-blooded animal cells by the protein according to (17) (1) or (2). A prophylactic / therapeutic agent for diseases indicated to be caused by M toxin by a drug containing a salt, (18) gastric inflammation, gastric ulcer, gastric cancer and the screening method described in (14) or the screening kit described in (15). Provide the medicine according to (17). Best mode for carrying out the invention A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the present invention a bacterial strain in Helicobacter pylori, for example, NCTC 11637, NCTC 11916, DT 61A, NCTC 11639, R85-13 6P. , R85-13-12F, R85-13-11P, T81213-NTB, J99, 4, U2-1, 85D08, MC903, MC123, Tx30a, 26695, UA 1182, etc. May be. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 70% or more, preferably about 80% or more, more preferably about 90% or more, still more preferably about 70% or more, more preferably about 90% or more, the amino acid sequence represented by SEQ ID NO: 1. Examples include amino acid sequences having about 95% or more homology. As a protein having substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the present invention, for example, the protein has substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 and has a sequence. A protein having substantially the same activity as the protein having the amino acid sequence represented by No. 1 is preferable. Examples of substantially homogeneous activities include cell growth inhibitory activity, cytotoxic activity, and activity that causes cell death. Substantially homogeneous means that their activities are homogeneous in nature (eg, physiologically or pharmacologically). Therefore, it is preferable that the activities such as cytotoxicity are the same (eg, about 0.1 to 100 times, preferably about 0.5 to 10 times, more preferably about 0.5 to 2 times), but the degree of these activities and the degree of protein. Quantitative factors such as molecular weight may be different. The cell growth inhibitory activity, cytotoxic activity, or activity that causes cell death can be measured according to a method known per se, but can be measured, for example, according to a screening method described later. The protein of the present invention includes, for example, an amino acid sequence in which 1 to 150 (preferably 1 to 50) amino acids in the amino acid sequence represented by SEQ ID NO: 1 are deleted; an amino acid represented by SEQ ID NO: 1. Amino acid sequence in which 1 to 100 (preferably 1 to 50 (more preferably 1 to 30)) amino acids are added to the sequence; 1 to 50 (preferably, preferably) to the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequence in which 1 to 30) amino acids are inserted; Amino acid sequence in which 1 to 50 (preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1 are replaced with other amino acids; Alternatively, so-called mutin such as a protein containing an amino acid sequence combining them is also included. When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited. The proteins herein are N-terminal (amino-terminal) at the left end and C-terminal (carboxyl-terminal) at the right end according to the convention of peptide marking. In the protein of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, the C-terminal is usually a carboxyl group (-COOH) or a carboxylate (-COO-), but the C-terminal is an amide (-COO-). -CONH₂) Or ester (-COOR). Here, as R in the ester, for example, C such as methyl, ethyl, n-propyl, isopropyl or n-butyl_1-6Alkyl groups such as cyclopentyl, cyclohexyl, etc._3-8C of cycloalkyl groups such as phenyl, α-naphthyl, etc._6-12Aryl groups, such as phenyl-C such as benzyl, phenethyl, etc._1-2Α-naphthyl-C such as alkyl group or α-naphthylmethyl_1-2C such as alkyl group_7-14It is an aralkyl group. When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminal, the protein of the present invention also includes an amidated or esterified carboxyl group. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, in the protein of the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) is a protecting group (eg, C formil group, acetyl group, etc.)._1-6C such as alkanoyl_1-6Those protected by an acyl group (such as an acyl group), those in which the N-terminal glutamine residue produced by cleaving in vivo is pyroglutamine-oxidized, and substituents on the side chain of amino acids in the molecule (for example, -OH, -SH) , Amino group, imidazole group, indol group, guanidino group, etc. are suitable protective groups (eg, formyl group, acetyl group, etc. C_1-6C such as alkanoyl group_1-6It also includes those protected by (acyl groups, etc.) or complex proteins such as so-called glycoproteins to which sugar chains are bound. The partial peptide of the protein of the present invention is the partial peptide of the protein of the present invention described above, and preferably has the same activity as the above-mentioned protein of the present invention (eg, cell growth inhibition or cell growth inhibition). Anything can be used as long as it is. For example, it has at least 20% or more, preferably 50% or more, more preferably 70% or more, more preferably 90% or more, and most preferably 95% or more amino acid sequences in the constituent amino acid sequences of the protein of the present invention. Peptides having cell growth inhibitory activity, cytotoxic activity, or cell death-causing activity are used. In addition, the partial peptide of the present invention lacks 1 to 5 (preferably 1 to 3) amino acids in its amino acid sequence, or 1 to 10 (preferably 1 to 3) in its amino acid sequence. Five (preferably 1-3) amino acids have been added, or 1-5 (preferably 1-3) amino acids have been inserted into the amino acid sequence, or in the amino acid sequence. 1 to 5 (preferably 1 to 3) amino acids may be replaced with other amino acids. In addition, the C-terminal of the partial peptide of the present invention is usually a carboxyl group (-COOH) or a carboxylate (-COO-), but like the protein of the present invention described above, the C-terminal is an amide (-CONH).₂) Or ester (-COOR) (R has the same meaning as described above). Further, in the partial peptide of the present invention, similarly to the above-mentioned protein of the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protective group, and the N-terminal side is raw. Glutamine residues produced by cleaving in the body are pyroglutamine-oxidized, substituents on the side chains of amino acids in the molecule are protected by appropriate protective groups, or so-called glycopeptides to which sugar chains are bound. Complex peptides and the like are also included. Further, since the partial peptide of the present invention can be used as an antigen for producing an antibody, it does not necessarily have to have cell growth inhibitory activity, cytotoxic activity or the like. As the salt of the protein or partial peptide of the present invention, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used, and particularly physiologically acceptable. The acid addition salt to be added is preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. The protein of the present invention or a salt thereof can be produced from the cells of various strains of Helicobacter pylori described above by a method for purifying a protein known per se, or a transformant containing DNA encoding a protein described later can be produced. It can also be produced by culturing. It can also be produced according to the peptide synthesis method described later. When produced from cells of various strains of Helicobacter pylori, the cells are centrifuged by ultrasonic crushing, etc., and then extracted by sulfide precipitation, etc., and the extract is subjected to ion exchange chromatography and hydrophobicity. It can be purified and isolated by combining chromatography such as chromatography. A commercially available resin for protein synthesis can be usually used for the synthesis of the protein, partial peptide, or salt thereof of the present invention, or an amide compound thereof. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamide methyl resin, polyacrylamide resin, 4- (2', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resin, etc. can be mentioned. it can. Using such a resin, amino acids appropriately protected with an α-amino group and a side chain functional group are condensed on the resin according to the sequence of the target protein according to various condensation methods known per se. At the end of the reaction, proteins are excised from the resin and various protecting groups are removed at the same time, and an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the desired protein or an amide compound thereof. Regarding the condensation of the protected amino acids described above, various activation reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N'-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid was added directly to the resin together with the racemization inhibitor (for example, HOBt, HOOBt), or the protected amino acid was activated in advance as a symmetric acid anhydride or HOBt ester or HOObt ester. It can be added to the resin later. As the solvent used for activating the protected amino acid and condensing with the resin, a solvent known to be usable for the protein condensation reaction can be appropriately selected. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide and the like. Sulfoxides, ethers such as pyridine, dioxane, tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or appropriate mixtures thereof are used. The reaction temperature is appropriately selected from the range known to be used for the protein bond formation reaction, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. Activated amino acid derivatives are usually used in 1.5-4 fold excesses. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated with acetic anhydride or acetylimidazole so as not to affect the subsequent reaction. Examples of the protecting group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl and trifluoroacetyl. , Phtaloyl, formyl, 2-nitrophenylsulphenyl, diphenylphosphinoti oil, Fmoc, etc. are used. The carboxyl group is, for example, an alkyl esterified (eg, a linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazidation, t-butoxy It can be protected by carbonyl hydrazideization, tritylhydrazideation, etc. The hydroxyl groups of serines can be protected, for example, by esterification or etherification. As a group suitable for this esterification, for example, a lower (C1-6) alkanoyl group such as an acetyl group, an aloyl group such as a benzoyl group, a group derived from carbon dioxide such as a benzyloxycarbonyl group and an ethoxycarbonyl group, etc. are used. Be done. Further, examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, a t-butyl group and the like. As the protecting group for the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl2-Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used. As the protecting group for imidazole of histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Bos, Trt, Fmoc and the like are used. Activated carboxyl groups of the raw material include, for example, the corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenols, 2,4,5-trichlorophenols, 2,4-dinitrophenols, Esters with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccimid, N-hydroxyphthalimide, HOBt)] and the like are used. As the activated amino group of the raw material, for example, the corresponding phosphate amide is used. Protecting group removal (desorption) methods include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, and trifluo. Acid treatment with lomethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine and the like, reduction with sodium in liquid ammonia and the like are also used. The desorption reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C, but in the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1 It is effective to add a cation trapping agent such as 4-butanedithiol, 1,2-ethanedithiol and the like. The 2,4-dinitrophenyl group used as the imidazole protecting group for histidine is removed by thiophenol treatment, and the formyl group used as the indol protecting group for tryptophan is the above-mentioned 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkaline treatment with dilute sodium hydroxide solution, dilute ammonia or the like. The protection of functional groups that should not be involved in the reaction of the raw material, the protecting groups, the elimination of the protecting groups, the activation of the functional groups involved in the reaction, and the like can be appropriately selected from known groups or known means. As another method for obtaining an amide form of a protein, for example, first, the α-carboxyl group of the carboxy-terminal amino acid is amidated to protect it, and then the peptide (protein) chain is extended to the desired chain length on the amino group side. , A protein from which only the protective group of the α-amino group at the N-terminal of the peptide chain was removed and a protein from which only the protective group of the carboxyl group at the C-terminal was removed were produced, and both proteins were placed in the mixed solvent as described above. Condensate with. The details of the condensation reaction are the same as described above. After purifying the protective protein obtained by condensation, all protecting groups can be removed by the above method to obtain a desired crude protein. This crude protein is purified by making full use of various known purification means, and the main fraction is freeze-dried to obtain an amide form of the desired protein. To obtain an ester of a protein, for example, the α-carboxyl group of a carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester of the desired protein is obtained in the same manner as the amide of a protein. be able to. The partial peptide of the present invention or a salt thereof can be produced according to a method for synthesizing a peptide known per se, or by cleaving the protein of the present invention with a suitable peptidase. As the peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can constitute a partial peptide of the present invention with a residual portion and removing the protecting group when the product has a protecting group. Examples of known condensation methods and desorption of protecting groups include the methods described below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966); Schroeder and Luebke, The Peptide, Academic Press, New York (1965); Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. ) (1975); Haruaki Yajima and Shunpei Sakakibara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977); Supervised by Haruaki Yajima, Development of Follow-up Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten .. After the reaction, the partial peptide of the present invention can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method or a method similar thereto, and conversely, when it is obtained by a salt, a known method or a method similar thereto. Can be converted to free form or other salts. The DNA encoding the protein of the present invention may be any DNA as long as it contains the above-mentioned base sequence encoding the protein of the present invention. Further, any of the genomic DNA, the genomic DNA library, the above-mentioned cDNA derived from the cell / tissue, the above-mentioned cDNA library derived from the cell / tissue, and the synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, total RNA or mRNA fraction prepared from the above-mentioned cells / tissues can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method). The DNA encoding the protein of the present invention includes, for example, a DNA containing the base sequence represented by SEQ ID NO: 2 or a base sequence that hybridizes with the base sequence represented by SEQ ID NO: 2 under high stringent conditions. However, any DNA that encodes a protein having substantially the same activity as the protein of the present invention (eg, cytotoxic activity, etc.) may be used. More specifically, as the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 1, DNA having the base sequence represented by SEQ ID NO: 2 or the like is used. The DNA encoding the partial peptide of the present invention may be any DNA as long as it contains the above-mentioned base sequence encoding the partial peptide of the present invention. Further, any of the genomic DNA, the genomic DNA library, the above-mentioned cDNA derived from the cell / tissue, the above-mentioned cDNA library derived from the cell / tissue, and the synthetic DNA may be used. The DNA encoding the partial peptide of the present invention is, for example, a DNA having a partial base sequence of a DNA having a base sequence represented by SEQ ID NO: 2, or a DNA having a base sequence represented by SEQ ID NO: 2 and high stringent conditions. A DNA having a base sequence to hybridize and having a partial base sequence of DNA encoding a protein having substantially the same activity as the protein of the present invention is used. Cloning of DNA that completely encodes the protein or partial peptide of the present invention (hereinafter, these proteins and the like may be simply abbreviated as the protein of the present invention in the description of cloning and expression of DNA encoding these proteins and the like). As a means of, a part or the whole region of the protein of the present invention is amplified by a PCR method known per se using a synthetic DNA primer having a partial base sequence of the protein of the present invention, or DNA incorporated into an appropriate vector is used. It can be sorted by hybridization with a DNA fragment encoding the above or one labeled with synthetic DNA. Hybridization methods include, for example, Molecular Cloning, 2nd, J. Sambrook et al., Cold Spring Harbor Lab. Press, It can be carried out according to the method described in 1989. When using a commercially available library, it can be performed according to the method described in the attached instruction manual. The conversion of the base sequence of DNA is carried out by using a known kit, for example, MutanTM-G (manufactured by Takara Shuzo Co., Ltd.), MutanTM-K (manufactured by Takara Shuzo Co., Ltd.), etc. It can be carried out according to the method of the above or a method similar thereto. The DNA encoding the cloned protein can be used as it is for the purpose, digested with a restriction enzyme if desired, or added with a linker. The DNA may have ATG, GTG, TTG as translation initiation codons on its 5'end and TAA, TGA or TAG as translation termination codons on its 3'end. These translation start codons and translation stop codons can also be added using a suitable synthetic DNA adapter. The expression vector of the protein of the present invention is, for example, (a) cutting out a target DNA fragment from the DNA encoding the protein of the present invention, and (b) linking the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by. Vectors include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13, pET30), bacillus-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage. In addition to bacteriophage such as bacteriophage, retrovirus, vaccinia virus, animal virus such as baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when animal cells are used as a host, SRα promoter, SV40 early promoter, HIV / LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. If the host is Escherichia, the trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter, etc., if the host is Bacillus, SPO1, SPO2 promoter, penP promoter, etc. When the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter, or the like is preferable. In addition to the above, an expression vector containing an enhancer, a selectable marker, an SV40 replication origin (hereinafter, may be abbreviated as SV40ori) and the like can be used. Examples of the selectable marker include a dihydrofolate reductase (hereinafter, sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], an ampicillin resistance gene (hereinafter, sometimes abbreviated as Ampr), and a neomycin resistance gene (hereinafter, may be abbreviated as Ampr). Hereinafter, G418 resistance), which may be abbreviated as Neor, canamycin resistance gene and the like can be mentioned. In particular, when the dhfr gene is used as a selectable marker using dhfr gene-deficient Chinese hamster cells, recombinant cells can also be selected using a thymidine-free medium. If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. If the host is a bacterium of the genus Escherichia, the PhoA signal sequence, OmpA, the signal sequence, etc., if the host is a bacterium of the genus Bacillus, the α-amylase signal sequence, the subtilisin signal sequence, etc. In some cases, the MFα / signal sequence, SUC2 / signal sequence, etc., and when the host is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used. A transformant can be produced using a vector containing DNA encoding the protein of the present invention constructed in this manner. As the host, for example, Escherichia spp., Bacillus spp., Yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia spp. Are, for example, Escherichia coli K12, DH1, DH5α (Proc.Natl.Acad.Sci.USA, Vol.60,160 (1968)), JM103 (Nucleic Acids Research, Vol. Used by 9,309 (1981)), JA221 (Journal of Molecular Biology, Vol.120,517 (1978)), HB101 (Journal of Molecular Biology, Vol.41,459 (1969)), C600 (Genetics, Vol.39,440 (1954)), etc. Be done. As the Bacillus spp., For example, Bacillus subtilis MI114 (Gene, Vol.24,255 (1983), 207-21 (Journal of Biochemistry, Vol.95,87 (1984))) is used as the yeast. For example, Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris, etc. .. Insect cells include, for example, when the virus is AcNPV, Spodoptera frugiperda cells (Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, and High FiveTM cells derived from eggs of Trichoplusia ni. , Cells derived from Mamestra brassicae or cells derived from Estigmena acrea, etc. are used. When the virus is BmNPV, silk moth-derived cell lines (Bombyx mori N cells; BmN cells) are used. As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf21 cells (above, Vaughn, JL et al., In Vivo, 13,213-217 (1977)) and the like are used. As insects, for example, silk moth larvae are used [Maeda et al., Nature, Vol.315,592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr-) cell), and mouse L. Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used. In addition, various normal human cells such as hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangium cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrous cells, Muscle cells, fat cells, immune cells (eg macrophages, T cells, B cells, natural killer cells, obese cells, neutrophils, basal spheres, eosinophils, monospheres), macronuclear cells, synovial cells, cartilage It is also possible to use cells, bone cells, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.). To transform Escherichia spp., For example, Proc.Natl.Acad.Sci.USA, Vol.69, Transformation of Bacillus can be carried out, for example, according to the method described in Molecular &amp; General Genetics, Vol. 168, 111 (1979). To transform yeast, for example, the method described in Methods in Enzymology, Vol.194,182-187 (1991), Proc.Natl.Acad.Sci.USA, Vol.75,1929 (1978), etc. can be performed. it can. Transformation of insect cells or insects can be carried out, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). To transform animal cells, for example, the method described in Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, Vol.52, 456 (1973) can be performed. it can. In this way, a transformant transformed with an expression vector containing DNA encoding a protein can be obtained. When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as the medium used for culturing, and a carbon source necessary for the growth of the transformant is included in the medium. Nitrogen source, inorganic substances and others are contained. Carbon sources include, for example, glucose, dextrin, soluble starch, sucrose, etc. Nitrogen sources include, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. Examples of the inorganic or organic substance and the inorganic substance of the above include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8. As a medium for culturing Escherichia spp., For example, M9 medium containing glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. Agents such as 3β-indrill acrylic acid can be added here to allow the promoter to work more efficiently if necessary. If the host is Escherichia, culturing is usually carried out at about 15-43 ° C for about 3-24 hours, and aeration and agitation can be added if necessary. If the host is a Bacillus bacterium, the culture is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing transformants whose host is yeast, the medium may be, for example, Burkholder minimal medium (Bostian, KL et al., Proc.Natl.Acad.Sci.USA), Vol.77,4505 (1980). )) And SD medium containing 0.5% casamino acid (Bitter, GA et al., Proc.Natl.Acad.Sci.USA, Vol.81,5330 (1984)). The pH of the medium is preferably adjusted to about 5-8. Culturing is usually carried out at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added as necessary. When culturing a transformant in which the host is an insect cell or an insect, an additive such as 10% bovine serum immobilized on Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) is appropriately used as a medium. The added one is used. The pH of the medium is preferably adjusted to about 6.2-6.4. Culturing is usually carried out at about 27 ° C for about 3 to 5 days, and aeration and stirring are added as necessary. When culturing a transformant in which the host is an animal cell, the medium includes, for example, MEM medium (Science, Vol.122,501 (1952)) containing about 5 to 20% of fetal bovine serum, DMEM medium (Virology, Vol). .8,396 (1959)), RPMI 1640 medium (The Jounal of the American Medical Association, Vol.199,519 (1967)), 199 medium (Proceeding of the Society for the Biological Medicine, Vol.73,1 (1950)), etc. Used. The pH is preferably about 6-8. Culturing is usually carried out at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary. As described above, the protein of the present invention can be produced extracellularly of the transformant. The protein of the present invention can be separated and purified from the culture by, for example, the following method. When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and ultrasonically, lysozyme and / or freeze-thaw, etc. A method of obtaining a crude protein extract by centrifugation or filtration after destroying cells or cells is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100TM. When the protein is secreted into the culture broth, after the culture is completed, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. The protein contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining a separation / purification method known per se. These known separation and purification methods include mainly methods using solubility such as salting out and solvent precipitation, dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis. A method that utilizes the difference in charge, a method that utilizes the difference in charge such as ion exchange chromatography, a method that utilizes the difference in hydrophobicity such as hydrophobic chromatography, a method that utilizes specific affinity such as affinity chromatography, A method that utilizes the difference in hydrophobicity such as reverse-phase high-speed liquid chromatography, a method that utilizes the difference in isoelectric focusing such as isoelectric focusing, and the like are used. When the protein thus obtained is obtained as a free form, it can be converted into a salt by a method known per se or a method similar thereto, and conversely, when it is obtained with a salt, a method known per se or a method similar thereto. Can be converted to free form or other salts. The protein produced by the recombinant can be optionally modified or the polypeptide can be partially removed by allowing an appropriate protein-modifying enzyme to act before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The presence or activity of the protein of the present invention thus produced or a salt thereof can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like. The antibody against the protein or partial peptide of the present invention or a salt thereof may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the protein or partial peptide of the present invention or a salt thereof. An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these proteins and the like may be simply abbreviated as the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen and is known per se. It can be produced according to the method for producing an antibody or antiserum of. Preparation of monoclonal antibody (a) Preparation of monoclonal antibody-producing cells: The protein of the present invention is administered to a warm-blooded animal at a site where antibody production is possible by itself, a carrier, or a diluent. A complete Freund's adjuvant or an incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability at the time of administration. The administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. When producing monoclonal antibody-producing cells, warm-blooded animals immunized with the antigen, for example, individuals with antibody titers from mice were selected, and the spleen or lymph node was collected 2 to 5 days after the final immunization and contained in them. Monoclonal antibody-producing hybridomas can be prepared by fusing these antibody-producing cells with allogeneic or heterologous animal myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described later with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to known methods, such as the Koehler and Milstein method [Nature, 256, 495 (1975)]. Examples of the fusion accelerator include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, and P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) used to the number of myeloma cells is 1: 1 to 20: It is about 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%, and cells are efficiently incubated at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes. Fusion can be carried out. Various methods can be used to screen for monoclonal antibody-producing hybridomas, for example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) adsorbed with a protein antigen directly or with a carrier, followed by a radioactive substance or A method of detecting a monoclonal antibody bound to a solid phase by adding an enzyme-labeled anti-immunoglobulin antibody (when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, anti-immunity Examples thereof include a method of adding a hybridoma culture supernatant to a solid phase on which a globulin antibody or protein A is adsorbed, adding a protein labeled with a radioactive substance or an enzyme, and detecting a monoclonal antibody bound to the solid phase. The selection of the monoclonal antibody can be carried out according to a method known per se or similar. It can usually be carried out in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As the selection and breeding medium, any medium can be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture. A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culture temperature is usually 20-40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be carried out under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-mentioned measurement of the antibody titer in antiserum. 1640 medium, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20-40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be carried out under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-mentioned measurement of the antibody titer in antiserum. 1640 medium, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20-40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be carried out under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above-mentioned measurement of the antibody titer in antiserum. (b) Purification of monoclonal antibody: Separation and purification of monoclonal antibody is performed by a method known per se, for example, a method for separating and purifying immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, etc. Only the antibody is collected by an adsorption / desorption method using an ion exchanger (eg, DEAE), an ultracentrifugation method, a gel filtration method, an antigen-binding solid phase, or an active adsorbent such as protein A or protein G, and the binding is dissociated to obtain the antibody. Specific purification method to obtain] can be performed. [Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced according to a method known per se or similar. For example, an immune antigen (protein antigen) itself or a complex thereof is formed and a warm-blooded animal is immunized in the same manner as in the above-mentioned method for producing a monoclonal antibody, and the immune animal contains an antibody against the protein of the present invention. It can be produced by collecting a substance and separating and purifying the antibody. Regarding the complex of the immune antigen and the carrier protein used for immunizing warm-blooded animals, the type of carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody efficiently responds to the hapten immunized by cross-linking the carrier. If possible, any substance may be crosslinked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. may be cross-linked in a weight ratio of about 0.1 to 20, preferably about 1 to 5 with respect to hapten 1. The method of coupling at the ratio of is used. Further, various condensing agents can be used for the coupling of the hapten and the carrier, and an active ester reagent containing a glutaraldehyde, a carbodiimide, a maleimide active ester, a thiol group, a dithioviridyl group or the like is used. The condensation product is administered to a warm-blooded animal at a site where antibody production is possible, either by itself or with a carrier or a diluent. A complete Freund's adjuvant or an incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability at the time of administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 3 to 10 times. The polyclonal antibody can be collected from blood of a warm-blooded animal immunized by the above method, ascites, etc., preferably from blood. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the above-mentioned measurement of the antibody titer in the antiserum. Separation and purification of polyclonal antibody can be carried out according to the same method for separation and purification of immunoglobulin as the above-mentioned separation and purification of monoclonal antibody. Since the therapeutic agent containing the protein or partial peptide of the present invention and the protein of the present invention have cancer cytotoxic activity, disease tissue excision (including both total excision and partial excision, but partial excision is preferable. ) In particular, it can be used as a therapeutic drug for fixed cancer. When the protein or the like of the present invention is used as the above-mentioned therapeutic / preventive agent, it should be purified to at least 90%, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more. Is preferable. The cancer cell growth inhibitory activity of the protein or the like of the present invention can be measured according to a method known per se, or an activity that causes cytotoxic activity or cell death according to a method similar to the method known per se, etc. It is preferable to carry out by the method described in 1. Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., even if these compounds are novel compounds. It may be a known compound. The compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is a test compound described above, for example, a peptide, a protein, a non-peptide compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract. , Animal tissue extract, plasma, etc., and is a compound having an activity of inhibiting cytotoxic activity such as the protein of the present invention or an activity of inhibiting cancer cell growth inhibition. As the salt of the compound, the same salt as the above-mentioned salt of the protein of the present invention is used. When the compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic agent, it can be carried out according to conventional means. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used. The formulations thus obtained are safe and low toxicity, and therefore, for example, in humans or warm-blooded animals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, birds, cats, dogs, monkeys, etc.). Can be administered to. The dose of the compound or its salt varies depending on its action, target disease, administration target, administration route, etc., but in general, in adults (assuming a body weight of 60 kg), the compound is about 0.1 to 100 mg per day. , Preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose of the compound varies depending on the subject, target disease, etc., but when administered in the form of an injection to an adult (as 60 kg), the compound is usually administered per day. It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered. Screening of drug candidate compounds for diseases: Since the proteins of the present invention have cytotoxic activity, compounds that promote the functions of the proteins of the present invention (eg, cytotoxic activity, etc.) or salts thereof are, for example, therapeutic agents for cancer. On the other hand, the compound that inhibits the function of the protein or the like of the present invention or a salt thereof can be used as a medicine for treating / preventing gastric inflammation and gastric ulcer, for example. Therefore, the protein or the like of the present invention is useful as a reagent for screening a compound or a salt thereof that promotes or inhibits the function of the protein or the like of the present invention. That is, the present invention comprises (1) the function of the protein of the present invention or a partial peptide thereof or a salt thereof (for example, cytotoxic activity), which is characterized by using the protein of the present invention or a partial peptide thereof or a salt thereof. The function of a promoting compound or a salt thereof (sometimes abbreviated as a promoter in "(3) Screening of drug candidate compounds for diseases"), or a protein of the present invention or a partial peptide thereof or a salt thereof (for example, cytotoxic activity). Screening method for compounds that inhibit (such as) (may be abbreviated as inhibitor in "(3) Screening of drug candidate compounds for diseases"); A screening kit for an accelerator or an inhibitor, which comprises the protein of the present invention or a partial peptide thereof or a salt thereof (in "(3) Screening of a drug candidate compound for a disease", the screening kit of the present invention More specifically, for example, (2) (i) a protein of the present invention or a partial peptide thereof or a salt thereof and a cell (eg, various warm-blooded animal tissues described above (preferably human)) are provided. When contacted with normal cells containing blood cell cells derived from (such as) or the above-mentioned cancer cells) and (ii) cells (eg, the above-mentioned various warm-blooded animal tissues (eg, the above-mentioned various warm-blooded animal tissues) to the protein of the present invention or a partial peptide thereof or a salt thereof A method for screening an accelerator or an inhibitor, which comprises contacting normal cells containing blood cell cells derived from humans or the like or the above-mentioned cancer cells) with a test compound; the present invention. It is characterized by containing a protein or a partial peptide thereof or a salt thereof and cells (eg, normal cells including blood cells derived from various warm-blooded animal tissues (preferably humans) described above or cancer cells described above). Kits for screening for accelerators or inhibitors are provided. Specifically, the screening method is characterized in that, for example, the cytotoxic activity of the protein or the like of the present invention in the cases (i) and (ii) is measured and compared. The cytotoxic activity, cell growth inhibitory activity, or cell death-inducing activity of the protein of the present invention can be measured according to a method known per se or a method similar thereto. However, more specifically, using an established cell line or the like, all three of the substrate containing the test compound, the negative control which is the substrate not containing the test compound, and the positive control as the substrate containing the M toxin. , Or by a method using two of these, the cell number is compared under conditions that can satisfy statistical significance, and in the presence or absence of these activities, or in the increase or decrease, the inhibitory activity of cytotoxic activity or cell proliferation activity Alternatively, a specific sample having an inhibitory activity on cell proliferation activity or cytotoxic activity can be detected. Examples of the cells to be used include normal cells including blood cell cells derived from the above-mentioned various warm-blooded animal tissues (preferably humans, etc.) or the above-mentioned various warm-blooded animal cancer cells (eg, uterine body cancer, endometrial tumor, breast cancer). , Gastric cancer, liver cancer, pancreatic cancer, bile sac cancer, colon cancer, prostate cancer, lung cancer, kidney cancer, neuroblastoma, bladder cancer, malignant melanoma, tongue cancer, gingival cancer, mouse fibroblasts, green monkey kidney cells, rats Liver cancer, etc.) is used. Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., even if these compounds are novel compounds. It may be a known compound. To carry out the above screening method, a preparation of the protein or the like of the present invention is prepared by suspending the protein or the like of the present invention in a buffer suitable for screening. The buffer can be any buffer that does not inhibit the reaction between the protein of the present invention and the test compound, such as a phosphate buffer having a pH of about 4 to 10 (preferably a pH of about 6 to 8) and a Tris-hydrochloric acid buffer. It may be. Specific screening methods include 1 a method of directly observing cell changes under a microscope and counting using a hemocytometer, etc., 2 potassium, hemoglobin, etc. that are eluted from the cells into the solution due to cell death. 3 Method of measuring the number of viable cells remaining after the reaction using tetrazolium salt, etc., 4 Method of measuring the number of viable cells remaining after the reaction, 5 Cell Examples thereof include a method of confirming cell death by inducing apoptosis in the cell. For example, a test compound that increases the cytotoxic activity in the case of (ii) above by about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case of (i) above. On the other hand, the cytotoxic activity in the case of (ii) above is about 20% or more, preferably 30% or more, more preferably about 50, as compared with the case of (i) above. A test compound that inhibits% or more can be selected as a compound that inhibits cytotoxic activity such as the protein of the present invention. These can also be performed as a wide variety of large-scale screening methods. Below, using the hemoglobin measurement method by hemolytic reaction of erythrocytes as 2 and the WST method as 3 , activated carbon, CM cellulose, and calcium alginate were selected as compounds showing anti-M toxin action as adsorbents. Shown. It is also possible to examine and compare solutions containing these negative controls, positive controls and test compounds in animal models to confirm the effects of anti-M toxin substances at the animal level, in which case a widespread warm-blooded animal It can be used. In particular, mice, rats, dogs, and monkeys are used, but gerbils, mice, and monkeys are useful as infection models. When the compound obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be carried out according to conventional means. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used in the same manner as the above-mentioned pharmaceuticals containing the protein of the present invention. The formulations thus obtained are safe and low toxicity, and therefore, for example, in humans or warm-blooded animals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, birds, cats, dogs, monkeys, etc.). Can be administered to. The dose of the compound or a salt thereof varies depending on its action, target disease, administration target, administration route, etc., but for example, it promotes the function of the protein of the present invention as a tissue regenerating agent after removal of diseased tissue. When the compound is orally administered, generally, in an adult (assuming a body weight of 60 kg), the compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day. In the case of other animals, the amount converted per 60 kg can be administered. Quantification of the protein of the present invention or a partial peptide thereof or a salt thereof: An antibody against the protein or the like of the present invention (hereinafter, may be abbreviated as the antibody of the present invention) can specifically recognize the protein or the like of the present invention. Therefore, it can be used for quantification of the protein or the like of the present invention in the test solution, particularly quantification by the sandwich immunoassay method. That is, in the present invention, (i) the antibody of the present invention is competitively reacted with the test solution and the labeled protein of the present invention, and the labeled protein of the present invention is bound to the antibody. A method for quantifying a protein or the like of the present invention in a test solution, which comprises measuring the ratio of the above, and (ii) an antibody of the present invention insolubilized on the test solution and a carrier, and a label of the present invention. Provided is a method for quantifying a protein or the like of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after simultaneously or continuously reacting with the antibody of the above. In the quantification method of (ii) above, one antibody is an antibody that recognizes the N-terminal portion of the protein or the like of the present invention, and the other antibody is an antibody that reacts with the C-terminal portion of the protein or the like of the present invention. desirable. Further, the monoclonal antibody against the protein or the like of the present invention (hereinafter, may be referred to as the monoclonal antibody of the present invention) can be used to quantify the protein or the like of the present invention, or can be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, or the F (ab') 2, Fab', or Fab fraction of the antibody molecule may be used. The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and the antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (for example, the amount of protein) in the test solution is not particularly limited. Any measurement method may be used as long as it is a measurement method in which the amount of the antigen is detected by chemical or physical means and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, the nephrometry, the competitive method, the immunometric method and the sandwich method are preferably used, but the sandwich method described later is particularly preferable in terms of sensitivity and specificity. As the labeling agent used in the measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes include, for example, [¹²⁵I], [¹³¹I], [³H], [¹⁴C] etc. are used. The enzyme is preferably stable and has a large specific activity, and for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescamine isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding the antibody or antigen to the labeling agent. In insolubilizing an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing a protein or enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicon, and glass. In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and another labeled monoclonal antibody of the present invention is reacted (secondary reaction), and then on the insolubilized carrier. The amount of the protein of the present invention in the test solution can be quantified by measuring the activity of the labeling agent. The primary reaction and the secondary reaction may be carried out in the reverse order, at the same time, or at different times. Labeling agents and insolubilization methods can be similar to those described above. Further, in the immunoassay method by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody does not necessarily have to be one type, and a mixture of two or more types of antibodies is used for the purpose of improving the measurement sensitivity. You may. In the method for measuring a protein or the like of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used for the primary reaction and the secondary reaction is preferably an antibody having a different binding site such as the protein of the present invention. Be done. That is, as for the antibody used in the primary reaction and the secondary reaction, for example, when the antibody used in the secondary reaction recognizes the C-terminal portion of the protein of the present invention, the antibody used in the primary reaction is preferable. An antibody that recognizes an N-terminal other than the C-terminal is used. The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competitive method, an immunometric method, or nephrometry. In the competitive method, the antigen in the test solution and the labeled antigen are competitively reacted with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), measure the labeled amount of either B or F, and quantify the amount of antigen in the test solution. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the above antibody, and a immobilized antibody is used as the first antibody, or A soluble antibody is used as the first antibody, and a solid phase method using a solid phase antibody as the second antibody is used. In the immunometric method, the antigen in the test solution and the immobilized antigen are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test solution is separated. Is reacted with an excess amount of the labeled antibody, then a immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the labeled amount of any phase is measured to quantify the amount of antigen in the test solution. In nephrometry, the amount of insoluble sediment produced as a result of the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry or the like utilizing laser scattering is preferably used. In applying these individual immunological measurement methods to the quantification method of the present invention, it is not necessary to set special conditions, operations, or the like. The protein and the like measurement system of the present invention may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, review articles, books, etc. can be referred to. For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Continued Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1979), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (Published in 1993), Eiji Ishikawa et al., "Enzyme immunoassay" (2nd edition) (Medical Shoin, published in 1982), Eiji Ishikawa et al., "Enzyme immunoassay" (3rd edition) (Medical Shoin, Showa) Published in 1987), "Methods in ENZY MOLOGY" Vol.70 (Immunochemical Techniques (Part A)), the same book Vol.73 (Immunochemical Techniques (Part B)), the same book Vol.74 (Immunochemical Techniques (Part C)), the same book Vol. .84 (Immunochemical Techniques (Part D: Selected Immunoassays)), the same book Vol.92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), the same book Vol.121 (Immunochemical Techniques (Part I:)) Hybridoma Technology and Monoclonal Antibodies)) (above, published by Academic Press), etc. can be referred to. As described above, by using the antibody of the present invention, the protein or the like of the present invention can be quantified with high sensitivity. Furthermore, when an increase in the concentration of the protein or the like of the present invention is detected in a person infected with Helicobacter pylori by quantifying the concentration of the protein or the like of the present invention using the antibody of the present invention, for example, gastric inflammation, gastric ulcer, gastric cancer. , Cardiac valvular disease, diabetes, various cancers (eg, uterine body cancer, endometrial tumor, breast cancer, colon cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, bile sac cancer, kidney cancer, neuroblastoma, bladder cancer, It can be diagnosed as having a disease such as melanoma) or having a high possibility of developing it in the future. In addition, the antibody of the present invention can be used to detect a protein or the like of the present invention present in a subject such as a body fluid or tissue. In addition, preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction during purification, analysis of the behavior of the protein of the present invention in a test cell, etc. Can be used for. Drugs Containing the Antibody of the Present Invention The antibody of the present invention (neutralizing antibody) having an action of neutralizing the activity of the protein of the present invention is, for example, gastric inflammation, gastric ulcer, gastric cancer, cardiovalvular disease, diabetes, various cancers ( For example, use as a drug for treating / preventing diseases such as endometrial cancer, endometrial tumor, breast cancer, colon cancer, prostate cancer, lung cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.) Can be done. The humanized antibody of the present invention against the protein of the present invention includes, for example, gastric inflammation, gastric ulcer, gastric cancer, valvular heart disease, diabetes, various cancers (eg, endometrial cancer, endometrial tumor, breast cancer, colon cancer, prostate cancer, etc. It can be used as a medicine for treating / preventing diseases such as lung cancer, kidney cancer, neuroblastoma, bladder cancer, melanoma, etc.). The humanized antibody was prepared according to the method described in Nat Biotechnol, 14,845-851. (1996), Nat Genet.15,146-156. (1997), PNAS, 97 (2), 722-727 (2000) and the like. can do. Hereinafter, in "(5) Drug containing the antibody of the present invention", the neutralizing antibody and the humanized antibody of the present invention are collectively referred to as the antibody of the present invention. The therapeutic / prophylactic agent for the above-mentioned diseases containing the antibody of the present invention can be used as it is as a liquid preparation or as a pharmaceutical composition having an appropriate dosage form, such as humans or mammals (eg, rats, rabbits, sheep, pigs, cows, cats, etc.). It can be administered orally or parenterally to dogs, monkeys, etc.). The dose varies depending on the administration target, target disease, symptom, administration route, etc., but for example, when used for the treatment / prevention of an adult endometrial tumor patient, a single dose of the antibody of the present invention is used. Usually, it is about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, and more preferably 0. It is convenient to administer about 11 to 5 mg / kg body weight by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibodies of the invention can be administered by themselves or as suitable pharmaceutical compositions. The pharmaceutical composition used for the above administration comprises the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration. That is, for example, the composition for oral administration includes solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, and capsules (soft capsules). Included), syrups, emulsions, suspensions, etc. Such compositions are produced by methods known per se and contain carriers, diluents or excipients commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets. The sequence numbers in the sequence listing described later indicate the following sequences. [SEQ ID NO: 1] The amino acid sequence of the protein (M toxin) derived from Helicobacter pylori 60190 of the present invention is shown. [SEQ ID NO: 2] The nucleotide sequence of DNA encoding the Helicobacter pylori 60190-derived protein (M toxin) of the present invention having the amino acid sequence represented by SEQ ID NO: 1 is shown. [SEQ ID NO: 3] The base sequence of the primer (synthetic) DNA used in Example 3 is shown. [SEQ ID NO: 4] The base sequence of the primer (synthetic) DNA used in Example 3 is shown. The transformant Escherichia coli M toxin / pET30EK / LIC / DH5α obtained in Example 3 described later has been available from October 17, 2002, 1-1-1, Higashi, Tsukuba-shi, Ibaraki, Japan, Central 6th. It has been deposited at the National Institute of Advanced Industrial Science and Technology (IPOD) Patent Organism Depositary Center under the deposit number FERM BP-8218. In addition, the hybridoma clone No. 4 obtained in Example 4 described later was designated as BALB-c / P3U1 / 004-1G9 from October 23, 2002, 1-1-1, Higashi, Tsukuba City, Ibaraki Prefecture, Japan. It has been deposited under the deposit number FERM BP-8222 at the National Institute of Advanced Industrial Science and Technology (IPOD) Patent Organism Depositary, Central No. 6. In addition, hybridoma clone No. 101 is BALB-c / P3U1 / 101-1C10, and from October 23, 2002, 1-1-1, Higashi, Tsukuba-shi, Japan, Central 6th National Institute of Advanced Industrial Science and Technology It has been deposited at the National Institute of Advanced Industrial Science and Technology (IPOD) Patent Organism Depositary as deposit number FERM BP-8223. In addition, hybridoma clone No. 116 is BALB-c / P3U1 / 116-5D7, and from October 23, 2002, 1-1-1, Higashi, Tsukuba-shi, Japan, Central 6th National Institute of Advanced Industrial Science and Technology It has been deposited at the National Institute of Advanced Industrial Science and Technology (IPOD) Patent Organism Depositary as deposit number FERM BP-8224. Example Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. The gene manipulation method using Escherichia coli followed the method described in Molecular Cloning. Example 1 A method for purifying and extracting the toxin of the present invention from Helicobacter pylori. Helicobacter pylori can be obtained from already established isolates (eg, from the American Type Culture Collection) or isolated from clinical specimens. In this example, a isolate of Helicobacter pylori 60190 was used. This isolate was first subjected to microaerophile conditions at a temperature of 37 ° C and a humidity of 90% or more using an agar medium obtained by adding 5% bovine serum (manufactured by Sigma) to a Brain Heart Infusion agar medium (manufactured by Difco). (CO₂2-5 passages (about 1-2 weeks) were cultured in 5-10%). After confirming that it was not dead or cocoidized under a microscope and confirmed good growth, 5% 2,6-di-O-methyl- was added to Brain Heart Infusion agar medium (manufactured by Difco) without adding serum. It was transplanted to the one supplemented with β-cyclodextrin. After confirming the culture and growth condition under the same conditions as described above, the cells are transplanted to those having 2,6-di-O-methyl-β-cyclodextrin concentrations gradually adjusted to 2%, 1%, and 0.5%. Then, after shaking culture in Brain Heart Infusion liquid medium containing 0.5% 2,6-di-O-methyl-β-cyclodextrin at a temperature of 37 ° C and microaerobic conditions at 100-120 rpm for about 16 hours. , 10000 × g, centrifuged for 20 minutes to obtain pellet-shaped cells. This is 10 mM Tris-HCl After ultrasonically crushing in a buffer solution of pH 7.7 (hereinafter referred to as A buffer solution. Since the target protein is pI 6.08, the pH may be about pH 6.1 or higher) and storing at -80 ° C for 24 hours. Again, the ultrasonically crushed material was ultracentrifuged at 100,000 × g for 60 minutes, and only the uppermost layer separated into three layers was extracted. This extract was crudely purified with 70% ammonium sulfate. The resulting extract was subjected to ion exchange chromatography (DEAE Sephacel of Amersham Pharmacia Biotech AB) using an anion exchange resin having beads having a relatively large particle size. The equilibrium buffer was A buffer, and the eluate was A buffer plus 0.3 M salt NaCl. Concentration gradient extraction was performed using these, and an appropriate amount of each fraction was added dropwise to the wells in which HeLa cells were planted to evaluate the viability of the cells in each well. Evaluation is WST Reagent Co., Ltd. Dojin Chemical Research Institute Cell Counting Kit) was used. A fraction showing a significantly lower survival rate than the control and a fraction having a relatively consistent increase curve in protein content are evaluated together with the results of electrophoresis and used in the next purification step. It was used as a sample fraction. Next, hydrophobic chromatography (Phenyl Sepharose CL-4B from Amersham Pharmacia Biotech AB), which is a different chromatography of the separation system from the next time, was selected. As the equilibrium buffer solution, a 10 mM phosphate buffer solution containing 1 M ammonium sulfate was used, and as the elution buffer solution, 40% ethylene glycol was used as a 10 mM phosphate buffer solution. After elution with a concentration gradient, each sample was evaluated in the same manner as described above. Furthermore, the sample extracted from the above step is again subjected to anion exchange chromatography (RESOURCE of Amersham Pharmacia Biotech AB) having beads having a relatively small particle size. Extracted in Q). The equilibrium buffer was A buffer, and the elution buffer was A buffer plus 1M salt NaCl. From these, a band of a single protein having a molecular weight of about 41000 was finally obtained. The type and order of chromatography can be different from those described above, and can be further added. The obtained single band was stained with Coomassie Brilliant Blue, transferred to a nitrocellulose membrane or a (polyvinylidene difluoride) membrane by a blotting device, and analyzed by an amino acid sequencer. As a result, as described above, it was in agreement with the N-terminal amino acid sequence of the locus HP1037 of the registration database (Helicobacter pylori 22695) at a ratio of 95% (19 bases out of 20 bases). (Figure 1) Example 2 Amino acid sequence and DNA sequence by genetic engineering adjustment method In this example, an isolate of Helicobacter pylori 60190 was used, but as described above, all gene analysis of Helicobacter pylori 22695 with different strains has already been performed, and TIGR (The Institute for Genomic) has already been performed. Homological loci can be inferred by searching the Research) database. This revealed that the locus HP1037 of Helicobacter pylori 22695 encodes a homologous protein. Based on this, the toxin of the present invention was cloned. That is, using Helicobacter pylori 60190 as a template, first, for convenience, multiple sets (5'side and 3'side) of appropriate primers based on locus HP1037, upstream locus HP1036 and downstream locus HP1038 were prepared. Then the sequence was performed. The DNA polymerase used is a proofreading one with a proofreading function, and each primer set is configured to sufficiently include each other's primer portions, and the sequence is performed multiple times from the 5'side and the 3'side. I did. The DNA sequence thus obtained is shown in SEQ ID NO: 2 in the sequence listing. Moreover, this amino acid sequence is shown in SEQ ID NO: 1. Example 3 Expression experiment of toxin protein by genetic recombination, Escherichia coli was used for the gene recombination experiment. The pET-30EK / LIC vector (Novagen) was used as the vector, and BL21 (DE3) was used as the expression Escherichia coli. The inserted gene encodes the toxin protein derived from Helicobacter pylori 60190 cloned in Example 2, using SEQ ID NO: 3 with GACGACGACAAG added to the 5'side of the sense strand as a primer, and the 5'side of the antisense strand. Was prepared by PCR using SEQ ID NO: 4 to which GAGGAGAAGCCCGGTTA was added as a primer. The prepared inserted gene was scraped by T4 DNA polymerase in the presence of 25 mM dATP and 100 mM DTT to match the LIC site of the vector, and then heated in the presence of 25 mM EDTA. The prepared recombinant was sequenced again and confirmed to be identical to SEQ ID NO: 1, then transformed into Escherichia coli BL21 (DE3) for expression and cultured in LB medium containing 30 μg / ml kanamycin. OD₆₀₀Incubate under shaking at 37 ° C. and 250 rpm until the concentration reaches about 0.4, add isopropyl-beta-thiogalactoside to a final concentration of 1 mM, and shake for another 2 hours. Since the fusion protein formed an inclusion body, Escherichia coli was centrifuged from this culture solution, and an inclusion body of the protein was obtained by BugBuster reagent and Benzonase Nuclease (both by Novagen). The protein was separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and silver stained to identify a single applicable band before refolding to healer cells and other warm-blooded animal cells. Using WST reagent (Cell Counting Kit, Dojin Chemical Laboratory Co., Ltd.) was used for comparison with the control. As a result, a significant difference was observed in the survival rate, and the expression protein also showed the same activity as the purified protein. Furthermore, it was confirmed that it has similar activity not only in HeLa cells, which are human cervical cancer, but also in normal human gastric cells. (Fig. 2) It was also confirmed that mammalian cells generally have a wide range of activity as well as other human tissues. (Fig. 3, Fig. 4) Example 4 Preparation of monoclonal anti-M toxin antibody: 240 μg of BALB / C mouse, immunogenic refolded expressed M toxin, was subcutaneously administered twice in several places. The spleen was removed from the mouse 4 days after the final immunization, and the spleen was compressed and filtered through a stainless mesh and suspended in Eagles Minimum Essential Medium (MEM) to obtain a pancreatic cell suspension. As cells used for cell fusion, BALB / C mouse-derived myeloma cells P3-X63.Ag 8. Using U1 (P3U1) [Current Topics in Microbiology and Immunology, 81, 1 (1978)]. Cell fusion was performed according to the original method [Nature, 256, 495 (1975)]. That is, the spleen cells and P3U1 were washed three times with a serum-free MEM, the spleen cells and the P3U1 number ratio were mixed so as to be 6.6: 1, and the cells were precipitated by centrifugation at 750 rpm for 15 minutes. It was. After sufficiently removing the supernatant, loosen the precipitate lightly, add 0.3 ml of 45% polyethylene glycol (PEG) 6000 (Wako Pure Chemical Industries, Ltd.), and let stand in a 37 ° C warm water tank for 7 minutes for fusion. Was performed. After fusion, MEM was gradually added to the cells, a total of 15 ml of MEM was added, and the cells were centrifuged at 750 rpm for 15 minutes to remove the supernatant. This cell precipitate is added to GIT medium (Wako Junyaku Co., Ltd.) (GIT-10% FCS) containing 10% fetal bovine serum with 1 ml of P3U1 2 × 10⁵Floating so as to be individual, 1 well 1 ml was sown in 168 wells in a 24-hole multi-dish (manufactured by Iwaki Co., Ltd.). After seeding, cells were cultured at 37 ° C in a 5% carbon dioxide incubator. 24 hours later HAT (hypoxanthine 1 × 10)^-4M, Amino Pterin 4 × 10^-7M, thymidine 1.6 × 10^-3HAT selective culture was started by adding 1 ml of GIT-10% FCS medium (HAT medium) containing M) per well. HAT selective culture was continued by discarding 1 ml of the old solution 4 to 7 days after the start of the culture and then adding 1 ml of HAT medium. Hybridoma growth was observed 9 days after cell fusion, and the supernatant was collected. The antibody titer in the culture supernatant was measured as follows. That is, 100 μl of the culture supernatant and 100 μl of HRP-labeled M toxin diluted 200-fold with buffer C were added to each well of the anti-mouse immunoglobulin antibody-bound microplate, and the mixture was reacted overnight at 4 ° C. After washing the plate with PBS, in order to prepare an anti-mouse immunoglobulin antibody-bound microplate, first, 0.1 M carbonate buffer containing 100 μg / ml of goat anti-mouse immunoglobulin antibody (IgG fraction, manufactured by Dako), pH 9 The .6 solution was dispensed into 96-well microplates in 100 μl increments and left at 4 ° C for 24 hours. Next, the plate was washed with phosphate buffered saline (PBS, pH 7.4), followed by 25% Block Ace (Snow Brand Milk Products) and 0.1% NaN to block the excess binding sites in the wells.₃300 μl of PBS containing, pH 7.2 was dispensed and treated at 4 ° C. for at least 24 hours. Buffer EC [0.2% BSA, 0.4M NaCl, 0.4% Block Ace, 0.05% CHAPS [3-[(colamidpropyl) dimethylammonio] propanesulfonic acid] in each well of the above anti-mouse immunoglobulin antibody-bound microplate. , 2 mM EDTA and 0.1% NaN₃A 0.02 M phosphate buffer containing 100 μl of mouse antiserum diluted with pH 7.0] was added, and the mixture was reacted at 4 ° C for 16 hours. Next, the plate was washed with PBS and pH 7.4, 100 μl of HRP-labeled TGC-839 peptide (26-34) was added, and the mixture was reacted at room temperature for 7 hours. The HRP-labeled refolding toxin protein prepared in Example 3 above was prepared 100-fold diluted with 0.02 M phosphate buffer containing buffer C [1% BSA, 0.4 M NaCl, and 2 mM EDTA, PH 7.0]. .. Next, after washing the plate with PBS and pH 7.4, the enzyme activity on the solid phase was determined by the TMB microwelper oxidase substrate system (KIRKEGAARD &amp; PERRY). LAB. Funakoshi Chemicals Handling) 100 μl was added and the reaction was carried out at room temperature for 10 minutes. After stopping the reaction by adding 100 μl of 1 M phosphoric acid, the absorbance at 450 nm (Abs.450) was measured with a plate reader (MTP-120, manufactured by Corona Publishing Co., Ltd.). The enzyme activity on the solid phase was measured according to these methods. As a result, 18 wells having antibody titers were selected from 123 wells, and hybridomas were cryopreserved. Furthermore, 6-well hybridomas, No. 4, No. 53, No. 61, No. 76, No. 101 and No. 116, were cloned by the dilution method. Upon cloning, BALB / C mouse thymocytes were used as feeder cells in a well 5 × 10⁵Added to be individual. After cloning, the antibody titer in the culture supernatant was measured according to the same method. The positive clones were No. 4, No. 101 and No. 116. These were designated as antibody-producing hybridomas of the expressed M toxin. Example 5 Determination of monoclonal antibody class / subclass: Anti-rabbit IgG antibody-bound microplates were prepared according to the method described in Example 4. That is, 100 μl of 0.1 M carbonate buffer containing 100 μg / ml of goat anti-rabbit immunoglobulin antibody (IgG fraction, manufactured by Dako) and pH 9.6 solution were dispensed into 96-well microplates at 4 ° C for 24 hours. I left it. Next, the plate was washed with phosphate buffered saline (PBS, pH 7.4), followed by 25% Block Ace (Snow Brand Milk Products) and 0.1% NaN to block the excess binding sites in the wells.₃300 μl of PBS containing, pH 7.2 was dispensed and treated at 4 ° C. for at least 24 hours. Next, 50 μl of buffer EC and 100 μl of the subtype-specific antibody included in the Isotype typing kit manufactured by Biorad were added to the anti-rabbit IgG antibody-bound microplate and reacted at 4 ° C for 1 day. After washing the plate with PBS and pH 7.4, the above hybridoma culture supernatant was added, and the mixture was reacted at 4 ° C for 1 day. The plate was washed with PBS pH 7.4, 100 μl of the HRP-labeled refolding toxin protein prepared in Example 3 above diluted 100-fold with buffer C was added, and the mixture was reacted at room temperature for 6 hours. After washing the plate with PBS and pH 7.4, the enzyme activity on the solid phase was measured according to the method described in Example 4. As a result, the classes and subclasses of the monoclonal antibodies produced by these hybridomas were No. 4 (IgG1), No. 101 (IgG2b), and No. 116 (IgG2a). Example 6 Mouse ascites of hybridomas: Mouse ascites was performed on hybridomas, No. 4, No. 101 and No. 116. 1 ~ 3 × 10 to mice (BALB / C, female) intraperitoneally administered 0.5 ml of mineral oil in advance⁶After intraperitoneal administration of the above hybridoma cells / animal, antibody-containing ascites was collected 6 to 20 days later. The monoclonal antibody was repurified from the obtained ascites with a reprotein-A column. That is, about 25 ml of ascites was added to an equal volume of binding buffer (3.5 M NaCl, 0.05% NaN).₃Dilute with 1.5 M glycine containing, pH 9.0) and then apply to a recombinant protein-A-agarose (Repligen) column pre-equilibrium with binding buffer to give the specific antibody to elution buffer (0.05% NaN).₃Elute with 0.1 M citrate buffer containing pH 3.0). The eluate was dialyzed against PBS and pH 7.4 at 4 ° C for 2 days, sterilized and filtered through a 0.22 μm filter (manufactured by Millipore), and stored at 4 ° C or -80 ° C. Example 7 Preparation of polyclonal anti-M toxin antibody 4.1 mg of the cytotoxic protein M toxin of the present invention was mixed with Freund's complete adjuvant and subcutaneously immunized to rabbits, and one week later, the same amount was further mixed with Freund's incomplete adjuvant and subcutaneously immunized. After immunization, the collected blood was centrifuged to remove blood cell components to obtain antiserum. Example 8 Analysis of M toxin by Western Blotting: SDS-Sample Buffer containing 2-mercaptoethanol was added to the culture supernatant obtained in Example 3 and electrophoresed on Peptide-PAGE (TEFCO), which was electrically transferred to a PVDF membrane (Amersham). As the primary antibody, each antibody (2 μg / ml) obtained in Example 4 and Example 6 was used, and as the secondary antibody, HRP (Horseradish peroxidase) -labeled anti-rabbit and anti-mouse IgG antibody (2000-fold dilution; Dako) was used. Using. Color development was performed using the ECL Western Blot Detection System (Amersham). As a result, it was confirmed that each primary antibody recognizes the expressed protein. (Fig. 5) Example 9 The column was filled with 0.5 ml each of activated carbon (diameter 0.2 to 0.1 mm), CM cellulose, and calcium alginate, and the Tris buffer solution was 10 mM. After equilibration at pH 7.7, the columns were closed with 0.5 ml of M toxin 400 nM having refolding activity added to each column, and the cells were allowed to stand at 25 ° C for 60 minutes. During that time, only the Tris buffer solution and the Tris buffer solution containing the same M toxin were allowed to stand under the same conditions. Then, all the columns were opened, and the drops of 100 μl each were collected, and 0.5 ml of Tris buffer was further added to each column to collect 100 μl each. For erythrocytes, the whole blood of a healthy adult was centrifuged 2-3 times at 800 × g for 10 minutes until the supernatant became clear, and 10 μl of the precipitated erythrocytes was added to 990 μl of 10 mM Tris buffer as a positive control. A negative control was obtained by adding 10 μl to 0.9% saline 10 mM Tris buffer, and the sample was compared with a sample prepared from the above column containing 1/100 volume of erythrocytes. The comparison was made by measuring the relative concentration of hemoglobin eluted with a multiple plate reader (Biorad) with an absorbance of 415 nm. (Fig. 6) Example 10 HeLa cells (human cervical cancer cells) were exposed to the fractions from each column obtained in Example 9, and the cell viability was measured by the WST method, which is one of the measurement methods using a tetrazolium salt. HeLa cells are cultured in 96-well plates for 24 hours, 10,000 per well. Add to each hole of the negative control of the culture solution only and each test solution, and the positive control including the concentration of 20 nM, and incubate at 37 ° C for 12 hours. Then, using the Cell Counting Kit (Dojin Research Institute Co., Ltd.), the cell viability detected by the WST method was measured by the absorbance at the wavelength of 415 nm. (Fig. 7, Fig. 8) Possibility of industrial use The protein of the present invention or a partial peptide thereof can be used, for example, as a therapeutic agent for cancer. In addition, the antibody of the present invention can be used for identification of the protein of the present invention in blood, tissue, urine, and stool collected from a test patient, and for proof of infection by Helicobacter pylori. It can also be used for quantification of the protein of the present invention. In addition, the proteins of the invention are useful as reagents for screening compounds that promote or inhibit the activity of the proteins of the invention. [Sequence list]<img file="JP4776166B2_D0001.tif" /><img file="JP4776166B2_D0002.tif" /><img file="JP4776166B2_D0003.tif" /><img file="JP4776166B2_D0004.tif" />[Simple explanation of drawings] FIG. 1 shows the results of anion chromatography of the sample obtained in Example 1. a is the activity of M toxin in each fraction and the absorbance of its eluted protein. b is SDS-PAGE by silver staining of fractions 16 to 22 during the chromatography of a. c is a band of protein toxins transferred to the PVDF membrane by electroblotting. d and e show the phenotypic changes in HeLa cells after 24 hours of control and 1 nM extracted cytotoxic solution. The scale is 50 μm. FIG. 2 shows the changes in cell morphology due to the recombinant toxin obtained in Example 3. a is 6 hours after negative control of HeLa cells. b and c are HeLa cells 3 hours and 6 hours after the addition of recombinant M toxin 5nM. d is CRL7407 (ATCC) fetal normal gastric cells 6 hours after negative control. e and f are CRL7407 cells 3 hours and 6 hours after the addition of recombinant M toxin 5nM. FIG. 3 shows the susceptibility of various cancer cells to M toxin in Example 3. It was measured using the WST method. The X-axis shows the concentration of M toxin in the substrate, and the Y-axis shows the absorbance by light with a wavelength of 415 nm directly or as the phase ratio when the negative control is 100%. HLF: Rat liver cancer, colon26: Mouse colorectal cancer. Similar to FIG. 3, FIG. 4 shows T24: human bladder cancer, OVK18: human ovarian cancer, KLM-1: human pancreatic cancer, A-549: human lung cancer, Ca9-22: human gingival cancer, and CRL1500: human breast cancer. FIG. 5 shows Western blotting of the monoclonal antibody performed in Example 8. The dilution ratio of the culture supernatant of all hybridoma cells is 20 times. FIG. 6 shows the M toxin activity when calcium alginate was used as an adsorbent in Example 9. The negative control contains only 10 mM Tris pH 7.7 as a substrate, and the positive control contains a substrate and M toxin 10 mM. The X-axis is each fraction that passes through the calcium alginate column. The M toxin concentration in each fraction averages 10 nM, one of which exceeds at least 10 nM. When erythrocyte destruction occurs, the hemoglobin concentration in the solution rises, showing its 415 nm absorbance on the Y-axis. FIG. 7 shows the% survival rate of activated carbon showing M toxin inhibitory activity due to the adsorptive action obtained in Example 10 in HeLa cells. The X-axis shows the eluate of each fraction, with an average M toxin concentration of 10 nM, one of which exceeds at least 10 nM. The Y-axis is a percentage of the solution of each fraction measured by the WST method divided by a positive control. Similar to FIG. 7, FIG. 8 shows the% survival rate of calcium alginate and CM-cellulose showing M toxin inhibitory activity in HeLa cells by the adsorption action obtained in Example 10.