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Cytotonic protein and utlization thereof

Cytotonic protein and utlization thereof description/claims

The present application is a divisional of application Ser. No. 10/496,384, which is the national stage under 35 U.S.C. 371 of PCT/JP02/12752, filed Dec. 5, 2002, which claims priority from JP 2001-371210, filed Dec. 5, 2001. The entire contents of prior application Ser. No. 10/496,384 and PCT/JP02/12752 are herein incorporated by reference.

This invention relates to a new cytotoxic protein (M toxin, mucous layer devastating toxin) produced by Helicobacter pylori and the use.

It has been considered that the human gets many gastritis, gastric ulcer and gastric cancer by Helicobacter pylori. A distinct direct cytotoxic factor causing destruction of gastric epithelial cells and irreversible cell death at the beginning of these diseases has not been specified. A factor changing pH environment and immunity reaction in the stomach, a factor adhering to gastric epithelial cells by Helicobacter pylori, or move properties of bacteria themselves has been indicated as a factor developing such diseases. However, it has been unclear hitherto that gastric mucosal destruction triggering gastritis, gastric ulcer and gastric cancer is injured in any process, or what is a direct responsible factor to the gastric mucosal destruction. The only is that vacuolating toxin having cytotoxicity is isolated, but it has week cytotoxic activity and reversible cytotoxic activity. As a fatal cytotoxic factor of a pathogenic factor, it has not been found in vivo and in vitro.

Many researchers estimate that Helicobacter pylori, as described in the above, in the environment stomach in vivo, secretes a direct cytotoxic factor for gastric mucosal cells. Considering the importance of diseases, all sequences of the gene is confirmed in 1996. However, in spite of the use of serum, it is impossible to isolate and identify the presumable toxin due to separation conditions, with difficult culture conditions and purification conditions and unfixed valuation systems of the toxin.

One problem is that responsible protein causing gastritis, gastric ulcer and gastric cancer by Helicobacter pylori infection is found, a mass production method of the toxic protein is established, new M toxin is identified, and diagnosis and screening method is established. Using these methods, it is desired that responsible toxin of a cytotoxic factor for gastric mucosal cells is controlled, and preventive and treating agents of gastritis, gastric ulcer, gastric cancer and the like are developed and a method for using the toxin is found.

Inventors of this invention have studied earnestly to resolve the above-mentioned problem, and identified a new toxin that causes irreversible cell death when Helicobacter pylori is cultured under serum-free conditions that is similar to environment in the stomach different from usual. It has been found that this toxin has a toxic power 1000 to 100000 times per unit of above-mentioned vacuolating toxin, and it causes irreversible cell death for various warm-blooded animal cells comprising not only gastric epithelial cells but also immunocytes. The inventors of this invention have repeatedly researched on these views to attain to the present invention.

Namely, this invention provides the following. (1) A cytotoxic protein comprising a protein having at least 70% or more identity for the amino acid sequence represented by SEQ ID No. 1. (2) A partial peptide of the cytotoxic protein described in claim 1, characterized in that the protein has the same cytotoxic activity as that of the amino acid sequence represented by SEQ ID No. 1. (3) The cytotoxic protein of claim 1 or claim 2 wherein the protein is produced with Helicobacter pylori. (4) The cytotoxin protein of claim 1 or claim 2 wherein the protein is obtained by culturing a transformant which is transformed with a recombination vector containing DNA of SEQ ID No. 2 coding the cytotoxin protein of claim 1 or 2. (5) The cytotoxin protein of claim 4 wherein the transformant is deposited by deposition No. FERM BP-8218 at National Institute of Advanced Industrial Science and Technology (IPOD).

(6) An antitumor agent which contains the cytotoxic protein of claim 1 or 2. (7) A monoclonal antibody specific against the cytotoxic protein which is obtained by immunization of the cytotoxic protein of claim 1 or 2 against a mammal. (8) The monoclonal antibody of claim 7 which is produced with a hybridoma clone of deposition No. FERM BP-8222. (9) The monoclonal antibody of claim 7 which is produced with a hybridoma clone of deposition No. FERM BP-8223. (10) The monoclonal antibody of claim 7 which is produced with a hybridoma clone of deposition No. FERM BP-8224. (11) A polyclonal antibody specific against the cytotoxic protein which is obtained by immunization of the cytotoxic protein of claim 1 or 2 against a mammal. (12) A method for detecting and diagnosing the cytotoxic protein of claim 1 or 2 wherein the monoclonal antibody or polyclonal antibody described in any one of claims 7-11 is used. (13) An agent for preventing and treating gastric cancer, gastritis and gastric ulcer triggered by the cytotoxic protein of claim 1 or 2, wherein the monoclonal antibody or polyclonal antibody described in any one of claims 7-11 are used. (14) A method for screening a compound promoting or inhibiting activity of the protein of claim 1 or 2, wherein cell multiplication inhibition activity, cytotoxic activity or cell death is judged by comparison between negative or positive control groups with a warm-blooded animal cell. (15) A kit for screening a compound or its salt promoting or inhibiting activity of the protein of claim 1 or 2, wherein the protein of claim 1 or 2 is comprised. (16) A compound or its salt promoting or inhibiting activity of the protein of claim 1 or 2, wherein the compound is obtained by the screening method of claim 14 or with the kit for screening of claim 15. (17) A medicine containing a compound or its salt, wherein the compound has activity inhibiting cytotoxic activity of warm-blooded animal cells with the protein of claim 1 or 2. (18) A medicine of claim 17, which is an agent for preventing or treating gastritis, gastric ulcer, gastric cancer and a disease showing that it is caused with M toxin by the screening method of claim 14 or with the screening kit of claim 15.

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