WO2007037555A1 - Therapeutic or diagnostic application of dusp15 gene - Google Patents

Abstract

It is intended to provide a therapeutic agent for cancer containing an expression inhibitor or activity inhibitor of DUSP15 protein; a method for screening a compound which can be used as an active ingredient of such a therapeutic agent; an antibody against the DUSP15 protein; a diagnostic agent for cancer/a diagnostic method for cancer using the antibody and the like.

Description

 Specification

Technical field for therapeutic or diagnostic use of D U S P 15 gene

 The present invention relates to the gene 15 gene specifically amplified in cannabis and its therapeutic or diagnostic use. Background art

As a characteristic of malignant tumors, growth, invasion and metastasis can be killed. Surgical excision or radiation Is local therapy sufficient for the treatment of metastatic recurrence? Development of phytotherapy that has been adequately addressed? To improve future treatment outcomes. It was killed by direct and / or RNA action leading to cell death. Many other examples, such as bone marrow cells, gastrointestinal epithelial cells and dividing normal cells, had strong side effects. On the other hand, the mechanism of molecular cell invasion, proliferation, and metastasis of molecular cells in recent years has been regarded as a molecule that acts specifically on the specific mechanism of the canine cells. As a representative example, treatment of non-small cell pulmonary canal GFR (epidermal growth factor receptor) tyrosine kinase inhibitor (generic name: Kehuitinif) (WO 9 6 Z 3 3 9 8 0) Next to third place. By age group, they are in the order of 60's or 1's. Cause of increase in large intestine Can it be considered as an environmental factor? It has been pointed out that it may be a cause for the westernization of dietary habits. The development of the large intestine or cocoon leaves is awaited. One used for diagnosis (CEA, CA 19-9) is a diagnostic agent with a higher ft ability that shows advanced colorectal disease and has no organ specificity. Disclosure of the invention

 It is recommended to treat or treat mandarin under the above conditions.

 Especially for drugs with high specificity for cans and Z

 In view of the situation as described above, the present inventors have found that there is a U S P 15 gene that has been intensively studied (especially the large intestine) and is frequently amplified. The present inventors have been able to suppress the growth of cancer cells by inhibiting the expression of D US P 15 in cancer cell lines, particularly cervical cancellous cell lines, and have completed the present invention. That is, the present invention provides a screening method for a screening agent, a quinotocan for diagnosis of a candidate substance having an inhibitory action on a therapeutic agent.

(1) A substance that inhibits the expression of DU SP 15 gene as an active ingredient (c) Te to DU SP 15 gene or a part thereof (d) DU SP 15 gene variant acting in a tiff manner on DU SP 15 gene or part thereof

 (e) Nucleic acid that specifically cleaves the transcript of the DUS P 15 gene

And

 (f) Compounds that inhibit DUS P 15 gene transcription or DUS P 15 translation (excluding nucleic acids above)

(3) A therapeutic agent that effectively comprises a DU S P 15 protein inhibitory substance, comprising a substance selected from the group consisting of:

 (4) Is the above DU S P 1 5 protein active ft inhibitor (a) Antibody against the DU S P 1 5 protein

 (b) a DU S P 1 5 protein variant having a dominant quality relative to the DU S P 15 protein;

 (c) Compounds that bind to the DU S P 15 protein (excluding mutants)

Including a substance selected from the group consisting of

(5) The above-mentioned cannula agent according to any one of (4), which is a large intestine or cervical epilepsy.

 (6) A screening agent for the expression inhibitor of DU S P 15 gene,

(a) Test on cells expressing DUS P 15 gene (a) Contact between DU SP 1 5 protein and test compound

(b) the process of binding the DUS P 15 protein to the test compound and

 (c) A screening method comprising a compound that binds to the DU S P 15 protein.

 (8) The method described in (7) above.

 (9) Antibody against DU S P 15 protein.

 (10) A therapeutic agent for epilepsy comprising the antibody according to (9) above. (1 1) Radioisotope Therapeutic protein A low-molecular-weight drug The above-mentioned (10 therapeutic agent further comprising a hector carrying a gene.

 (1 2) The above-mentioned epilepsy or cervical epilepsy or (1 1)

 (1 3) A can diagnosis agent comprising the antibody according to (9) above.

(1 4) DUS P 15 gene or a part of the nucleotide sequence Candidate diagnostic agent containing high fritase under unfavorable conditions

 (1 5) The above-mentioned canine or large intestine can also be used.

 (16) for can diagnosis comprising the antibody described in (9) above

(17) DUSP 15 gene or a part of its base sequence The method described in 9).

 (2 1) DU S P 15 tan haku or quantified using mass spectrometer Described in (1 9) or (2 0) above

(2 2) The method according to (19) to (2 1) above, wherein the anti-DUSP 15 antibody is used to perform DUSP 15 or Z or quantitative determination.

 (2 3) (a) A step of contacting a biological sample derived from a subject with a DU S P 15 antibody

 (b) Step of detecting or quantifying the antibody and DUS P 15 tank in the above test

The method described in (2 2) above.

 (24) (a) A biological test derived from a test subject and a step of generating a nucleonuclease consisting of a base sequence of a stringent high-flip tyre and a high-fly tyre to the base sequence of a DU S P 15 fragment.

 (b) A step of detecting a high-frequency event between the above-mentioned hollow nucleotide and DU S or a fragment thereof during the above test.

The method according to (19) or (20) above.

 (25) The method described in (19) to (24) above for use in diagnosis of cans.

 (2 6)

(2 7) DUS P 15 gene expression inhibitor Use of cassava activity inhibitors.

 (3 1) Holinuku having the nucleotide sequence of SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7 SEQ ID NO: 9 or SEQ ID NO: 10 (3 2) DU SP 15 gene expression inhibitor is an active ingredient SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 8 SEQ ID NO: 9 or SEQ ID NO: 10 A canine therapeutic agent containing a chito having the nucleotide sequence of SEQ ID NO: 10 According to the present invention, a novel drug useful for the treatment of cans (eg, colon cane) If it is a quinoto and method, a method of screening a candidate compound is provided.

Brief description of the 0 side

 01 is a histogram showing the frequency of the DU S P 15 gene with respect to the degree of amplification of two genes derived from colonic epilepsy.

 02 is an optical micrograph (phase contrast image) showing the results when s 1 RNA of the large intestine cane cell line Caco 2 and RKO E 65 gene is transfected.

 03 is a graph showing the results of quantitative evaluation of the RNA 1 effect when DU S P 15 residue A was transfected into the large intestinal cane cell line C a c o 2.

04 shows the results of transfection of the s1 RNA of the large intestinal cancell lines Caco 2 and RKO E 6 genes. The graph shows the results verified by cell count determination.

 Figure 7 is a photograph showing the results of a no-sense experiment performed using various normal organ tissues.

 08 shows optical micrographs (fluorescence images) of some of the specimen cells (six cells) from colorectal cancer patients analyzed by the FISH method.

09 A and 09 B are the krafts of sera derived from (A) and (B) sera from healthy subjects analyzed by mass spectrometry.

 01 0 A to C show the correspondence between the amino acids (or amino acid sequences) determined by MS / MS analysis

01 1 is a graph showing the results of verifying the RNA i effect when D US P 1 RNA was transfected into the cervical cancer cell line HeLa cell line.

01 2 is an optical micrograph showing the results of detailed observation of the experiment of 01 1 according to the time series under a microscope (differential varieties, best mode for carrying out the invention). Specimen amplification was performed using specimens, and the number of genes specific to the large intestine was increased 15% (Dualspecificitypnosplike 1 5) of the regions where amplification occurred frequently in the specimen It was found that the sample was. Therefore, it is called dual specificity phosphatase (Dualsptyphosphatase (DSP)). Frotin phosphatase (P roteinphose) is divided into serine threonine specific phosphatase (P and) and thyronphosphatase (PTP) containing nostin residues. NK & N eel Β G (1 9 9 6) C e 1 1 8

3 6 8 M u s t e 1 l n, T e t a 1 (2 0 0 t B i o s c i 7 8 5-14 2 M u s t e 1 ι a 1 (2 0 0 3) I mm u nol R e v 1 9 2, 7). Recently, metal io-P (T o o t 1 e T L t a 1 (2 0 0 3))

42 6 2 9 9-3 0 2 R a a p u r e d d l J (2 0 0 3) N at u r e 4 6, 2 9 5-2 9 8 t a 1 (2 0 0 3) N at r e 4 2 6 2 4 7 P T P is P T 1 B (C ha r b o n n e t a 1 (1 9 8 9) P r C N at l A c a d

5 A 8 6 5 2 5 2-5 2 6), and 5 (Hermiston ML eta 1 (2 0 0 R ev I mmu nol 2 1 1 0 7-1 3 7) Flotine Tyronon Phosphate activity and non-receptive with both onion-specific phosphatide activity

(Yu van 1 y ama J eta 1 (1 9 9 6 1 8 6-1 9 2 My ers, MP eta 1 roc Natl A cad S ci USA 9 9 0 5 7) There are 9 6 PTPs on the human genus, of which 26 are non-receptor types and 4 are 1 type of receptor type. (B haduridh am ini R (2 0 0 3) P rotein E 8 1-8 8 8).

 A representative example of DSP is MAP kinasephose (MK P), which is dephosphorylated and inactivated from erogen activated protein Erk J nkp 3 8 (S axena, M & n T (2 0 0 0) Semin I mmu nol 1 2 9 6 A lonso A eta 1 (2 0 0 3) r Genet 5 3 3 3— 3 5 8).

 In addition to typical DSPs such as MKP, there is a MKP kinase (MK P kinase — targeting mo ti roof. VH 1 derived from vaccinia virus (V accinis) or DSP (Guan KL (1 9 9 1) N ature 3 5 0, 3 5 9-3 6 2) 耒 DU SP 3 (VHR) (I shibashi T

(1 9 9 2) P roc Natl A cad S ci 9 1 2 1 7 0-1 2 1 7 4) DU SP 2 2 (VHX) I am confused.

 DU S P 1 5 (VH Y) is characterized by testis in normal anorexia. Detailed analysis shows that it is expressed in spermatocytes. In addition, it is localized to the membrane at the acid or binding site (clean residue) or at the N-terminus and is localized in the cell. Not directly related to DU S P 15

 The present inventors can suppress the expression of the DU S P 15 gene by suppressing the expression of the DU S P 15 gene by suppressing the expression of the DU S P 15 gene by inhibiting RNA l. It is also possible to make a diagnosis of can by measuring DU S P 15, which will be described in detail below.

1 Drug with anti-cane action

First, the present invention provides (1) a cannula therapeutic agent containing DU SP 15 gene expression inhibitor and (2) a cane therapeutic agent containing a DU SP 15 tft inhibitor as an active ingredient. Middle “DU SP 1 5 gene” means that it means a human DU element (SEQ ID NO: 1) consisting of 1 3 8 3 bases registered as Accession No 0 6 1 1 in NC Chitote (A lonso A The “DU SP 15 gene” to be used in the middle and the high-rise chain are known methods or examples such as molecular cloning (Molecullng T hird E dition JS a mb ra 1 C old S pr ing Harbor L as 2 0 0 1) According to the method described in the above or according to the instruction manual attached when using a commercially available life rally. Here, “stringent conditions can be any of moderate conditions and conditions can be selected.“ Low stringent conditions are 5 XSSC 5 X Tenhard solution 0 5% SDS murmite 3 The condition is 2 ° C. Also, “medium string is 5 XSSC 5 X Tenhard ^ liquid 0 5 0% formamide 卜 4 2 ° C.“ High string condition ”is 5 XSSC 5 X Tenhard solution 0

There is a condition at 5 0% formamide. We can efficiently obtain a DNA having a high degree of homology with these conditions. However, it is possible that there are multiple factors, such as temperature, flow concentration, flow time, salt concentration, and other factors. is there

F In this specification, “inhibition of gene expression” refers to a gene that inhibits any one of a series of events (including transcription (mRNA generation) quality generation) from the gene. Inhibits the production of coated hazelnut.

 In the present specification, “DUS P 1 5 protein” refers to P 1 5 protein (SEQ ID NO: 2) from 2 3 5 amino acid residues registered in Aceticion N 542 1 7 8 ) And the activity of the tannins (for example, the flothiin mouth non-phosphatase activity 14 selected from the threonine-specific phosphatase activity) and 1 amino acid residue relative to this amino acid sequence. Deletion, substitution, insertion, insertion, or addition / lifetime mutant protein.

Is the amino acid mutation site in the above-mentioned mutant tanned habitous or substantially the same quality as that of the original tanned habitat, with no particular restrictions? Number of mutations, for example 1 to 50 0 1 to 3 0 1 to 2 5 1 to 2 0 1 to 1 5 1 9 9 1 to 8 1 to 7 1 to 6 (1 to several) 1 1 to 3 1 to 2 1 The number of mutations is generally good. In addition, such a mutant protein is composed of two rows of SEQ ID NO: about 70% or more 75% or more 80% or more 85% or more 9 1% or more 9 2% or more 9 3% or more 9 4% or more 9 Any portion having the same activity as the activity ft of the DUSP 15 protein described above may be used for the site consisting of the amino acid sequence that follows. For example, as represented by SEQ ID NO: 2, it consists of at least 20 amino acids residues, preferably at least 50 or at least 70, more preferably at least 1 or at least 200 amino acids. A. Preferably this contains an acid sequence in the part involved in the activity of DUSP 15 protein. In addition, the amino acid residue in the amino acid sequence used in the present invention is 1 or about 1 to 20 in the amino acid sequence, more preferably about 1 to 10 or about 1 to 5). Or deletion, addition, substitution, or modification.

The DUSP 15 protein used in the present invention can be prepared from the cells and tissues. In addition, this material can be synthesized by a known hematopoietic synthesizer, or can be prepared by reversion using an appropriate host cell selected from eukaryotes. DUSP 15 used in the present invention may be derived from either species. Preferably, it is derived from human. “Substantially the same quality of active life” indicates their activity or property. Therefore, the enzyme activity (flotinthyronone activity tt and serine threonine-specific phosphatase activity (for example, about 0 0 1 to 100 0 times preferably about 0 5 1) Whether it should be performed in accordance with the known method described in the document “Nanato” is measured according to the screening method described later. The identity of the amino acid sequence and the base sequence is determined by the alcoholic B LAST T (proc N atl S ci U SA 8 7 2 2 64-2 2 6 8 1 9 9 0 at 1 A cad S ci USA 9 0 5 8 7 3 Determined based on B LAS T alcoholism A fukuro crumb called BLASTX has been developed hu 1 SF, eta 1 J Mol Biol 0 3 1 9 9 0). The base sequence is resolved using B LAS TN. For example, score = 1 0 0 wordl 1 2 is used. Also, using BL AS TX, the amino acid sequence is halometer, for example, score = 50 wo rd = 3. B LAST and G apped BLAST When using the tephortohalameter of each flocrum In this specification, the term “cane treatment agent” is used as an anticancer agent, a cancer cell aphotonos inducer, a cancer cell growth inhibitor, a cancer agent, an antiepileptic agent Used to mean including the like. Note that the terms “cancer” and “tumor” in this application have the same meaning.

1 1 DUS Ρ 1 5 containing an expression inhibitor of the gene The present invention is, in one embodiment, DUS Ρ 1 5 Examples of substances from DU SP 15 gene to DU SP 15 mRNA

 (a) An DU S P 15 gene or part of it

(b) leverage for DU S P 15 gene or part thereof (c) DU S P 15 gene variant that acts as a mimic for DU S P 15 gene or part thereof or

 (d) Other transcription inhibitor compounds

Somehow included.

 Examples of substances that inhibit DUS P 1 5 tan from DUS P 1 5 mRNA

 (e) Horinucleo 卜 possessed by DU S P 15 mRNA or a part thereof (eg s 1 RNA)

 (f) Renucleotide for DU S P 15 mRNA or a part thereof

 (g) Horinucleotipox having L for DU S P 15 mRNA or a part thereof or

 (h) Other translation inhibitor compounds

Somehow included.

In the present specification, “nucleic acid” means RNA or DNA. “Nucleic acid” includes such modifications as those having other heterocyclic bases decorated with furin and hydrin bases. The product consists of methylated and / or liminated furin and / or lyminylated and furanized furin. 1 Nucleic acids that have an inhibitory effect are used as active ingredients. What is RNA i? When a double-stranded RNA that is the same as or has the target gene sequence is introduced into a cell, the introduced external endogenous I gene is inhibited. Double-stranded RNA that generates 9 to 30 bases For example, ds RNA (doublnd RA) siRNA (small inter RNA) or sh RNA (shorthairpin). Such RNA is localized to the desired site. It can also be delivered and expressed locally using a hector that is generated above How to prepare such a double-stranded RNA (dsRNA siRNA A) Known from

0 0 2 r-

― Ο 1 6 0 6 2 US 囯 Open Permit 2 0 0 2. / 0 Ν a tur ge gen tic s 2 4 (2),

1 8 0 1 1 8 3 G e ne s ι s 2 6 (4)

2 4 0 1 2 4 4 N at u r e S p e 2 1 4 0 7

3 1 9 1 2 0 G e n e s & D e v V o 1

A p r 1 6 94 8-9 5 8 P r o c N at 1

S c 1 U S A 9 9 (8) 1 6 Ap r 5

2 0 S C l e n c e, 2 9 6 (5 5 6 7), 1 9

5 5 0-5 5 3 P r o c N at 1 A c a d

SAAP r 3 0 9 9 9, 6 04 7-6 0 5 2 ˜25 bases Most preferably 2 1-2 bases. Specifically, using the following s 1 RNA (used in Example 3)

(table 1 )

 s 1 R N A sequence

In this specification, “antisense nucleic acid” or “anti-leocyto” refers to having at least a small number of horinucleotide in a target DNA region, and having a high frequency with the horinucleotide or part of it. The antisense nucleic acid, which is a nucleic acid, is RNA DNA or modified NADA) The antisense nucleic acid of the present invention is alternatively a modified nucleic acid (RNA DNA). NA Single-stranded DNA Double-stranded RNA — Double-stranded RNA may be NA hyphenated. Modified nucleic acid ingredients Sulfur derivatives of nucleic acids thiophosphite derivatives It does not need to be a sequence complementary to the endogenous gene or a part of it, as long as it effectively suppresses the expression of the gene.

 For example, design of a complementary antisense sequence near the 5 'end of mRNA of DU S P 15 gene is gene translation. Complement to the coat region or 3 'untranslated region. About 70% or more, more preferably about 90% or more, and most preferably about 95% or more of the transcript of the antisense gene effective for inhibiting gene translation.

 Effective expression of target gene using antisense nucleic acid The length of antisense nucleic acid is at least about 10 bases (about 40), preferably 15 bases or more, more preferably 0 bases or more. Is it possible to design a nucleic acid with a length of about 500 bases or less by referring to the publicly known literature? Hirashima and Inoue New Chemistry Laboratory 2 Nucleic acid IV gene Japanese Biochemical Society Tokyo Chemical Dojin 1 9 9 3 p 3 1 9 K awakam 1 eta 1 P harm T ech V ol 8 p 2 4 7 1 9 9 2 V ol 8 p 3 2 STC rookeeta 1 edse Research and Ap plicatio Press 1 9 9 3

In addition, in the cane remedy of the present invention, DU SP 15 Some have active domains (tankaku nucleus 0 3 5 p 2 1 9 1). Note: Reversing note type FEBSL ett, 1 9 8 8, 2 2 8, p SL ett 1 9 8 8 2 3 9 p 2 8 5 Tanha 1 9 9 0 3 5 p 2 1 9 1 N υ c 1 A cids 8 9 1 7 p 7 0 5 9 For example, N type 1 p 3 4 9 Nuc 1 A cids R es 1 9 9 1 7 5 1 Hiroshi Kikuchi Chemistry and Biology 1 9 9 2 3 0 p 1 Using such a rehothyme, it is possible to specifically inhibit the transcript of the original SP 15 gene.

 Furthermore, the present invention can use the transcription activity of the DU S P 15 gene as an active ingredient. For example, it is a factor involved in the expression and transcription of the DUS P 15 gene. Such a compound is a natural product, but a compound such as a synthetic compound can be obtained by the screening method described later. 1 2 DU S P 1 5 Contains protein inhibitor of protein activity M

In another embodiment, the present invention provides a therapeutic agent for epilepsy comprising a DU SP 15 inhibitory substance Excluding foreign bodies)

 Somehow included.

 As used herein, “antibody” means the full length of a protein or an antibody. The form of the antibody of the present invention is not particularly limited as long as it binds to DU S P 15 protein, but also includes a human antibody, a recombinant antibody, an antibody fragment, and an antibody modification product in addition to the above-mentioned monoclonal antibody. Antibodies that bind to D protein (anti-DU S P 15 antibody) can be prepared by those skilled in the art. Anti-DU S P 15 will be described later.

 In this specification, “DUS P 1 5 protein mutant with Tiff properties against DUS P 1 5 protein” By expressing a glanced gene, the activity of endogenous ft field 5 protein is lost or reduced. (Kunihiro Tsuchida, Gene Activity Inhibition Experiment Method Tahira (2 0 0 1) 2 6-3 2 Natto).

 Further, in the present invention, a compound other than a mutant that binds to DU SP 1 5 protein can be used as an active ingredient as a substance to obtain DU SP 1 5 protein. Is a compound that binds to Such compounds may be natural products. Such compounds can be obtained by the screening method described later.

Also provide.

 One preferred embodiment is a method using DU S P 15 protein and exposure as indicators. In general, DU S P 15 tantalum compounds are expected to be effective in inhibiting the activity of DU S P 15 protein. Here, the compound is preferably bound to the DU S P 15 position. In this method, the protein is further brought into contact with the test compound. DU SP 1 5 In response to an indicator for detecting binding to a test compound Example 1 5 Purified form of protein Intracellular or extracellular or bound to an affinity column For example, radiation knowledge, fluorescence knowledge, etc. can be given as necessary for the knowledge of compounds used as needed. In this method, the binding of DU SP 15 protein is detected next.

 There is no restriction | limiting in particular as a test compound used for this method. Compound Organic compound Inorganic compound Tanhaku Hefti Compound As well as Compound life rally Gene library Cell extract On cell culture / blue Fermentation microbial product Sea / sheep extract Extracts etc. are not limited to these.

The binding of DU SP 1 5 protein to the test compound depends on the knowledge attached to the test compound bound to Example 1 5 protein. In addition, DU SP 1 is generated by the binding of the test compound to the protein that is expressed intracellularly or extracellularly. Select a test compound that inhibits activity.

 The compounds separated by this method have an anti-epileptic effect and are useful as therapeutic agents.

 In another aspect of the present invention, the screen linking method is based on D U S P current.

 In this method, the DUSP 15 gene is first expressed and contacted with a test compound. It is not limited to the origin of the “cell” used, whether it is a cell that can be used as a source of the cell. As a “cell expressing a gene”, a cell having an endogenous DUSP 15 or a cell expressing or expressing an exogenous DUSP 15 gene can be used. Cells expressing an exogenous gene are usually prepared by introducing the expression vector left in D U S P 15 into the Sukuo cells. The expression vector can be produced by general genetic engineering techniques.

Natural or compounds organic compounds ^to motor Compound Tanhaku compound off compound Namihi to quality life Larry Gene Raifurari cell extract Cell culture凊products of fermenting microorganisms marine raw extract or the like is not particularly limited test compounds to be used in the process Used.

Test compound in cells expressing DUSP 15 gene Normal culture of cells expressing DUSP 15 gene I will come. For example, it is possible to extract the cells expressing DUS P 15 gene according to a conventional method, and measure transcriptional reherence by carrying out the Noisen-Yon method or RT_PCR method using this mRNA as a cocoon. Or, separate the D motor's floor motor region according to the usual method, and connect it below (for example, a gene that can be detected using Lunferase, GFP, and other colors as an indicator). It is possible to measure transcriptional rehering by observing the activity. It is also possible to collect the protein fraction from DUS-expressing cells and to measure the expression of D-molecules by electro-peristalsis such as SDS-PAGE. Furthermore, it is also possible to measure the translational reher by detecting the expression of the porphyra by Western blotting using an antibody against the porphyra. The antibodies used for DU SP 15 may be detectable antibodies, specifically monoclonal antibodies or polyclonal antibodies.

In this method, the compound selected as a compound that decreases the expression level compared to a roll that is not contacted with a test compound is 3 1 Anti-DUS P 1 5 antibody

As used herein, “anti-DU SP 15 antibody” includes antibodies that are DU SP 1 (including fragments (partial edge) or salts thereof). The anti-DU SP 1 reclonal antibody used in the present invention may be a monoclonal antibody. The class of the antibody is not particularly limited, and anti-preferably having IgA type such as IgGIgMD or IgE is preferably IgG or IgM, and more easily IgG, more preferably IgG. The term “antibody” used herein is meant to include any antibody fragment or derivative, and includes, for example, Fab ₂ CDR-humanized antibody, multifunctional antibody, and single-chain antibody. Antibody production methods of the present invention are well known in the art, such as the production of antibodies by known methods (see, for example, E & Lane Danantibody Collaborative Laboratory Laboratory 8). .

 (1) Preparation of antigen

In the present invention, the tanned USP 15 used as a sensitizing anti-IT or a salt thereof. The above DUSP quality also includes a margin in its part. This is limited. For example, it is a fragment of the amino acid sequence of SEQ ID NO: 2, eg, 40 or more 60 or more 80 or more 100 or more amino acids The part has a sequence part. these Salt or organic acid (for example, acetic acid, quinic acid, flohy, etc.) Used as a sensitizing antigen for antibody acquisition, SP 15 is restricted to the animal species from which it is derived. Tanja or preferred Tancho or preferred.

 (2) DU S P 1 5 Monoclonal protein

(I) Collection of antibody-producing cells

 The above-mentioned DUPSP 15 protein is administered to a mammal mouse as an antigen using the portion of the protein (in this specification, these are collectively referred to as 15 protein) as an antigen. When no hunt is used per animal of antigen, 0 1 to 10 Omg is used, and when using no hunt, 1 to 100 ^ g is used. Quantum immunity is limited by insertion into the subcutaneous or intraperitoneal cavity as an example of aluminum hydroxide cultivated. Not every few days to several weeks Good weekly intervals 1 to 10 times, preferably 2 to 5 times Immunization 1 to 60 days after the last immunization day, preferably 1 to 14 days after is collected. Can spleen cells or lymph cells be listed as antibody-producing cells? Spleen cells or local lymph node cells

(I I) Cell fusion

Antibody-producing cells and myelo to obtain hyphenoma NS I / 1 -A g 4-1 S 0/1 and mouse mye YB 2 0 and myroma myeloma cell line are listed Next, cell fusion between the above myeloma cells and antibody producing cells Serum-free DMEM R PM I — 1 6 40 1 1 0 ⁶ to 1 1 0 ⁷ cells 111 1 and 2 X 1 0 ⁵ to 2 X 1 0 ⁶ cells and production of Zm l myeloma cells Cell ratio of cells to myeloma cells 2 1 to 3 1 Carry out fusion reaction in the presence of a cell fusion promoter Cell fusion promotion Average molecular weight 1 0 0 0 to 6 0 0 0 . It is also possible to fuse antibody-producing cells using commercially available cell fusion devices that utilize electrical stimulation (eg, electon).

(ill) Selection of hybridomas and clones As a method of target hybridomas from cells after cell fusion treatment, cells ¹ 1 Suspension, for example, R 0 medium containing urchin fetal clot After appropriate dilution Micro Thailand evening frame Individual we 1 We fire about 1 1 Add selective medium to each well and change the ground to culture. As a result, the cells that grow after the start of culture in the selective medium are obtained as hyphritoma. Next, whether or not antibodies that react with the protein are present in the culture medium of the proliferated hyphritoma. Screening ning is not good following normal methods. For example, it grows as a hyphenoma A normal cell culture method or ascites formation method or the like is used as a method for monochromating from the thus obtained hy- toroma. In the cell culture method, PM I-16 40 medium containing hyphritoma 10 medium culture medium or charming culture medium culture medium (for example, 3 7 ° C 5% 7 to 14 days) to be obtained from the culture supernatant antibody. If ascitic fluid is taken approximately 1 X 1 0 ⁷ or administered Haifuri Tomah re Thoma mammalian the same species animal-derived myeloma cells to atmospheric and 1 after 2 weeks Collecting the above-mentioned antibodies If purification of the antibody is required, the ammonium sulfate salting-out method, ion raffi-el filtration, affinity chromatography method can be selected as appropriate, or these can be combined.

(3) Horizon for DU SP 15 matrix First, the above antigens are administered to mammals, such as Rantoumau. The dose of the antigen per animal is 0 l to 100 mg when used, and 10 g is used for ananhunt. Freund Hunt (F CA) Freund 卜 Incomplete Hanu Hunt (FI Aluminum Hanu Hunt, etc.) can be listed as an immun hunt. Immunization is performed by injection into the king skin or intraperitoneal cavity. Every few weeks, preferably 2 to 1 to 10 times, preferably 2 to 5 times and Collect the antibody that reacts with D protein (column adsorbed fraction) on an affinity column that is fixed with protein. Reaction of polyclonal antibody in anti-blood clots to algae.

 (4) Antibody fragments

F ab or F ab, ₂ fragments can be prepared by digestion using conventional methods. Humanized antibodies are described, for example, by Riec hma nn et al. (R in J Mol Biol Oct 5 20 3 (3) 1 9 8 8) and J ones et al. (J ones et al. Nat 1 5 2 2- 5 2 5 1 9 8 6) It is possible to use one of the methods described in 1).

Chimeric antibodies are, for example, “Experimental Medicine (Special issue 16, No 1 0 1 9 8 8”). Equity 3— 7 3 2 8 0 Human antibodies are, for example, “Nature Genetic 1 5, p 1 `` 4 6-1 5 6 1 9 9 7 '' `` Naturelcs Vol 7 p 1 3-2 1 1 9 9 4 '' Special 3 6 5 Gazette International Application Publication W 0 9 4 2 5 5 8 5 G Monthly Nos. 40 to 50, 1 9 9 5 "V ol 3 6 8, p 8 5 6-8 5 9, 1 9 9 4" An antibody that binds to SP 15 protein may be used for the purpose of suppressing cancer cell metastasis, etc. In the same way, the antibody DU SP 15 can be obtained using bioluminescent compounds as well as phycoerythrin and fluoride. The presence of bioluminescent protein is measured by fluorescence. This knowledge is important for the biologically generated renoferrin, linofelase, and aequorin. The antibody of the present invention is used to specifically detect P 15 protein etc. in a sample of body fluid. Used and also used to purify DU SP 1 5 protein etc. DU SP 1 5 protein in each fraction during purification Analyzes the behavior of DUS P 1 5 protein in cells I will come.

3 2 Anti-DUS P 15 antibody complex or anti-DU SP 15 antibody used in the present invention is an agent or a diagnostic agent itself or an agent having a neutralizing activity. Therefore, the present invention is used in another embodiment for use as an addictive therapy or addictive drug in the US. Complexes of P 15 antibody and other glazes are also provided. According to such an embodiment, the anti-DU SP 15 antibody of the present invention is used as a labeling agent for the determination of the therapeutic effect. Iodine-1 2 5 C ^{1 25} I) and iodine-1 3 1 As with the above elements, these radiohalogen elements can be widely used as diagnostic agents for radiation therapy and knowledge of antibodies and hefty. For example, ^{1 25} I or ¹ can be conjugated to an antibody by a known method such as the chloramine T method. In addition, for diagnostic purposes, tech m indium 1 1 1 and potassium 1 6 7 ( ^{6 7} Ga for treatment use indium 1 90 ( ^{9 0} Y) rhenium ⁶ R e) or rhenium 1 1 8 8 ( ^{1 88} R e) Something used Ordinarily when antibodies are labeled using radioactive isotopes. As the metal chelating agent, EDTA D nonionic compound cyclam and DOTA or other known chelating agents can be bound to the antibody in advance and then released, or the radioactive metal chelate can be formed and then bound to the antibody.

In the present invention, as an example of “therapeutic protein”, a cytokine that activates is preferable. It is possible to use linonyanph toxin to directly kill colonic canal cells. For example, for therapeutic proteins, coat the antibody or antibody fragment. C. Coat the DNA. C. Connect the DNA to coat the fusion antibody. Cyclophosphine alkylating agent 5—Full meso trexase Antimetabolic agent Yunomainon Mitomycin C Yunolhinone Toxorhinone Hinkristin Hinfrastin Hintesin-like hormone agent Bed oncology (Japan Clinical Oncology Research Group 1 9 9 6 Years Cancer and / or Height Mouth Cochisson Fretonison's Steroline Intome Yunon's Non-Steroit Agent Gold Tiolamin's Immunomodulator Cyclophos Anti-inflammatory agents such as chlorfenilamine maleate, antihistamines, etc. (Inflammation and anti-inflammatory therapy, Shoyaku Shuppan Co., Ltd.) For example, as a method of binding unomata Kurtar al Bond between amino acid of antibody and antibody through Law water Karuho like or be opened Karuhokinru 棊 amino and antibodies of evening Unomainon Te.

 As an example of “virus hector”, a virus hector modified so as to be capable of binding to anti-DU of the present invention is used. Watenovirus hector (Wang P et 5) Somatic Celland Molec 2 1, 4 2 9- 44 1) Retrovirus Hector (RK eta 1 (1 9 9 6) JV ir

5 7 0 1-5 7 0 5) Lentiviral Hector (N a For those who need gene therapy together with anti-DU SP 15 antibodies Anti-DU SP 15 antibodies can be recognized as antigens (ie, D).

 The anti-DUS P 15 antibody and the other drug can be conjugated to chemical W or. Here, “chemical bonds” include ionic bonds, covalent bonds, intermolecular forces, and hydrophobic interactions, and “genetic engineering bonds” include, for example, a fusion protein consisting of a milk protein. In the alternative, the antibody and the therapeutic protein will be bound.

4 Formulation and administration method

 Activity of DU SP 1 5 proteins containing the DU SP 15 gene expression inhibitor of the present invention [4 Anti-DUS containing anti-DUS P 15 antibody containing the inhibitor or anti-DUS of the present invention P 15 antibody or radioisotope therapy Tancha drug and virus hector carrying a therapeutic gene or any of these, or any of these native or genetically engineered therapeutic agents are known It can be formulated.

In formulating the therapeutic agent of the present invention, a chemically acceptable carrier can be added according to a conventional method. For example, excipient coloring +4 flavor storage +4 stabilizer buffer f Latin Medium-chain fatty acid trichryslate Horio kinoechi

/ Ya 6 0 Saccharose Calhoquine methylcellulose Cones

 Examples of the dosage form of the therapeutic agent of the present invention are oral powders, pills, powders, condylar agents, fine granules, soft cuffs, tans, helenotes, sublinguals, stings, etc. Non-propellant suppositories, transdermal Agents Ointments Plasters For liquids for external use, etc., use the optimal dosage form according to the route of administration and the subject of administration. The activity of DUS P 15 protein as an active ingredient (15 gene expression) Inhibitors can be determined from 0 1 to 9 9 in the drug product.

The dosage of the active ingredient of the drug of the present invention varies depending on the subject of administration Subject administration method Oral administration In general (as 60 kg) More preferably, it is about 1 to 100 mg. When administered parenterally, the single dose varies depending on the organ symptoms, administration method, etc. For example, the normal person (for example, 6 kg) About 30 mg per day, preferably about 0 About 1 to 20 mg About 0 1 to 1 Omg should be administered by intravenous vaginal discharge However, ultimately, the type of dosage form Administration method 崈 Considering the symptoms of the deaf, etc. It will come out. Bladder canister Uterine canister (eg cervical canine uterine canopy) Glandular pancreas Knee canopy Brain tumor Blood tumor etc. Preferably used for the prevention and treatment of large intestine.

 The leaf preparation of the present invention contains a DUSP 15 protein activity inhibitor SP 15 gene expression inhibitor as an active ingredient. Cancer agent Cancer metastasis inhibitor Cancer cell phohotosin inducer, etc. The type of cancer is not specific. The drug of the present invention contains both a DUSP 15 protein and a substance that inhibits the expression of the DUSP 15 gene. In the therapeutic agent of the present invention, the sense nucleic acid is used as a monochromic or retrovirus vector. After insertion into a viral hector, it should be administered according to known means.Antisense nucleic acid should be administered alone or physiologically, and administered via a gene-like catheter Come.

In the present invention, a combination of a combination of a recombinant fatenovirus particle and an anti-DUSP 15 antibody may be used independently in the treatment of cancer. Generally, it is used together with a pharmaceutical carrier. As such a carrier, water, physiological saline, krucose-hytoalf isotonic solution or the like is preferably used as a carrier. Like that It may vary depending on the nature of the cell tissue. The number of administrations is 1 and the administration period may be 1 day to several months or more. In addition, the viral hectare nucleic acid molecule used in the present invention can be used for diagnosis of a specific cell and / or disease state. For example, by incorporating a detectable marker gene into the nucleic acid molecule of C, it is possible to detect and diagnose tumor cells by combining with the virus Hector USP 15 antibody obtained by transfecting cells or anti-DUSP 1 5 Can be used to detect and diagnose tumor cells by allowing detection by antibodies

5 Diagnostic agents and diagnostic methods for cancer

 The present invention also provides a diagnostic agent. One preferred diagnostic agent of the present invention is (a) a DUSP 15 antibody or (b) a DUSP 15 gene or a part of it. Diagnostic agents and diagnostics using 5 1 anti-DUSP 15 antibody containing

Since antibodies against DUSP 15 proteins can specifically recognize DUSP 15, it is possible to quantify DUS proteins in the test solution. Specifically, the anti-antibody of the present invention Detection of binding between the fragment and anti-DUSP 15 antibody

 In the present specification, “subject-derived biological test” includes subject-related or body fluid (for example, blood (including whole blood, plasma, serum, etc.), liquid, saliva, perspiration, elliptical fluid, etc.). A “subject” is a human subject who is or is suspected of receiving or desired to receive a human subject. Examples of such cans include large intestine, stomach, and lungs. Gland or esophageal or liver or biliary or splenic or uterine canal (eg cervical or uterine canal) testis or spleen or urinary canal tumor Tumor or blood tumor is preferred.

 The immunoassay for issuing DUSP 1 in a subject's unexplained biological test as described above is based on the binding of a single antibody from a biological sample collected from a subject with a risk of kan (eg, large intestine). Antibody binding, including measuring anti-DUSP 15 antibody and the amount of immunospecific binding by the antibody, under conditions to detect whether DUSP 15 protein or increased expression is detected. In this case, detection of increased D halite expression or disease state indications. If necessary, you can compare the DUSP 15 tanned reher to that without a cane.

One embodiment of the above immunoassay is, for example, a blood clot test +4 It can be determined appropriately by those skilled in the art.

One method of detectably labeling anti-DUS P 15 antibodies is to bind the antibody to an enzyme such as the enzyme Imnoanose (EIA) [Vo 1 1 er, immunoadsorbent by A] ('The E nz yme Li mu nosorbent A ssay) (ELISA) lagnostlc Horizons 2 1-7 JC lin Pathol 3 1 1 9 7 8 B utler by ological A ssociates Quartblication, Wa lkersvi 1 le MD r A Meth E 7 3 4 8 2 to 5 2 3 1 9 8 1] by JE. Fermentation with antibody Appropriate substrate Fluorophotometry is used to produce a chemical molecule that can be detected by fluorescence measurement with visible means. Enzymes that can be used to provide detectable knowledge to antibodies include, but are not limited to, herokinase and alkaline properties. This detection can also be achieved by a colorimetric method using a chromogenic substrate. Other methods that can be used in the present invention include the race (RIA) Santoi immunoassay method, the immunometric method, the immunofluorescence assay method ( FIA) Time-resolved fluorescence RFIA) Enzyme immunoassay (EIA) Luminescence immunoassay Electrochemiluminescence immunoassay (ECLIA) Latinox As described above, it is possible to diagnose various diseases related to DUS P 15 dysfunction by utilizing the in vivo D-quantitative method using the antibody of the present invention. Example 1 If an increase in the concentration of brown sea urchin is detected, whether it is likely due to overexpression of the brown sea urchin (for example, kan) or is likely to be affected in the future? The

 It should be noted that the anti-DU S P 15 antibody of the present invention may be i n v i v. The preparation of antibody preparations that can be used here is well known in the art. For example, the antibody—Kireet NuclMedb Biol 1 9 9 0 1 7 2 is described. An example of an antibody having a paramagnetic ion used for magnetic resonance is described in, for example, M ag e e s e a n c e e n Me c i c e e e 1 9 9 1 -342.

5 2 Horinucleocyto (eg DNA) pro-blocking and diagnostic methods

In the diagnostic method of the present invention, whether the DUS P 15 gene or the flow designed for the DUS P 15 gene is used is, for example, (a) the subject-based DUS P 15 gene or its fragment base A salt that can be swallowed up under the conditions of a streaky sunset in the array. And z or quantify. The base sequence used as the flow can be 12 bases or more 15 bases or more 18 bases or more 2 14 bases or more 2 7 bases or more 30 bases or more, or even a nucleotide fragment. High and low stringent conditions may be used. Included in the base sequence of DU SP 15 gene or its fragment is also included in the base sequence of DU SP 15 gene or its fragment. For example, international publication number 8 9 Z0 6 6 9 8 EP—A 0 2 US Pat. No. 2 9 1 5 0 8 EP—A 0 0 6 3 8 7

0 1 7 3 2 5 1 EP— described in A 0 1 2 8 0 1 8 In the diagnostic method of the present invention, the DU SP 1 5 gene is publicly detected by using a nucleocytofluorine or a flymer. Or come to quantify. As an example, Sasan Hai Furi Yusen Noun No Isei No Yoon RT—PCR method PCR—SSCP method (G s Vol. 5 8 7 4-8 7 9 (1 9 8 9)) P rogsofthe National A cade my lencesofthe Un ited Statemerlca Volume 8 6 2 7 6 6〜 2 7 7 0 (1 9 8

1 SH method DNA Chinov or Array C GH (C omp Hang the NA fragment (BAC, PAC YAC, etc.) at high density with a separate dye using S Achinov, and simultaneously perform hyonation on the chemome DNA fragment on the slide to determine its binding state. There is a method to detect anomalous cohesion number with high resolution by detecting (P 1 neta 1 (1 9 9 8)

1) o

 In the present invention, in order to detect whether or not the DUS P 15 gene is expressed, the mR quasigene of the cellular DU SP 15 (housekeeping gene (for example, Shape et al., JM mmary G land B iol N la 3 (1 9 9 8) 3 1 5-3 2 4 Wu YY s JLA cta der venere (2 0 0 0) 2-3) mRNA reherence, preferably by R, can also be compared.

 Is it possible that the target sequence (duration of DNA mRN was confirmed by overexpression of DUSP 15 gene by the above-mentioned method is a disease caused by overexpression of DUS P 15 (for example)? High or possible to be jealous.

5 3 Diagnosis method using mass spectrometer

Another embodiment of the diagnostic method of the present invention is a sputum during the test. By determining the intensity of the sample, it is possible to identify the individual amino acids of the protein and hemiform from the results of mass spectrometry.

 There are various types of ionization, such as matrix antheno tracer tetherization (MAL D I), electrosfray ionization, phase (E I C I) and field desorption (FD). For ion separation, ion fraction compatible with ionization method For example MAL DI, time-of-flight type (ti me ght TºF) Mass spectrometer For ESI, quadruple ion trough type Magnetic type mass An analyzer or a quantity analyzer may be used in tandem. For example, I M S / M S Q-T O F M S MALD I — T〇 It is also possible to determine the amino acid sequence by another method for determining amino acid sequence, for example, one (eg, gas phase sequencer).

5 4 Diagnostic kinoto

The present invention also provides a quinoto for quantifying or quantifying a subject DU SP 15 protein or fragment thereof containing an anti-DU SP 15 antibody. In addition, the base sequence of the gene or a part of it contains a base sequence that can be hyfitized under stringent conditions. The D US P 15 gene or a fragment thereof in an unexamined biological sample. Ovarian can brain tumor or blood tumor is included.

 In the present specification, the term “can marker” refers to a subject's body fluid (such as urine phosphorus eight fluids, saliva, sweat, semen, etc.) or a cell that is not derived from cells or normal tissues, or is a molecule that is alternatively enhanced in expression. This suggests the presence of the molecule in the subject or in the tissue.

 The quinoto of the first embodiment contains antigens (DUSP 15 protein and its components and edge or Z or components to be quantified in the body fluid test from the subject. For example, DUS or ELISA. For example, tissue sections or body fluids such as blood or urine, such as blood or urine, are used to detect and Z or quantify antibodies such as radioactivity, fluorescence ratio The quinoto of the present invention contains a labeled secondary antibody The quinoto of the above second embodiment is a stringent high hyphenation sequence in the DUSP 15 gene or base sequence. Article containing holynucleocytos having a base sequence that can be used as a chair. The quinoto of the present invention may contain the above-mentioned holy fixed on DNA chinofu.

The quinoto of the present invention is a highly efficient anti-DUSP 15 antibody DUSP 15 having a stringent base sequence. Example

 Example 1 Colonic Canine Specific Amplification by Array C GH Method In this example, a colonic specimen specific gene amplification region was prepared in a sample of 20 samples of large intestine cannula and array C verification was performed.

 As a result, it was frequently amplified in large intestine specimens. As a result of scrutiny using public DB gene information (NC BI http // wn 1 mn ι hgo vZ), C lone RP 1 1 — 2 4 3 J 1 6 (Dualspecificityphosphatke 1 5) (NCBIA ccession No 6 1 1) genes or colon colonists were frequently elevated (01 Table 2). 01 is a histogram showing the frequency of amplification of the DU SP 15 gene for large samples. Table 2 shows the frequency of DU SP 15 gene colonic epilepsy (GZR value) and frequency. Show. The average value is the GZR value or the average value of one individual.

 As shown in Table 2, DU S P 1 5 gene was found to be amplified in 5 60% of 2 0 0 individuals.

1 7 The maximum value was 2 4 and it happened very frequently.

 (Table 2)

In this example, the frequency of the colon cane S was high and the amplification degree of the colon cane-derived cultured cell line was verified.

 <Sanflu preparation>

 The cultured cell line used was Caco 2 E 6 which is a cell line derived from the large intestine. Kenome DNA was extracted from the cultured cells according to the key phrase using BLOOD & CELLEDENAQ (Q IAGEN).

 Array C GH>

 In order to confirm the amplification of the gene region, the details of the array GH were carried out in the same manner as in Example 1. Result

 Table 3 shows the DU S P15 gene colony cell line ZR value). As shown, it was found that colonic undifferentiated cells BAC Clone R P 1 1-2 4 3 J 16 located in P 1 5 gene was amplified.

(Table 3)

<Quantitative PCR> did. Framer synthesized the following sequence (○ PE RO used

 Framer array

 5-C C GCAC TGATC TAAAGGCATTC No. 3)

 5, — GGTC TCAAAC T CATGGC C TTTG No. 4) Results

 Table 4 shows the cell lines of DU S P15 gene with large intestine. The values are relative to the DNA of Akira (normal). In addition, we found that DUS P 15 inheritance was also observed in the colon cell line (Table 4).

Based on these results, the DUS P 15 gene was also amplified in the colon colony cell line (C aco 6), so that the cells were selected for functional analysis of the DUS P 15 gene in the canine. RNA i analysis using KOE 6) and its phenotype RNA 1 analysis>

 Cell lines were purchased from ATCC and attached to the front collage. s 1 RN A synthesized s l RNA that selectively selects specific 21 mer within the gene (Q I AGEN

(Table 5)

 s 1 R N A sequence

The introduction of s 1 RNA into Caco 2 cells was performed using ○ 1 igolne (Invltrogen) and introduced into the cells according to the protocol with Ι Ο Μ ηΜ ^. For introduction into the sl RNA vesicle, RNA i FE CT (QI AGEN) was introduced into the cells following the flow of the nM s 1 RN A ^ Observed under a microscope CDNA was synthesized according to the call using First-Strand Synthesis SyrRT-PCR (Invitrogen).

 Quantitative RT-PCR using this cDNA as a saddle type Real PCR is performed using SYBRG reen RT-PCRRe (Applied Biosystems). led biosystems), the realmer synthesized the following sequence (consigned to OPER ON)

 5 '-AC C A C GAT T GT GA C A G C G TAT G No. 1 1)

 5 -AA C T C T T C AAG C T G C T G C C TAA Column No. 1 2)

 For the canonical gene for calculating the relative ratio, G l y c e r e — 3 — p h o s p h a t e d e h y d r o g e n a s H)

s 1 Number of viable cells after introduction of RNA using A 1 amar bluerce) according to the attached protocol, Wal 20 Multllabel / Luminescence AR VO (Perkin Elmer) The observation image on the 4th day is shown (in each 0, the upper row X 4 0 0). a, b and c have 3 DU SP 15 inheritances, respectively, NC shows inactivity control, and the phenotypes are the ab and c 3 types of sl RN tlve control trol (NC). It was noticeably finer (02 A, B).

 03 shows the result of performing CR after carrying out slan sfefe t tion of DU S P15 gene to Caco2 cell and collecting the cell 24 hours later. The relative amount to NC was shown using contrast as the intrinsic 14 control. As a result of confirming the effect of rehel shown by quantitative RT—PCR, the expression level of NA was significantly reduced (03).

For 4A and B, the number of viable cells was measured by measuring leaf counts using the Cranco2 cells and cells with sl RNA of DUS P 15 gene T ransfec on the 4th day. Relative amounts are shown. As a result of the determination, it was found that in Caco 2 cells, 1 type of DU SP 1 l RNA (c) was found in all 3 types of s 1 RNA of 15 genes (ab, c) in RKOE 6 cells N Compared to egative control (NC), it was a little less (04 AB). It was observed whether the number of cells was low. All the sl RNAs of the SP 15 gene were subjected to t test. By carrying out this study, we verified the specific effects of the suppressive effect of epilepsy genes.

 RNA 1 analysis

 The cell line used was CCD 18 Co, which was cleared from AT CC. The s 1 RNA c sequence used was used. For introduction of s1 RNA into the cell, L1 am ine 2200 (Invitrogen) was used. The slRNA was introduced into the cell according to the attached protocol. g atl v e C o n t r o ls ι RNA (Q I AG). After introduction, the cells were observed under an inverted microscope for 5 days.

 <Quantitative RT—PCR analysis>

 The same procedure as described in Example 3 was performed.

 <Measurement of the number of living cells>

 The same procedure as described in Example 3 was performed.

 ϋ.

 The effect of RNA 1 of cell line C derived from normal large intestine of the large intestine of DU S Ρ 15 gene is as follows.

05 shows the observed image obtained on day 5 after sransfection of the DUS P 15 gene in C CD 1 8 Co cells (lower 0 X 2 0 0). DU SP 15 c indicates one type of DU SP 15 RNA (c sequence of Example 3), and NC indicates no call. As shown, no NC effect was observed by phenotypic observation (05). 55

〇E 6 is DUSP 15 gene due to RNA i effect. Is the proliferation or suppression of the peritoneal cell markedly suppressed? Large intestinal vesicle strain C CD 1 8 It has been found that it can be a pharmacological gene with little effect on normal cells. Example 5 Evaluation of organ specificity

 Whether the DUS P 15 gene is an organ such as a normal tissue or not was evaluated by the North Sun-Hifuri known in the art.

 MTN Multiple Tissue Northern Blots (Clontech) according to the flow 25m (5 -cagacaccgggggagg column number 1 3)) Oil go DNA Probe

Specifically, 23 types of organs (Heart (HearO M (Brain) Placenta (Lung) Liver (Liver) Skeletal muscle (Skeletal (Kidney)) Knee (Pancreas) Spleen (Spleen) ) Thoracic Gland (Prostate) Testis Ovary Intestine Colon Colon Peripheral Blood Leukocyte Stomach Thyroid Cord Lymph Node Trachea Gland ) About Bone Marrow et al (2004) J Biol Chem 279 (31) 32586-32591) was found to be a hunt in the vicinity of about 15 kb in the testis, confirming its precise expression. In this analysis, a new hunt was found around 13 kb in the heart, suggesting the occurrence of Olfactory Futhrian sputum. Other organs were confirmed to have developed. It is highly possible that the specific expression of the testis and the visceral expression of the testis are due to the influence of the drug that made this gene anxious or limited. Example 6 Evaluation of gene amplification

 The gene amplification degree (G / R) or more than 1 or 2 tissue according to the FISH method known in the art using the array CGH method. DUSP 15 gene or Clone RP11-243J16 DNA probe

 08 shows a part of the canine cells observed in each specimen tissue (A to J) (Analytical photomicrograph (fluorescence image). As shown, it was found that more than 3 horns were found. 1 2 It was confirmed that DU SP 1 5 remains in 10 specimens, and it was shown that gene amplification occurred in the early stage of the disease state stasis.

A large amount of protein was removed from the alpha-chromium using a fur solution (10 mM Tns HC1 ph 74 4 + 150 mM NaCl) 500 ProteomeLab IgY-12 SC Fronto-Hearty Noyo (BECKMAN COULTER A24618). To the obtained fraction, final concentration of 10 mM nothiothreitol (Wako 049-08972) was added, and reduction reaction was performed. After the reaction was completed, the alkylation reaction was carried out at room temperature by adding ョ (SIGMA 144-48-9) to a final concentration of 10 mM. After completion of the reaction, 4 times the volume of the reaction solution was added, and the mixture was allowed to stand at _80 ° C for 1 hour, and then centrifuged for 30 minutes to collect the protein. A part of the regenerated material was dissolved in 80 x L 2M urea + 100 mM ammonium bicarbonate solution, and a part of it was used for the measurement of the concentration of tanchus by the BCA method. The digestion reaction was carried out for 16 hours at 37 ° C (Promega V51) so that the quality was 1/50 (w / w).

2) Analysis using Nano-HPLC direct mass spectrometer

The heftito fragment 0 1 was separated by a -Precolumn cartridge (C 5 ΠΙ 300 ΠΙ 300 ΠΙ id χ 5 mm LC PACKINGS 163589) Nano column (C18 PepMap 3 m 100 A 75 mid PACKINGS 160321). The HPLC apparatus was Ultim PACKINGS. The flow rate is 200nL / min. 0 1% Chinoic acid (Wak containing 2% Acetonitrile (MERCK 1287229) and 0 1% Chitol containing concentration gradient or 0 57% / min linear clone is installed. A complete MS / MS analysis was performed. 3) Analysis

 All analysis data obtained from the mass spectrometer was output via Data Analysis (Bruker Daltonics). Analyzes of all the output molecules were extracted only for the analysis of MS / MS data in the extracted events, and analyzed for the presence of ion hekes with masses of fractional molecules 9 A and 09 B shows (A) serum derived from a person with large intestine and (B) serum derived from a healthy subject, analyzed and analyzed as described above.

 01 0 A ~ (: indicates the correspondence between 09 determined by MS / MS analysis and amino acid (or amino acid sequence).

09 Ayohi 01 As shown in 0 0, Ion heeks corresponding to the target molecule fracts or blood clots derived from colonic epilepsy. That is, at least a partial sequence called C WGF EE is determined as shown in 0 0 B, and this corresponds to columns 136 to 142 in the amino acid sequence of DUS P (SEQ ID NO: 2). Thus, as shown in 01 OA, the amino-to-145 positional heftofragment (SEQ ID NO: 14) Was the growth-suppressing effect observed? Is it confirmed in the cervical cane cell line? RNAi analysis was performed using the above-mentioned 3 using the HeLa cell line of cervical cane-derived cells evaluated by the RNAi analysis method described above. Introducing gofect amine (Invitrogen) into the cells according to the float call with nM s1 RNA, we used negative control RNA (QI).

01 1 shows the results of BiJ assay (MTT assay) in HeLa cells using cells on day 4 after sl RN fectlon of D US P 15 gene. The craft indicated relative amounts to NC (Neg siRNA (Qiagen)). 3 types of siRNA (abc) of DUSP 15 gene shown in 0 (abc) showed 26% growth-inhibiting effect (p <0 01 t, further time-sequentially observed differential icing images under microscope) Specifically, HeLa cells were subjected to siRNA (13,4 days after ab fuc- noon), and the results were shown in 01 2. The growth rate of NC is higher than that of NC using Negative control s lRNA. (Indicated by the sign of inhibition and cell death). The present invention provides a therapeutic agent for cans, a diagnostic agent, a diagnostic method, and a kinoto used for treatment. Therefore, it is useful in fields such as the present invention or acupuncture.

Claims

 The scope of the claims

1 DU S P 1 5 Therapeutic agent containing an inhibitor of gene expression as an active ingredient.

 2 Is it a substance that inhibits the expression of the DUS P 15 gene?

 (a) Nucleic acid having the RNA effect on the expression of the DUS P 15 gene

 (b) DUS P 15 gene transcript or part thereof sense nucleic acid

And

 (c) Nucleic acid that specifically cleaves the transcript of DU S P15 gene

A substance selected from the group consisting of: 3 DU S P 1 5

 4 DUS P 1 5

 Antibody to the DUS P 15 protein

The agent for treatment of epilepsy according to claim 3.

 5 The above-mentioned cannula or the cervical canal.

 6 Screening for DU S P 15 gene expression inhibitor

(a) Test on cells expressing DUS P 15 gene (a) Contact DUSP 15 protein with test compound

(b) The process of connecting the DU S P 15 protein to the test compound

 (C) A screening method comprising a compound that binds to the DU S P 15 protein.

 8. The method according to claim 1, wherein the colorectal or cervical canal is used.

 Antibody against 9 DU S P 1 5 protein.

 1 0 contract * Therapeutic agent comprising the antibody according to item 9.

 1 1 Radioisotope Therapeutic protein Low-molecular-weight drug Drug further containing a hectare carrying a gene.

 1 2 The agent for treating epilepsy according to 1 1 above, wherein the epilepsy or cervical epilepsy is.

 1 3 A can diagnostic agent comprising the antibody according to claim 9.

 1 4 DU S P 15 A diagnostic agent that contains a high-definition chair under a high-definition condition under a gene or a part of its base sequence.

 1 5 The aforementioned canine or large intestine can be detected. Claims 1 3 or 1 4 Diagnostic agent.

1 6 Canine diagnosis kit containing the antibody of claim 9 1 7 DU SP 1 5 gene or a part of its nucleotide sequence can be used under high hyphenation conditions The method described.

 21. The method according to claim 19 or 20, wherein DU S Ρ 1 5 protein or quantitative determination is performed using a mass spectrometer. 2 2 Anti-DU S タ ン 1 5 DU SP 1 5 Quantify and / or quantitate ** SP 1 1 Any of 9 to 2 1 2 3 (a) Subject-derived biological test and DU SP 1 A step of contacting with 5 antibodies.

 (b) Detecting or quantifying the antibody and DU S P 15 tan in the test

The method according to claim 22.

 2 4 (a) Subject-derived biological test 4 and DU S P 1 5

 (b) a step of detecting a high-frequency event between the hollow nucleotide and DU or a fragment thereof during the test 4;

The method according to claim 19 or 20.

 25. A method according to claims 19 to 24 for use in the diagnosis of cans.

 2 6 The aforementioned large intestine. * Indicated in item 25. 2 7 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 SEQ ID NO: 10