JP2003277289A - Preventive/remedial agent for cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preventive/remedial agent or the like for cancer. <P>SOLUTION: A compound or its salt for adjusting the activity of protein having an amino acid sequence the same as or substantially the same as that represented by sequence number: 1, antisense nucleotide containing at least a part of a base sequence complementary or substantially complementary to the base sequence of DNA which codes protein or its partial peptide having an amino acid sequence the same as or substantially the same as that represented by sequence number 1, etc., can be used as preventive/remedial agents for cancer, etc. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preventive / therapeutic agent for cancer, a diagnostic agent, and the like.

[0002]

2. Description of the Related Art In chemotherapy for cancer, the life-prolonging effect is improved by the development of new anticancer agents, and the number of cases toward cure is increasing. However, most of the currently used anticancer agents have strong cytotoxicity that damages DNA or inhibits cell division, and therefore damages normal cells to some extent, especially Strong side effects often appear in the bone marrow where the division is active. In order to comprehensively analyze gene expression, a microarray method in which cDNA or an oligonucleotide is immobilized has been developed, and a technique for finding a disease-specific change in gene expression has spread and its usefulness has been confirmed. For example, Affyme
Trix's GeneChip system is being widely used for diagnosing diseases such as cancer and discovering target genes for drug discovery. When introduced into a cell, the antisense oligonucleotide hybridizes with RNA having a complementary sequence to produce RNase.
It also induces RNA degradation by H to inhibit protein translation, or it also leads to direct protein synthesis inhibition by hybridization. Since it is possible to specifically suppress the target gene function, it has been frequently used as a means for analyzing the function of a gene, and some antisense oligonucleotides are being developed for clinical application. Recently, it was revealed that the Toll receptor acts as a receptor involved in innate immunity in insects in which the adaptive immune system does not exist. Toll-lik, a molecule showing homology to this Toll receptor
E receptors (Toll-like receptors; TLRs) also exist in mammals and are also said to be involved in innate immunity. Up to now, nearly 10 types of TLRs have been cloned. For example, TLR2 and TLR6 recognize lipoproteins and glycolipids, TLR4 recognizes lipopolysaccharides, TLR5 recognizes Flagellin, and TLR9 recognizes CpG.
It has been reported that it is involved in DNA recognition (Nature Immunology 2 (8), 675-680, 2001).
In addition, TLR1 (Proc Natl Acad Sci USA 95 (2) volume,
588-593, 1998) is involved in microbial product recognition in cooperation with TLR2 (Non-patent document 1: J Immunol. 165).
(12), pages 7125-7132, 2000), TLR2 and TL
Inhibits the action of R6 hetero-receptor (Non-patent document 2:
J Immunol. 166 (1), 15-19, 2001). Furthermore, recently, TLR1 knockout mice were analyzed, and it was reconfirmed that TLR1 coacts with TLR2 in ligand recognition, and at the same time, a new triacyl peptide such as Pam ₃ CSK ₄ as a TLR1 ligand (non-patent document) Reference 3: J Immunol. 169 (1), 1
0-14, 2002) and OspA (Non-patent document 4: Nat).
Med. 8 (8), 878-884, 2002).
In addition, research on signal transduction factors has also been conducted on TLR2 and TLR4.
Is being analyzed, for example Receptor-inter
Analysis of acting serine / threonine kinase-2 (RIPK2) knockout mice has shown that this kinase acts in signal transduction of TLR2 and TLR4 (Non-patent Document 5: Nature. 416 (6877), 190-194. Page, 2002
Years), but the knowledge about TLR1 signal is very poor. On the other hand, paclitaxel, which is frequently required as an anticancer agent, exhibits a lipopolysaccharide-like action on mouse macrophages, which Kawasaki et al.
And MD2 signals (Non-patent document 6: J Biol Chem. 275 (4), 2251-2254, 2000.
Year). However, both TLR4 and mouse MD2 are required, and this MD2 has species specificity,
It has been reported that paclitaxel has no LPS-like action in human MD2. Most of these studies on the TLR1 gene relate to cells of the immune system, and there is no report on the relationship between the TLR1 gene and cancer (solid cancer).

[Non-Patent Document 1] J Immunol. 165 (12), 7125-7132
Page, 2000

[Non-Patent Document 2] J Immunol. 166 (1), 15-19, 2001.
Year

[Non-Patent Document 3] J Immunol. 169 (1), 10-14, 2002.
Year

[Non-Patent Document 4] Nat Med. 8 (8), 878-884, 2002.

[Non-Patent Document 5] Nature. 416 (6877), 190-194, 20.
02 years

[Non-Patent Document 6] J Biol Chem. 275 (4), 2251-2254.
Page, 2000

[0003]

A drug that targets a molecule specifically expressed in cancer cells and can inhibit the growth of cancer cells or induce apoptosis is desired. Also,
There is also a strong demand for effective treatments for existing anticancer drug-resistant cancers and drugs that increase sensitivity to existing anticancer drugs from the viewpoint of reducing side effects.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found a gene whose expression is significantly increased in cancer tissues, and based on this finding, As a result of further studies, the present invention has been completed.

That is, the present invention provides (1) SEQ ID NO: 1
A preventive / therapeutic agent for cancer, which comprises a compound or a salt thereof that inhibits the activity of a protein or a partial peptide thereof or a salt thereof that contains the same or substantially the same amino acid sequence as the amino acid sequence represented by 2) It comprises a compound or its salt that inhibits gene expression of a protein or its partial peptide or its salt, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Cancer preventive / therapeutic agent,
(3) A base sequence complementary or substantially complementary to the base sequence of a DNA encoding a protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or its An antisense nucleotide containing a part thereof, (4) a prophylactic / therapeutic agent for cancer comprising the antisense nucleotide according to (3) above, (5) the same as the amino acid sequence represented by SEQ ID NO: 1 or A prophylactic / therapeutic agent for cancer, which comprises an antibody against a protein containing a substantially identical amino acid sequence or a partial peptide thereof or a salt thereof,
(6) Cancer is colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovary Cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, the preventive / therapeutic agent according to (1) to (5) above, (7) the amino acid sequence represented by SEQ ID NO: 1, A cancer diagnostic agent comprising an antibody against a protein containing the same or substantially the same amino acid sequence, or a partial peptide thereof or a salt thereof, (8) the same or substantially the same amino acid sequence represented by SEQ ID NO: 1. D encoding a protein having a partially identical amino acid sequence or a partial peptide thereof
Diagnostic agent for cancer containing NA, (9) cancer is colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney Cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or hematological tumor, (10) Toll according to the above (7) or (8) Or therapeutic agent for cancer containing a compound having an activity of inhibiting the activity of a receptor like a receptor, (11) Cancer comprising a compound having the activity of inhibiting the expression of a Toll-like receptor or a salt thereof (12) A cancer characterized by using a protein or a partial peptide thereof or a salt thereof which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Screening method for preventive / therapeutic agents, (13) A kit for screening a preventive / therapeutic agent for cancer, which comprises a protein having the same or substantially the same amino acid sequence as that represented by the number 1 or a partial peptide thereof or a salt thereof, (14) Screening of a preventive / therapeutic agent for cancer, which comprises using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof Method, (15) SEQ ID NO: 1
(16) A screening kit for a preventive / therapeutic agent for cancer, which comprises a DNA encoding a protein or a partial peptide thereof having an amino acid sequence identical or substantially identical to the amino acid sequence represented by A preventive / therapeutic agent for cancer obtainable by using the screening method according to (12) or (14) or the screening kit according to (13) or (15),
(17) Induction of apoptosis comprising a compound or its salt that inhibits the activity of a protein or its partial peptide or its salt, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. (18) A compound or a salt thereof, which inhibits the gene expression of a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. (19) An apoptosis inducer characterized by using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. (20) the same as the amino acid sequence represented by SEQ ID NO: 1 Or a method for screening an apoptosis inducer, which comprises using a DNA encoding a protein having substantially the same amino acid sequence or a partial peptide thereof, (21) the same as the amino acid sequence represented by SEQ ID NO: 1 or An anticancer drug sensitivity enhancer comprising a compound or a salt thereof, which inhibits the activity of a protein or a partial peptide thereof or a salt thereof, which contains substantially the same amino acid sequence, and is represented by (22) SEQ ID NO: 1. Anticancer drug sensitivity enhancer comprising a compound or a salt thereof that inhibits the expression of a gene of a protein or a partial peptide thereof or a salt thereof that contains the same or substantially the same amino acid sequence as the amino acid sequence of ) For mammals, an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1. Administering an effective amount of a compound or a salt thereof that inhibits the activity of a protein or a partial peptide thereof or a salt thereof containing an acid sequence, or a compound or a salt thereof that inhibits the expression of a gene of the protein or a partial peptide thereof or a salt thereof. Cancer prevention and treatment methods characterized by
(24) The activity of a protein, a partial peptide thereof, or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 for producing a preventive / therapeutic agent for cancer The use of a compound or a salt thereof that inhibits, or a compound or a salt thereof that inhibits the expression of a gene of the above protein or a partial peptide thereof or a salt thereof is provided.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter also referred to as the protein of the present invention or the protein used in the present invention. ) Is a human or warm-blooded animal (eg, guinea pig, rat, mouse,
Chicken, rabbit, pig, sheep, cow, monkey, etc.) cells (eg, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet) Cell, endothelial cell, smooth muscle cell, fibroblast, fiber cell, muscle cell, adipocyte, immune cell (eg, macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, Eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells Etc.) or any tissue in which those cells are present, such as the brain, each part of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, inferior Pituitary Stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung,
Digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen,
Submandibular gland, peripheral blood, prostate, testicles, ovaries, placenta, uterus,
It may be a protein derived from bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

The amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 6% of the amino acid sequence represented by SEQ ID NO: 1.
An amino acid sequence having a homology of 0% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is included. A protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 above. , A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable.

[0008] Examples of substantially the same activity include signal transduction activity (eg, Toll-like receptor (preferably T
Intracellular signaling activity of LR1), ligand binding activity (eg Toll-like receptor (preferably TLR1))
And a ligand or a low molecule binding activity). The term “substantially the same” means that those properties are qualitatively (eg, physiologically or pharmacologically) the same. Therefore, the signal transduction activity and the ligand binding activity are equivalent (eg, about 0.01 to 100).
Times, preferably about 0.1 to 10 times, more preferably 0.
However, the quantitative factors such as the degree of activity and the molecular weight of the protein may be different. Examples of the ligand include lipopeptides derived from bacteria (preferably triacylated lipopeptides, etc.) or N-palmitoyl-S- obtained by chemical synthesis.
dipalmitoylglyceryl (Pam ₃ ) Cys- Ser- (Lys) ₄ (CS
K ₄ ), N-palmitolyl-S-dilaurylgryceryl (N-Pam-S-Lau
₂ ) Triacyl peptides such as CSK ₄ , Borrelia burgdorf
An outer-surface lipoprotein (OspA) derived from eri is used. The signal transduction activity and the ligand binding activity are measured by known methods, for example, J. Immunol. 166 (1).
Vol., Pp. 15-19, 2001, or a method similar thereto. The TLR family activates the NF-κB signal by ligand binding. Therefore, for example, the activity of the protein [eg, Toll-like receptor (eg, TLR1 etc.)] used in the present invention is the Introduced into cells with
(A) a liquid containing a ligand such as a microbial cell lysate, a microbial culture supernatant, a eukaryotic cell lysate, a eukaryotic cell culture supernatant,
A substance having a binding activity equivalent to that of (b) the ligand itself or (c) the natural ligand is added, and the expression level of the reporter is measured by a known method. The reporter expression level can be measured by using the reporter protein activity using luciferase activity, alkaline phosphatase activity, etc. as an index. In addition, the signal transduction activity can be measured by measuring and comparing the apoptotic activity against an anti-cancer agent (eg, paclitaxel) with or without the expression of the protein (eg, TLR1) used in the present invention by a known method. . The apoptotic activity against an anticancer drug (eg, paclitaxel) in the presence or absence of a ligand can be measured and compared by a known method to measure the signal transduction activity. Furthermore, in order to measure the signal transduction of TLR1 used in the present invention with higher sensitivity, a TLR1 signal-related molecule (eg, TLR2, MD) is used.
Cells having the ability to express TLR1 signal-related molecules (eg, TLR2, MD) may be used.
2 etc.) and the above activity may be measured.

The protein used in the present invention is, for example, 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 30 in the amino acid sequence represented by SEQ ID NO: 1. Is an amino acid sequence in which about 1 to 10 amino acids, more preferably a number (1 to 5) amino acids have been deleted, 1 or 2 or more (for example, about 1 to 100) in the amino acid sequence represented by SEQ ID NO: 1. 1 or 2 to the amino acid sequence represented by SEQ ID NO: 1, preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5) amino acids. Amino acid sequence in which one or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5)) amino acids are inserted, SEQ ID NO: 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5)) amino acids in the amino acid sequence represented by A so-called mutein such as a protein containing an amino acid sequence in which is substituted with another amino acid, or an amino acid sequence obtained by 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 protein in the present specification is a peptide
According to the convention of the mark, the left end is the N-terminal (amino terminal), the right end
Is the C terminal (carboxyl terminal). SEQ ID NO: 1
Including proteins containing the represented amino acid sequences
The protein used in the present invention has a C-terminal
Ruboxyl group (-COOH), carboxylate (-C
OO^-), Amide (-CONH₂) Or ester (-C
OOR). Here at Esther
Examples of R include, for example, methyl, ethyl, n-propy
C such as toluene, isopropyl and n-butyl_1-6Alkyl
Groups, eg C, such as cyclopentyl, cyclohexyl, etc.
_3-8Cycloalkyl groups such as phenyl, α-naphthyl
C such as le_6-12Aryl groups such as benzyl, phenene
Phenyl-C such as chill_1-2Alkyl group or α-na
Α-Naphthyl-C such as futylmethyl_1-2Alkyl group
Which C_7-14Aralkyl group, pivaloyloxymethyl group
Which is used. The protein used in the present invention is C-terminal
Carboxyl group (or carboxylate) other than the end
If so, the carboxyl groups are amidated or
Proteins that are telylated are also used in the present invention
include. Examples of the ester in this case include those described above.
C-terminal ester and the like are used. Furthermore, this
The protein used in Ming has N-terminal amino acid residues
The amino group of (eg, a methionine residue) is a protecting group (eg,
C such as formyl group and acetyl group _1-6Alkanoyl, etc.
C_1-6Protected by an acyl group, etc., in vivo
The N-terminal glutamine residue produced by cleavage with
Oxidized lutamin, a position on the side chain of an amino acid in the molecule
Substituents (eg, -OH, -SH, amino group, imidazole
Groups, indole groups, guanidino groups, etc.) are suitable protecting groups
(For example, C such as formyl group and acetyl group._1-6Arca
C such as noyl group_1-6Protected by an acyl group)
Or a so-called glycoprotein in which sugar chains are bound
Any complex protein etc. are included. Used in the present invention
Specific examples of the protein include, for example, SEQ ID NO: 1
A protein containing the amino acid sequence represented by (TLR
1) and the like.

The partial peptide of the protein used in the present invention is a partial peptide of the protein used in the present invention, and preferably has the same properties as the protein used in the present invention. Any one will do. Specifically, for the purpose of preparing the antibody of the present invention, which will be described later, a peptide having the 27th to 578th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 and the like can be mentioned. For example, it has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the protein used in the present invention. A peptide or the like is used. The partial peptide used in the present invention has 1 or 2 or more (preferably about 1 to 10 and more preferably a number (1 to 1 in the amino acid sequence.
5) amino acids are deleted, or 1 or 2 or more (preferably about 1 to 20) in the amino acid sequence,
More preferably about 1 to 10, more preferably several (1 to 5) amino acids are added, or 1 or 2 or more (preferably about 1 to 20 and more preferably) amino acid sequence is added. About 1 to 10, more preferably several (1 to 5) amino acids are inserted, or 1 or 2 or more (preferably 1) in the amino acid sequence is inserted.
-10 amino acids, more preferably several amino acids, still more preferably 1 to 5 amino acids) may be substituted with other amino acids.

[0012] In the partial peptide used in the present invention C-terminal carboxyl group (-COOH), a carboxylate (-COO ^-), may be any of an amide (-CONH ₂₎ or an ester (-COOR) . Further, the partial peptide used in the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, like the above-mentioned protein used in the present invention,
Amino acid residue of N-terminal amino acid residue (eg, methionine residue) protected with a protecting group, glutamine residue cleaved in vivo at the N-terminal side is pyroglutamine-oxidized, amino acid in molecule Those in which the substituent on the side chain of is protected by a suitable protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound are included.
The partial peptide used in the present invention can also be used as an antigen for antibody production.

The salt of the protein or partial peptide used in the present invention is a physiologically acceptable acid (eg,
A salt with an inorganic acid, an organic acid) or a base (eg, an alkali metal salt) is used, and a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid),
Alternatively, organic acids (eg, acetic acid, formic acid, propionic acid,
Fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid,
Malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like salts are used. The protein or its partial peptide or its salt used in the present invention can be produced from the cells or tissues of humans or warm-blooded animals described above by a known method for purifying a protein, or a protein containing a DNA encoding the protein. It can also be produced by culturing the transformant.
It can also be produced according to the peptide synthesis method described below. When producing from human or mammalian tissues or cells, after homogenizing human or mammalian tissues or cells, extraction is performed with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated by combining

For the synthesis of the protein or partial peptide used in the present invention, its salt, or its amide, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin,
PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4-
Examples thereof include (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin and 4- (2', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein. At the end of the reaction, the protein or partial peptide is cleaved from the resin, at the same time various protecting groups are removed, and then an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or amide of them. To do. Regarding the condensation of the above-mentioned protected amino acids, various activation reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As carbodiimides, D
CC, N, N'-diisopropylcarbodiimide, N-
Ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is directly added to the resin together with a racemization-inhibiting additive (eg, HOBt, HOOBt), or a symmetrical acid anhydride or HOBt ester or HOOBt is added.
It can be added to the resin after previously performing activation of the protected amino acid as an ester.

The solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol,
Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from the range known to be applicable to protein bond-forming reactions, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. 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 cannot be obtained by repeating the reaction, it is possible to acetylate the unreacted amino acid with acetic anhydride or acetylimidazole so that the subsequent reaction is not affected.

Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc and the like are used. The carboxyl group is, for example, alkyl esterified (for example,
Linear, branched or cyclic alkyl esterification such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl and the like, aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazideation, t-butoxycarbonylhydrazideation, tritylhydrazideation, etc. You can The hydroxyl group of serine can be protected by, for example, esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group. Used. Moreover, examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of the protective group for the phenolic hydroxyl group of tyrosine include Bz
1, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z,
t-Butyl or the like is used. Examples of the protecting group for imidazole of histidine include Tos and 4-methoxy-
2,3,6-trimethylbenzenesulfonyl, DNP,
Benzyloxymethyl, Bum, Boc, Trt, Fm
oc or the like is used.

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinide, N
-Hydroxyphthalimide, ester with HOBt)]
Are used. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used. Examples of the method for removing (eliminating) the protecting group include Pd
-Catalytic reduction in a hydrogen stream in the presence of a catalyst such as black or Pd-carbon, or acid with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. Treatment, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used. The elimination reaction by the acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol,
The addition of cation trapping agents such as para-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol is effective. Further, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane group. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

Protection of a functional group which should not be involved in the reaction of the raw material, removal of the protective group, removal of the protective group, activation of the functional group 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 or partial peptide, for example, first, the α-carboxyl group of the carboxy-terminal amino acid is amidated and protected, and then the peptide (protein) chain is added to the amino group side up to the desired chain length. After the extension, a protein or partial peptide in which only the N-terminal α-amino protecting group of the peptide chain is removed and a protein or partial peptide in which only the C-terminal carboxyl protecting group is removed are produced. The protein or peptide is condensed in a mixed solvent as described above. The details of the condensation reaction are the same as above. After purifying the protected protein or peptide obtained by condensation, all protecting groups can be removed by the above method to obtain the desired crude protein or peptide. The crude protein or peptide can be purified by utilizing various known purification means, and the main fraction can be lyophilized to obtain the amide of the desired protein or peptide. To obtain an ester form of a protein or peptide, for example, α- of the carboxy terminal amino acid is used.
After condensing the carboxyl group with a desired alcohol to form an amino acid ester, the desired protein or peptide ester can be obtained in the same manner as the protein or peptide amide.

The partial peptide used in the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein used in the present invention with an appropriate peptidase. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing the partial peptide or amino acid that can form the partial peptide used in the present invention with the residual portion and removing the protective group when the product has a protective group. it can. Examples of known condensation methods and removal of protective groups include the methods described in the following (1) to (3). M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten After that, the partial peptide used in 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 analogous thereto, and conversely, when a partial peptide is obtained, a known method or a method similar thereto is used. It can be converted into the educt or other salt by the method.

The polynucleotide encoding the protein used in the present invention may be any polynucleotide containing the nucleotide sequence encoding the protein used in the present invention. Preferably DNA
Is. As DNA, genomic DNA, genomic DNA
Library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, synthetic D
Any of NA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse Transcriptase Polymer can be directly prepared by using a total RNA or mRNA fraction prepared from the cells and tissues described above.
It can also be amplified by ase Chain Reaction (hereinafter abbreviated as RT-PCR method). The DNA encoding the protein used in the present invention is, for example, a DNA containing the base sequence represented by SEQ ID NO: 2, or a hybrid with the base sequence represented by SEQ ID NO: 2 under high stringent conditions. Contains soybean base sequence,
Any DNA may be used as long as it encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1.

DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 2 under high stringent conditions
Is, for example, about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% with the base sequence represented by SEQ ID NO: 2. As described above, most preferably, a DNA containing a nucleotide sequence having a homology of about 95% or more is used. Hybridization can be carried out by a known method or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al.,
Cold Spring Harbor Lab. Press, 1989) and the like. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions. The high stringent condition is, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM.
And the temperature is about 50-70 ° C, preferably about 60-65 ° C.
The conditions of are shown. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically,
As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, DNA containing the nucleotide sequence represented by SEQ ID NO: 2 is used.

The DNA encoding the partial peptide used in the present invention may be any DNA as long as it contains the base sequence encoding the partial peptide used in the present invention. In addition, genomic DNA, genomic DNA library, c derived from the cells / tissues described above
It may be DNA, a cDNA library derived from the cells / tissues described above, or synthetic DNA. Examples of the DNA encoding the partial peptide used in the present invention include, for example, DNA having a part of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or the nucleotide sequence represented by SEQ ID NO: 2 and hystrin. A DNA containing a base sequence that hybridizes under a gentle condition and containing a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention is used. SEQ ID NO: 2
DNA capable of hybridizing with the nucleotide sequence represented by
Same meaning as above. The same hybridization method and high stringent conditions as described above are used.

D which completely encodes the protein or partial peptide used in the present invention (hereinafter, in the description of cloning and expression of the DNA encoding them, these may be simply referred to as the protein of the present invention)
As a means for cloning NA, a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention is used for amplification by the PCR method, or DNA incorporated into an appropriate vector is used as a part of the protein of the present invention. Alternatively, it can be selected by hybridization with a DNA fragment coding for the entire region or one labeled with a synthetic DNA. The hybridization method is, for example, Molecular Cloning 2nd (J. Sambrook et al.,
Cold Spring Harbor Lab. Press, 1989) and the like. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. The conversion of the nucleotide sequence of DNA is performed by PCR, a known kit such as Mutan ^™ -superExp.
ress Km (Takara Shuzo), Mutan ^TM -K (Takara Shuzo)
Etc., etc., and the known methods such as the ODA-LAPCR method, the Gapped duplex method, the Kunkel method, and the like, or methods similar thereto can be used. The DNA encoding the cloned protein can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA may have ATG as a translation initiation codon at the 5′-terminal side and TAA, TGA or TAG as a translation termination codon at the 3′-terminal side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. The protein expression vector of the present invention is
For example, (a) DNA encoding the protein of the present invention
A desired DNA fragment is cut out from the
It can be produced by ligating the fragment downstream of the promoter in an appropriate expression vector.

As the vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pBR322)
UC13), a Bacillus subtilis-derived plasmid (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, 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 an animal cell is used as a host, SRα promoter, SV40 promoter, L promoter
TR promoter, CMV promoter, HSV-TK
Examples include promoters. Of these, CMV
It is preferable to use (cytomegalovirus) promoter, SRα promoter and the like. When the host is a bacterium belonging to the genus Escherichia, the trp promoter, the lac promoter, the recA promoter, the λP _L promoter,
lpp promoter, T7 promoter, etc., when the host is a Bacillus genus, SPO1 promoter, S
When the host is yeast such as PO2 promoter and penP promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. Examples of selection markers include dihydrofolate reductase (hereinafter, dhfr).
The gene [methotrexate (M
TX) resistance], ampicillin resistance gene (hereinafter referred to as Amp
^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r , G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells lacking the dhfr gene, the target gene can also be selected by a thymidine-free medium. In addition, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention, if necessary. When the host is Escherichia, the PhoA signal sequence, OmpA signal sequence, etc., when the host is Bacillus, the α-amylase signal sequence, subtilisin signal sequence, etc., the host is yeast. In some cases, MFα · signal sequence, SUC2 · signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used, respectively. Using the vector containing the DNA encoding the protein of the present invention thus constructed, transformants can be produced.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include:
For example, Escherichia coli K1
2. DH1 [Proc. Natl. Acad. Sci. USA], Volume 60, 1
60 (1968)], JM103 [Nucleic Acids Research], Volume 9, 30.
9 (1981)], JA221 [Journal of Molecular Biolo (Journal of Molecular Biolo
gy), 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41.
, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gene,
24, 255 (1983)], 207-21 [Journal of Biochemis
try), Vol. 95, 87 (1984)] and the like are used.
Examples of the yeast include Saccharomyces cerevisiae AH22 and AH22.
^{R -, NA87-11A, DKD-5D} , 20B-1
2. Schizosaccharomy
ces pombe) NCYC1913, NCYC2036, Pichia pastoris KM71 and the like are used.

As the insect cell, for example, the virus A
In the case of cNPV, larvae of the night-thief moth (Spodop
tera frugiperda cell; Sf cell), Trichoplusia ni
MG1 cells from the midgut of the mouse, H from the eggs of Trichoplusia ni
igh Five ^TM cells, cells from Mamestra brassicae or
Cells derived from Estigmena acrea are used. When the virus is BmNPV, the silkworm-derived cell line (Bombyx mor
i N cells; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf
21 cells (above Vaughn, JL et al., In Vivo
ivo), 13, 213-217, (1977)) is used. Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (198).
5)]. Animal cells include, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter, abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dhf
r ^-) cell), mouse L cells, mouse AtT-2
0, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells and the like are used. For transforming Escherichia, for example, Proc. Natl. Aca (Proc. Natl. Aca)
d. Sci. USA), 69, 2110 (1972) and Gene, 17: 107 (1982).

For transforming Bacillus, for example, Molecular & General Genetics, Vol. 168,
111 (1979) and the like. For transforming yeast, for example, Methods in Enzymolog
y), 194, 182-187 (1991), Procedures of the National Academy of Sciences of the USA (Proc. Na).
tl. Acad. Sci. USA), 75, 1929 (1978).
And the like. Transformation of insect cells or insects can be performed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988) and the like. To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, Volume 52, 456
It can be performed according to the method described in (1973).
In this way, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained. When the host is culturing a transformant that is a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source necessary for the growth of the transformant, A nitrogen source, an inorganic substance, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal and potato extract. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. Also,
Yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York1
972] is preferable. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter so as to work efficiently. When the host is a bacterium belonging to the genus Escherichia, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, 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 a transformant whose host is yeast, examples of the medium include Burkholder's minimum medium [Bostian, KL
Et al., Proc. Natl. Acad. Sci. USA, 77, 4505.
(1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Procedures of the National Academy of Sciences of
The USA (Proc. Natl. Acad. Sci. US
A), 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 agitation are added if necessary. When culturing a transformant whose host is an insect cell or an insect, the medium is Grac
e's Insect Medium (Grace, TCC, Nature (Natur
e), 195, 788 (1962)) to which appropriate additives such as immobilized 10% bovine serum have been added. PH of medium
Is preferably adjusted to about 6.2-6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added if necessary. When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science, 12
Volume 2, 501 (1952)], DMEM medium [Virology, Volume 8, 396 (1959)], RPMI
1640 medium [Journal of the American
Medical Association (The Journal of the A
merican Medical Association) 199, 519 (19
67)], 199 medium [Proceeding of the Society for the Biological Medi
cine), 73, 1 (1950)] and the like. p
H is preferably about 6-8. Culture is usually about 30 ℃
Perform at about -40 ° C for about 15-60 hours, adding aeration and agitation as needed. As described above, the protein of the present invention can be produced inside the transformant, inside the cell membrane or outside the cell.

The protein of the present invention can be separated and purified from the above culture by the following method, for example. When the protein of the present invention is extracted from cultured bacterial cells or cells, after the culture, the bacterial cells or cells are collected by a known method, suspended in an appropriate buffer solution, and then subjected to ultrasonic waves, lysozyme and / or freeze-thawing. After destroying the bacterial cells or cells by the method, a method of obtaining a crude protein extract by centrifugation or filtration 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-100 ^™ . When the protein is secreted into the culture medium, after the culture is completed, the cells or cells are separated from the supernatant by a known method, and the supernatant is collected. The protein contained in the thus obtained culture supernatant or the extract can be purified by appropriately combining known separation / purification methods. Known separation and purification methods for these are mainly methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. The difference in charge, the difference in charge such as ion exchange chromatography, the method using specific affinity such as affinity chromatography, and the difference in hydrophobicity such as reversed-phase high performance liquid chromatography A method utilizing a difference in isoelectric points such as a method and an isoelectric focusing method is used.

When the protein thus obtained is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto, and conversely, when it is obtained as a salt, a known method or a method similar thereto is used. Depending on the method, it can be converted into the educt or other salt. In addition, the protein produced by the recombinant can be optionally modified or the polypeptide can be partially removed by acting an appropriate protein-modifying enzyme before or after the purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The presence of the protein of the invention thus produced is
It can be measured by an enzyme immunoassay using a specific antibody or Western blotting.

The antibody against the protein or partial peptide or salt thereof used in the present invention 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 or salt thereof used in the present invention. Good. Antibodies against the protein or partial peptide or a salt thereof used in the present invention (hereinafter, these may be simply referred to as the protein of the present invention in the description of the antibody) are known by using the protein of the present invention as an antigen. It can be produced according to the method for producing an antibody or antiserum. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal at a site where antibody production is possible by itself, or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells, warm-blooded animals immunized with the antigen, for example, individuals with antibody titers are selected from mice, and spleens or lymph nodes are collected 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by fusing the resulting antibody-producing cells with myeloma cells of the same or different species. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation is a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1
975)]. Examples of the fusion accelerator include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

Examples of myeloma cells include NS-1,
Examples include myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000-PEG6000)
Is added at a concentration of about 10 to 80%, and 20 to 40 ° C.,
Cell fusion can be efficiently performed by preferably incubating at 30 to 37 ° C. for 1 to 10 minutes. Although various methods can be used for screening a monoclonal antibody-producing hybridoma, for example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed with a carrier, and then a radioactive substance or Anti-immunoglobulin antibody labeled with an enzyme (when the cells used for cell fusion are mice, anti-mouse immunoglobulin antibody is used) or protein A
And detecting the monoclonal antibody bound to the solid phase, adding the hybridoma culture supernatant to the solid phase adsorbing the anti-immunoglobulin antibody or protein A, adding the protein labeled with a radioactive substance or enzyme, Examples include a method of detecting a monoclonal antibody bound to a phase. Selection of the monoclonal antibody can be performed according to a known method or a method similar thereto. Normal HAT
(Hypoxanthine, aminopterin, thymidine) can be added to the medium for animal cells. As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1-2
RP containing 0%, preferably 10-20% fetal bovine serum
MI 1640 medium, G containing 1-10% fetal bovine serum
IT medium (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 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of Monoclonal Antibody Monoclonal antibody can be separated and purified by a known method, for example, a method for separating and purifying immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, Adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen binding solid phase or active adsorbent such as protein A or protein G to collect only antibody and dissociate the binding Specific Purification Method to Obtain]]. [Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, immune antigen (protein antigen)
As such, or by forming a complex thereof with a carrier protein, immunize a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody, collect an antibody-containing substance against the protein of the present invention from the immunized animal, It can be produced by separation and purification. Regarding the complex of the immunizing antigen and the carrier protein used for immunizing warm-blooded animals, the type of carrier protein and the mixing ratio of carrier and hapten are such that the antibody is effective against the hapten immunized by crosslinking with the carrier. If possible, any kind may be cross-linked at any ratio, but, for example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. in a weight ratio of about 0.1 to 2 with respect to 1 hapten.
A method of coupling at a rate of 0, preferably about 1 to 5, is used. Further, for the coupling of the hapten and the carrier, various condensing agents can be used, but glutaraldehyde, carbodiimide, maleimide active ester,
An active ester reagent containing a thiol group or a dithiopyridyl group is used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. Administration is usually about 2
~ Once every 6 weeks, about 3 to 10 times in total.
The polyclonal antibody can be collected from the blood, ascites, etc., of the warm-blooded animal immunized by the above method, preferably from the blood. To measure the polyclonal antibody titer in antiserum,
It can be measured in the same manner as the above-mentioned measurement of antibody titer in antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

Complementary to the nucleotide sequence of the DNA encoding the protein or partial peptide used in the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of antisense nucleotides),
Alternatively, the antisense nucleotide having a substantially complementary nucleotide sequence or a part thereof may be the DNA of the present invention.
Any antisense nucleotide as long as it contains a base sequence complementary to or substantially complementary to the base sequence of or a part thereof and has an action capable of suppressing the expression of the DNA. Good, but antisense DNA is preferred. The term “substantially complementary nucleotide sequence to the DNA of the present invention” refers to, for example, a total nucleotide sequence or a partial nucleotide sequence of a nucleotide sequence complementary to the DNA of the present invention (that is, a complementary strand of the DNA of the present invention) and about 70 nucleotides. % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 9% or more.
Examples include base sequences having a homology of 5% or more.
In particular, among all the base sequences of the complementary strand of the DNA of the present invention,
(A) In the case of an antisense nucleotide directed to translational inhibition, about 70% or more of the complementary strand of the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the nucleotide sequence near the start codon), Preferably about 80% or more, more preferably about 90% or more, most preferably about 9%.
When the antisense nucleotide having a homology of 5% or more is (b) an antisense nucleotide that directs RNA degradation by RNaseH, about 70% or more of the complementary strand of the entire base sequence of the DNA of the present invention including an intron, Preferably about 80% or more, more preferably about 90% or more,
Most preferably, antisense nucleotides each having a homology of about 95% or more are suitable. In particular,
An antisense polynucleotide having a base sequence complementary to or substantially complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a part thereof, preferably, for example: An antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof (more preferably, SEQ ID NO: 2 or SEQ ID NO: 3). An antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by or a part thereof. The antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases. Phosphate residue (phosphate) of each nucleotide constituting antisense DNA to prevent degradation by hydrolase such as nuclease
May be substituted with, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, and phosphorodithionate. The sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base moiety (pyrimidine, purine) is also chemically modified. May have a DNA having the nucleotide sequence represented by SEQ ID NO: 2.
Any substance may be used as long as it hybridizes to. These antisense nucleotides are known as DN
It can be manufactured using an A synthesizer or the like.

According to the present invention, an antisense nucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is used as a nucleotide sequence information of a DNA encoding a cloned or determined protein. It can be designed based on and synthesized. Such a polynucleotide (nucleic acid) can hybridize with RNA of the protein gene of the present invention, can inhibit the synthesis or function of the RNA, or can interact with the protein-related RNA of the present invention. The expression of the protein gene of the present invention can be regulated / controlled. A polynucleotide complementary to a selected sequence of a protein-related RNA of the invention, and a protein-related RNA of the invention
The polynucleotide capable of specifically hybridizing with is useful for regulating and controlling the expression of the protein gene of the present invention in vivo and in vitro, and is useful for treating or diagnosing diseases and the like. The term "corresponds"
The term “has homology with or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide (base sequence or nucleic acid) and a peptide (protein) usually refers to an amino acid of a peptide (protein) in a command derived from the nucleotide (nucleic acid) sequence or its complement. . 5'end hairpin loop of protein gene, 5 '
Terminal 6-base pair repeat, 5'terminal untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3'terminal untranslated region, 3'terminal palindrome region, and 3'terminal hairpin loop Can be selected as a preferred region of interest, but any region within the protein gene can be selected as a region of interest. Regarding the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, when the target nucleic acid can hybridize with the target region, the target nucleic acid is compared with the polynucleotide of the target region. Can be said to be "antisense". Antisense (poly) nucleotides are
Polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or others having non-nucleotide backbones Polymers (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages (provided that the polymers have base pairings and base pairs such as those found in DNA and RNA). Containing a nucleotide having a configuration that allows attachment) and the like. They are double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA,
In addition it can be a DNA: RNA hybrid,
Furthermore, an unmodified polynucleotide (or an unmodified oligonucleotide), and an addition of a known modification,
For example, one having a label known in the art, one having a cap, one having a methylation, one having one or more natural nucleotides substituted with an analog, one having an intramolecular nucleotide modification, for example, a non-nucleotide Those having a charged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate,
Having a phosphorodithioate, etc., such as a protein (nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.)
Or those having side chain groups such as sugars (eg, monosaccharides), those having intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, radioactive metals, boron) , An oxidizing metal, etc.), an alkylating agent, or a modified bond (eg, α-anomer type nucleic acid). As used herein, the term "nucleoside", "nucleotide" and "nucleic acid" may include those having not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. The modified nucleotide and the modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen, an aliphatic group, or the like,
Alternatively, it may be converted into a functional group such as ether or amine. The antisense (poly) nucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, it makes the antisense nucleic acid more stable in the cell, increases the cell permeability of the antisense nucleic acid, has a greater affinity for the target sense strand, and if it is toxic, Make sense nucleic acid less toxic. Thus many modifications are known in the art, for example J. Kawakami et al., Pharm Tech Japan, Vo.
l. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Cr
ooke et al. ed., Antisense Research and Applicatio
ns, CRC Press, 1993 etc. The antisense nucleic acid of the present invention has an altered or modified sugar,
It may contain a base or a bond, and may be provided in a special form such as liposome, microsphere, applied by gene therapy, or provided in an added form. Thus, as an additive form, polycationic substances such as polylysine that act to neutralize the charge of the phosphate group, lipids that enhance the interaction with cell membranes and increase the uptake of nucleic acids ( Examples thereof include hydrophobic ones such as phospholipids and cholesterol). Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'-end or 5'-end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include
A capping group specifically arranged at the 3'-end or the 5'-end of a nucleic acid, which includes a group for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various known methods.

Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as the DNA of the present invention and Abbreviated), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the antibody of the present invention), and an antisense nucleotide of the DNA of the present invention (hereinafter referred to as the anti-nucleotide of the present invention). (Sometimes abbreviated as “sense nucleotide”).

Since the protein of the present invention has increased expression in cancer tissues, it can be used as a disease marker. That is, it is useful as a marker for early diagnosis in cancer tissues, determination of severity of symptoms, and prediction of disease progression. Therefore, a drug containing an antisense nucleotide of a gene encoding the protein of the present invention, a compound that inhibits the activity of the protein of the present invention or a salt thereof, or an antibody against the protein of the present invention is, for example, colon cancer, breast cancer, lung cancer. , Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer , A prophylactic / therapeutic agent for cancer such as brain tumor or blood tumor, and an apoptosis inducer.

(1) Screening for Drug Candidate Compounds against Diseases Since the protein of the present invention has increased expression in cancer tissues and has an apoptosis inhibitory action, it regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention. ) Compound or a salt thereof, for example, colon cancer, breast cancer, lung cancer,
Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer,
Can be used as a prophylactic / therapeutic agent for cancers such as spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor. it can. It can also be used as an apoptosis action regulator, preferably as an apoptosis inducer. Moreover, since the protein of the present invention has an activity of suppressing apoptosis induced by an anticancer agent (eg, paclitaxel), a compound that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention or The salt can also be used as an anticancer drug sensitivity enhancer. Therefore, the protein of the present invention is useful as a reagent for screening a compound or its salt that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention. That is, the present invention is characterized by using the protein of the present invention (eg, ligand binding activity, signal transduction activity, etc.)
A method for screening a compound or its salt that regulates (promotes or inhibits, preferably inhibits) is provided. More specifically, for example, (i) the ligand binding activity or signal transduction activity of cells having the ability to produce the protein of the present invention, and (ii) the cells having the ability to produce the protein of the present invention and test compounds A method for screening a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention, which is characterized by comparing the ligand binding activity or the signal transduction activity of a mixture is used. In the above screening method,
For example, in the cases (i) and (ii), the ligand binding activity or the signal transduction activity can be determined by a known method, for example, J. Imm.
Unol. 166 (1), pp. 15-19, 2001 or a method analogous thereto, and measure and compare. Specifically, in the case of screening a compound having an action of promoting ligand binding or intracellular signal transduction, (i)
When the protein expression vector of the present invention was introduced into a cell together with a reporter gene containing a binding sequence of NF-κB in a promoter or enhancer and cultured, (ii) the protein expression vector of the present invention was NF-
The reporter expression level in the case where the binding sequence of κB is introduced into a cell together with a reporter gene contained in a promoter or an enhancer and cultured in the presence of a test compound is compared. The reporter expression level is the luciferase activity,
It can be measured by using reporter protein activity such as alkaline phosphatase activity as an index. Specifically, when screening a compound having a ligand binding inhibitory effect or an intracellular signal transduction inhibitory effect,
(I) The protein expression vector of the present invention is introduced into a cell together with a reporter gene containing a binding sequence of NF-κB in a promoter or an enhancer, and if necessary,
(A) a liquid containing a ligand such as a microbial cell lysate, a microbial culture supernatant, a eukaryotic cell lysate, a eukaryotic cell culture supernatant,
(B) When the ligand itself or (c) a substance having a binding activity equivalent to that of the natural ligand is added and cultured,
i) The protein expression vector of the present invention is introduced into cells together with a reporter gene containing a binding sequence of NF-κB in a promoter or enhancer, and (a) a microbial cell disruption solution, a microbial culture supernatant, and Culture in the presence of a test compound by adding a substance that has a binding activity equivalent to that of a lysate of nuclear cells, a eukaryotic cell culture supernatant, etc., (b) the ligand itself or (c) the natural ligand. In this case, the reporter expression level is measured and compared. Examples of the ligand include lipopeptides derived from bacteria (preferably triacylated lipopeptides, etc.) or N-palmitoyl-S- obtained by chemical synthesis.
dipalmitoylglyceryl (Pam ₃ ) Cys- Ser- (Lys) ₄ (CS
K ₄ ), N-palmitolyl-S-dilaurylgryceryl (N-Pam-S-Lau
₂ ) Triacyl peptides such as CSK ₄ , Borrelia burgdorf
An outer-surface lipoprotein (OspA) derived from eri is used. The reporter expression level can be measured by using the reporter protein activity such as luciferase activity and alkaline phosphatase activity as an index.
In addition, the signal transduction activity can be measured by measuring and comparing the apoptotic activity against an anti-cancer agent (eg, paclitaxel) with or without the expression of the protein (eg, TLR1) used in the present invention by a known method. . The apoptotic activity against an anticancer drug (eg, paclitaxel) in the presence or absence of a ligand can be measured and compared by a known method to measure the signal transduction activity. Furthermore, in order to measure the signal transduction of TLR1 used in the present invention with higher sensitivity, cells having an ability to express a TLR1 signal-related molecule (eg, TLR2, MD2, etc.) may be used, A TLR1 signal-related molecule (eg, TLR2, MD2, etc.) may be forcibly expressed and the above activity may be measured. As the cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with the vector containing the DNA encoding the protein of the present invention is used. As the host, for example, animal cells such as COS7 cells, CHO cells and HEK293 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on the cell membrane by culturing by the method described above is preferably used. The method of culturing cells capable of expressing the protein of the present invention is the same as the method of culturing the transformant of the present invention described above. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like. For example, the ligand binding activity or signal transduction activity in the case of (ii) above is about 20% or more as compared with the case of (i) above,
The test compound, which preferably decreases by 30% or more, more preferably about 50% or more, is used as a compound that inhibits the activity of the protein of the present invention, and the ligand binding activity or signal transduction activity in the case of (ii) above is A test compound that increases by about 20% or more, preferably by 30% or more, more preferably by about 50% or more compared to the case can be selected as a compound that promotes the activity of the protein of the present invention.

The compound having an activity of inhibiting the activity of the protein of the present invention is a safe and low-toxic drug for suppressing the physiological activity of the protein of the present invention, such as colon cancer,
Breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, It is useful as a prophylactic / therapeutic agent for cancer such as pancreatic cancer, brain tumor or blood tumor, apoptosis inducer, anti-cancer drug sensitivity enhancer and the like. The compound having the activity of promoting the activity of the protein of the present invention is useful as a safe and low toxic pharmaceutical for enhancing the action of the protein of the present invention. Compounds or salts thereof obtained using the screening method or screening kit of the present invention, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products,
It is a compound selected from cell extract, plant extract, animal tissue extract, plasma and the like. As the salt of the compound, those similar to the salts of the peptide of the present invention described above are used.

Furthermore, since the expression of the gene encoding the protein of the present invention is also increased in cancer tissues, the compound or its salt that regulates the expression of the gene encoding the protein of the present invention, such as breast cancer, colon cancer, etc. , Breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer It is also useful as a prophylactic / therapeutic agent for cancer such as pancreatic cancer, brain tumor or blood tumor, an apoptosis inducer, and an anticancer drug sensitivity enhancer. Therefore, the DNA of the present invention regulates (promotes or inhibits the expression of the gene encoding the protein of the present invention,
It is useful as a reagent for screening a compound or its salt that inhibits preferably. The screening method includes (iii) culturing cells having the ability to produce the protein of the present invention and (iv) culturing cells having the ability to produce the protein used in the present invention in the presence of a test compound. A screening method is characterized in that it is compared with the case. In the above method, in the cases of (iii) and (iv), the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured and compared. Examples of the cell having the ability to produce the test compound and the protein of the present invention include the same ones as described above. The amount of protein is measured by a known method, for example, using an antibody that recognizes the protein of the present invention, the protein present in a cell extract or the like is subjected to Western analysis,
It can be measured according to a method such as an ELISA method or a method similar thereto. The amount of mRNA can be measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a probe, or PCR using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a primer. It can be measured according to the method or a method similar thereto. For example, the gene expression level in the case of (iv) is about 20% or more, preferably 30% or more, as compared with the case of (iii).
More preferably, the test compound that inhibits by about 50% or more is used as a compound that suppresses the expression of the gene encoding the protein of the present invention, and the gene expression level in the case of (iv) is higher than that in the case of (iii). , A test compound that increases by about 20% or more, preferably by 30% or more, more preferably by about 50% or more can be selected as a compound that promotes the expression of the gene encoding the protein of the present invention.

The screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide used in the present invention. The compound or its salt obtained by using the screening method or the screening kit of the present invention 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, a plant extract. , An animal tissue extract, plasma or the like, or a salt thereof, and a compound or a salt thereof that regulates the activity (eg, ligand binding activity or signal transduction activity) of the protein of the present invention. As the salt of the compound, those similar to the salts of the protein of the present invention described above are used. The compound or its salt that regulates (preferably inhibits) the activity of the protein of the present invention, and the compound or its salt that regulates (preferably inhibits) the expression of the gene encoding the protein of the present invention are each, for example, colon cancer, Breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, It is useful as a prophylactic / therapeutic agent for cancer such as pancreatic cancer, brain tumor or blood tumor, apoptosis inducer, anti-cancer drug sensitivity enhancer and the like. When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned medicine, it can be formulated according to a conventional method.

For example, as a composition for oral administration, solid or liquid dosage forms, specifically tablets (sugar-coated tablets,
Film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets. As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, intraarticular injection. Includes dosage forms such as agents. Such an injection is prepared according to a known method, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like are used. Suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg polysorbate 8
0, HCO-50 (polyoxyethylene (50mol) adduct
of hydrogenated castor oil)] and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is usually filled in a suitable ampoule. The suppository used for rectal administration is prepared by mixing the above compound or a salt thereof with a usual suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form suitable for the dose of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg per dosage unit dosage form, especially 5 to 10 injections.
It is preferable that the above antibody is contained in 0 mg, and in other dosage forms, 10 to 250 mg. Each composition described above may further contain other active ingredients as long as it does not cause an unfavorable interaction with the above compound. Since the preparation thus obtained is safe and has low toxicity, for example, humans or warm-blooded animals (for example, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog,
It can be administered orally or parenterally to monkeys, chimpanzees, etc.). The dose of the compound or its salt varies depending on its action, target disease, administration subject, administration route, etc., but for example, for the purpose of treating breast cancer, the compound or its salt that regulates the activity of the protein of the present invention When administered orally, adults (body weight 60 kg
In the above), about 0.1 to 100 mg, preferably about 1.0 to 50 m of the compound or a salt thereof per day.
g, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound or its salt varies depending on the administration subject, target disease, etc., but for example, a compound that regulates the activity of the protein of the present invention for the purpose of treating breast cancer, or When the salt is usually administered in the form of an injection to an adult (body weight is 60 kg), the compound or salt thereof is about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably About 0.1
It is convenient to administer about 10 mg to the cancer lesion by injection. Also in the case of other animals, the dose converted per 60 kg of body weight can be administered. In addition, the above compounds can be used in combination with existing anticancer agents (eg, paclitaxel, docetaxel, etc.), and side effects of the anticancer agents that damage normal cells are reduced. At this time, the timing of administration is not limited,
It may be administered at the same time or at different times. The dose can be appropriately selected based on the dose clinically used. The compounding ratio of the compound to the anticancer agent can be appropriately selected depending on the administration subject, administration route, target disease, symptom, combination and the like.

(2) Quantification of the protein of the present invention, its partial peptide or its salt An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) specifically recognizes the protein of the present invention. Therefore, it can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention is
(I) An antibody of the present invention is allowed to competitively react with a test solution and a labeled protein of the present invention, and the ratio of the labeled protein of the present invention bound to the antibody is measured. And a method for quantifying the protein of the present invention in a test solution, and (ii) the test solution and the antibody of the present invention insolubilized on a carrier and another labeled antibody of the present invention are simultaneously or consecutively There is provided a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction. In the quantification method of (ii) above, it is desirable that one antibody recognizes the N-terminal portion of the protein of the present invention and the other antibody reacts with the C-terminal portion of the protein of the present invention.

In addition to quantifying the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as "monoclonal antibody of the present invention"), detection by tissue staining or the like can also be performed. . For these purposes, the antibody molecule itself may be used, or 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 may include an antibody, an antigen or an antibody-antigen complex corresponding to the amount of antigen (for example, the amount of protein) in the liquid to be measured. Any measuring method may be used as long as it is a method of detecting the amount by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used, but from the viewpoint of sensitivity and specificity, the sandwich method described later is particularly preferably used. 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, a lanthanide element or the like is used. Examples of radioactive isotopes include [ ¹²⁵ I],
[ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably stable and has a large specific activity,
For example, β-galactosidase, β-glucosidase,
Alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. As the luminescent substance, for example,
Luminol, luminol derivative, luciferin, lucigenin and the like are used. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing and immobilizing proteins or enzymes 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, a test solution is reacted with the insolubilized monoclonal antibody of the present invention (first reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. The amount of the protein of the present invention in the test liquid 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, may be carried out simultaneously, or may be carried out at different times. The labeling agent and the method of insolubilization can be the same as those described above. In addition, 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 may be used for the purpose of improving the measurement sensitivity. In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibodies of the present invention used in the primary reaction and the secondary reaction are preferably antibodies having different binding sites for the protein of the present invention. That is, 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 and the secondary reaction is preferably an antibody used in the primary reaction. 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 measuring 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 reacted competitively with the antibody, and then the unreacted labeled antigen is reacted.
(F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), the labeled amount of either B or F is measured, and the amount of antigen in the test liquid is quantified. In this reaction method, a soluble antibody is used as the antibody, a liquid phase method using polyethylene glycol, a second antibody against the antibody is used for B / F separation, and a solid phase antibody is used as the first antibody, or The first antibody is soluble and the second antibody is a solid-phased antibody. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are competitively reacted with a fixed amount of the labeled antibody and then the solid phase and the liquid phase are separated, or the antigen in the test liquid is separated. After reacting with an excess amount of the labeled antibody and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated.
Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry 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 and the like. The protein measurement system of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like. For example, Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. "Enzyme Immunoassay" (Medical Shoin, Showa Showa) 1993), Eiji Ishikawa et al., "Enzyme Immunoassay" (second edition) (Medical Shoin, published in 1982), Eiji Ishikawa, et al. "Enzyme Immunoassay" (3rd edition) (Medicine Shoin, Showa) 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunochem
ical Techniques (Part A)), ibid Vol. 73 (Immunochem
ical Techniques (Part B)), ibid Vol. 74 (Immunochem
ical Techniques (Part C)), ibid Vol. 84 (Immunochem
ical Techniques (Part D: Selected Immunoassays)),
Ibid Vol. 92 (Immunochemical Techniques (Part E: Mono
clonal Antibodies and General Immunoassay Method
s)), ibid Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodie
s)) (above, published by Academic Press). As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, when an increase (or decrease) in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, colorectal cancer, breast cancer, lung cancer, prostate cancer,
Esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor It can be diagnosed as having cancer or having a high possibility of contracting in the future. Further, when the concentration of the protein of the present invention is high, it can be diagnosed that the effect of administering the anticancer agent (eg, paclitaxel) is low. Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as body fluid or tissue. In addition, for 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.

(3) Genetic Diagnostic Agent The DNA of the present invention can be used as a probe, for example, to prepare human or warm-blooded animals (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow,
Since it is possible to detect an abnormality (gene abnormality) of the DNA or mRNA encoding the protein of the present invention or its partial peptide in horses, cats, dogs, monkeys, chimpanzees, etc., for example, the DNA or mRNA can be detected.
It is useful as a gene diagnostic agent for damage, mutation or decrease in expression, increase in DNA or mRNA, or excessive expression. The above-mentioned gene diagnosis using the DNA of the present invention is carried out, for example, by known Northern hybridization or PCR-SSCP method (Genomics,
Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences of USA.
heNational Academy of Sciences of the United State
s of America, 86, 2766-2770 (1
989)) and the like. For example, when overexpression or reduction is detected by Northern hybridization or when a DNA mutation is detected by PCR-SSCP method, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer ,
Liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer,
It can be diagnosed as being likely to have a cancer such as a brain tumor or a blood tumor.

(4) Medicament Containing Antisense Nucleotide The antisense nucleotide of the present invention, which can bind to the DNA of the present invention complementarily and suppress the expression of the DNA, has low toxicity. Since the function of the protein of the present invention or the DNA of the present invention (eg, ligand binding activity or signal transduction activity) can be suppressed, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver Cancer, biliary tract cancer, spleen cancer, kidney cancer,
Bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain / blood cancer, and other cancer preventive / therapeutic agents, apoptosis inducers, anticancer drug sensitivity enhancers, etc. Can be used as When the above antisense nucleotide is used as the above preventive / therapeutic agent,
It can be formulated and administered according to known methods.
In addition, for example, after inserting the antisense nucleotides described above alone or in a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc., according to a conventional method, a human or mammal (eg, rat, rabbit) , Sheep, pigs, cattle, cats, dogs, monkeys, etc.) orally or parenterally. The antisense nucleotide can be administered as it is or in the form of a formulation with a physiologically acceptable carrier such as an auxiliary agent for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant. Furthermore, for the purpose of improving pharmacokinetics, prolonging half-life, and improving intracellular uptake efficiency, the above antisense nucleotides are formulated alone or in combination with a carrier such as liposome (injection), and then intravenously, subcutaneously or intraarticularly Alternatively, it may be administered to a cancer lesion or the like. Although the dose of the antisense nucleotide varies depending on the target disease, the administration subject, the administration route, etc., for example, when the antisense nucleotide of the present invention is administered for the purpose of treating breast cancer, an adult (body weight 60 kg) is generally used. In (), about 0.1 to 100 mg of the antisense nucleotide is administered per day. Furthermore, the antisense nucleotide can also be used as a diagnostic oligonucleotide probe for investigating the presence or expression of the DNA of the present invention in tissues or cells. Similar to the above antisense nucleotides, double-stranded RNA containing a part of RNA encoding the protein of the present invention, RNA encoding the protein of the present invention
A ribozyme containing a part of the above can also suppress the expression of the gene of the present invention, and can suppress the function of the protein used in the present invention or the DNA used in the present invention in vivo. ,Colorectal cancer,
Breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, It can be used as a prophylactic / therapeutic agent for cancer such as pancreatic cancer, brain tumor or blood tumor, apoptosis inducer, anticancer drug sensitivity enhancer and the like. Double-stranded RNA can be prepared by known methods (eg, Natu
re, 411, 494, 2001), and can be designed and manufactured based on the sequence of the polynucleotide of the present invention. Ribozymes can be prepared by known methods (eg, TRENDS in Molecu
lar Medicine, 7: 221, 2001), and can be designed and manufactured based on the sequence of the polynucleotide of the present invention. For example, R encoding the protein of the present invention
It can be produced by linking a known ribozyme to a part of NA. As a part of RNA encoding the protein of the present invention, a portion (RNA fragment) close to the cleavage site on the RNA of the present invention which can be cleaved by a known ribozyme can be mentioned. When the above double-stranded RNA or ribozyme is used as the above-mentioned preventive / therapeutic agent,
Formulated in the same manner as antisense polynucleotides,
It can be administered. Similar to the antisense nucleotide, the protein of the present invention or an aptamer of DNA encoding the protein of the present invention can suppress the expression of the gene of the present invention, and is used in vivo in the present invention or the present invention. DNA used in
Since it can suppress the function of, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer,
Liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer,
It can be used as a preventive / therapeutic agent for cancers such as brain tumors or blood tumors, an apoptosis inducer, and an anticancer drug sensitivity enhancer. The aptamer can be produced according to a known method.

(5) Medicament Containing the Antibody of the Present Invention The antibody of the present invention having the activity of neutralizing the activity of the protein of the present invention is, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, Cancers such as gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor It can be used as a safe drug with low toxicity such as a prophylactic / therapeutic agent, an apoptosis inducer, and an anticancer drug sensitivity enhancer. The preventive / therapeutic agent for the above diseases containing the antibody of the present invention has low toxicity, and as a liquid formulation as it is, or as a pharmaceutical composition in an appropriate dosage form, a human or mammal (eg, rat, rabbit, sheep, pig) , Cow, cat,
Orally or parenterally (eg, in dogs, monkeys, etc.)
Intra-articular administration). The dose varies depending on the administration subject, target disease, symptom, administration route, etc., but when it is used for treatment / prevention of adult breast cancer, for example, the antibody of the present invention is usually used as a single dose. .
01 to 20 mg / kg body weight, preferably 0.1 to 1
0 mg / kg body weight, more preferably 0.1-5 m
g / kg body weight about 1 to 5 times a day, preferably 1
It is convenient to administer by powder inhalation about once to three times a day. In the case of other parenteral administration and oral administration, an amount similar to this can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a composition is provided as a dosage form suitable for oral or parenteral administration (eg, intravenous injection administration). Each composition described above may contain other active ingredients as long as the composition does not cause an unfavorable interaction with the antibody.

(6) "Preventive / therapeutic agent for cancer comprising a compound having a Toll-like receptor activity-regulating activity or a salt thereof" of the present invention and a "Toll-like receptor expression-modulating activity "A compound having a Toll-like receptor activity-regulating action" for "a prophylactic / therapeutic agent for cancer containing a salt thereof"
May be any compound as long as it has a Toll-like receptor activity-regulating action, and examples thereof include colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, and spleen. Cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain cancer or hematological cancer, etc. Used as a drug sensitivity enhancer. "Compound having a Toll-like receptor expression-regulating action"
May be any compound as long as it has a Toll-like receptor expression regulating action, and examples thereof include colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, and spleen. Cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain cancer or hematological cancer, etc. Used as a drug sensitivity enhancer. The prophylactic / therapeutic agent is produced in the same manner as above.

(7) DNA Transfer Animal The present invention provides D which encodes an exogenous protein of the present invention.
Provided is a non-human mammal having NA (hereinafter abbreviated as exogenous DNA of the present invention) or a mutant DNA thereof (may be abbreviated as exogenous mutant DNA of the present invention). That is, the present invention provides (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, (2) an animal according to (1), wherein the non-human mammal is a rodent, (3) (2) An animal according to (2), wherein the rodent is a mouse or rat; and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof, which can be expressed in a mammal. . The non-human mammal having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter, abbreviated as the DNA-transferred animal of the present invention) is used for embryo cells including unfertilized eggs, fertilized eggs, sperms and their progenitor cells. Preferably, at the stage of embryogenesis in development of non-human mammals (more preferably at the stage of single cells or fertilized egg cells and generally before the 8-cell stage), calcium phosphate method, electric pulse method, lipofection method, agglutination method, Micro injection method, particle gun method, DEA
It can be produced by transferring the target DNA by the E-dextran method or the like. Also, the DN
The foreign DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, etc. by the A transfer method and can be used for cell culture, tissue culture, etc., and these cells can be used for the above-mentioned embryo. The DNA-transferred animal of the present invention can also be produced by fusing cells with a known cell fusion method. Examples of non-human mammals include cows,
Pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Among them, rodents, especially mice (for example, C57 as a pure line), which have relatively short ontogeny and biological cycle from the viewpoint of creating a pathological animal model system, and are easy to reproduce.
BL6 line, DBA2 line, etc.
C3F ₁ system, BDF ₁ system, B6D2F ₁ system, BAL
B / c strain, ICR strain, etc.) or rat (eg,
(Wistar, SD, etc.) and the like are preferable. "Mammal" in a recombinant vector that can be expressed in a mammal
Examples of the non-human mammals include humans and the like.

The exogenous DNA of the present invention means the DNA of the present invention once isolated and extracted from mammals, not the DNA of the present invention originally possessed by non-human mammals.
The mutant DNA of the present invention is a DNA having a mutation (eg, mutation) in the base sequence of the original DNA of the present invention,
Specifically, DNA in which a base is added, deleted, or replaced with another base is used, and abnormal DNA is also included. The abnormal DNA means a DNA that expresses the abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used. The exogenous DNA of the present invention is
It may be derived from mammals of the same or different species from the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter that can be expressed in animal cells. For example, when transferring the human DNA of the present invention, expression of DNA derived from various mammals (for example, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) having the DNA of the present invention having high homology with the human DNA The DNA construct of the present invention is highly expressed by microinjecting a DNA construct (eg, a vector) having the human DNA of the present invention bound downstream of various promoters capable of causing the fertilized egg of a target mammal, for example, a mouse fertilized egg. A DNA-transferred mammal that produces

Examples of the expression vector of the protein of the present invention include Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage such as λ phage, retrovirus such as Moloney leukemia virus, vaccinia virus or baculovirus. The animal viruses and the like are used. Of these, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, and the like are preferably used. DN above
Examples of the promoter that regulates A expression include:
DNA promoters derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, (Eg, mouse) -derived promoters such as albumin, insulin I
I, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP dependence Protein kinase βI subunit, dystrophin, tartrate-resistant alkaline phosphatase,
Atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 phosphatase (Na, K-ATPas)
e), neurofilament light chain, metallothionein I
And IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1
α), β actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, H chain variable region (VN
P), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A, vasopressin and other promoters are used. Among them, cytomegalovirus promoter, human peptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoter, which can be highly expressed systemically, are preferable. The vector preferably has a sequence (generally called a terminator) that terminates the transcription of the desired messenger RNA in a DNA-transferred mammal. For example, DNAs derived from viruses and various mammals
Can be used, and preferably the SV40 terminator of simian virus is used.

In addition, in order to further highly express the desired foreign DNA, the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, and the like are provided 5 ′ upstream of the promoter region, between the promoter region and the translation region. Alternatively, it may be linked to the 3'downstream of the translation region depending on the purpose. The normal translation region of the protein of the present invention includes human or various mammals (for example, rabbit, dog, cat, guinea pig, hamster, rat,
Known as all or part of genomic DNA from liver, kidney, thyroid cells, fibroblast-derived DNA derived from mice etc.) and various commercially available genomic DNA libraries, or from liver, kidney, thyroid cells, fibroblast-derived RNA It is possible to obtain the complementary DNA prepared by the above method as a raw material. In addition, an exogenous abnormal DNA can be prepared by mutating a translation region of a normal protein obtained from the above-mentioned cells or tissues by a point mutagenesis method. The translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering method in which it is linked to the downstream of the promoter and, if desired, the upstream of the transcription termination site. The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the embryonic cell of the produced animal after DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of its embryonic cells and somatic cells. means. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells and somatic cells. Exogenous normal DNA of the present invention
Non-human mammals that have translocated
After confirming that A is stably retained, the DNA-carrying animal can be subcultured in a normal breeding environment. Transfer of foreign DNA of the present invention at the fertilized egg cell stage is
It is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the producing animal after DNA transfer means that all the progeny of the producing animal have the exogenous DNA of the present invention in all of their germinal cells and somatic cells. Means The offspring of this type of animal that inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in excess in all of their germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all offspring can be passaged so that the DNA has an excess.

In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally obtained by promoting the function of the endogenous normal DNA. It may develop hyperactivity and can be used as a model animal for its pathological condition.
For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases related to the protein of the present invention, and to investigate treatment methods for these diseases. It is possible. In addition, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, a preventive / therapeutic agent for diseases associated with the protein of the present invention, such as colon cancer, Breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer,
It can also be used for screening tests for preventive / therapeutic agents for cancers such as bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor. On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured as a DNA-carrying animal in a normal breeding environment after confirming that the exogenous DNA is stably retained by mating. Furthermore, the foreign DNA of interest can be incorporated into the above-mentioned plasmid and used as a protozoa. The DNA construct with the promoter can be prepared by a usual DNA engineering technique.
The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is
It is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the producing animal after DNA transfer means that all the progeny of the producing animal have the abnormal DNA of the present invention in all of its germinal cells and somatic cells. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the progeny can be passaged so that they have the DNA.

In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally obtained by inhibiting the function of the endogenous normal DNA. It may become inactive refractory and can be used as a model animal for its pathological condition. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the functionally inactive refractory disease of the protein of the present invention and to examine the method for treating this disease. In addition, as a specific applicability, the animal with high expression of the abnormal DNA of the present invention shows the functional inhibition (dominant negative action) of the normal protein by the abnormal protein of the present invention in the functionally inactive refractory disease of the protein of the present invention. It becomes a model to elucidate. In addition, the foreign abnormal DNA of the present invention
Since the mammal having the metastasis of the present invention has an increased symptom of the released protein of the present invention, a preventive / therapeutic agent for the protein of the present invention or a functionally inactive refractory disease, for example, colon cancer, breast cancer, lung cancer, or prostate Cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or It can also be used for screening tests for preventive and therapeutic agents for cancer such as blood tumors. Further, other applications of the above-mentioned two kinds of the DNA-transferred animals of the present invention include, for example, use as a cell source for tissue culture, DNA in tissues of the DNA-transferred animals of the present invention, or R.
Analysis of the relationship with peptides specifically expressed or activated by the protein of the present invention by directly analyzing NA or analyzing peptide tissues expressed by DNA, cells of tissues having DNA are analyzed. The cells are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture, screening for agents that enhance the function of cells by using the cells described above, and It is conceivable to isolate and purify the mutant protein and produce its antibody.

Furthermore, the DNA-transferred animal of the present invention can be used to examine the clinical symptoms of diseases related to the protein of the present invention, including functionally inactive refractory diseases of the protein of the present invention. More detailed pathological findings in each organ of the disease model related to the protein of the invention can be obtained, which can contribute to the development of new therapeutic methods and further to the research and treatment of secondary diseases caused by the diseases. Further, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain released DNA-transferred cells, culture the cells, or systematize the cultured cells. . Furthermore, it is possible to investigate the specification of the protein-producing cell of the present invention, its relation to apoptosis, differentiation or proliferation, or the signal transduction mechanism in them, and to investigate their abnormalities. It becomes an effective research material for Furthermore, the DNA of the present invention
In order to develop a therapeutic agent for a disease related to the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention using a metastatic animal, using the above-mentioned test method and quantification method, etc., It is possible to provide an effective and rapid screening method for the therapeutic drug for the disease. In addition, D of the present invention
DNA of a disease associated with the protein of the present invention using the NA-transferred animal or the foreign DNA expression vector of the present invention
It is possible to study and develop treatment methods.

(8) Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a DNA expression-deficient non-human mammal of the present invention. That is, the present invention provides (1) a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated,
(2) The embryonic stem cell according to item (1), wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), (3) neomycin-resistant item (1) The embryonic stem cell according to item (4), the embryonic stem cell according to item (1), wherein the non-human mammal is a rodent, (5) the embryonic stem cell according to item (4), wherein the rodent is a mouse, ( 6) The DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated, (7) The DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), The non-human mammal according to item (6), wherein the reporter gene can be expressed under the control of a promoter for the DNA of the present invention,
(8) The non-human mammal according to item (6), wherein the non-human mammal is a rodent, (9) the non-human mammal according to item (8), wherein the rodent is a mouse, and (10) ) D of the present invention, which comprises administering a test compound to the animal according to item (7) and detecting the expression of a reporter gene.
Provided is a screening method for a compound or its salt that promotes or inhibits the promoter activity against NA.

The non-human mammal embryonic stem cells in which the DNA of the present invention is inactivated means that the expression ability of the DNA is suppressed by artificially mutating the DNA of the present invention possessed by the non-human mammal. Alternatively, the DNA does not substantially have the ability to express the protein of the present invention by substantially losing the activity of the protein of the present invention encoded by the DNA (hereinafter, referred to as the knockout DNA of the present invention. Occasionally) non-human mammalian embryonic stem cells (hereinafter ES
Abbreviated as cell). As the non-human mammal, the same ones as described above are used. Examples of the method for artificially mutating the DNA of the present invention include, for example, deletion of a part or all of the DNA sequence by a genetic engineering method, other DNA
By inserting or replacing. Due to these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the reading frame of the codon or disrupting the function of the promoter or exon.

Specific examples of the non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated (hereinafter abbreviated as the DNA inactivated ES cell of the present invention or the knockout ES cell of the present invention) include, for example, The DNA of the present invention contained in the target non-human mammal is isolated, and a drug resistance gene represented by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloram) is present in the exon portion thereof. A DNA sequence (eg, pol) that disrupts exon function by inserting a reporter gene typified by phenicol acetyl transferase gene or terminates transcription of the gene in the intron portion between the exons.
DN having a DNA sequence constructed so as to disrupt the gene by inserting a yA addition signal or the like) and thereby making it impossible to synthesize a complete messenger RNA.
A chain (hereinafter abbreviated as targeting vector)
Is introduced into the chromosome of the animal by, for example, a homologous recombination method, and the obtained ES cells are targeted to a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe. It can be obtained by selecting the knockout ES cells of the present invention by analysis by the PCR method using the DNA sequence of the neighboring region other than the DNA of the present invention used for vector preparation as a primer. Further, as the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like, for example, those already established as described above may be used, and known Evans
It may be newly established according to the method of Kaufman. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used. However, since the immunological background is not clear, an alternative pure line immunological genetic background is used. For the purpose of obtaining ES cells whose expression is clear, for example, C57BL / 6 mouse and C57BL / 6
BDF ₁ mice a lack of egg collection number was improved by crossing with DBA / 2 of (F ₁ of C57BL / 6 and DBA / 2)
Those established by using can also be used favorably. BDF ₁
In addition to the advantage that the number of eggs collected is large and the eggs are strong, the mouse has C57BL / 6 mice in the background. Therefore, the ES cells obtained by using these mice have C57BL / 6 It can be advantageously used in that its genetic background can be changed to that of C57BL / 6 mice by backcrossing with / 6 mice. Also, ES
When establishing cells, blastocysts on the 3.5th day after fertilization are generally used, but in addition to this, 8-cell stage embryos are ovulated and cultured up to the blastocysts for efficient use of many early embryos. Can be obtained. Either male or female ES cells may be used, but male ES cells are usually more convenient for producing a germ line chimera. In addition, it is desirable to distinguish males and females as soon as possible in order to reduce troublesome culturing.

As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in the sex determining region on the Y chromosome by the PCR method can be mentioned. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture It is possible to perform the primary selection of ES cells in the early stage by discriminating males and females, and the early selection of male cells can significantly reduce the labor required in the initial stage of culture.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of ES cells obtained is 100% of the normal number
However, if it is difficult because of physical manipulations during establishment, the gene of ES cell is knocked out, and then cloned again into normal cell (for example, cell having 2n = 40 chromosomes in mouse). Is desirable. The embryonic stem cell line thus obtained usually has a very good proliferative property, but since it is easy to lose the ability to develop ontogeny, it is necessary to carefully subculture. For example, on a suitable feeder cell such as STO fibroblast, LIF (1-
Incubate in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% carbon dioxide, 90% air) in the presence of 10,000 U / ml at about 37 ° C. However, at the time of subculture, for example, trypsin / EDTA
Solution (usually 0.001-0.5% trypsin / 0.1-5
mM EDTA, preferably about 0.1% trypsin / 1m
(M EDTA) treatment to obtain single cells and seeding on newly prepared feeder cells. Such passage is usually carried out every 1 to 3 days. At this time, it is desirable to observe the cells and to discard the cultured cells when morphologically abnormal cells are found. ES cells are differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by performing monolayer culture until reaching a high density or suspension culture until forming a cell clump under appropriate conditions. Is possible [MJ Evans and MH
Kaufman, Nature Volume 292, 154, 1981.
Year; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1
981; TC Doetschman et al., Journal of Embiology and Experimental Morphology, Vol. 87, p. 27, 1985], deficient DNA expression of the present invention obtained by differentiating ES cells of the present invention. The cells are useful in in vitro cell biology studies of the proteins of the invention.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from normal animals by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level. is there. As the non-human mammal,
The same as the above is used. The non-human mammal deficient in DNA expression of the present invention can be obtained by, for example, introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells, and inactivating the DNA of the present invention in the targeting vector. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination. A cell in which the DNA of the present invention has been knocked out is a DNA on or near the DNA of the present invention.
A DNA sequence on a Southern hybridization analysis or targeting vector using the sequence as a probe,
It can be determined by analysis by the PCR method using a DNA sequence of a region other than the mouse-derived DNA of the present invention used as the targeting vector as a primer. When a non-human mammal embryonic stem cell is used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cell is cloned at an appropriate time, for example, at the 8-cell stage. It is injected into an animal embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has the mutated DNA locus of the present invention, all tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of the cells having the added DNA locus of the present invention, for example, by judging the coat color. The individual thus obtained is usually an individual deficient in heteroexpression of the protein of the present invention,
Individuals who do not express the protein of the present invention can be crossed to obtain individuals who do not express the protein of the present invention.

When egg cells are used, for example, a transgenic non-human mammal having a targeting vector introduced into its chromosome can be obtained by injecting a DNA solution into the nucleus of an egg cell by the microinjection method. It can be obtained by selecting those having a mutation in the DNA locus of the present invention by gene homologous recombination as compared with a transgenic non-human mammal. In this way, an individual whose DNA of the present invention has been knocked out can be passaged in a normal breeding environment after confirming that the DNA of the animal individual obtained by mating is also knocked out. Furthermore, the acquisition and maintenance of the germ line may be carried out according to the conventional method. That is, by mating male and female animals carrying the inactivated DNA,
A homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The homozygous animals obtained are
It can be efficiently obtained by breeding a mother animal in a state where there are one normal individual and a plurality of homozygotes. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred. DNA of the present invention
The non-human mammal embryonic stem cells inactivated by the method are very useful for producing the non-human mammal deficient in DNA expression of the present invention. Further, the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, and thus is a model of diseases caused by inactivation of the biological activity of the protein of the present invention. Therefore, it is useful for investigating the causes of these diseases and examining therapeutic methods.

(8a) Method for Screening Compound Having Curative / Preventive Effect Against Disease Caused by DNA Deficiency or Damage of the Present Invention The non-human mammal deficient in DNA expression of the present invention is D
It can be used for screening a compound having a therapeutic / preventive effect on diseases caused by NA deficiency or damage. That is, the present invention results from a defect or damage of the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in DNA expression of the present invention and observing and measuring the change in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against diseases such as cancer. Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include the same ones as described above. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc., and these compounds are novel compounds. Or a known compound may be used. Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound and compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / prophylactic effect of a compound can be tested. Examples of the method for treating a test animal with a test compound include oral administration and intravenous injection,
It can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound is
It can be appropriately selected depending on the administration method, the properties of the test compound, and the like.

For example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, When screening a compound having a therapeutic / prophylactic effect against cancer such as testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, the test compound is administered to the non-human mammal deficient in DNA expression of the present invention Then, the difference in the degree of cancer development and the difference in the degree of cancer cure from the test compound-non-administered group is observed with time in the above tissues. In the screening method, when a test compound is administered to a test animal, the test compound is improved when the disease symptoms of the test animal are improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more. It can be selected as a compound having a therapeutic / prophylactic effect against the above diseases. The compound obtained by using the screening method is a compound selected from the above-mentioned test compounds and has a therapeutic / prophylactic effect against a disease caused by a deficiency or damage of the protein of the present invention. It can be used as a medicine such as a safe and low toxic preventive / therapeutic agent. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well. The compound obtained by the screening method may form a salt, and the salt of the compound may be a physiologically acceptable acid (eg, inorganic acid, organic acid, etc.) or base (eg, alkali metal, etc.). ) And the like are used, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid,
Benzenesulfonic acid, etc.) and the like are used. The drug containing the compound or its salt obtained by the screening method can be manufactured in the same manner as the drug containing the protein of the present invention. Since the preparation thus obtained is safe and has low toxicity, for example,
It can be administered to humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). The dose of the compound or its salt varies depending on the target disease, the administration subject, the administration route, etc. However, for example, when the compound is orally administered, it is generally an adult (weight 60 kg).
In breast cancer patients, the amount of the compound is about 0.
1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg is administered. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, the compound is usually administered in the form of an injection to an adult (as 60 kg) breast cancer patient. In this case, about 0.01 to 30 m of the compound per day
It is convenient to administer about g, 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 dose converted per 60 kg can be administered.

(8b) Method for Screening Compound for Promoting or Inhibiting Promoter Activity for DNA of the Present Invention The present invention provides a non-human mammal deficient in expression of DNA of the present invention
Provided is a method for screening a compound or its salt that promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene. In the above screening method, the non-human mammal deficient in DNA expression of the present invention, among the non-human mammal deficient in DNA expression of the present invention, the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used. Examples of the test compound include the same ones as described above. As the reporter gene, the same ones as described above are used, and the β-galactosidase gene (lacZ), the soluble alkaline phosphatase gene, the luciferase gene and the like are preferable. DN of the present invention in which the DNA of the present invention is replaced with a reporter gene
In a non-human mammal deficient in A expression, the reporter gene exists under the control of the promoter for the DNA of the present invention, and thus the activity of the promoter can be detected by tracing the expression of the substance encoded by the reporter gene. For example, when a part of the DNA region encoding the protein of the present invention is replaced with β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue of the protein of the present invention is originally expressed instead of the protein of the present invention. Β-galactosidase is expressed in. Thus, for example, 5-bromo-4-chloro-3-indolyl-β-
By staining with a reagent that serves as a substrate for β-galactosidase such as galactopyranoside (X-gal), the expression state of the protein of the present invention in an animal body can be easily observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, and a phosphate buffered saline (PB
After washing with S), it is reacted with a staining solution containing X-gal at room temperature or around 37 ° C. for about 30 minutes to 1 hour, and then the tissue specimen is washed with 1 mM EDTA / PBS solution to give β-galactosidase. The reaction may be stopped and the color may be observed. In addition, mRNA encoding lacZ may be detected according to a conventional method. The compound or its salt obtained by using the above screening method is a compound selected from the above-mentioned test compounds, and is a DNA of the present invention.
A compound that promotes or inhibits the promoter activity against The compound obtained by the screening method may form a salt, and the salt of the compound may be a physiologically acceptable acid (eg, inorganic acid) or base (eg, organic acid) or the like. Are used, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (for example, acetic acid, formic acid,
Propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) and the like are used.

The compound or its salt that promotes or inhibits the promoter activity for the DNA of the present invention can regulate the expression of the protein of the present invention and the function of the protein, and therefore, for example, colon cancer, breast cancer, lung cancer. , Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer It is also useful as a prophylactic / therapeutic agent for cancer such as brain tumor or blood tumor. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well. The drug containing the compound or its salt obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or its salt. Since the preparation thus obtained is safe and has low toxicity, for example, humans or mammals (for example, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat,
Dogs, monkeys, etc.). The dose of the compound or its salt varies depending on the disease to be treated, the subject to be administered, the route of administration and the like.
When a compound that inhibits the promoter activity for NA is orally administered, the compound is generally administered to an adult (with a body weight of 60 kg) in an amount of about 0.1 per day in a breast cancer patient.
~ 100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease and the like, but for example, a compound that inhibits the promoter activity against the DNA of the present invention is usually in the form of an injection ( When administered to a breast cancer patient (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered by intravenous injection per day. It is convenient to do this. In the case of other animals, the dose converted per 60 kg can be administered. As described above, the non-human mammal deficient in the expression of the DNA of the present invention is the DNA of the present invention.
It is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter against Escherichia coli, and greatly contributes to the investigation of the cause of various diseases caused by defective DNA expression of the present invention or the development of prophylactic / therapeutic agents. it can. In addition, by using DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream of the DNA, and this is injected into an egg cell of an animal to obtain a so-called transgenic animal (transgenic animal). Once created, it is possible to specifically synthesize the protein and study its action in the living body. Further, by linking an appropriate reporter gene to the above promoter part and establishing a cell line in which it is expressed,
It can be used as a search system for low-molecular compounds having an action of specifically promoting or suppressing the production ability of the protein of the present invention itself in the body.

In the present specification, when an abbreviation is used for bases, amino acids, etc., IUPAC-IUB Commission on Bioche
It is based on the abbreviations by mical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecylsulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine: Threon: Threon: Threon: Thres. : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe Phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Sec: selenocysteine (selenocysteine)

The substituents, protecting groups and reagents commonly used in the present specification are represented by the following symbols. Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine -4 (R) - carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: Benzyloxymethyl Z: Benzyloxycarbonyl Cl-Z: 2-Chlorobenzyloxycarbonyl Br-Z: 2-Bromobenzyloxycarbonyl Boc: t-Butoxycarbonyl DNP: Dinitrophenyl Trt: Trityl Bum: t-Butoxymethyl Fmoc. : N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-no Bornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in this specification show the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of TLR1. [SEQ ID NO: 2] This shows the base sequence of DNA encoding TLR1 having the amino acid sequence represented by SEQ ID NO: 1. [SEQ ID NO: 3] This shows the base sequence of DNA containing the full-length gene encoding TLR1. [SEQ ID NO: 4] This shows the base sequence of the antisense used in Example 2. [SEQ ID NO: 5] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 6] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 7] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 8] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 9] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 10] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 11] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 12] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 13] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 14] DN inserted into vector in Example 3
The base sequence of the A fragment is shown.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[Examples] Example 1 In order to clarify genes specifically upregulated in breast cancer tissue, total RNA (Table 1) extracted from 2 cases of breast cancer tissue and 23 normal tissues was used as a material. , Oli
gonucleotide microarray (Human Genome U95A, U95B,
Gene expression analysis was performed using U95C, U95D, U95E; Affymetrix). The experimental method was in accordance with the experimental manual (Expression analysis technical manual) of Affymetrix. As a result, the expression of the TLR1 gene was detected only in the breast cancer tissue. All normal tissues analyzed were below the detection limit (Table 2).

[0075]

[Table 1]

[Table 2]

Example 2 In order to analyze the effect of TLR1 gene whose expression was observed specifically in breast cancer on apoptosis, a TLR1 antisense oligonucleotide introduction experiment was carried out. First,
After designing an antisense (SEQ ID NO: 4) for the base sequence represented by SEQ ID NO: 3, a phosphorothioate-oligonucleotide was synthesized, purified by HPLC and used in an introduction experiment (Amersham Pharmacia Biotech). As a control oligonucleotide, the reverse sequence (SEQ ID NO: 5) of the base sequence shown in SEQ ID NO: 4 was similarly phosphorized.
It was converted to othioate and purified by HPLC before use (Amersham Pha
rmacia Biotech). Breast cancer cell line MDA as test cell
-MB-231 (In Vitro, 14 (11), 911-915, 197.
8 years, purchased from ATCC), using 18 × 10 ³ cells in a 24-well plate (Fal
con)). Oli for oligonucleotide introduction
gofectAMINE (manufactured by Invitrogen) was used and its protocol was followed. RNeasy mini kit (Qia
RNA is extracted according to the
Using the rmoScriptRT-PCR system (GIBCO BRL) c
DNA was prepared. The RCR reaction consists of two primers (SEQ ID NO: 6 and SEQ ID NO: 7), and QuantumR
NA 18S Internal Standards (Ambion) 18S r
Multiplex PCR was performed by simultaneously adding RNA primers and competitors according to the manual. After the PCR reaction, the reaction solution was electrophoresed, the intensity of the band stained with ethidium bromide was corrected by the band intensity of 18S rRNA by BioImage IQ software (manufactured by Genomic Solutions), and then compared. On the other hand, the effect on apoptosis is 3 after the introduction of antisense oligonucleotide.
On the day, Cell death detection ELISA (Roche) was used to compare with the control oligo-introduced sample. As a result, expression of TLR1 (R
NA) was reduced to 55% compared to the time when the control (reverse) oligonucleotide was introduced (100%). After introduction 3
On day 1, apoptosis increased to 186% compared to control (100%). From this, it was revealed that TLR1 is not only specifically expressed in cancer, but also involved in proliferation and apoptosis of cancer cells.

Example 3 (1) Preparation of TLR1 and MD2 Expression Vector In order to prepare a TLR1 expression system, the full length gene of TLR1 was cloned. Marathon ready using 2 kinds of primers (SEQ ID NO: 8 and SEQ ID NO: 9)
Using a cDNA library (Clontech) as a template, P
PCR was performed according to the protocol attached to fu polymerase (Straragene). PCR is 10xPfubuffer 2μ
l, 10 mM each dNTP mixture (Clontech) 0.4 μl, 20 mM
20 μl reaction solution containing 0.8 μl each of the above-mentioned two primers prepared above, 0.5 μl of template cDNA solution, and 0.1 μl of Pfu polymerase at 94 ° C. for 2 minutes, followed by 94 ° C., 30 seconds, 55 ° C. 30
Second, 72 ℃ · 3 minutes reaction after 42 cycles, 72
The reaction was carried out at ℃ for 7 minutes. After the PCR reaction, the products were separated by agarose gel electrophoresis, the band of interest was cut out, and the pCR4Blunt-TOPO vector (Invitroge
(manufactured by company n). As a result of confirming the nucleotide sequence of the inserted fragment, it was found to match with the human TLR1 nucleotide sequence. Therefore, PCR was performed using the above-obtained vector as a template using two more primers (SEQ ID NO: 10 and SEQ ID NO: 11). went. PCR is 10xPfu buffer 2μ
l, 10 mM each dNTP mixture (Clontech) 0.4 μl, 50 mM
0.2 μl of each of the above-mentioned two types of primers prepared as described above, template cDNA
Solution 0.5 μl, 20 μl containing 0.4 μl Pfu polymerase
After pretreatment of the reaction solution of 94 ° C for 2 minutes, 94 ° C for 30 seconds, 55 ° C
-The reaction was performed for 30 seconds at 72 ° C for 3 minutes for 25 cycles, and then at 72 ° C for 7 minutes. Pf
After PCR using u polymerase (Straragene), A was added to both ends of the PCR product according to the protocol attached to pCR4-TOPO vector (Invitrogen), and the product was separated by agarose gel electrophoresis to obtain the desired product. The band was cut out and cloned into pCR4-TOPO vector (Invitrogen). After confirming that there is no error in the sequence,
Ligation kit ver. Was added to p3XFLAG-CMV-14 (SIGMA) digested with BclII (Nippon Gene) and similarly treated with BclII.
2 (manufactured by Takara) was used for insertion. It was confirmed again that there was no error in the sequence, and the obtained TLR1 expression vector was used in the following experiments. On the other hand, two kinds of primers (SEQ ID NO:
12 and SEQ ID NO: 13), and derived from human spleen c
K using DNA library (Clontech) as a template
PCR was performed using OD polymerase (manufactured by Toyobo). PCR is 10x KOD buffer 2 μl, 2.5 mM each dNTP
mixture (manufactured by Clontech) 1.6 μl, each of the above two primers prepared in 50 mM 0.2 μl, template cDNA solution 1 μl, 20 μl reaction solution containing 0.2 μl KOD polymerase, 94 ° C., after pretreatment for 2 minutes, 94 The reaction was performed by carrying out 42 cycles of reaction at 30 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 3 minutes, and then for 72 minutes at 72 ° C. for 7 minutes. After the PCR reaction, the products were separated by agarose gel electrophoresis, the target band was cut out, digested with restriction enzymes KpnI and NotI, and then digested with the same restriction enzymes.
It was ligated to pcDNA3.1 / Hyg (+) (manufactured by Invitrogen) to obtain an MD2 expression vector. The nucleotide sequence of the fragment inserted into this vector is shown in SEQ ID NO: 14. As a result of confirming the nucleotide sequence of the inserted fragment, it was found to be identical with the nucleotide sequence of human MD2.

(2) Effect on reporter activity of TLR1 Next, in order to see the activity of TLR1 in cancer cells,
The expression vector obtained in (1) above was used in NCI-H1299 cells (A
(Purchased from TCC) and examined the effect on various reporter activities. On the day before the transfection of the expression vector shown below, 2 × 10 ⁴ NCI-H1299 strains were seeded on a 24-well plate (manufactured by Falcon), and transfection was performed using Fugene6 (manufactured by Roche) according to the attached protocol. The TLR1 expression vector obtained in Example 3 (1) above and the pcDNA3.1 vector used as a control were 0.19.
μg / well was used. As a reporter, a vector having an AP1 site or an NF-κB site upstream of the secretory alkaline phosphatase gene (all are Clonte
ch) or upstream of the firefly luciferase gene
0.19 μg of vector (Clontech) having E site /
Wells, pRL-TK used to correct reporter activity
(Manufactured by Toyo Ink Co., Ltd.) was used at 0.13 μg / well. The reporter secretory alkaline phosphatase activity or firefly luciferase activity was measured by Great EscAPe SEAP Fluorescence Detecti 2 days after transfection, respectively.
on Kit kit (Clontech) and Piccagene dual sea pansy double luciferase assay system (Toyo Ink).
The sea shiitake luciferase activity expressed by pRL-TK was measured according to the protocol of Picagene dual sea pansy double luciferase assay system (Toyo Ink), and the reporter activity value was corrected. As a result, due to the expression of TLR1, the AP1 reporter activity was 5. compared to the pcDNA3.1 vector used as a control.
6-fold, NF-κB reporter activity was 3.3-fold, and ISRE reporter activity was 1.7-fold. Than this,
By expressing TLR1, lung cancer-derived NCI-H1
It was found that strain 299 activates AP1 signal, NF-κB signal and ISRE signal without adding a ligand. Therefore, by carrying out these promoter-reporter assays, compounds having a TLR1 signal regulatory action can be screened.

(3) Effect on Induction of Apoptosis by Paclitaxel Next, in order to see the effect of TLR1 signaling on cancer, the effect on the induction of apoptosis by paclitaxel was observed. On the day before transfection with the expression vector, 5 × 10 ⁵ HCT116 (purchased from ATCC) were seeded in a 10 cm dish (manufactured by Falcon), and transfection was performed according to the protocol of Fugene6 (manufactured by Roche). The combinations shown in Table 3 such that the TLR1 expression vector and MD2 expression vector obtained in Example 3 (1) above or the pcDNA3.1 vector (manufactured by Invitrogen) used as a control were each 11 μg per dish Used in. The cells were collected by trypsinization 4 hours after the transfection, seeded in 96-well plates (manufactured by Falcon) at 3 × 10 ³ cells each, and N-palmitoyl-S-dilauryl-glyceryl CSK ₄ (Pam ₃ ), which is a TLR1 ligand. CS
K ₄ ) (Peptide Institute) was added (1 μg / ml) and /
Alternatively, the anticancer drug paclitaxel (Sigma) was added (0.125 μM) to see the effect on apoptosis. 2 days after culturing in 96-well plate, Cell Death
Using the Detection ELISA plus kit (Roche), apoptosis was detected according to the attached protocol. As shown in FIG. 1, the absorbance was higher in the presence of paclitaxel than in the absence thereof, confirming that apoptosis was induced by paclitaxel (white bar vs. gray bar). In contrast, addition of Pam ₃ CSK ₄ decreased the absorbance, indicating that addition of TLR1 ligand decreased apoptosis (gray bar vs. shaded bar). Furthermore, it was found that transfection of TLR1 and MD2 without addition of Pam ₃ CSK ₄ reduced paclitaxel-induced apoptosis (gray bar vs black bar). From this, it was clarified that the TLR1 signal has an effect of reducing apoptosis caused by the anticancer agent.

[0080]

INDUSTRIAL APPLICABILITY The protein used in the present invention is a diagnostic marker for cancer, and therefore, a compound or its salt that regulates (preferably inhibits) the activity of the protein,
Regulate (preferably inhibit) expression of the gene of the protein
Examples of the compound or salt thereof, the antibody of the present invention, the antisense nucleotide of the present invention include colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, and spleen cancer. , Safe as a prophylactic / therapeutic agent for cancer such as renal cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, and apoptosis promoter It can be used as a medicine and also as a therapeutic agent for cancer that is resistant to anticancer agents. In addition, since the sensitivity to an anticancer drug (eg, paclitaxel) is increased, the dose of the existing anticancer drug can be reduced to reduce side effects.

[0081]

[Sequence list] SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Preventing and treating agent for cancer <130> P02-0147 <150> JP2001-398165 <151> 2001-12-27 <160> 14 <210> 1 <211> 786 <212> PRT <213> Human <400> 1 Met Thr Ser Ile Phe His Phe Ala Ile Ile Phe Met Leu Ile Leu Gln                  5 10 15 Ile Arg Ile Gln Leu Ser Glu Glu Ser Glu Phe Leu Val Asp Arg Ser              20 25 30 Lys Asn Gly Leu Ile His Val Pro Lys Asp Leu Ser Gln Lys Thr Thr          35 40 45 Ile Leu Asn Ile Ser Gln Asn Tyr Ile Ser Glu Leu Trp Thr Ser Asp      50 55 60 Ile Leu Ser Leu Ser Lys Leu Arg Ile Leu Ile Ile Ser His Asn Arg  65 70 75 80 Ile Gln Tyr Leu Asp Ile Ser Val Phe Lys Phe Asn Gln Glu Leu Glu                  85 90 95 Tyr Leu Asp Leu Ser His Asn Lys Leu Val Lys Ile Ser Cys His Pro             100 105 110 Thr Val Asn Leu Lys His Leu Asp Leu Ser Phe Asn Ala Phe Asp Ala         115 120 125 Leu Pro Ile Cys Lys Glu Phe Gly Asn Met Ser Gln Leu Lys Phe Leu     130 135 140 Gly Leu Ser Thr Thr His Leu Glu Lys Ser Ser Val Leu Pro Ile Ala 145 150 155 160 His Leu Asn Ile Ser Lys Val Leu Leu Val Leu Gly Glu Thr Tyr Gly                 165 170 175 Glu Lys Glu Asp Pro Glu Gly Leu Gln Asp Phe Asn Thr Glu Ser Leu             180 185 190 His Ile Val Phe Pro Thr Asn Lys Glu Phe His Phe Ile Leu Asp Val         195 200 205 Ser Val Lys Thr Val Ala Asn Leu Glu Leu Ser Asn Ile Lys Cys Val     210 215 220 Leu Glu Asp Asn Lys Cys Ser Tyr Phe Leu Ser Ile Leu Ala Lys Leu 225 230 235 240 Gln Thr Asn Pro Lys Leu Ser Ser Leu Thr Leu Asn Asn Ile Glu Thr                 245 250 255 Thr Trp Asn Ser Phe Ile Arg Ile Leu Gln Leu Val Trp His Thr Thr             260 265 270 Val Trp Tyr Phe Ser Ile Ser Asn Val Lys Leu Gln Gly Gln Leu Asp         275 280 285 Phe Arg Asp Phe Asp Tyr Ser Gly Thr Ser Leu Lys Ala Leu Ser Ile     290 295 300 His Gln Val Val Ser Asp Val Phe Gly Phe Pro Gln Ser Tyr Ile Tyr 305 310 315 320 Glu Ile Phe Ser Asn Met Asn Ile Lys Asn Phe Thr Val Ser Gly Thr                 325 330 335 Arg Met Val His Met Leu Cys Pro Ser Lys Ile Ser Pro Phe Leu His             340 345 350 Leu Asp Phe Ser Asn Asn Leu Leu Thr Asp Thr Val Phe Glu Asn Cys         355 360 365 Gly His Leu Thr Glu Leu Glu Thr Leu Ile Leu Gln Met Asn Gln Leu     370 375 380 Lys Glu Leu Ser Lys Ile Ala Glu Met Thr Thr Gln Met Lys Ser Leu 385 390 395 400 Gln Gln Leu Asp Ile Ser Gln Asn Ser Val Ser Tyr Asp Glu Lys Lys                 405 410 415 Gly Asp Cys Ser Trp Thr Lys Ser Leu Leu Ser Leu Asn Met Ser Ser             420 425 430 Asn Ile Leu Thr Asp Thr Ile Phe Arg Cys Leu Pro Pro Arg Ile Lys         435 440 445 Val Leu Asp Leu His Ser Asn Lys Ile Lys Ser Ile Pro Lys Gln Val     450 455 460 Val Lys Leu Glu Ala Leu Gln Glu Leu Asn Val Ala Phe Asn Ser Leu 465 470 475 480 Thr Asp Leu Pro Gly Cys Gly Ser Phe Ser Ser Leu Ser Val Leu Ile                 485 490 495 Ile Asp His Asn Ser Val Ser His Pro Ser Ala Asp Phe Phe Gln Ser             500 505 510 Cys Gln Lys Met Arg Ser Ile Lys Ala Gly Asp Asn Pro Phe Gln Cys         515 520 525 Thr Cys Glu Leu Gly Glu Phe Val Lys Asn Ile Asp Gln Val Ser Ser     530 535 540 Glu Val Leu Glu Gly Trp Pro Asp Ser Tyr Lys Cys Asp Tyr Pro Glu 545 550 555 560 Ser Tyr Arg Gly Thr Leu Leu Lys Asp Phe His Met Ser Glu Leu Ser                 565 570 575 Cys Asn Ile Thr Leu Leu Ile Val Thr Ile Val Ala Thr Met Leu Val             580 585 590 Leu Ala Val Thr Val Thr Ser Leu Cys Ile Tyr Leu Asp Leu Pro Trp         595 600 605 Tyr Leu Arg Met Val Cys Gln Trp Thr Gln Thr Arg Arg Arg Ala Arg     610 615 620 Asn Ile Pro Leu Glu Glu Leu Gln Arg Asn Leu Gln Phe His Ala Phe 625 630 635 640 Ile Ser Tyr Ser Gly His Asp Ser Phe Trp Val Lys Asn Glu Leu Leu                 645 650 655 Pro Asn Leu Glu Lys Glu Gly Met Gln Ile Cys Leu His Glu Arg Asn             660 665 670 Phe Val Pro Gly Lys Ser Ile Val Glu Asn Ile Ile Thr Cys Ile Glu         675 680 685 Lys Ser Tyr Lys Ser Ile Phe Val Leu Ser Pro Asn Phe Val Gln Ser     690 695 700 Glu Trp Cys His Tyr Glu Leu Tyr Phe Ala His His Asn Leu Phe His 705 710 715 720 Glu Gly Ser Asn Ser Leu Ile Leu Ile Leu Leu Glu Pro Ile Pro Gln                 725 730 735 Tyr Ser Ile Pro Ser Ser Tyr His Lys Leu Lys Ser Leu Met Ala Arg             740 745 750 Arg Thr Tyr Leu Glu Trp Pro Lys Glu Lys Ser Lys Arg Gly Leu Phe         755 760 765 Trp Ala Asn Leu Arg Ala Ala Ile Asn Ile Lys Leu Thr Glu Gln Ala     770 775 780 Lys Lys 785 <210> 2 <211> 2358 <212> DNA <213> Human <400> 2 atgactagca tcttccattt tgccattatc ttcatgttaa tacttcagat cagaatacaa 60 ttatctgaag aaagtgaatt tttagttgat aggtcaaaaa acggtctcat ccacgttcct 120 aaagacctat cccagaaaac aacaatctta aatatatcgc aaaattatat atctgagctt 180 tggacttctg acatcttatc actgtcaaaa ctgaggattt tgataatttc tcataataga 240 atccagtatc ttgatatcag tgttttcaaa ttcaaccagg aattggaata cttggatttg 300 tcccacaaca agttggtgaa gatttcttgc caccctactg tgaacctcaa gcacttggac 360 ctgtcattta atgcatttga tgccctgcct atatgcaaag agtttggcaa tatgtctcaa 420 ctaaaatttc tggggttgag caccacacac ttagaaaaat ctagtgtgct gccaattgct 480 catttgaata tcagcaaggt cttgctggtc ttaggagaga cttatgggga aaaagaagac 540 cctgagggcc ttcaagactt taacactgag agtctgcaca ttgtgttccc cacaaacaaa 600 gaattccatt ttattttgga tgtgtcagtc aagactgtag caaatctgga actatctaat 660 atcaaatgtg tgctagaaga taacaaatgt tcttacttcc taagtattct ggcgaaactt 720 caaacaaatc caaagttatc aagtcttacc ttaaacaaca ttgaaacaac ttggaattct 780 ttcattagga tcctccagct ggtttggcat acaactgtat ggtatttctc aatttcaaac 840 gtgaagctac agggtcagct ggacttcaga gattttgatt attctggcac ttccttgaag 900 gccttgtcta tacaccaagt tgtcagcgat gtgttcggtt ttccgcaaag ttatatctat 960 gaaatctttt cgaatatgaa catcaaaaat ttcacagtgt ctggtacacg catggtccac 1020 atgctttgcc catccaaaat tagcccgttc ctgcatttgg atttttccaa taatctctta 1080 acagacacgg tttttgaaaa ttgtgggcac cttactgagt tggagacact tattttacaa 1140 atgaatcaat taaaagaact ttcaaaaata gctgaaatga ctacacagat gaagtctctg 1200 caacaattgg atattagcca gaattctgta agctatgatg aaaagaaagg agactgttct 1260 tggactaaaa gtttattaag tttaaatatg tcttcaaata tacttactga cactattttc 1320 agatgtttac ctcccaggat caaggtactt gatcttcaca gcaataaaat aaagagcatt 1380 cctaaacaag tcgtaaaact ggaagctttg caagaactca atgttgcttt caattcttta 1440 actgaccttc ctggatgtgg cagctttagc agcctttctg tattgatcat tgatcacaat 1500 tcagtttccc acccatcagc tgatttcttc cagagctgcc agaagatgag gtcaataaaa 1560 gcaggggaca atccattcca atgtacctgt gagctaggag aatttgtcaa aaatatagac 1620 caagtatcaa gtgaagtgtt agagggctgg cctgattctt ataagtgtga ctacccggaa 1680 agttatagag gaaccctact aaaggacttt cacatgtctg aattatcctg caacataact 1740 ctgctgatcg tcaccatcgt tgccaccatg ctggtgttgg ctgtgactgt gacctccctc 1800 tgcatctact tggatctgcc ctggtatctc aggatggtgt gccagtggac ccagacccgg 1860 cgcagggcca ggaacatacc cttagaagaa ctccaaagaa atctccagtt tcatgcattt 1920 atttcatata gtgggcacga ttctttctgg gtgaagaatg aattattgcc aaacctagag 1980 aaagaaggta tgcagatttg ccttcatgag agaaactttg ttcctggcaa gagcattgtg 2040 gaaaatatca tcacctgcat tgagaagagt tacaagtcca tctttgtttt gtctcccaac 2100 tttgtccaga gtgaatggtg ccattatgaa ctctactttg cccatcacaa tctctttcat 2160 gaaggatcta atagcttaat cctgatcttg ctggaaccca ttccgcagta ctccattcct 2220 agcagttatc acaagctcaa aagtctcatg gccaggagga cttatttgga atggcccaag 2280 gaaaagagca aacgtggcct tttttgggct aacttaaggg cagccattaa tattaagctg 2340 acagagcaag caaagaaa 2358 <210> 3 <211> 2806 <212> DNA <213> Human <400> 3 acagactgcc aaatggaaca gacaagcagg ttgtcttgtg ttaaagaaaa tgagatatga 60 gtcagttact cccggaggca atgctgctgt tcagctcttg tgtttttgtg gccagggtct 120 tcatgaacac taataggggt accaggccct cttccttgtt agaagaaatc aggataacaa 180 aggtatattg ggcaccccta caaaaggaat ctgtatctgt atcaagatga tctgaagaac 240 agcttctacc tttaggaatg tctagtgttc caaaatgact agcatcttcc attttgccat 300 tatcttcatg ttaatacttc agatcagaat acaattatct gaagaaagtg aatttttagt 360 tgataggtca aaaaacggtc tcatccacgt tcctaaagac ctatcccaga aaacaacaat 420 cttaaatata tcgcaaaatt atatatctga gctttggact tctgacatct tatcactgtc 480 aaaactgagg attttgataa tttctcataa tagaatccag tatcttgata tcagtgtttt 540 caaattcaac caggaattgg aatacttgga tttgtcccac aacaagttgg tgaagatttc 600 ttgccaccct actgtgaacc tcaagcactt ggacctgtca tttaatgcat ttgatgccct 660 gcctatatgc aaagagtttg gcaatatgtc tcaactaaaa tttctggggt tgagcaccac 720 acacttagaa aaatctagtg tgctgccaat tgctcatttg aatatcagca aggtcttgct 780 ggtcttagga gagacttatg gggaaaaaga agaccctggg ggccttcaag actttaacac 840 tgagagtctg cacattgtgt tccccacaaa caaagaattc cattttattt tggatgtgtc 900 agtcaagact gtagcaaatc tggaactatc taatatcaaa tgtgtgctag aagatagcaa 960 atgttcttac ttcctaagta ttctggcgaa acttcaaaca aatccaaagt tatcaagtct 1020 taccttaaac aacattgaaa caacttggaa ttctttcatt aggatcctcc agctggtttg 1080 gcatacaact gtatggtatt cctcaatttc aaacgtgaag ctacagggtc agctggactt 1140 cagagatttt gattattctg gcacttcctt gaaggccttg tctatacacc aagttgtcag 1200 cgatgtgttc ggttttccgc aaagttatat ctatgaaatc ttttcgaata tgaacatcaa 1260 aaatttcaca gtgtctggta cacgcatggt ccacatgctt tgcccatcca aaattagccc 1320 gttcctgcat ttggattttt ccaataatct cttaacagac acggtttttg aaaattgtgg 1380 gcaccttact gagttggaga cacttatttt acaaatgaat caattaaaag aactttcaaa 1440 aatagctgaa atgactacac agatgaagtc tctgcaacaa ttggatatta gccagaattc 1500 tgtaagctat gatgaaaaga aaggagactg ttcttggact aaaagtttat taagtttaaa 1560 tatgtcttca aatatactta ctgacactat tttcagatgt ttacctccca ggatcaaggt 1620 acttgatctt cacagcaata aaataaagag cattcctaaa caagtcgtaa aactggaagc 1680 tttgcaagaa ctcaatgttg ctttcaattc tttaactgac cttcctggat gtggcagctt 1740 tagcagcctt tctgtattga tcattgatca caattcagtt tcccacccat cagctgattt 1800 cttccagagc tgccagaaga tgaggtcaat aaaagcaggg gacaatccat tccaatgtac 1860 ctgtgagcta ggagaatttg tcaaaaatat agaccaagta tcaagtgaag tgttagaggg 1920 ctggcctgat tcttataagt gtgactaccc ggaaagttat agaggaaccc tactaaagga 1980 ctttcacatg tctgaattat cctgcaacat aactctgctg atcgtcacca tcgttgccac 2040 catgctggtg ttggctgtga ctgtgacctc cctctgcatc tacttggatc tgccctggta 2100 tctcaggatg gtgtgccagt ggacccagac ccggcgcagg gccaggaaca tacccttaga 2160 agaactccaa agaaatctcc agtttcatgc atttatttca tatagtgggc acgattcttt 2220 ctgggtgaag aatgaattat tgccaaacct agagaaagaa ggtatgcaga tttgccttca 2280 tgagagaaac tttgttcctg gcaagagcat tgtggaaaat atcatcacct gcattgagaa 2340 gagttacaag tccatctttg ttttgtctcc caactttgtc cagagtgaat ggtgccatta 2400 tgaactctac tttgcccatc acaatctctt tcatgaagga tctaatagct taatcctgat 2460 cttgctggaa cccattccgc agtactccat tcctagcagt tatcacaagc tcaaaagtct 2520 catggccagg aggacttatt tggaatggcc caaggaaaag agcaaacgtg gccttttttg 2580 ggctaactta agggcagcca ttaatattaa gctgacagag caagcaaaga aatagattac 2640 acatcaagtg aaaaatattc ctcctgttga tattgctgct tttggaagtt ccaacaatga 2700 ctttattttg catcagcata gatgtaaaca caattgtgag tgtatgatgt aggtaaaaat 2760 atataccttc gggtcgcagt tcaccattta aaaaagaaaa aaaaaa 2806 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gtttcgccag aatacttagg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 ggattcataa gaccgctttg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 gggtcagctg gacttcagag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 aaaatccaaa tgcaggaacg 20 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 gccagggtct tcatgaacac taat 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 agcagcaata tcaacaggag gaat 24 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 caagatctga tgactagcat cttccatttt gccat 35 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 ctagatctct atttctttgc ttgctctgtc agctt 35 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 aaaaggtacc tgatgattag ttactgatcc tc 32 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 aaagcggcgc ctcaatttat tctaatttga attag 35 <210> 14 <211> 624 <212> DNA <213> Human <400> 14 ggcgggccgc tcccacttcg gcacgagggg cacgaggtaa atcttttctg cttactgaaa 60 aggaagagtc tgatgattag ttactgatcc tctttgcatt tgtaaagctt tggagatatt 120 gaatcatgtt accatttctg tttttttcca ccctgttttc ttccatattt actgaagctc 180 agaagcagta ttgggtctgc aactcatccg atgcaagtat ttcatacacc tactgtgata 240 aaatgcaata cccaatttca attaatgtta acccctgtat agaattgaaa ggatccaaag 300 gattattgca cattttctac attccaagga gagatttaaa gcaattatat ttcaatctct 360 atataactgt caacaccatg aatcttccaa agcgcaaaga agttatttgc cgaggatctg 420 atgacgatta ctctttttgc agagctctga agggagagac tgtgaataca acaatatcat 480 tctccttcaa gggaataaaa ttttctaagg gaaaatacaa atgtgttgtt gaagctattt 540 ctgggagccc agaagaaatg ctcttttgct tggagtttgt catcctacac caacctaatt 600 caaattagaa taaattgagt attt 624

[Brief description of drawings]

FIG. 1 shows the results of examining the effect of paclitaxel on the induction of apoptosis.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 35/00 4C086 A61P 35/00 43/00 105 43/00 105 C12Q 1/68 A C12N 15/09 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 A61K 37/02 ZNA 33/50 C12N 15/00 A (72) Inventor Yuichi Sugi 1-10 Tokodai, Tsukuba, Ibaraki Prefecture 6 (72) Inventor Satoshi Nishizawa 3-12-1, Matsushiro Tsukuba, Ibaraki 1 Takeda Matsushiro Residence No. 301 F term (reference) 2G045 AA26 AA29 AA40 BB03 BB20 CA26 CB01 CB17 CB21 DA12 DA13 DA14 DA36 DA78 FB02 CA01 A12 A12 A12 A12 A12 A12 HA20 4B063 QA19 QQ08 QQ53 QQ79 QQ96 QR32 QR48 QR55 QS32 QX01 4C084 AA01 AA13 AA16 BA22 BA35 NA14 ZB26 4C085 AA13 AA14 BB11 CC21 EE01 4C086 AA01 EA16 MA0 1 NA14 ZB21 ZB26

Claims (24)

[Claims] 1. A compound or a salt thereof which inhibits the activity of a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Cancer preventive / therapeutic agent. 2. A compound or a salt thereof which inhibits the expression of a gene containing a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. A preventive / therapeutic agent for cancer. 3. A base sequence complementary or substantially complementary to the base sequence of a DNA encoding a protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Or an antisense nucleotide containing a part thereof. 4. A preventive / therapeutic agent for cancer, which comprises the antisense nucleotide according to claim 3. 5. A preventive / therapeutic agent for cancer, which comprises an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof. . 6. The cancer is colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer. 6. The preventive / therapeutic agent according to claims 1 to 5, which is ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor. 7. A diagnostic agent for cancer, which comprises an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof. 8. A diagnostic agent for cancer, which comprises a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. 9. The cancer is colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer. The diagnostic agent according to claim 7 or 8, which is ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor. 10. A preventive / therapeutic agent for cancer, which comprises a compound having a Toll-like receptor activity-inhibiting activity or a salt thereof. 11. A preventive / therapeutic agent for cancer, which comprises a compound having a Toll-like receptor expression inhibitory action or a salt thereof. 12. A preventive / therapeutic agent for cancer, which comprises using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. Screening method. 13. Prevention / treatment of cancer, which comprises a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. Agent screening kit. 14. A preventive / therapeutic agent for cancer, which comprises using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. Screening method. 15. Prevention / treatment of cancer, which comprises a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. Agent screening kit. 16. The screening method according to claim 12 or claim 14 or claim 13 or claim 1.
A preventive / therapeutic agent for cancer, which can be obtained using the screening kit according to 5. 17. A compound or its salt, which inhibits the activity of a protein or its partial peptide or its salt, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Apoptosis inducer. 18. A compound or its salt, which inhibits the expression of a gene of a protein or its partial peptide or its salt, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. An apoptosis-inducing agent. 19. A method for screening an apoptosis-inducing agent, which comprises using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. 20. A method for screening an apoptosis inducer, which comprises using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. 21. A compound or a salt thereof, which inhibits the activity of a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Anticancer drug sensitivity enhancer. 22. A compound or a salt thereof which inhibits gene expression of a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. An anti-cancer drug sensitivity enhancer. 23. A compound which inhibits the activity of a protein or a partial peptide thereof, or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 in mammals, or a compound thereof salt,
Alternatively, a method for preventing / treating cancer, which comprises administering an effective amount of a compound or a salt thereof that inhibits the expression of a gene of the above protein or a partial peptide thereof or a salt thereof. 24. A method for producing a preventive / therapeutic agent for cancer, which comprises a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. Use of a compound or a salt thereof, which inhibits the activity, or a compound or a salt thereof, which inhibits the expression of a gene of the above protein or its partial peptide or its salt.