JP2003277288A - 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, an 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. To comprehensively analyze gene expression, a microarray method with immobilized cDNA or oligonucleotide was developed,
The spread of technology for finding changes in disease-specific gene expression
Its usefulness has been confirmed. For example, from Affymetrix
The 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, induces RNA degradation by RNaseH to inhibit protein translation, or inhibits direct protein synthesis by hybridization. Also brings. 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. It has become increasingly clear in recent years that chromatin structures are deeply involved in the control of cell division, proliferation, or gene transcription. Of the histones that compose chromatin, the histone tail, in particular, is known to contribute to changing the structure of chromatin by modifications such as acetylation, phosphorylation, and methylation (edited by Nakatani et al.). , October 2001, Experimental Medicine). Suppressor of variegation 3-9 homolog 1 (hereinafter sometimes referred to as SUV39H1) is a position effect variegation (PE) of Drosophila.
V) A gene isolated as a human homolog of the suppressor gene Su (var) 3-9 (Non-patent document 1 EMBO.J., 18: 1923).
-1938, 1999). SUV39H1 is a histone methyltransferase that specifically donates a methyl group to lysine at the 9th position from the amino-terminal side of histone H3 protein, and is involved in the formation of a chromatin structure. Lysine, which undergoes methyl group transfer by SUV39H1, undergoes acetylation modification in addition to methylation, and both transferases (histone methyl group transfer enzyme and histone acetyltransferase) or detransferase function, and both are antagonistic. Is said to bind to this lysine residue (Non-Patent Document 2 Nature, Volume 406, 593-
599, 2000). On the other hand, transgenic mice (Non-patent document 3, Mech Dev 107 (1-2), 141-153, 200
1 year), knockout mouse (Non-patent document 4 Cell 107
Vol., Pp. 323-337, 2001), but no major change in phenotype was reported except that the former had abnormal erythroblast differentiation and some growth retardation. Also, to investigate the function of SUV39H1, Nielsen et al.
The effect of the E promoter on the Cyclin E promoter activity of SUV39H1 was examined using a vector inserted upstream of the reporter gene. As a result, it was reported that SUV39H1 suppressed CyclinE promoter activity by co-expressing it with Rb1 (Non-Patent Document 5, Nature 412, 561-565).
Page, 2001). From these results, until recently, the role of SUV39H1 in cancer was rather recognized as a tumor suppressor gene (Non-Patent Document 6 Nature Reviews Canc
er 2, pp. 469-476, 2002), SUV39H1 in carcinogenesis
Are not believed to be actively involved. In recent years, development of an anticancer agent targeting histone deacetylase and inhibiting its function has been advanced, but a targeting histone methyltransferase has not been reported so far.

[Non-Patent Document 1] EMBO.J., 18: 1923-1938, 1999

[Non-Patent Document 2] Nature, 406, 593-599, 2000

[Non-Patent Document 3] Mech Dev 107 (1-2), 141-153, 20.
01 years

[Non-Patent Document 4] Cell 107, pages 323-337, 2001

[Non-Patent Document 5] Nature 412, 561-565, 2001

[Non-Patent Document 6] Nature Reviews Cancer Volume 2, 469-476.
Page, 2002

[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.

[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:
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, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by 1. (2) Containing a compound or its salt that inhibits the 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) Complementary or substantial to the base sequence of a DNA encoding a protein or its partial peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. An antisense nucleotide containing a base sequence complementary to or a part thereof (4)
A preventive / therapeutic agent for cancer comprising the antisense nucleotide according to (3) above, (5) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or Cancer preventive / therapeutic agent comprising an antibody against the partial peptide or a salt thereof, (6) cancer is colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, Biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor (1) to (5) above The preventive / therapeutic agent described in (7) A cancer comprising an antibody against a protein containing 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. Diagnostic of, ( ) SEQ ID NO: Protein or diagnostic agent for cancer comprising a DNA encoding the partial peptide having an amino acid sequence identical or substantially identical to the amino acid sequence represented by the (9) cancer,
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, The diagnostic agent according to (7) or (8) above, which is thyroid cancer, pancreatic cancer, brain tumor or blood tumor, (10) comprising a compound having a histone methyltransferase activity-regulating activity or a salt thereof. (11) A preventive / therapeutic agent for cancer, (11) A preventive / therapeutic agent for cancer, comprising a compound having a histone methyltransferase expression-regulating action or a salt thereof, (12) SEQ ID NO:
A method for screening 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 1 or a partial peptide thereof or a salt thereof, (13) sequence 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, (1
4) A method for screening 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. (15) A preventive / therapeutic 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. (16) The screening method according to (12) or (14) above or (13) above
Alternatively, a preventive / therapeutic agent for cancer obtainable by using the screening kit described in (15) above, (17) containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. An apoptosis inducer containing a compound or a salt thereof that inhibits the activity of a protein or a partial peptide thereof or a salt thereof, (1
8) Apoptosis comprising a compound or its salt that inhibits the gene expression of a protein or its partial peptide or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 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 or a partial peptide thereof or a salt thereof. (20) A method for screening an apoptosis-inducing agent, 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) For mammals,
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, or the above-mentioned protein or its partial peptide or its A method for preventing or treating cancer, which comprises administering an effective amount of a compound that inhibits the expression of a salt gene or a salt thereof, (22) SEQ ID NO: for producing a preventive or therapeutic agent for cancer 1. A compound or salt thereof 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 1, or the gene of said protein or its partial peptide or its salt Use of a compound or its salt that inhibits the expression of

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION SEQ ID NO: 1 used in the present invention
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by (hereinafter sometimes referred to as the protein of the present invention or the protein used in the present invention) is a human or warm-blooded animal (for example, 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, epidermis) Cell, epithelial cell, 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 Sphere, basophil, eosinophil, monocyte), megakaryocyte, synovial cell, chondrocyte, osteocyte, osteoblast, osteoclast, mammary cell, hepatocyte or stroma Cells, or their precursor cells, stem cells or cancer cells) or any tissue in which these cells are present, such as the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, thalamus) Lower part, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessel It may be a protein derived from heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, 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.

Examples of substantially the same activity include histone methyltransferase activity and the like. Substantially homogenous means that their properties are qualitatively (eg, physiologically,
Or (pharmacologically) indicate homogeneity. Therefore, histone methyltransferase activity is equivalent (eg, about 0.
It is preferably 0.1 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times), but the quantitative factors such as the degree of activity and the molecular weight of the protein are different. Good. Histone methyltransferase activity can be measured by a method known per se, for example, Nature, 406, 593-.
It can be measured according to the method described on page 599, 2000 or a method similar thereto. Specifically, the protein of the present invention and S-adenosyl-L radiolabeled with a methyl group are used.
-Reacting with methionine and a histone protein or a polypeptide having the N-terminal sequence of histone H3,
The radioactivity of histone H3 or polypeptide by transmethylation is measured. This reaction is carried out in an appropriate buffer. After the enzymatic reaction, the reaction product is separated by, for example, SDS-PAGE or the like, and first compared with the mobility of histone H3 or the like serving as a standard control and identified. The radioactivity in the quantification is measured according to a known method using a scintillation counter, fluorography or the like.

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 (SUV39H
1) and so on.

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 described later, a peptide having the amino acid sequence of 42nd to 366th 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 20, more preferably about 1 to 10 and still more preferably several (1 to 5)) amino acid sequences in the amino acid sequence. 2) is deleted, or 1 or 2 or more (preferably about 1 to 20 and more preferably 1 to 1) is added to the amino acid sequence.
About 0, more preferably several (1 to 5) amino acids are added, or 1 or 2 or more (preferably about 1 to 20, more preferably 1) amino acid sequence is added.
-10, more preferably several (1-5) amino acids are inserted, or 1 or 2 or more (preferably about 1-20, more preferably 1-10) in the amino acid sequence. About one, more preferably a number (1-5)
Amino acids) may be replaced 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, 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 method known per se for protein purification, or contains a DNA encoding the protein. It can also be produced by culturing a 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 the sequence of the target protein according to various condensation methods known per se. 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 the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, additives for suppressing racemization (for example, HOBt, HO
The protected amino acid can be directly added to the resin together with OBt), or can be added to the resin after previously performing activation of the protected amino acid as a symmetrical acid anhydride or HOBt ester or HOOBt 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 peptide synthesis method known per se, 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 method known per se or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al.
l., Cold Spring Harbor Lab. Press, 1989). 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 means, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20.
mM, temperature about 50-70 ° C, preferably about 60-6
The conditions of 5 ° C. are shown. Especially, the sodium concentration is about 19 mM
Most preferably, the temperature is about 65 ° C. More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, DNA containing the base 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. can be used according to a method known per se such as ODA-LAPCR method, Gapped duplex method, Kunkel method or the like, or a method analogous thereto. 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 has ATG as a translation initiation codon at the 5'terminal side, and TAA, TGA or TAG as a translation stop codon at the 3'terminal side.
May have. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. Examples of the expression vector of the protein of the present invention include (a) D encoding the protein of the present invention.
The desired DNA fragment is cut out from NA, and (b) the D
It can be produced by ligating the NA fragment downstream of the promoter in a suitable 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 a bacterium of the genus Escherichia, the PhoA signal sequence, OmpA signal sequence, etc., and when the host is a bacterium of the genus Bacillus, the α-amylase signal sequence, the subtilisin signal sequence, etc., the host is yeast. In some cases, the MFα signal sequence,
When the host is an animal cell such as SUC2 signal sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, etc. can be used. 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. Schizosaccharomys pombe
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 method known per se, and the supernatant is collected. The protein contained in the thus obtained culture supernatant or the extract can be purified by appropriately combining separation / purification methods known per se. 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 as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or The free form or other salt can be converted by a method analogous thereto. 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. An antibody 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) uses the protein of the present invention as an antigen and is known per se. 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. The monoclonal antibody can be selected according to a method known per se or a method analogous thereto. Normal H
It can be performed in a medium for animal cells supplemented with AT (hypoxanthine, aminopterin, thymidine). As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1
RPMI 1640 medium containing -20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM) -101, Nissui Pharmaceutical Co., Ltd., etc. 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 method known per se, for example, a method for separating and purifying immunoglobulin [eg salting out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method]. , Adsorption / desorption method with ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as protein A or protein G, etc. The specific purification method for obtaining [Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, an immune antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-mentioned method for producing a monoclonal antibody. It can be produced by collecting the substance and separating and purifying the antibody. 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 the carrier and the 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 in any ratio, but, for example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. in a weight ratio of about 0.1 to 20, preferably about 1 to 1 hapten. A method of coupling at a rate of 5 is used. Although various condensing agents can be used for coupling the hapten and carrier, glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing thiol group, dithiopyridyl group, etc. are 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. The administration is usually about once every 2 to 6 weeks, about 3 to 1 in total.
It is performed about 0 times. 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. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the 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 has a base sequence complementary to or substantially complementary to the base sequence of or a part thereof and has an action 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, a polynucleotide corresponding to the gene of the present invention, which is capable of inhibiting the replication or expression of the gene, such as an antisense polynucleotide (nucleic acid), has been cloned or determined. It can be designed and synthesized based on the nucleotide sequence information of the DNA encoding the protein. 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. The polynucleotide complementary to the selected sequence of the protein-related RNA of the present invention and the polynucleotide capable of specifically hybridizing with the protein-related RNA of the present invention can be used as a protein gene of the present invention in vivo and in vitro. It is useful for regulating / controlling the expression of erythrocyte, and for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide, a nucleotide sequence or a 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′-end 6-base pair repeat, 5′-end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3′-end untranslated region, 3 ′ The end palindromic region and the 3 ′ end hairpin loop can be selected as the preferred target region, but any region within the protein gene can be selected as the target. 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)
The nucleotide is a polynucleotide containing 2-deoxy-D-ribose, a polynucleotide containing D-ribose, an N- of a purine or pyrimidine base.
Other types of polynucleotides that are glycosides,
Alternatively, other polymers having non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that such polymers are found in DNA or RNA. Containing a nucleotide having a configuration that allows pairing of bases and attachment of bases) and the like. They are double-stranded DNA, single-stranded DNA, double-stranded R
NA, single-stranded RNA, DNA: RNA hybrid, unmodified polynucleotide (or unmodified oligonucleotide), and known modification can be added, for example, a label known in the art. , Capped, methylated, substituted with one or more natural nucleotides by analogs, modified intramolecularly with nucleotides such as uncharged bonds (eg methylphosphonate, With phosphotriesters, phosphoramidates, carbamates, etc., charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.)
Those having side chain groups such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (for example, monosaccharides), intercurrents Compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), alkylating agents, modified It may have a bond (for example, an α-anomeric nucleic acid). Where "nucleoside" and "nucleotide"
The term "nucleic acid" may include those having not only purine and pyrimidine bases but also other modified heterocyclic bases. These modifications are
It may contain 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, or a functional group such as ether or amine is added. It may have been converted. The antisense (poly) nucleotide (nucleic acid) of the present invention includes RNA and DN.
A 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, Vol. 8, pp.247, 1992; Vo
l. 8, pp. 395, 1992; ST Crooke et al. ed., Antis
ense Research and Applications, CRC Press, 1993 etc. The antisense nucleic acid of the present invention may contain altered or modified sugars, bases, linkages, provided in a special form such as liposomes and microspheres, or applied by gene therapy, It can be provided in 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 are 3'of the nucleic acid
It can be attached to the end or 5'end, and can be attached to bases, sugars,
It may be attached via an intramolecular nucleoside bond. Other groups include 3'end or 5'end of nucleic acid.
Examples of the cap group specifically arranged at the end include those 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 antisense nucleic acid is
It can be examined by 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 methods known per se.

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 of Drug Candidate Compounds against Diseases Since the protein of the present invention has an increased expression in cancer tissues and an inhibitory effect on apoptosis, the activity of the protein of the present invention is regulated (promoted or inhibited, preferably inhibited) ) 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. 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 in that the protein of the present invention is used for the activity of the protein of the present invention (for example,
Provided is a method for screening a compound or its salt that regulates (promotes or inhibits, preferably inhibits) histone methyltransferase activity and the like. More specifically, for example, it is characterized in that (i) the histone methyltransferase activity of the protein of the present invention is compared with (ii) the histone methyltransferase activity of the mixture of the protein of the present invention and a test compound. 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 is used. In the screening method, for example, in the cases of (i) and (ii), the histone methyltransferase activity is a method known per se,
For example, it is measured and compared according to the method described in Nature, 406, pages 593-599, 2000 or a method similar thereto. Specifically, (i) a case where the protein of the present invention is reacted with S-adenosyl-L-methionine radiolabeled with a methyl group and a histone protein or a polypeptide having an N-terminal sequence of histone H3, (ii) When the protein of the present invention is reacted with S-adenosyl-L-methionine radiolabeled with a methyl group and a histone protein or a polypeptide having an N-terminal sequence of histone H3 in the presence of a test compound Of histone by methyl group transfer
The radioactivity of H3 or the polypeptide is measured, and the compound or its salt that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention is screened. This reaction is carried out in an appropriate buffer. After the enzymatic reaction,
For example, the reaction product is separated by SDS-PAGE or the like,
First, compare with the mobility of histone H3 etc. as a standard control,
Identify. The radioactivity in the quantification is measured according to a known method using a scintillation counter, fluorography or the like. Radioactively labeled methyl group
S-adenosyl-L-methionine, histone proteins and polypeptides having the N-terminal sequence of histone H3 may be reacted with a protein of the invention after mixing with a test compound, and the Is contacted with a radiolabeled S-adenosyl-L-methionine, a histone protein and a polypeptide having the N-terminal sequence of histone H3, and then a test compound may be added.
In addition, (i ') a case where a protein of the present invention, S-adenosyl-L-methionine and a histone protein or a polypeptide having an N-terminal sequence of histone H3 is reacted,
(ii ′) Methylation (dimethyl / trimethyl) of a protein of the present invention, S-adenosyl-L-methionine, and a polypeptide having an N-terminal sequence of histone protein or histone H3 in the presence of a test compound. )
The lysine residue is, for example, anti-Histone H3 (dimethyl / trimethy
lysine9) antibody or the like to measure each, and screen for a compound or its salt that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention. Further, (i ') the protein of the present invention, S-adenosyl-L-
Methionine and histone proteins or histone H3
And (ii ') the protein of the present invention, S-adenosyl-L-methionine and a histone protein or a polypeptide having an N-terminal sequence of histone H3, was used as a test compound. In existence,
By appropriately purifying the reaction product of the reaction, and measuring the mass spectrometry (eg, using TOF-MS, etc.), the activity of the protein of the present invention can be determined using the change in molecular weight associated with methylation as an index. Compounds or salts thereof that modulate (enhance or inhibit, preferably inhibit) are screened. The above-mentioned protein of the present invention is preferably produced by culturing a transformant containing a DNA encoding the protein of the present invention. Further, cells capable of expressing the protein of the present invention may be similarly reacted to measure the radioactivity of histone H3 or polypeptide by transmethylation. Examples of cells having the ability to produce the protein of the present invention include:
A host (transformant) transformed with the vector containing the above-mentioned DNA encoding the protein of the present invention is used. Examples of the host include COS7 cells, CH
Animal cells such as O cells and HEK293 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed 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, a test compound which reduces the histone methyltransferase activity in the case of (ii) by about 20% or more, preferably by 30% or more, more preferably by about 50% or more as compared with the case of (i) above is used. As a compound that inhibits the activity of the protein of the invention, the histone methyltransferase activity in the case of (ii) above is about 20% or more, preferably 30% or more, more preferably about as compared with the case of (i) above. A test compound that increases by 50% or more can be selected as a compound that promotes the activity of the protein of the present invention.

The compound having the 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 hematological tumor, and an apoptosis inducer. 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 by using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts. , A compound selected from 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 , A prophylactic / therapeutic agent for cancer such as pancreatic cancer, brain tumor or blood tumor, and an apoptosis inducer. Therefore, the DNA of the present invention is useful as a reagent for screening a compound or its salt that regulates (promotes or inhibits, preferably inhibits) the expression of the gene encoding the protein of the present invention. As a screening method,
(Iii) A comparison between the case of culturing cells having the ability to produce the protein of the present invention and (iv) the case of culturing cells having the ability to produce the protein used in the present invention in the presence of a test compound. The screening method is characterized by carrying out. In the above method, (ii
In the cases of i) 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 can be measured by a known method, for example, by using an antibody that recognizes the protein of the present invention, the protein present in the cell extract or the like is analyzed by Western analysis or ELISA.
It can be measured according to a method such as a 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, a test compound which inhibits the gene expression level in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii), is used in the present invention. As a compound that suppresses the expression of the gene encoding the protein of (1), the gene expression level in the case of (iv) above is about 20% or more, preferably 3% compared to the case of (iii) above.
A test compound that increases by 0% 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. Compounds or salts thereof selected from animal tissue extract, plasma and the like, and compounds or salts thereof that regulate the activity of the protein of the present invention (eg histone methyltransferase activity etc.). 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 hematological tumor, and an apoptosis inducer. 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 prophylactic / therapeutic agent, it can be formulated in a conventional manner.

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 method known per se and contains a carrier, a diluent or an excipient which is 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 method known per se, 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 auxiliary agents, and the like,
Suitable solubilizing agents such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant [eg polysorbate 80, HCO-50 (polyoxyethylene).
(50 mol) 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 antibody or a salt thereof with an ordinary 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 contain other active ingredients as long as the composition does not cause an unfavorable interaction with the antibody. 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, regulates (preferably inhibits) the activity of the protein of the present invention for the purpose of treating breast cancer.
When the compound or a salt thereof is orally administered, generally in an adult person (60 kg body weight), the compound or a salt thereof is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably, per day. Is about 1.0 to 20
administer 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, it regulates the activity of the protein of the present invention for the purpose of treating breast cancer (preferably Compound or its salt that inhibits) in the form of injection usually for adults (body weight 60 kg
As a compound), about 0.01 to 30 mg, preferably about 0.1 to 30 mg of the compound or a salt thereof per day is administered.
It is convenient to administer about 20 mg, more preferably about 0.1-10 mg by injection to the cancer lesion. Also in the case of other animals, the dose converted per 60 kg of body weight can be administered. In addition, the above-mentioned compounds are existing anticancer agents [eg, alkylating agents (eg, cyclophosphamide, ifosfamide, nimustine, ranimustine, carbocon, etc.), antimetabolites (eg, methotrexate, 5-fluorouracil, tegafur, Carmofur, UFT, doxyfluridine, cytarabine, enocitabine, mercaptopurine, mercaptopurine riboside, thioguanine, etc.), anticancer antibiotics (eg, mitomycin, adriamycin, daunorubicin, epirubicin, pirarubicin, idarubicin, bleomycin, peplomycin, actinomycin, etc.) , Plant-derived anticancer agents (eg, vincristine, vinblastine, vindesine,
Etoposide, camptothecin, irinotecan, etc.), cisplatin, carboplatin, nedaplatin, paclitaxel, docetaxel, estramustine, etc.]. At this time, the timing of administration is not limited, and these may be administered to the administration subject 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. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I],
[ ³ H], [ ¹⁴ C], etc. 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 is not necessarily 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, 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 blood It can be diagnosed as having a cancer such as a tumor or having a high possibility of contracting in the future. 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 can be carried out, for example, by known Northern hybridization or PCR-SSCP method (Genomics (Genomics)).
s), Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences of USA.
s of the National Academy of Sciences of the United
States of America), Vol. 86, 2766-2770
Page (1989)) 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, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer Cancers such as, 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 Can be diagnosed as likely.

(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, histone methyltransferase 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,
It can be used as a prophylactic / therapeutic agent for cancers such as brain tumors or blood tumors, and an apoptosis inducer. When the above antisense nucleotide is used as the above-mentioned preventive / therapeutic agent, it can be formulated and administered according to a method known per se. Further, for example, the antisense nucleotides alone or retroviral vector,
After inserting into an appropriate vector such as adenovirus vector, adenovirus associated virus vector, etc., according to a conventional method, for humans or mammals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) Can be administered 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. ), The amount of the antisense nucleotide is about 0.1 to 100 m per day.
g. Further, the antisense nucleotide is
It can also be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA of the present invention or its expression in tissues or cells. RNA encoding the protein of the present invention, similar to the above antisense nucleotides
Double-stranded RNA containing a part of RNA, a ribozyme containing a part of RNA encoding the protein of the present invention, and the like,
Since the expression of the gene of the present invention can be suppressed and the function of the protein used in the present invention or the DNA used in the present invention can be suppressed in vivo, 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 used as a preventive / therapeutic agent for cancer, apoptosis inducer 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.

(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 prophylactic / therapeutic agent and an apoptosis inducer. 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 a suitable dosage form, human or mammal (eg, rat, rabbit, sheep, pig, Cow, cat,
Orally or parenterally (eg, in dogs, monkeys, etc.)
Intravenous injection). Although the dose varies depending on the administration subject, target disease, symptom, administration route, etc., 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. .0
1 to 20 mg / kg body weight, preferably 0.1 to 10
mg / kg body weight, more preferably 0.1-5 mg
/ Kg body weight is conveniently administered about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an 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).
It is preferably provided as an inhalant. Each composition described above may contain other active ingredients as long as the composition does not cause an unfavorable interaction with the antibody.

(6) "A preventive / therapeutic agent for cancer comprising a compound having a histone methyltransferase activity regulating activity or a salt thereof" of the present invention and "a histone methyltransferase expression regulating activity""A compound having a histone methyltransferase activity-regulating action" for "a cancer preventive / therapeutic agent containing a compound or a salt thereof" is any compound having a histone methyltransferase activity-regulating action. Well, 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 used as a prophylactic / therapeutic agent for cancers such as brain tumors and blood tumors, and an apoptosis inducer. The “compound having a histone methyltransferase expression-regulating action” may be any compound as long as it has a histone methyltransferase expression-regulating action, and examples thereof include colon cancer, breast cancer, lung cancer, prostate cancer, and esophagus. 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, etc. It is used as a prophylactic / therapeutic agent for cancer and an apoptosis inducer. The preventive / therapeutic agent is
It is manufactured 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 cell fusion method known per se. Examples of the non-human mammal include cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like.
Among them, rodents, especially mice (for example, C5 as a pure line), which have relatively short ontogeny and biological cycle in terms of creating a pathological animal model system and are easy to reproduce.
7BL / 6 strain, DBA2 strain, etc.
6C3F ₁ system, BDF ₁ system, B6D2F ₁ system, BA
LB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD etc.) and the like are preferable. Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.

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 prophylactic / therapeutic agents for cancers such as bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, and apoptosis inducer. .. 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. DNA with promoter
The construct can be prepared by a usual DNA engineering method. The transfer of the abnormal 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 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 prophylactic / therapeutic agents for cancers such as blood tumors, and apoptosis inducers. 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 its exon portion has a neomycin resistance gene, a drug resistance gene represented by a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloram). A DNA sequence (eg, pol) that disrupts the function of an exon by inserting a reporter gene typified by a phenicol acetyltransferase gene or terminates transcription of the gene at an 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, for example, those already established as described above may be used.
It may be newly established according to the method of Kaufma. For example, in the case of mouse ES cells, it is currently generally 12
Although 9 lines of ES cells have been used, their immunological background is not clear. For example, C57BL is used for the purpose of obtaining an alternative ES cell whose immunological genetic background is clear. BDF which improved the number of eggs collected from / 6 mice and C57BL / 6 by crossing with DBA / 2
Those established by using ₁ mouse (F _{1 of} C57BL / 6 and DBA / 2) can also be favorably used. The BDF ₁ mouse has the advantage that the number of eggs collected is large and the eggs are strong, and since it has the C57BL / 6 mouse as a background, the ES cells obtained using this can be used as a pathological model mouse. , C57BL / 6 mice can be advantageously used in that its genetic background can be changed to that of C57BL / 6 mice. In addition, when ES cells are established, blastocysts on the 3.5th day after fertilization are generally used, but other than this, 8-cell stage embryos are ovulated and cultured up to the blastocysts for efficient and large numbers. The early embryos of 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 suitable feeder cells such as STO fibroblasts, LIF (1 to
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 subculture is usually performed every 1 to 3 days, but it is desirable to observe the cells at this time 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 preventive / therapeutic agent for cancers such as brain tumors or blood tumors, and an apoptosis inducer. 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, it can be used, for example, in humans or mammals (for example, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered. The dose of the compound or salt thereof is
Although there are differences depending on the target disease, administration subject, administration route, etc., for example, in the case of oral administration of the compound of the present invention that inhibits the promoter activity for DNA, in general, in an adult (60 kg body weight) breast cancer patient, About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound per day
Administer. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc.
For example, when a compound that inhibits the promoter activity for the DNA of the present invention is administered in the form of an injection to an adult (as 60 kg) breast cancer patient, the compound is administered in an amount of about 0.01 to 30 mg, preferably about 30 mg per day. 0.1-2
It is convenient to administer about 0 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. As described above, the non-human mammal deficient in DNA expression of the present invention is extremely useful for screening a compound or its salt that promotes or inhibits the activity of the promoter for the DNA of the present invention, and It can greatly contribute to the investigation of the causes of various diseases caused or the development of preventive / therapeutic agents. 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 binding an appropriate reporter gene to the above promoter portion and establishing a cell line capable of expressing this, a low molecular weight compound having an action of specifically promoting or suppressing the production ability of the protein of the present invention itself in the body. Can be used as a search system for.

In this specification, when an abbreviation is used for a base or amino acid, IUPAC-IUB Commission
Based on the abbreviations based on on Biochemical Nomenclature or abbreviations commonly used in this field, 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 SUV39H1. [SEQ ID NO: 2] This shows the base sequence of DNA encoding SUV39H1 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 SUV39H1. [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 probe used in EXAMPLE 2. [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 4. [SEQ ID NO: 12] This shows the base sequence of the primer used in EXAMPLE 4. [SEQ ID NO: 13] This shows the base sequence of the primer used in EXAMPLE 4. [SEQ ID NO: 14] This shows the base sequence of the oligonucleotide for inserting myc tag used in EXAMPLE 4. [SEQ ID NO: 15] This shows the base sequence of the oligonucleotide for inserting myc tag used in EXAMPLE 4. [SEQ ID NO: 16] This shows the base sequence of the primer used in EXAMPLE 5. [SEQ ID NO: 17] This shows the base sequence of the probe used in EXAMPLE 5. [SEQ ID NO: 18] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 19] This shows the base sequence of the primer used in EXAMPLE 3.

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 a gene group whose expression is specifically upregulated in a breast cancer tissue, total RNA (Table 1) extracted from 1 breast cancer tissue and 23 other 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 analysis technical manual of Affymetrix. As a result, the expression of SUV39H1 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 An SUV39H1 antisense oligonucleotide introduction experiment was carried out to analyze the effect of the SUV39H1 gene whose expression was observed specifically in breast cancer on apoptosis. First, after designing an antisense (SEQ ID NO: 4) for the full-length sequence shown in 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 of the nucleotide sequence represented by SEQ ID NO: 4 (SEQ ID NO: 5) was similarly phosphorothi.
oate, HPLC purified and used (Amersham Pharmac
iaBiotech). Breast cancer cell line MDA-MB-231 as test cells
(In Vitro, Volume 14 (11), pages 911-915, 1978, purchased from ATCC), using 18 × 10 ^{3 on the} day before oligonucleotide introduction.
Individual cells were seeded on a 24-well plate (Falcon). OligofectAMINE (Inv
Itrogen) was used and the protocol was followed.
RNA was extracted 7 hours after the introduction according to the protocol of RNeasy mini kit (manufactured by Qiagen), and TaqMan Reverse Transc
Using ription Reagents (Applied Biosystems)
cDNA was prepared. Quantitative PCR was carried out using two primers (SEQ ID NO: 6 and SEQ ID NO: 7) and a probe (SEQ ID NO: 8; Amersham Pharmacia Biotech), ABI PR
It was performed by ISM ^™ 7700 Sequence Detector (manufactured by Applied Biosystems). On the other hand, the effect on apoptosis is as follows: Cell 3 days after introduction of antisense oligonucleotide
It compared with the control oligo introduction sample using death detection ELISA (made by Roche). As a result, 7 hours after the introduction of antisense, the expression (RNA) of SUV39H1 was 100% when the control (reverse) oligonucleotide was introduced.
It was decreased to 70% compared to the control, and apoptosis was increased to 213% at 3 days after the transfer compared to the control (100%). From this, it was revealed that SUV39H1 is not only specifically expressed in cancer, but also involved in cancer cell proliferation and apoptosis.

Example 3 (1) SUV39H1 Expression Vector In order to carry out an expression experiment of SUV39H1, a full-length gene was cloned. PCR was performed using two types of primers (SEQ ID NO: 18 and SEQ ID NO: 19), using the Marathon ready cDNA library (Clontech) as a template and Pfu polymerase (Straragene). PCR
10xPfu buffer 2 μl, 10 mM each dNTP mixture (Clonte
ch) 0.4 μl, each of the above two primers prepared to 20 mM
0.8 μl, template cDNA solution 0.5 μl, Pfu polymerase 0.1
Pre-treat 20 μl of the reaction solution containing μl at 94 ° C for 1 min.
The reaction was carried out by carrying out the reaction at 42 ° C for 7 minutes after 42 cycles of the reaction at 1 ° C for 1 minute, 57 ° C for 1 minute and 72 ° C for 2 minutes. After the PCR reaction, the products are separated by agarose gel electrophoresis, the desired band is cut out, and the Gel Extraction kit is used.
(Qiagen) was used to purify the reaction product. This purified product was used as the Zero Blunt TOPO PCR cloning kit (Invit
(made by rogen) pCR-Blunt I based on the attached protocol
Cloned into I-TOPO vector (Invitrogen),
Vector 1 was obtained. Next, two primers (SEQ ID NO:
9 and SEQ ID NO: 10) and using the vector 1 obtained above as a template, Pfu polymerase (Straragene
PCR was performed. PCR is 10xPfu buffer 2μ
l, 10 mM each dNTP mixture (Clontech) 0.4 μl, 20
0.8 μl of each of the above two primers prepared in mM, 0.5 μl of template plasmid solution, 0.1 μl of Pfu polymerase
Pretreat 1 μl of the reaction solution at 94 ° C for 1 minute, then
The reaction was carried out by performing a reaction at 72 ° C for 7 minutes after 25 cycles of reaction at 1 ° C for 1 minute and 2 minutes at 72 ° C. After the PCR reaction, the products are separated by agarose gel electrophoresis and the band of interest is cut out and Gel Extraction kit (Qiagen)
Was used to purify the reaction product. After this, pcDNA3.1 / V5 His TA Expression vector (Invi
(manufactured by trogen) was added with A according to the attached protocol, and cloned into the same vector. Check the nucleotide sequence of the insert and confirm that the sequence is correct.
An expression vector (Vector 2) was obtained.

(2) Effect on Tumor Suppressor Gene p53 In order to investigate the function of SUV39H1, SUV39H1 was forcibly expressed in cancer cells and its effect was observed. Colorectal cancer-derived HCT116 (ATCC
2 x 10 ⁴ pieces, 24 well plate (made by Falcon)
The SUV39H1 expression vector (Vector 2) obtained in (1) above after inoculating the cells and culturing at 37 ° C in a 5% CO ₂ environment for 1 day.
(0.13 μg) or pcDNA3.1 (Invitrogen
(Manufactured) (0.13 μg). SUV39H1
In order to see the action of p53 on p53, a reporter vector (manufactured by Clontech) (0.13 μg) having a p53 response element inserted upstream of the firefly luciferase gene was transfected together with the expression vector of (1) above, and further,
To compensate for transfection efficiency, pRL-TK (Pr
(Omega) (0.13 μg) was also co-transfected. As a transfection reagent, Fugene6 (Roche
Manufactured by K.K.) was used and the attached protocol was followed. 2 days after transfection, Dual-Luciferase Reporter Assa
The y-system (manufactured by Promega) was used to measure firefly luciferase activity and sea shiitake luciferase activity. The measured value of firefly luciferase activity was corrected by the measured value of sea oyster luciferase activity and compared.
When set to 0, it decreased to 65 when SUV39H1 was introduced, and SUV3
9H1 was found to suppress the function of the tumor suppressor gene p53

Example 4 (1) Preparation of expression vector with myc tag In order to facilitate purification of SUV39H1, a sequence was designed to attach a myc tag to the amino terminal side. Using the vector 1 obtained in Example 3 (1) as a template, Pfu polymerase (Strat
agene) and PCR was performed according to the attached protocol. Two kinds of primers (SEQ ID NO: 11 and SEQ ID NO: 12) and two kinds of primers (SEQ ID NO: 1) for the production of amino acid deletion mutants after the 181st position
1 and SEQ ID NO: 13) in the same manner as above
PCR was performed. PCR is 10xPfu buffer 2μl, 10mM each
dNTP mixture (manufactured by Clontech) 0.4 μl, 0.8 μl each of the above two primers prepared to 20 mM, template plasmid solution 0.5
20 μl of reaction solution containing μl and 0.1 μl of Pfu polymerase was pretreated at 94 ° C. for 1 minute, then 94 ° C. for 1 minute, 57 ° C. for 1 minute, 72
The reaction was performed at 42 ° C for 7 minutes after 42 cycles of 2 minutes at 72 ° C. After the PCR reaction, the products were separated by agarose gel electrophoresis, the desired band was cut out, and the reaction products were purified using Gel Extraction kit (manufactured by Qiagen). After digesting the purified product with restriction enzymes EcoRI and XhoI, the product is again separated by agarose gel electrophoresis, and the band of interest is cut out and the Gel Extraction kit is used.
Was used to purify the reaction product. including myc tag sequence,
Nucleotide fragments having the base sequence represented by SEQ ID NO: 14 or SEQ ID NO: 15 were mixed, heated and cooled. The reaction product was digested with restriction enzymes KpnI and EcoRI, and after agarose gel electrophoresis, Gel Extraction kit (Qiagen).
Was used to purify the enzymatic digestion product. The above-mentioned PCR product and the product obtained by digesting the myc tag sequence with the restriction enzyme were digested with
PcDNA3.1 (+) vector (Invitrogen digested with I and XhoI
Product). Check the nucleotide sequence of the inserted fragment, confirm that the sequence is correct, and add Myc to the amino terminus.
A full-length SUV39H1 expression vector to which a tag was added and a SUV39H1 expression vector in which the Myc tag was added to the amino terminus and amino acids at positions 181 and later were deleted were obtained. (2) Expression of myc-tagged SUV39H1 protein 1 × 10 ⁵ HeLa cells (purchased from ATCC) were seeded on a 35 mm dish (manufactured by Falcon), cultured for 1 day, and then cultured in Example 4 above.
SUV39H1 expression plasmid (1 with myc tag obtained in (1)
μg) was transfected using Fugene6 (Roche) according to the attached protocol. The day after transfection, the culture medium was replaced with a medium containing 500 μg / ml of G418 (manufactured by GIBCO). After that, the viable cells were selected by exchanging the culture medium with the G418-containing medium every 3 days. Then G4
The cells that became resistant to 18 were cultured in a 10 cm dish until they became confluent, and a nuclear extract was prepared using NE-PER (manufactured by Pierce). After addition of the CERII solution, the protocol attached to NE-PER was followed except that ultrasonic treatment was performed using an ultrasonic generator. The obtained nuclear extract protein solution (NER solution) was separated by SDS-PAGE and subjected to Western blotting. Anti-myc antibody (Invitroge
ne) was used as the secondary antibody with anti-mouse IgG alkaline phosphatase conjugate (Promega). As a result, a SUV39H1 protein with a myc tag of about 52 kDa was confirmed.

Example 5 (1) Preparation of His-tagged SUV39H1 expression vector Using the vector 2 obtained in the above Example 3 (1) as a template,
Two primers (SEQ ID NO: 16 and SEQ ID NO: 1)
Using 7), PCR was performed using Pfu polymerase (Stratagene) according to its attached protocol. PCR
10xPfu buffer 2μl, 2.5mM each dNTP mixture (Clontec
h) 1.6 μl, each of the above two primers prepared to 50 mM
20 μl of reaction solution containing 0.2 μl, template plasmid solution 0.5 μl, and Pfu polymerase 0.4 μl was pretreated at 94 ° C. for 1 min, then 94 ° C. for 1 min, 57 ° C. for 1 min, 72 ° C. for 2 min. After 25 cycles of the reaction, the reaction was carried out at 72 ° C for 5 minutes. After the reaction, Advantage2 polymerase (Clonte
ch.) was added and reacted at 72 ° C. for 15 minutes, and then electrophoresed on agarose gel to cut out the desired band. Then Gel Extractio
After purifying the reaction product using n kit (Qiagen), pQ
The E30UA vector (manufactured by Qiagen) was ligated according to the protocol attached thereto, and transformed into Escherichia coli JM109 (manufactured by Takara). It was confirmed that there was no error in the nucleotide sequence, and a transformant JM109 carrying the His-tagged SUV39H1 expression vector was obtained. (2) Expression of His-tagged SUV39H1 protein Using the transformant obtained in (1) above, 200 ml of CircleGrow
The SUV39H1 protein was expressed by culturing in a medium. When the expression was induced with 1 mM isopropylthiogalactopyranoside, SUV39H1 protein was not obtained in the soluble fraction.
It was purified using TA superflow (manufactured by Qiagen). As a result, the target recombinant SUV39H1 protein was
(A handbook for high-level expression and purificat
It was confirmed to be eluted with ion of 6xHis-tagged proteins, Qiagen). The eluate was then subjected to a molecular weight cut-off of 60 to refold the denatured SUV39H1.
Using a dialysis membrane of 00-8000 (Spectrum medical), 4
2 hours at C for buffer I, 2.5 hours for buffer II, 4 hours for buffer III, 14 hours for buffer IV, 2 hours for buffer V,
It dialyzed sequentially. The precipitate generated in the dialysis process was removed by centrifugation to obtain 0.04 mg of recombinant His-tagged SUV39H1 protein solution (dissolved in buffer solution V). Buffer I: 0.2 M Tris-HCl (pH 9.0), 3 mM 2-mercaptoethanol, 0.3 mM 2-hydroxyethyl disulfide, 3 M urea and 0.1% Triton X-100 Buffer II: 0.2 M Tris-HCl (pH 9.0) ), 3 mM 2-mercaptoethanol, 0.3 mM 2-hydroxyethyl disulfide, 0.8 M urea and 0.1% Triton X-100 buffer III: 0.1 M Tris-hydrochloric acid (pH 8.5), 3 mM 2-mercaptoethanol, 0.3 mM 2-hydroxy Ethyl disulfide, 0.2 M urea, 0.1 M sucrose, 20 mM potassium chloride and 10 mM magnesium chloride buffer IV: 50 mM Tris-HCl (pH 8.5), 1 mM 2-mercaptoethanol, 0.1 mM 2-hydroxyethyl disulfide, 50 mM urea, 10 mM chloride Magnesium, 20 mM potassium chloride and 250 mM saccharose buffer V: 50 mM Tris-HCl (pH 8.5), 20 mM potassium chloride, 10 mM magnesium chloride and 250 mM saccharose

Example 6 An inhibitor screening experiment is conducted using the protein solution obtained in Example 4 (2) or Example 5 (2) as an enzyme solution.
50 μl of reaction buffer containing 10 μl of enzyme solution (50 mM Tris-hydrochloric acid (pH 8.5), 20 mM potassium chloride, 10 mM magnesium chloride, 250 mM saccharose and 10 mM 2-mercaptoethanol) and 2 μl of a DMF solution containing a test compound,
Streptavidin-coated 96-well plate (Perkin-E
lmer) and leave at 37 ° C for 10 minutes. Then 1 ng of biotin-labeled histone H3 peptide (from Upstate) and [
³ H] S-adenosylmethionine (Amersham) (0.5 μCi)
The reaction is started by adding and mixing. 37 ° C
After 3 hours of reaction, the reaction solution was discarded and the washing buffer solution [0.05%
After washing with phosphate buffer containing Tween-20 three times, 100 μl of liquid scintillator (manufactured by Wako Pure Chemical Industries, Ltd.) is added, and the radioactivity is measured by a liquid scintillation counter (manufactured by Wallac). The 50% inhibitory concentration (IC ₅₀ value) can be determined by setting the activity when the DMF solution containing no test compound is added as 100. Compounds that give lower IC ₅₀ values are selected as compounds that more strongly inhibit the activity of the invention.

[0082]

INDUSTRIAL APPLICABILITY The protein used in the present invention is a diagnostic marker for cancer. A compound or a salt thereof that regulates (preferably inhibits) the activity of the protein, a compound or a salt thereof that regulates (preferably inhibits) the expression of a gene of the protein, the antibody of the present invention, the antisense nucleotide of the present invention, etc., 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 , A thyroid cancer, pancreatic cancer, a prophylactic / therapeutic agent for cancer such as brain tumor or blood tumor, and an apoptosis promoting agent, etc.

[0083]

[Sequence list] SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Preventing and treating agent for cancer <130> P02-0148PCT <150> JP2001-398220 <151> 2001-12-27 <160> 19 <210> 1 <211> 412 <212> PRT <213> Human <400> 1 Met Ala Glu Asn Leu Lys Gly Cys Ser Val Cys Cys Lys Ser Ser Trp                  5 10 15 Asn Gln Leu Gln Asp Leu Cys Arg Leu Ala Lys Leu Ser Cys Pro Ala              20 25 30 Leu Gly Ile Ser Lys Arg Asn Leu Tyr Asp Phe Glu Val Glu Tyr Leu          35 40 45 Cys Asp Tyr Lys Lys Ile Arg Glu Gln Glu Tyr Tyr Leu Val Lys Trp      50 55 60 Arg Gly Tyr Pro Asp Ser Glu Ser Thr Trp Glu Pro Arg Gln Asn Leu  65 70 75 80 Lys Cys Val Arg Ile Leu Lys Gln Phe His Lys Asp Leu Glu Arg Glu                  85 90 95 Leu Leu Arg Arg His His Arg Ser Lys Thr Pro Arg His Leu Asp Pro             100 105 110 Ser Leu Ala Asn Tyr Leu Val Gln Lys Ala Lys Gln Arg Arg Ala Leu         115 120 125 Arg Arg Trp Glu Gln Glu Leu Asn Ala Lys Arg Ser His Leu Gly Arg     130 135 140 Ile Thr Val Glu Asn Glu Val Asp Leu Asp Gly Pro Pro Arg Ala Phe 145 150 155 160 Val Tyr Ile Asn Glu Tyr Arg Val Gly Glu Gly Ile Thr Leu Asn Gln                 165 170 175 Val Ala Val Gly Cys Glu Cys Gln Asp Cys Leu Trp Ala Pro Thr Gly             180 185 190 Gly Cys Cys Pro Gly Ala Ser Leu His Lys Phe Ala Tyr Asn Asp Gln         195 200 205 Gly Gln Val Arg Leu Arg Ala Gly Leu Pro Ile Tyr Glu Cys Asn Ser     210 215 220 Arg Cys Arg Cys Gly Tyr Asp Cys Pro Asn Arg Val Val Gln Lys Gly 225 230 235 240 Ile Arg Tyr Asp Leu Cys Ile Phe Arg Thr Asp Asp Gly Arg Gly Trp                 245 250 255 Gly Val Arg Thr Leu Glu Lys Ile Arg Lys Asn Ser Phe Val Met Glu             260 265 270 Tyr Val Gly Glu Ile Ile Thr Ser Glu Glu Ala Glu Arg Arg Gly Gln         275 280 285 Ile Tyr Asp Arg Gln Gly Ala Thr Tyr Leu Phe Asp Leu Asp Tyr Val     290 295 300 Glu Asp Val Tyr Thr Val Asp Ala Ala Tyr Tyr Gly Asn Ile Ser His 305 310 315 320 Phe Val Asn His Ser Cys Asp Pro Asn Leu Gln Val Tyr Asn Val Phe                 325 330 335 Ile Asp Asn Leu Asp Glu Arg Leu Pro Arg Ile Ala Phe Phe Ala Thr             340 345 350 Arg Thr Ile Arg Ala Gly Glu Glu Leu Thr Phe Asp Tyr Asn Met Gln         355 360 365 Val Asp Pro Val Asp Met Glu Ser Thr Arg Met Asp Ser Asn Phe Gly     370 375 380 Leu Ala Gly Leu Pro Gly Ser Pro Lys Lys Arg Val Arg Ile Glu Cys 385 390 395 400 Lys Cys Gly Thr Glu Ser Cys Arg Lys Tyr Leu Phe                 405 410 <210> 2 <211> 1236 <212> DNA <213> Human <400> 2 atggcggaaa atttaaaagg ctgcagcgtg tgttgcaagt cttcttggaa tcagctgcag 60 gacctgtgcc gcctggccaa gctctcctgc cctgccctcg gtatctctaa gaggaacctc 120 tatgactttg aagtcgagta cctgtgcgat tacaagaaga tccgcgaaca ggaatattac 180 ctggtgaaat ggcgtggata tccagactca gagagcacct gggagccacg gcagaatctc 240 aagtgtgtgc gtatcctcaa gcagttccac aaggacttag aaagggagct gctccggcgg 300 caccaccggt caaagacccc ccggcacctg gacccaagct tggccaacta cctggtgcag 360 aaggccaagc agaggcgggc gctccgtcgc tgggagcagg agctcaatgc caagcgcagc 420 catctgggac gcatcactgt agagaatgag gtggacctgg acggccctcc gcgggccttc 480 gtgtacatca atgagtaccg tgttggtgag ggcatcaccc tcaaccaggt ggctgtgggc 540 tgcgagtgcc aggactgtct gtgggcaccc actggaggct gctgcccggg ggcgtcactg 600 cacaagtttg cctacaatga ccagggccag gtgcggcttc gagccgggct gcccatctac 660 gagtgcaact cccgctgccg ctgcggctat gactgcccaa atcgtgtggt acagaagggt 720 atccgatatg acctctgcat cttccggacg gatgatgggc gtggctgggg cgtccgcacc 780 ctggagaaga ttcgcaagaa cagcttcgtc atggagtacg tgggagagat cattacctca 840 gaggaggcag agcggcgggg ccagatctac gaccgtcagg gcgccaccta cctctttgac 900 ctggactacg tggaggacgt gtacaccgtg gatgccgcct actatggcaa catctcccac 960 tttgtcaacc acagttgtga ccccaacctg caggtgtaca acgtcttcat agacaacctt 1020 gacgagcggc tgccccgcat cgctttcttt gccacaagaa ccatccgggc aggcgaggag 1080 ctcacctttg attacaacat gcaagtggac cccgtggaca tggagagcac ccgcatggac 1140 tccaactttg gcctggctgg gctccctggc tcccctaaga agcgggtccg tattgaatgc 1200 aagtgtggga ctgagtcctg ccgcaaatac ctcttc 1236 <210> 3 <211> 2754 <212> DNA <213> Human <400> 3 ggcacgaggc gcgaggccgg ctaggcccga atgtcgttag ccgtggggaa agatggcgga 60 aaatttaaaa ggctgcagcg tgtgttgcaa gtcttcttgg aatcagctgc aggacctgtg 120 ccgcctggcc aagctctcct gccctgccct cggtatctct aagaggaacc tctatgactt 180 tgaagtcgag tacctgtgcg attacaagaa gatccgcgaa caggaatatt acctggtgaa 240 atggcgtgga tatccagact cagagagcac ctgggagcca cggcagaatc tcaagtgtgt 300 gcgtatcctc aagcagttcc acaaggactt agaaagggag ctgctccggc ggcaccaccg 360 gtcaaagacc ccccggcacc tggacccaag cttggccaac tacctggtgc agaaggccaa 420 gcagaggcgg gcgctccgtc gctgggagca ggagctcaat gccaagcgca gccatctggg 480 acgcatcact gtagagaatg aggtggacct ggacggccct ccgcgggcct tcgtgtacat 540 caatgagtac cgtgttggtg agggcatcac cctcaaccag gtggctgtgg gctgcgagtg 600 ccaggactgt ctgtgggcac ccactggagg ctgctgcccg ggggcgtcac tgcacaagtt 660 tgcctacaat gaccagggcc aggtgcggct tcgagccggg ctgcccatct acgagtgcaa 720 ctcccgctgc cgctgcggct atgactgccc aaatcgtgtg gtacagaagg gtatccgata 780 tgacctctgc atcttccgca cggatgatgg gcgtggctgg ggcgtccgca ccctggagaa 840 gattcgcaag aacagcttcg tcatggagta cgtgggagag atcattacct cagaggaggc 900 agagcggcgg ggccagatct acgaccgtca gggcgccacc tacctctttg acctggacta 960 cgtggaggac gtgtacaccg tggatgccgc ctactatggc aacatctccc actttgtcaa 1020 ccacagttgt gaccccaacc tgcaggtgta caacgtcttc atagacaacc ttgacgagcg 1080 gctgccccgc atcgctttct ttgccacaag aaccatccgg gcaggcgagg agctcacctt 1140 tgattacaac atgcaagtgg accccgtgga catggagagc acccgcatgg actccaactt 1200 tggcctggct gggctccctg gctcccctaa gaagcgggtc cgtattgaat gcaagtgtgg 1260 gactgagtcc tgccgcaaat acctcttcta gcccttagaa gtctgaggcc agactgactg 1320 agggggcctg aagctacatg cacctccccc actgctgccc tcctgtcgag aatgactgcc 1380 agggcctcgc ctgcctccac ctgcccccac ctgctcctac ctgctctacg ttcagggctg 1440 tggccgtggt gaggaccgac tccaggagtc ccctttccct gtcccagccc catctgtggg 1500 ttgcacttac aaacccccac ccaccttcag aaatagtttt tcaacatcaa gactctctgt 1560 cgttgggatt catggcctat taaggaggtc caaggggtga gtcccaaccc agccccagaa 1620 tatatttgtt tttgcacctg cttctgcctg gagattgagg ggtctgctgc aggcctcctc 1680 cctgctgccc caaaggtatg gggaagcaac cccagagcag gcagacatca gaggccagag 1740 tgcctagccc gacatgaagc tggttcccca accacagaaa ctttgtacta gtgaaagaaa 1800 gggggtccct gggctacggg ctgaggctgg tttctgctcg tgcttacagt gctgggtagt 1860 gttggcccta agagctgtag ggtctcttct tcagggctgc atatctgaga agtggatgcc 1920 cacatgccac tggaagggaa gtgggtgtcc atgggccact gagcagtgag aggaaggcag 1980 tgcagagctg gccagccctg gaggtaggct gggaccaagc tctgccttca cagtgcagtg 2040 aaggtaccta gggctcttgg gagctctgcg gttgctaggg gccctgacct ggggtgtcat 2100 gaccgctgac accactcaga gctggaacca agatctagat agtccgtaga tagcacttag 2160 gacaagaatg tgcattgatg gggtggtgat gaggtgccag gcactgggta gagcacctgg 2220 tccacgtgga ttgtctcagg gaagccttga aaaccacgga ggtggatgcc aggaaagggc 2280 ccatgtggca gaaggcaaag tacaggccaa gaattggggg tgggggagat ggcttcccca 2340 ctatgggatg acgaggcgag agggaagccc ttgctgcctg ccattcccag accccagccc 2400 tttgtgctca ccctggttcc actggtctca aaagtcacct gcctacaaat gtacaaaagg 2460 cgaaggttct gatggctgcc ttgctccttg ctcccccacc ccctgtgagg acttctctag 2520 gaagtccttc ctgactacct gtgcccagag tgcccctaca tgagactgta tgccctgcta 2580 tcagatgcca gatctatgtg tctgtctgtg tgtccatccc gccgaccccc cagactaacc 2640 tccaggcatg gactgaatct ggttctcctc ttgtacaccc ctcaacccta tgcagcctgg 2700 agtgggcatc aataaaatga actgtcgact gaaaaaaaaa aaaaaaaaaa aaaa 2754 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 atgcgtccca gatggctgcg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 gcgtcggtag accctgcgta 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 aaccttgacg agcggctg 18 <210> 7 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 tcgcctgccg gatg 14 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 8 ccgcatcgct ttctttgcca caa 23 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 ggaaagatgg cggaaaattt aaaa 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 gggctagaag aggtatttgc ggca 24 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 ccggaattcg cggaaaattt aaaaggctgc a 31 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 12 ccgctcgagc tagaagaggt atttgcggc 29 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 13 ccgctcgagc tagcccacag ccacctggtt ga 32 <210> 14 <211> 113 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 14 ggggtaccaa gatggagcag aaactcatct ctgaagagga tctggagcag aaactcatct 60 ctgaagagga tctggagcag aaactcatct ctgaagagga tctggaattc cgg 113 <210> 15 <211> 113 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 15 ccggaattcc agatcctctt cagagatgag tttctgctcc agatcctctt cagagatgag 60 tttctgctcc agatcctctt cagagatgag tttctgctcc atcttggtac ccc 113 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 16 tgtgtgcgta tcctcaagca gttc 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 17 ctagaagagg tatttgcggc agga 24 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 18 gtcgttagcc gtggggaaag atgg 24 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 19 atatgcagcc ctgaagaaga gacc 24

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 35/00 4C085 A61P 35/00 43/00 105 4C086 43/00 105 C12Q 1/02 C12N 15/09 1/48 Z C12Q 1/02 G01N 33/15 Z 1/48 33/50 Z G01N 33/15 C12N 9/10 33/50 9/99 // C12N 9/10 A61K 37/02 ZNA 9/99 C12N 15 / 00 A (72) Inventor Yuichi Suga 1-10-10 Tokodai, Tsukuba-shi, Ibaraki 6F term (reference) 2G045 AA26 AA29 AA40 BB03 BB20 CA26 CB01 CB17 CB21 DA12 DA13 DA14 DA36 DA78 FB02 FB03 4B024 AA01 CA10 DA80 CA80 DA05 DA11 EA02 EA03 EA04 FA02 GA11 HA11 4B050 CC03 CC04 DD11 FF04 FF11 FF12 FF14 LL03 4B063 QA01 QA18 QQ05 QQ13 QQ26 QR06 QR33 QR57 QR59 QR74 QR80 QS05 QS26 QS36 QX02 4C084 AA02 AA13 BA22 ZB21 ZB26 4C085 AA13 AA14 BB11 CC21 4C086 AA01 EA16 MA01 NA14 ZB21 ZB26

Claims (22)

[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 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. 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 histone methyltransferase activity-regulating action or a salt thereof. 11. A preventive / therapeutic agent for cancer, which comprises a compound having a histone methyltransferase expression-regulating 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 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 for 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. 22. A protein, a partial peptide thereof, or a salt thereof, which comprises 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. 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.