JP2004217634A - Preventing/treating agent for cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a medicinal agent useful as a preventing/treating agent for cancer, taking a molecule specifically expressed in cancer cells as a target and capable of performing the inhibition of propagation of the cancer cells or an apoptosis induction. <P>SOLUTION: A compound adjusting (preferably inhibiting) the activity of a protein having the same or substantially the same amino acid sequence with that of a histone methyl group transferase EZH2 of human origin, or its salt, a compound adjusting (preferably inhibiting) the expression of the protein, or its salt, an antisense polynucleotide containing a base sequence or a part of it, complementary to or substantially complementary to the base sequence of a polynucleotide encoding the protein or its partial peptide and an antibody against the protein can be used as the preventing/treating agent or an apoptosis-inducing agent, or the like, of the cancer, or the like. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a prophylactic / therapeutic agent and a diagnostic agent for cancer, a screening for a prophylactic / therapeutic agent for cancer, an apoptosis inducer, a screening for an apoptosis inducer, and the like.

In cancer chemotherapy, the development of new anticancer drugs has improved the survival benefit and is increasing the number of cases toward cure. However, most of the currently used anticancer drugs are highly cytotoxic, damaging DNA or inhibiting cell division. Strong side effects often appear in the dividing bone marrow.
In order to comprehensively analyze gene expression, a microarray method in which cDNA or oligonucleotides are immobilized has been developed, and techniques for finding disease-specific changes in gene expression have become widespread, and their usefulness has been confirmed. For example, Affymetrix's GeneChip system is increasingly being used to diagnose diseases such as cancer and to discover drug discovery target genes.
When introduced into a cell, an antisense oligonucleotide hybridizes with RNA having a complementary sequence and induces RNA degradation by RNaseH to inhibit protein translation, or inhibits direct protein synthesis by hybridization. Also bring. Since the target gene function can be specifically suppressed, it is frequently used as a means for analyzing the function of a gene, and some antisense oligonucleotides are being developed for clinical application.
In recent years, it has become increasingly clear that chromatin structure is deeply involved in the control of cell division, proliferation, or gene transcription. It is known that among the histones that make up chromatin, particularly the site called the histone tail undergoes modifications such as acetylation, phosphorylation, and methylation to contribute to the change of chromatin structure (Nakaya et al.) , Experimental Medicine, October 2001).
Drosophila Enhancer of zeste is a member of the Polycomb group, a protein that is known to suppress homeotic gene expression by regulating chromatin.
Chen et al. Used the exon trapping method from a chromosome 21 cosmid library to isolate genes associated with Down's syndrome (Genomics, 38, 30-37, 1996). One of the cloned exons shows high homology with the Drosophila Enhancer of zeste protein, and was subjected to full-length cloning and named ENHANCER OF ZESTE, DROSOPHILA, HOMOLOG 2 (hereinafter sometimes referred to as EZH2). (Genomics, 38, 30-37, 1996). Cardoso et al. Performed chromosome mapping of EZH2 and reported that it is present at 7q35.At the same time, since this region is abnormal in myeloid leukemia, EZH2 may be involved in the development of malignant bone marrow disease (Non-Patent Document 1 Europ. J. Hum. Genet., 8, 174-180, 2000). Varambally and colleagues found that EZH2 was up-regulated in hormone-insensitive prostate cancer, suggesting that it may be involved in malignant transformation of prostate cancer. Furthermore, they report that the growth of prostate cancer cell lines can be suppressed by designing and introducing siRNAs to EZH2 into cells (Non-Patent Document 2 Nature, 419, 624-629, 2002). . However, no report has been made on enhanced expression of EZH2 in cancer types other than the prostate. Furthermore, there is a report that the above-mentioned siRNA did not induce apoptosis in cancer cells (Non-patent Document 2, Nature, 419, 624-629, 2002).
Cao et al. Purified a complex of a protein called EED and EZH2 and reported that this complex methylated the 27th lysine residue of nucleosome histone H3 (Non-Patent Document 3 Science, vol. 298, 1039- 1043, 2002). In recent years, attention has been focused on the modification of histone proteins, particularly histone deacetylation and cancer, and the development of anticancer drugs targeting histone deacetylase and inhibiting its function has been promoted. Targets for methyltransferases have not been reported to date.
Europ. J. Hum. Genet., 8, 174-180, 2000 Nature, 419, 624-629, 2002 Science, 298, 1039-1043, 2002

  There is a keen need for drugs that target molecules that are specifically expressed in cancer cells and that can inhibit the growth of cancer cells or induce apoptosis.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, expression of EZH2 is enhanced in breast cancer, colon cancer, lung cancer, ovarian cancer, pancreatic cancer, etc. The present inventors have found that cancer cells undergo apoptosis by introducing sense oligos into cancer cells, and have completed the present invention.

That is, the present invention
(1) Cancer containing a compound or a salt thereof that inhibits the activity of 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 Prophylactic and therapeutic agents,
(2) 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 a compound thereof or a salt thereof which inhibits the expression of a gene thereof; Cancer prevention and treatment agents,
(3) a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of a polynucleotide 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; Or an antisense polynucleotide containing a part thereof,
(4) a prophylactic / therapeutic agent for cancer comprising the antisense polynucleotide according to the above (3),
(5) a prophylactic / therapeutic agent for cancer comprising 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 prophylactic / therapeutic agent according to (1), (2), (4) or (5), wherein the cancer is colorectal cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer.
(6a) the prophylactic / therapeutic agent according to the above (1), (2), (4) or (5), wherein the cancer is a hormone-independent cancer;
(6b) Cancer is colorectal cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid gland The prophylactic / therapeutic agent according to the above (1), (2), (4) or (5), which is a cancer, a pancreatic cancer, an ovarian cancer, a brain tumor, or a blood tumor;
(6c) Cancer is colon cancer, lung cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer The prophylactic / therapeutic agent according to the above (1), (2), (4) or (5), which is a pancreatic cancer, an ovarian cancer, a brain tumor, or a blood tumor.
(7) a diagnostic agent for cancer comprising 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 comprising a polynucleotide 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 diagnostic agent according to (7) or (8), wherein the cancer is colorectal cancer, breast cancer, lung cancer, pancreatic cancer, or ovarian cancer.
(9a) The diagnostic agent according to the above (7) or (8), wherein the cancer is a hormone-independent cancer,
(10) an agent for preventing or treating cancer comprising a compound having a histone methyltransferase activity inhibitory activity or a salt thereof,
(11) an apoptosis inducer comprising a compound having a histone methyltransferase activity inhibitory activity or a salt thereof,
(12) a prophylactic / therapeutic agent for cancer, comprising a compound having a histone methyltransferase expression inhibitory activity or a salt thereof;
(13) an apoptosis inducer comprising a compound having a histone methyltransferase expression inhibitory activity or a salt thereof,
(13a) the agent according to the above (10) to (13), wherein the histone methyltransferase is an enzyme that transfers a methyl group to the ninth and / or 27th lysine residue of histone H3;
(14) Screening of a prophylactic / therapeutic agent for cancer, characterized by using 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. Method,
(15) A prophylactic / therapeutic agent for cancer, comprising 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 kit,
(16) A prophylactic / therapeutic agent for cancer, characterized by using a polynucleotide 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,
(17) A prophylactic / therapeutic agent for cancer comprising a polynucleotide 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 kit,
(17a) A prophylactic / therapeutic agent for cancer obtainable using the screening method according to (14) or (16) or the screening kit according to (15) or (17).
(18) Induction of apoptosis comprising a compound which inhibits the activity of 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, or a salt thereof Agent,
(19) a protein or a partial peptide thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a compound which inhibits gene expression of a salt thereof, or a salt thereof Apoptosis inducer,
(20) a method for screening an apoptosis-inducing agent, which comprises using a protein having 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;
(21) 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,
(21a) an apoptosis inducer obtainable by using the screening method according to the above (20) or (21),
(22) Compounds that inhibit the activity of (i) a protein having 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 with respect to a mammal; A salt, (ii) a compound or a salt thereof that inhibits expression of the gene of the protein or its partial peptide or a salt thereof, (iii) an antibody against the protein or its partial peptide or a salt thereof, or (iv) an antibody to the protein or a portion thereof. Prevention and treatment of cancer, which comprises administering an effective amount of an antisense polynucleotide containing a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of a polynucleotide encoding a peptide or a part thereof. Law,
(23) a compound that inhibits the activity of (i) a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof, with respect to a mammal; A salt, (ii) a compound or a salt thereof that inhibits expression of the gene of the protein or its partial peptide or a salt thereof, (iii) an antibody against the protein or its partial peptide or a salt thereof, or (iv) an antibody to the protein or a portion thereof. A method for inducing apoptosis, which comprises administering an effective amount of an antisense polynucleotide containing a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of a polynucleotide encoding a peptide or a part thereof,
(24) inhibiting the activity of a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or inhibiting the activity of the protein or its partial peptide or a salt thereof A method for preventing and treating cancer, characterized by inhibiting gene expression,
(25) inhibits the activity of a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or inhibits the activity of a protein or a partial peptide thereof or a salt thereof Apoptosis induction method, characterized by inhibiting gene expression,
(26) For the production of a prophylactic / therapeutic agent for cancer, (i) a protein having 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 (Ii) a compound or its salt that inhibits the expression of the gene of the protein or its partial peptide or its salt, (iii) an antibody against the protein or its partial peptide or its salt, or ( iv) use of an antisense polynucleotide containing a nucleotide sequence complementary to or substantially complementary to a nucleotide sequence of a polynucleotide encoding the protein or a partial peptide thereof, or a part thereof;
(27) inhibiting the activity of (i) 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 for producing an apoptosis inducer; A compound or a salt thereof, (ii) a compound or a salt thereof that inhibits the expression of the gene of the protein or its partial peptide or a salt thereof, (iii) an antibody against the protein or a partial peptide or a salt thereof, or (iv) the protein Alternatively, there is provided use of an antisense polynucleotide containing a base sequence complementary to or substantially complementary to a base sequence of a polynucleotide encoding a partial peptide thereof or a part thereof.

  A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (protein used in the present invention) is a diagnostic marker for cancer. Compounds or salts thereof that regulate (preferably inhibit) the activity of the protein, compounds or salts thereof that regulate (preferably inhibit) gene expression of the protein, antibodies of the present invention, antisense polynucleotides of the present invention, etc. , For example, cancer (eg, colon, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, Testicular cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc.), preferably preventive / therapeutic agent for colon, breast, lung, pancreatic or ovarian cancer And can be used as a safe drug as an apoptosis inducer and the like.

A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter, also referred to as the protein of the present invention or the protein used in the present invention) may be human or warm-blooded. Animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) cells (eg, hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, obesity) Cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes Or stromal cells, or precursor cells of these 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, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, colon, Small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. Is also good.
The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 60% or more, more preferably about 70% or more as the amino acid sequence represented by SEQ ID NO: 1. And more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
Amino acid sequence homology was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gap allowed; matrix = BLOSUM62; filtering = OFF). Can be calculated.
Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 described above. And proteins having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1.

Examples of substantially the same activity include histone methyltransferase (eg, an enzyme that transfers a methyl group to lysine residues 9 and / or 27 of histone H3). Substantially homogenous indicates that the properties are homogenous (eg, physiologically or pharmacologically). Therefore, it is preferable that the histone methyltransferase activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Quantitative factors such as the degree of activity and the molecular weight of the protein may vary.
Histone methyltransferase activity can be measured by a method known per se, for example, the method described in Science, vol. 298, pp. 1039-1043, 2002 or a method analogous thereto. Specifically, (i) the protein-EED complex of the present invention, (ii) S-adenosyl-L-methionine radioactively labeled with a methyl group and (iii) histone protein, oligonucleosome or histone H3 around lysine 27 The polypeptide having the sequence is reacted, and the radioactivity of histone H3 or the polypeptide by methyl group transfer is measured. This reaction is performed in an appropriate buffer. After the enzymatic reaction, the reaction product is separated by, for example, SDS-PAGE or the like, and is first compared with the mobility of histone H3 or the like as a standard control and identified. Measurement of radioactivity for quantification is performed according to a known method using a scintillation counter, fluorography, or the like.

Examples of the protein used in the present invention include, for example, (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (eg, about 1 to 100, preferably about 1 to 30, Is an amino acid sequence in which about 1 to 10, more preferably a number (1 to 5) amino acids have been deleted, and (ii) one or two or more amino acids (for example, 1 to 5) in the amino acid sequence represented by SEQ ID NO: 1. An amino acid sequence to which about 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids have been added; (iii) represented by SEQ ID NO: 1 An amino acid in which one or more (eg, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids have been inserted into the amino acid sequence Array, (iv) 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5) in the amino acid sequence represented by SEQ ID NO: 1 ), Or so-called muteins, such as proteins containing (v) an amino acid sequence obtained by combining (v) an amino acid sequence with another amino acid.
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.

In the protein in this specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) in accordance with the convention of peptide labeling. The protein used in the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1 has a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), and an amide (—CONH ₂ ) at the C-terminus. Alternatively, any of ester (—COOR) may be used.
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl and n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl , C _6-12 aryl groups such as α-naphthyl, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or C _7- alkyl groups such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl. _{14 An} aralkyl group, a pivaloyloxymethyl group and the like are used.
When the protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein used in the present invention includes those in which the carboxyl group is amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Furthermore, the protein used in the present invention, the amino acid residue (eg, methionine residue) of the N-terminal amino group protecting group (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _{1- 6-} acyl group), N-terminal glutamine residue formed by cleavage in vivo, pyroglutamine-oxidized, substituent on the side chain of amino acid in the molecule (for example, -OH,- SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected with a suitable protecting group (eg, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group and acetyl group). Or complex proteins such as so-called glycoproteins to which sugar chains are bound.
Specific examples of the protein used in the present invention include, for example, a protein (EZH2) containing the amino acid sequence represented by SEQ ID NO: 1.

The partial peptide of the protein used in the present invention is a partial peptide of the protein used in the present invention described above, and is preferably any peptide having the same properties as the protein used in the present invention described above. It may be something.
Specifically, for the purpose of preparing the antibody of the present invention described later, a peptide having the 1st to 610th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 and the like can be mentioned. For example, it has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the protein used in the present invention. Peptides and the like are used.
The partial peptide used in the present invention has one or two or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably about 1 to 5) amino acid sequences in the amino acid sequence. Or one or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably a number (1 to 5)) amino acids in the amino acid sequence 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably about 1 to 5) amino acids are added to the amino acid sequence, or In the amino acid sequence, one or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are substituted with another amino acid. hand Good.

The partial peptide used in the present invention may have a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ) or an ester (—COOR) at the C-terminus.
Further, similar to the protein used in the present invention, the partial peptide used in the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, and the N-terminal amino acid residue (eg, , A methionine residue) in which the amino group is protected with a protecting group, a glutamine residue formed by cleavage of the N-terminal side in vivo, and pyroglutamine oxidation, and a substituent on the side chain of the amino acid in the molecule. Also included are those protected with an appropriate protecting group, and complex peptides such as so-called glycopeptides to which sugar chains are bonded.
The partial peptide used in the present invention can also be used as an antigen for preparing an antibody.

As the salt of the protein or partial peptide used in the present invention, a salt with a physiologically acceptable acid (eg, an inorganic acid, an organic acid) or a base (eg, an alkali metal salt) is used. Are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
The protein or its partial peptide or its salt used in the present invention can be produced from the aforementioned human or warm-blooded animal cells or tissues by a known protein purification method, or contains a DNA encoding the protein. It can also be produced by culturing a transformant. Further, it can be produced according to the peptide synthesis method described later.
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.

For the synthesis of the protein or partial peptide or a salt thereof, or an amide thereof used in the present invention, a commercially available resin for protein synthesis can be generally used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. 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 in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. I do.
For the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid was directly added to the resin together with a racemization inhibitor additive (for example, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetric acid anhydride or HOBt ester or HOOBt ester. It can be added later to the resin.

  The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Sulfoxides, 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 a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a 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 even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.

Examples of the protecting group for the amino group of the raw material include, for example, Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonylhydrazide, tritylhydrazide and the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for the esterification include lower (C _1-6 ) alkanoyl groups such as an acetyl group, aroyl groups such as a benzoyl group, and groups derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group. Used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
As the protecting group for the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.
As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.
As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above-mentioned acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol and the like is effective. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. 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.

The protection of the functional group that should not be involved in the reaction of the raw materials, the protective group, the elimination of the protective group, the 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 a partial peptide, for example, after amidating and protecting the α-carboxyl group of the carboxy-terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After the elongation, a protein or partial peptide from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the carboxyl group at the C-terminal has been removed, and these are produced. The protein or peptide is condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. This crude protein or peptide is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein or peptide.
In order to obtain an ester of a protein or peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein or An ester of the peptide can be obtained.

The partial peptide or a salt thereof used in the present invention 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. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the target peptide. it can. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (i) to (v).
(I) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments on Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)
(V) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten After the reaction, conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. Can be used to purify and isolate the partial peptide used in the present invention. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide obtained by a salt is a known method or analogous method It can be converted into a free form or another salt by a method.

The polynucleotide encoding the protein used in the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence encoding the protein used in the present invention. Preferably, it is DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be performed directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells and tissues described above.
The DNA encoding the protein used in the present invention includes, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence hybridized with the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions. Any DNA may be used as long as it contains a soybean base sequence and encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1 described above. Examples of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 include a DNA containing the nucleotide sequence represented by SEQ ID NO: 3.

Examples of DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 50% or more, preferably about 60% or more with the base sequence represented by SEQ ID NO: 2. More preferably, DNA containing a base sequence having a homology of about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used.
The homology of the nucleotide sequences was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gaps allowed; filtering = ON; match score = 1; It can be calculated by mismatch score = -3).
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., 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 conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, a DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used.

The DNA encoding the partial peptide used in the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide used in the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
As the DNA encoding the partial peptide used in the present invention, for example, a 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 a high string DNAs containing a base sequence that hybridizes under gentle conditions and a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention are used.
The DNA hybridizable to the base sequence represented by SEQ ID NO: 2 has the same significance as described above.
The same hybridization method and high stringency conditions as described above are used.

Means for cloning DNA that completely encodes proteins and partial peptides used in the present invention (hereinafter, in the description of cloning and expression of DNAs encoding them, these may be simply abbreviated as the protein of the present invention) Amplification by a PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or encoding a DNA incorporated in an appropriate vector into a part or the entire region of the protein of the present invention. And a DNA fragment labeled with a DNA fragment or a synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., 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.
The DNA base sequence can be converted using PCR, a known kit, for example, Mutan ^™ -super Express Km (Takara Shuzo Co., Ltd.), Mutan ^™ -K (Takara Shuzo Co., Ltd.), etc., using the ODA-LA PCR method. The method can be carried out according to a method known per se, such as the gapped duplex method and the Kunkel method, or a method analogous thereto.
The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
The expression vector for the protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by the following.

Examples of the vector include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.
The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter, etc. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used, if desired. it can. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r), neomycin resistant gene (hereinafter sometimes abbreviated as Neo ^r, G418 resistance). In particular, when the dhfr gene is used as a selection marker using dhfr gene-deficient Chinese hamster cells, the target gene can also be selected using a thymidine-free medium.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is Escherichia, PhoA signal sequence, OmpA signal sequence, etc., when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.
A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12.DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] Are used.
As the Bacillus bacterium, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036P, and so on. Used.

Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of night-moth (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, and High Five ^™ derived from egg of Trichoplusia ni. Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used.
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells and the like are used.
Natl. Acad. Sci. USA, 69, 2110 (1972) and Gene, 17, 107 (1982) can be used to transform Escherichia.

Transformation of Bacillus sp. Can be performed, for example, according to the method described in Molecular & General Genetics, vol. 168, 111 (1979).
The yeast can be transformed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). it can.
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
Transformation of animal cells can be performed, for example, according to the method described in Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can be.
Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.
When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and among them, a carbon source necessary for growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferable. . If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently.
When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed 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 belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
When culturing a transformant in which the host is yeast, for example, as a medium, Burkholder 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., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration or stirring is added.
When culturing a transformant in which the host is an insect cell or an insect, a medium such as 10% bovine serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) is appropriately used. The added one is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration and / or agitation are added.
When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], etc. Is used. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring are added.
As described above, the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.

The protein of the present invention can be separated and purified from the culture by, for example, the following method.
When extracting the protein of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.
The protein contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Using differences in charge, methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, and using differences in hydrophobicity such as reversed-phase high-performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.

When the protein thus obtained is obtained in a free form, it can be converted to 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 analogous thereto Can be converted to a free form or another salt.
The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
The presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody, Western blotting, or the like.

The antibody against the protein or partial peptide or a salt thereof used in the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention.
An antibody against a protein or a partial peptide or a salt thereof (hereinafter, these may be simply referred to as the protein of the present invention) used in the present invention uses the protein of the present invention as an antigen and is known per se. Can be produced according to the method for producing an antibody or antiserum described in (1).
[Preparation of monoclonal antibody]
(A) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal by itself or together with a carrier and a diluent at a site where antibody production is possible by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained therein. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently.
Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed together with a carrier, and then radioactive substances or A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with an enzyme or the like (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A; A method in which a hybridoma culture supernatant is added to a solid phase to which globulin antibodies or protein A is adsorbed, a protein labeled with a radioactive substance, an enzyme, or the like is added, and a monoclonal antibody bound to the solid phase is detected.
Selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of monoclonal antibody Monoclonal antibody can be separated and purified by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchange method Absorption / desorption method using body (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific antibody that collects only antibody by active adsorbent such as protein A or protein G, and dissociates the bond to obtain antibody Purification method].
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immunizing 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-described method for producing a monoclonal antibody. It can be produced by collecting a substance and separating and purifying the antibody.
Regarding the complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by crosslinking the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ~ 5 is used.
Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.
The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.

The nucleotide sequence of a polynucleotide (preferably 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 the antisense polynucleotide) As an antisense polynucleotide having a complementary or substantially complementary base sequence or a part thereof, a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof is used. Any antisense polynucleotide may be used as long as it has a portion and has an action of suppressing the expression of the DNA, but antisense DNA is preferable.
The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. In particular, in the case of an antisense polynucleotide directed to translation inhibition in the entire base sequence of the complementary strand of the DNA of the present invention, the base sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand of a base sequence near a codon, etc.) B) In the case of an antisense polynucleotide which directs RNA degradation by RNase H, it is about 70% or more, preferably about 80% or more, more preferably about 90% with the complementary strand of the entire base sequence of the DNA of the present invention including introns. As described above, most preferably, antisense polynucleotides each having about 95% or more homology are suitable.
Specifically, 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 the 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 an antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 3, or a part thereof.
The antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is replaced with a chemically modified phosphate residue such as phosphorothioate, methylphosphonate or phosphorodithionate. It may be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base portion (pyrimidine, purine) may also be chemically modified. Or any one that hybridizes to the DNA having the base sequence represented by SEQ ID NO: 2. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, the antisense polynucleotide (nucleic acid) corresponding to the protein gene of the present invention, which can inhibit the replication or expression of the gene, is cloned or the base of DNA encoding the determined protein is determined. It can be designed and synthesized based on sequence information. Such antisense polynucleotides can hybridize to the RNA of the protein gene of the present invention and inhibit the synthesis or function of the RNA, or through interaction with the protein-related RNA of the present invention. The expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of the protein-associated RNA of the present invention, and that can specifically hybridize with the protein-associated RNA of the present invention, can be used in vivo and in vitro to produce the protein gene of the present invention. It is useful for regulating and controlling the expression of, and also useful 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. The "correspondence" between a nucleotide, base sequence or nucleic acid and a protein usually refers to the amino acid of the protein (as indicated) 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 start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' Although a terminal palindrome region or a 3′-end hairpin loop can be selected as a preferable target region, any region within a protein gene can be selected as a target.
Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize with the target region, the target nucleic acid is Can be said to be "antisense." Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, non-polynucleotides, Other polymers having a nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymer is composed of bases such as those found in DNA or RNA. Pairing and nucleotides having an arrangement permitting base attachment) are included. They may be double stranded DNA, single stranded DNA, double stranded RNA, single stranded RNA, DNA: RNA hybrids, and may further comprise unmodified polynucleotides (or unmodified oligonucleotides), known modifications. Additions, e.g., those with labels known in the art, capped, methylated, substituted for one or more natural nucleotides with analogs, modified with intramolecular nucleotides Having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), having a charged or sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.) Such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Nal peptides, poly-L-lysine, etc.) or sugars (eg, monosaccharides, etc.) having side chain groups, interactive compounds (eg, acridine, psoralen, etc.), chelating compounds (eg, , Metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) Is also good. Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, or the like, or with functional groups such as ethers, amines, and the like. It may have been converted.
The antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include a sulfur derivative of a nucleic acid, a thiophosphate derivative, a nucleic acid which is resistant to degradation of polynucleoside amide and oligonucleoside amide, and the like. The antisense polynucleotide of the present invention can be designed, for example, as follows. That is, the antisense polynucleotide in a cell is made more stable, the cell permeability of the antisense polynucleotide is increased, the affinity for a target sense strand is increased, and if the toxicity is reduced. In some cases, the toxicity of the antisense polynucleotide is reduced. Many such modifications have been reported, for example, in Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Applications, CRC Press, 1993.
The antisense polynucleotides of the present invention may contain altered or modified sugars, bases, bonds, and may be provided in special forms such as liposomes, microspheres, or applied by gene therapy. , Can be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( Hydrophobic substances such as, for example, phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via bases, sugars, intramolecular nucleoside bonds. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of the antisense polynucleotide can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. .

  Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the protein of the present invention), a polynucleotide (eg, DNA) encoding the protein or partial peptide of the present invention (hereinafter, the present invention) Of the present invention), antibodies against the protein or partial peptide of the present invention or a salt thereof (hereinafter, sometimes abbreviated as the antibodies of the present invention), and antibodies of the polynucleotide (eg, DNA) of the present invention. The use of the sense polynucleotide (hereinafter, sometimes abbreviated as the antisense polynucleotide of the present invention) will be described.

  Since the expression of the protein of the present invention increases 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 symptom severity, and prediction of disease progression. Drugs containing an antisense polynucleotide 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 include, for example, cancer (eg, colorectal cancer , Breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid, pancreas , Ovarian cancer, brain tumor, blood tumor, etc.), preferably for colorectal, breast, lung, pancreatic or ovarian cancer, or for hormone-dependent cancer It can be used as an agent, an apoptosis inducer and the like.

(1) Screening of Candidate Drug Compounds for Diseases The protein of the present invention has increased expression in cancer tissues and has an apoptosis-suppressing action. Further, the antisense polynucleotide of the present invention has a cell death effect on cancer cells, an apoptosis induction / promotion effect, and the like. Therefore, compounds or salts thereof that regulate (enhance or inhibit, preferably inhibit) the activity of the protein of the present invention include, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer) , Liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid, pancreas, ovary, brain, blood, etc.) It can be used as a therapeutic agent, preferably as a preventive / therapeutic agent for colon cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer. It can also be used as an apoptosis inducer. Alternatively, it can be used as a prophylactic / therapeutic agent for hormone-dependent cancer.
Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention.
That is, the present invention provides a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity (eg, histone methyltransferase activity) of the protein of the present invention, characterized by using the protein of the present invention. Is provided.
More specifically, for example, the method is characterized by comparing (i) histone methyltransferase activity of the protein of the present invention and (ii) histone methyltransferase activity of a 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 (eg, histone methyltransferase activity) of the protein of the present invention.
In the above screening method, for example, in the cases of (i) and (ii), histone methyltransferase activity can be determined by a method known per se, for example, the method described in Science, vol. 298, pp. 1039-1043, 2002, or a method similar thereto. Measure according to the method and compare.
Specifically, (i) (a) the protein-EED complex of the present invention, (b) S-adenosyl-L-methionine radioactively labeled with a methyl group and (c) histone protein, oligonucleosome or histone H3 (A) in the presence of a test compound, (a) the protein-EED complex of the present invention, and (b) S-adenosyl radiolabeled with a methyl group in the presence of a test compound. When L-methionine and (c) a histone protein, an oligonucleosome or a polypeptide having a sequence around the lysine 27 of histone H3 are reacted, the radioactivity of histone H3 or polypeptide by methyl group transfer is measured, and the present invention A compound or a salt thereof that modulates (promotes or inhibits, preferably inhibits) the activity of the above protein is screened.
This reaction is performed in an appropriate buffer. After the enzymatic reaction, the reaction product is separated by, for example, SDS-PAGE or the like, and is first compared with the mobility of histone H3 or the like as a standard control and identified. Measurement of radioactivity for quantification is performed according to a known method using a scintillation counter, fluorography, or the like.
A methyl-radiolabeled S-adenosyl-L-methionine, a histone protein, an oligonucleosome or a polypeptide having a sequence around lysine 27 of histone H3 is mixed with a test compound, and then mixed with the protein-EED complex of the present invention. The protein-EED complex of the present invention may be reacted with S-adenosyl-L-methionine radioactively labeled with a methyl group, a histone protein, an oligonucleosome or a polypeptide having a sequence around the lysine 27 of histone H3. After contacting, the test compound may be added.
Further, (i ′) (a) the protein-EED complex of the present invention, (b) S-adenosyl-L-methionine and (c) a histone protein, an oligonucleosome or a polypeptide having a sequence around the lysine 27 of histone H3. (A) the protein-EED complex of the present invention, (b) S-adenosyl-L-methionine and (c) histone protein, oligonucleosome or histone H3 Methylated (monomethylated, dimethylated and / or trimethylated) lysine residue when a polypeptide having a lysine 27 peripheral sequence is reacted with, for example, an anti-Histone H3 (dimethyl / trimethyl lysine 27) antibody or the like. Each is measured, and a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention is screened.
Also, (i ″) (a) the protein-EED complex of the present invention, (b) S-adenosyl-L-methionine and (c) a histone protein, an oligonucleosome or a polypeptide having a sequence around lysine 27 of histone H3 And (ii '') in the presence of a test compound, (a) a protein-EED complex of the present invention, (b) S-adenosyl-L-methionine and (c) a histone protein, an oligonucleosome or When a polypeptide having a sequence around the lysine 27 of histone H3 was reacted, the change in molecular weight of each reaction product (purified as necessary) due to methylation was determined by mass spectrometry (eg, TOF- And the like, and a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention is 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. Furthermore, the same reaction may be carried out using cells capable of expressing the protein of the present invention, and the radioactivity of histone H3 or polypeptide by methyl group transfer may be measured.
As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as COS7 cells, CHO cells, and HEK293 cells are preferably used. For the screening, for example, a transformant that expresses the protein of the present invention by culturing by the method described above is preferably used. The method for culturing cells capable of expressing the protein of the present invention is the same as the above-described method for culturing the transformant of the present invention.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
For example, a test compound that reduces histone methyltransferase activity in the case of the above (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (i), As the compound that inhibits the activity of the protein of the present invention, the histone methyltransferase activity in the case of the above (ii) is about 20% or more, preferably 30% or more, more preferably as compared with the case of the above (i). A test compound that increases about 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 can be used as a safe and low toxic drug for suppressing the physiological activity of the protein of the present invention, for example, cancer (eg, 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, testis cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor , Blood tumors, etc.), preferably as a prophylactic / therapeutic agent for colorectal cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, an apoptosis inducer (of cancer cells), and the like.
The compound having the activity of promoting the activity of the protein of the present invention is useful as a safe and low-toxic drug for enhancing the action of the protein of the present invention.
Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts And compounds selected from plasma and the like. As the salt of the compound, those similar to the aforementioned salts of the peptide of the present invention are used.

Furthermore, the expression of the gene encoding the protein of the present invention also increases in cancer tissues. Further, the antisense polynucleotide of the present invention has a cell death effect on cancer cells, an apoptosis induction / promotion effect, and the like. Therefore, compounds that regulate the expression of the gene encoding the protein of the present invention or salts thereof include, for example, cancers (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, Prophylactic / therapeutic agents for biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc., preferably It can be used as a preventive / therapeutic agent for colorectal cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, an apoptosis inducer, and the like.
Therefore, the DNA of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the expression of a gene encoding the protein of the present invention.
The screening method includes (iii) culturing cells capable of producing the protein of the present invention and (iv) culturing cells capable of producing the protein used in the present invention in the presence of the test compound. A screening method characterized by performing a comparison with the case.
In the above method, the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases (iii) and (iv) is measured and compared.
Examples of the test compound and cells having the ability to produce the protein of the present invention include those described above.
The protein amount is measured by a known method, for example, using an antibody recognizing the protein of the present invention, and measuring the protein present in a cell extract or the like according to a method such as Western analysis or ELISA method or a method analogous thereto. can do.
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 analogous thereto.
For example, a test compound that 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), As a compound that suppresses the expression of the gene encoding the protein, the gene expression level in the case of the above (iv) is about 20% or more, preferably 30% or more, more preferably A test compound that increases 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.
Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention include the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, and plant extracts. A compound selected from animal tissue extract, plasma and the like, or a salt thereof, which regulates (preferably inhibits) the activity of the protein of the present invention (eg, histone methyltransferase activity and the like) or the expression of the gene of the protein. Or a salt thereof.
As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
Compounds or salts thereof that regulate (preferably inhibit) the activity of the protein of the present invention, and compounds or salts thereof that regulate (preferably inhibit) the expression of a gene encoding the protein of the present invention have low toxicity, for example, , Cancer (eg, colon, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis Cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc.), preferably colon, breast, lung, pancreatic or ovarian cancer, or It is useful as a medicament such as a prophylactic / therapeutic agent for hormone-independent cancer and an apoptosis inducer.
When the compound or a salt thereof 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 according to a conventional method.

For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As compositions for parenteral administration, for example, injections, suppositories and the like are used. Injections are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraarticular injection. Dosage forms such as agents. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, or the like is used, and benzyl benzoate, benzyl alcohol, or the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.

The above-mentioned oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like. Usually, each dosage unit dosage form has a dosage of 5 to 500 mg, especially 5 to 100 mg for an injection. Other dosage forms preferably contain 10 to 250 mg of the above antibody.
Each of the above-described compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the antibody.
The preparations obtained in this way are safe and low toxic, for example, human or warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Can be administered orally or parenterally.
The dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, administration route, and the like. For example, it modulates (preferably inhibits) the activity of the protein of the present invention for the purpose of treating breast cancer. When a compound or a salt thereof is administered orally, generally, in an adult (with a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 50 mg per day. About 1.0 to 20 mg is administered. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease and the like. For example, the activity of the protein of the present invention is regulated (preferably, for the purpose of treating breast cancer). When the compound or its salt is administered to an adult (with a body weight of 60 kg) usually in the form of an injection, the compound or its salt is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day. It is convenient to administer a dose, more preferably about 0.1 to 10 mg, to the cancerous lesion by injection. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
In addition, the above-mentioned compound is a known anticancer agent [eg, an alkylating agent (eg, cyclophosphamide, ifosfamide, nimustine, ranimustine, carbone, etc.), an antimetabolite (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, pepromycin, actinomycin) , Plant-derived anticancer agents (eg, vincristine, vinblastine, vindesine, etoposide, camptothecin, irinotecan, etc.), cisplatin, carboplatin, nedaplatin, paclitaxe It can be used in combination docetaxel, and estramustine, etc.]. At this time, the administration timing is not limited, and these may be administered to the administration subject simultaneously or at an interval. The dose can be appropriately selected based on the clinically used dose. The compounding ratio of the compound to the anticancer agent can be appropriately selected according to 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) can specifically recognize the protein of the present invention. Since it can be used, 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
(I) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody; And (ii) simultaneously or sequentially reacting the test solution with the antibody of the present invention insolubilized on a carrier and another antibody of the present invention labeled on a carrier. The present invention provides 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 (ii), it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.

The protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, or the F (ab ') ₂ , Fab' or Fab fraction of the antibody molecule may be used.
The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited. Any measurement method may be used as long as the amount is detected by chemical or physical means and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competition method, immunometric method and sandwich method are suitably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.
As a labeling agent used in a measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a lanthanide element and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, a cyanine fluorescent dye (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)), fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.
In the method for measuring the protein of the present invention by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different binding site to the protein of the present invention is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably An antibody that recognizes, for example, the N-terminal other than the C-terminal is used.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like.
In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. As the first antibody, a soluble antibody is used, and an immobilization method using an immobilized antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction against a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble sediment produced as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.
For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 70 (Immunochemical Techniques (Part C)) 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies). )) (The above is from Academic Press) ) And the like can be referred to.
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, cancer (eg, colon cancer, breast cancer , Lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid, pancreas, ovary Cancer, brain tumor, blood tumor, etc.), preferably colon cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, or can be diagnosed as having a high possibility of suffering 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 a body fluid or a tissue. Further, 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 at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.

(3) Gene Diagnostics The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog) , Monkeys, chimpanzees, etc.) can detect abnormalities (genetic abnormalities) in DNA or mRNA encoding the protein of the present invention or partial peptides thereof, for example, damage, mutation or reduced expression of the DNA or mRNA, It is useful as a diagnostic agent for a gene such as an increase or an overexpression of the DNA or mRNA.
The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879, 1989, Proceedings of the National Academy of Sciences of the United States). States of America, 86, 2766-2770, 1989).
For example, when overexpression or decrease is detected by Northern hybridization or when DNA mutation is detected by PCR-SSCP method, for example, cancer (eg, colorectal cancer, breast cancer, lung cancer, prostate cancer, Esophageal, gastric, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid, pancreas, ovary, brain, blood Etc.), preferably colon cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer.

(4) Pharmaceuticals Containing Antisense Polynucleotide The antisense polynucleotide of the present invention, which can complementarily bind to the DNA of the present invention and suppress the expression of the DNA, has a cell death effect on cancer cells and apoptosis. It has an inducing / promoting action, is low toxic, and can suppress the function of the protein of the present invention or the DNA of the present invention in vivo (eg, histone methyltransferase activity). (Eg, colorectal, breast, lung, prostate, esophageal, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid Cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc.), preferably preventive / therapeutic agent for colon, breast, lung, pancreatic or ovarian cancer, apoptosis inducer, etc. As Can be used. Further, it can be used as a prophylactic / therapeutic agent for hormone-independent cancer.
When the antisense polynucleotide is used as the prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se.
Also, for example, after inserting the antisense polynucleotide alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, a human or mammal (eg, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, and the like). The antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered using 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 the efficiency of intracellular uptake, the antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection), and is intravenously, subcutaneously, or joint cavity. In addition, it may be administered to a cancer lesion.
The dosage of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the antisense polynucleotide of the present invention is administered for the purpose of treating breast cancer, it is generally used in adults ( (Body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.
Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of expression thereof.
Similarly to the above-mentioned antisense polynucleotide, a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a ribozyme containing a part of the RNA encoding the protein of the present invention, and the like also include 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 in a living body can be suppressed, for example, cancer (eg, colon cancer, breast cancer, Lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid, pancreas, ovary Cancer, brain tumor, blood tumor, etc.), preferably as a prophylactic / therapeutic agent for colon cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, apoptosis inducer, etc. .
The double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
A ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known ribozyme to a part of RNA encoding the protein of the present invention. As a part of the RNA encoding the protein of the present invention, a portion (RNA fragment) close to a cleavage site on the RNA of the present invention, which can be cleaved by a known ribozyme, can be mentioned.
When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.

(5) Pharmaceutical Containing Antibody of the Present Invention The antibody of the present invention, which has the activity of neutralizing the activity of the protein of the present invention, is used for, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus) Cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc. ), Preferably a prophylactic / therapeutic agent for colon cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, or an apoptosis inducer. Further, it can be used as a prophylactic / therapeutic agent for hormone-independent cancer.
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rat, rabbit, sheep, pig, It can be administered orally or parenterally (eg, intravenously) to cattle, cats, dogs, monkeys, and the like. The dose varies depending on the administration subject, target disease, symptoms, administration route, and the like. For example, when used for the treatment or prevention of adult breast cancer, the antibody of the present invention is usually used in a dose of 0 to 1 dose. About 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 to 3 times a day It is convenient to administer about once. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration (eg, intravenous injection). It is preferably provided as an inhalant.
Each of the above-described compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the antibody.

(6) “a compound having an inhibitory activity on histone methyltransferase activity or an agent for preventing or treating cancer comprising a salt thereof” of the present invention, “a compound having an inhibitory activity on histone methyltransferase expression or a compound thereof” A cancer preventive / therapeutic agent containing a salt, an apoptosis-inducing agent containing a compound having a histone methyltransferase activity inhibitory activity or a salt thereof, and an inhibitor of histone methyltransferase expression "Apoptosis-inducing agent comprising a compound having an action or a salt thereof" Examples of the histone methyltransferase include enzymes that transfer a methyl group to the ninth and / or 27th lysine residue of histone H3. No.
The “compound having histone methyltransferase activity inhibitory activity” may be any compound as long as it has a histone methyltransferase activity inhibitory effect. Examples thereof include cancer (eg, 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, testis cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor , Blood tumors, etc.), preferably as a prophylactic / therapeutic agent for colon cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, an apoptosis inducer, and the like.
The “compound having an inhibitory effect on histone methyltransferase expression” may be any compound as long as it is a compound having an inhibitory effect on histone methyltransferase expression, and examples thereof include cancer (eg, 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, testis cancer, thyroid cancer, pancreatic cancer, ovarian cancer , Brain tumors, blood tumors, etc.), preferably as a prophylactic / therapeutic agent for colon cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, an apoptosis inducer, and the like.
The prophylactic / therapeutic agent and the inducer are produced in the same manner as described above.

(7) DNA-transferred animal The present invention relates to a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (the abbreviated as the exogenous mutant DNA of the present invention) Is provided.
That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) The 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 and capable of being expressed in a mammal. Things.
Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by transferring a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. In addition, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like by the DNA transfer method, and can be used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of the preparation of diseased animal model systems, and are easy to breed, particularly mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) As a system, _one B6C3F system, _one BDF system, _one B6D2F system, a BALB / c system, an ICR system, etc., or a rat (for example, Wistar, SD, etc.) is preferable.
"Mammals" in a 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 refers not to the DNA of the present invention originally possessed by non-human mammals, but to the DNA of the present invention once isolated and extracted from the mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA and the like are used, and also include abnormal DNA.
The abnormal DNA means a DNA that expresses an 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 may be derived from a mammal that is the same or different from the animal of interest. 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 capable of being expressed in animal cells. For example, when the human DNA of the present invention is transferred, DNA derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto is expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, a vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-transferring mammals can be created.

Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
Examples of the promoter for controlling the DNA expression include, for example, (i) a promoter of a DNA derived from a virus (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acid Protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen types I and II, cyclic AMP-dependent protein kinase βI subunit, dystrophy Fin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), 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-1α), β actin , Α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α Aku Down, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoters and the like are preferable.
The vector preferably has a sequence that terminates transcription of the target messenger RNA in a DNA-transferred mammal (generally called a terminator). For example, a sequence of each DNA derived from a virus and various mammals can be used. A simian virus SV40 terminator or the like is preferably used.

In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region or in the translation region for the purpose of further expressing the desired foreign DNA. It is also possible to connect 3 ′ downstream of the above depending on the purpose.
The normal translation region of the protein of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From the various genomic DNA libraries described above, or as a starting material, a complementary DNA prepared by a known method from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above-described cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA.
The translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which the translation region is ligated downstream of the promoter and, if desired, upstream of the transcription termination site.
Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal cells and somatic cells. means. The progeny of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as a DNA-carrying animal by confirming that the exogenous DNA is stably maintained by mating. .
Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage 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 germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess.

The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level and promotes the function of the endogenous normal DNA to eventually cause hyperactivity of the protein of the present invention. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.
In addition, since a mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the free protein of the present invention, a prophylactic / therapeutic agent for a disease associated with the protein of the present invention, for example, cancer ( For example, colon, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid Cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc.), preferably screening for colorectal, breast, lung, pancreatic or ovarian cancer, and apoptosis inducer It can also be used for testing.
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in an ordinary breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. Transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The offspring of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed the cells so that all offspring have the DNA.

The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of the endogenous normal DNA, and finally the functionally inactive form of the protein of the present invention. It can be refractory and can be used as a model animal for the disease. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
Also, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention can inhibit the abnormal protein of the present invention (dominant negative action) by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention. A model to elucidate.
In addition, since the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the free protein of the present invention, a prophylactic / therapeutic agent for the protein of the present invention or a functionally inactive refractory disease, for example, (E.g., colon, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, Thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc.), preferably colorectal cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer, and apoptosis inducer It can also be used for screening tests.
In addition, other possible uses of the above two kinds of DNA transgenic animals of the present invention include, for example,
(I) use as a cell source for tissue culture,
(Ii) a peptide specifically expressed or activated by the protein of the present invention, by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or analyzing a peptide tissue expressed by the DNA; Analysis of the relationship with
(Iii) culturing cells of DNA-bearing tissue by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture;
(Iv) Screening of a drug that enhances the function of the cell by using the cell described in (iii) above, and (v) Isolation and purification of the mutant protein of the present invention and production of an antibody thereof are conceivable.

Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention. More detailed pathological findings in each organ of a disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.
In addition, 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 the released DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to identify the protein-producing cell of the present invention, examine its relevance to apoptosis, differentiation or proliferation, or investigate the signal transduction mechanism thereof, and investigate their abnormalities. It is an effective research material for
Further, in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, using the DNA-transferred animal of the present invention, It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using a method or the like. In addition, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for diseases associated with the protein of the present invention.

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

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA which is artificially mutated to the DNA of the present invention possessed by the non-human mammal, thereby suppressing the expression ability of the DNA, or By substantially eliminating the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter sometimes referred to as the knockout DNA of the present invention). ) Non-human mammal embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same one as described above is used.
The method of artificially mutating the DNA of the present invention can be carried out, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene represented by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); Inability to synthesize complete messenger RNA Thus, a DNA strand having a DNA sequence constructed so as to eventually destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cells PCR using a DNA sequence on or near the DNA of the present invention as a probe and a DNA sequence on a targeting vector and a DNA sequence in a neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as a primer And can be obtained by selecting the knockout ES cells of the present invention.
As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known Evans and Kaufma method. It may be a newly established one. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. for example the purpose of acquiring the obvious ES cells, the BDF ₁ mice (C57BL / 6 and DBA / 2 for the egg collection number of lack of C57BL / 6 mice and C57BL / 6 were improved by crossing with DBA / 2 And those established using F ₁ ) can also be used favorably. BDF ₁ mice are often egg number, and, in addition to the advantage that the egg is tough, since with C57BL / 6 mice in the background, when the ES cells obtained therefrom, which were generated pathological model mice , C57BL / 6 mice can be advantageously used in that their genetic background can be replaced with C57BL / 6 mice by backcrossing.
In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. However, a large number of blastocysts can be efficiently obtained by collecting embryos at the 8-cell stage and culturing them up to blastocysts. Early embryos can be obtained.
Although either male or female ES cells may be used, male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.

As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a 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 The primary selection of ES cells in the early stage can be performed by discriminating between male and female, and the early stage of culture can be greatly reduced by enabling the selection of male cells at an early stage.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the normal cells (for example, chromosomes in mice) are knocked out. It is desirable to clone again into cells (number 2n = 40).
The embryonic stem cell line obtained in this way usually has very good growth properties, but it must be carefully subcultured because it tends to lose its ontogenic ability. For example, on a suitable feeder cell such as STO fibroblast, in the presence of LIF (1 to 10,000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, Preferably, a method is used in which cells are made into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on newly prepared feeder cells. Such passage is usually carried out every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985]. The DNA-deficient cells of the present invention obtained by differentiation are useful in in vitro cell biology studies of the proteins of the present invention.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
As the non-human mammal, those similar to the aforementioned can be used.
The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into a mouse embryonic stem cell or a mouse egg cell, and the DNA of the targeting vector of the present invention is inactivated by the introduction. 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 gene homologous recombination.
Cells in which the DNA of the present invention has been knocked out can be used as 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, and the DNA of the present invention derived from the mouse used for the targeting vector. The determination can be made by analysis by a PCR method using a DNA sequence of a nearby region other than DNA as a primer. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is cultured at an appropriate time, for example, at the 8-cell stage of a non-human mammalian stem cell. The chimeric embryo is injected into an animal embryo or a 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 the tissues are artificially mutated from the population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individual thus obtained is usually an individual with a heterozygous expression of the protein of the present invention, which is crossed with an individual with a heterozygous expression of the protein of the present invention, and homozygous expression of the protein of the present invention from their offspring. Defective individuals can be obtained.

When an egg cell is used, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method, and these transgenic non-human mammals can be obtained. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
In this manner, the individual in which the DNA of the present invention has been knocked out can be bred in an ordinary breeding environment after confirming that the DNA has been knocked out in the animal individual obtained by mating.
Furthermore, the germline can be obtained and maintained in accordance with a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.
In addition, since 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, a model of a disease 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 treatment methods.

(8a) Screening method for compounds having therapeutic / preventive effects against diseases caused by DNA deficiency or damage of the present invention Non-human mammals deficient in DNA expression of the present invention are deficient or damaged of the DNA of the present invention Can be used for screening for a compound having a therapeutic / preventive effect on diseases caused by the above.
That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / preventive effect on a disease, for example, cancer.
Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same ones as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like. These compounds are novel compounds. Or a known compound.
Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and tested using the change in each organ, tissue, disease symptoms, etc. of the animal as an index. Compounds can be tested for their therapeutic and prophylactic effects.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and 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 can be appropriately selected according to the administration method, the properties of the test compound, and the like.

For example, cancer (eg, 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, testis Cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc.), when screening for a compound having a therapeutic / preventive effect, a test compound is used in a non-human mammal deficient in expressing the DNA of the present invention. After the administration, the difference in the degree of onset of cancer and the degree of healing of the cancer from the group not administered with the test compound are observed over time in the above tissue.
In the screening method, when a test compound is administered to a test animal, the disease symptom of the test animal is 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 / preventive effect on the above diseases.
The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect on a disease caused by deficiency or damage of the protein of the present invention. It can be used as a drug for safe and low toxic prophylactic / therapeutic agents. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). ) Is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.
The preparation obtained in this way is safe and low toxic, and thus can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered, in general, an adult (as a body weight of 60 kg) 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 is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound is usually administered in the form of an injection to an adult (60 kg) breast cancer patient. In such a case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound by intravenous injection per day. In the case of other animals, the amount can be administered in terms of 60 kg.

(8b) Screening method for compound that promotes or inhibits the activity of a promoter for DNA of the present invention The present invention comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and detecting the expression of a reporter gene. The present invention provides a method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for DNA of the present invention.
In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expression of DNA of the present invention in which 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 compounds as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, a tissue that expresses the protein of the present invention should be used instead of the protein of the present invention. Β-galactosidase is expressed. Therefore, for example, the present invention can be easily performed by staining with a reagent serving as a substrate for β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal). Can be observed in the animal body. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.
The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt, and the salt of the compound may be a salt with a physiologically acceptable acid (eg, an inorganic acid) or a base (eg, an alkali metal). Are used, and especially preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.

The compound or its salt that promotes or inhibits the promoter activity of the DNA of the present invention can regulate the expression of the protein of the present invention and regulate the function of the protein. Breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid, pancreas, It is useful as a prophylactic / therapeutic agent for ovarian cancer, brain tumor, blood tumor, etc .;
Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
A drug containing the compound or a salt thereof 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 a salt thereof.
The preparation obtained in this way is safe and low toxic, and thus can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the subject to be administered, the administration route, and the like. For breast cancer patients (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. When administered to a breast cancer patient (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg of the compound per day is intravenously injected. It is convenient to administer. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to the investigation of the cause of various diseases caused or the development of preventive / therapeutic agents.
Further, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene-transferred animal). Once created, it will also be possible to specifically synthesize the protein and study its effects in living organisms. Furthermore, if a suitable reporter gene is bound to the above promoter portion, and a cell line that expresses the reporter gene is established, a low-molecular compound having an action of specifically promoting or suppressing the production ability of the protein itself of the present invention in the body. Can be used as a search system.

In the present specification, when bases, amino acids, and the like are indicated by abbreviations, they are based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated 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 triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threin Crythine : 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)

Further, substituents, protecting groups and reagents frequently 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: dinitro Phenyl 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-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
2 shows the amino acid sequence of EZH2.
[SEQ ID NO: 2]
2 shows the nucleotide sequence of DNA encoding EZH2.
[SEQ ID NO: 3]
2 shows the nucleotide sequence of DNA containing the full length gene encoding EZH2.
[SEQ ID NO: 4]
3 shows the base sequence of a primer used in Example 2.
[SEQ ID NO: 5]
3 shows the base sequence of a primer used in Example 2.
[SEQ ID NO: 6]
The base sequences of the primers used in Examples 2, 4 and 5 are shown.
[SEQ ID NO: 7]
The base sequences of the primers used in Examples 2, 4 and 5 are shown.
[SEQ ID NO: 8]
7 shows the base sequences of the probes used in Examples 2, 4, and 5.
[SEQ ID NO: 9]
4 shows the base sequence of the antisense oligonucleotide used in Example 3.
[SEQ ID NO: 10]
4 shows the base sequence of a control oligonucleotide used in Example 3.
[SEQ ID NO: 11]
14 shows the nucleotide sequence of the siRNA antisense strand of EZH2 used in Example 5.
[SEQ ID NO: 12]
14 shows the base sequence of the siRNA sense strand of EZH2 used in Example 5.
[SEQ ID NO: 13]
14 shows the base sequence of siRNA used in Example 5.
[SEQ ID NO: 14]
14 shows the base sequence of siRNA used in Example 5.

Hereinafter, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto.
Example 1
To identify genes that are specifically upregulated in breast cancer tissues, they were extracted from three normal breast tissues, four breast cancer tissues, two lung normal tissues, two lung cancer tissues, and 23 other normal tissues. Using total RNA (Tables 1 and 2) as a material, gene expression analysis was performed using an oligonucleotide microarray (Human Genome U95A, U95B, U95C, U95D, U95E; Affymetrix).
The experimental method was in accordance with the experimental manual (Expression analysis technical manual) of Affymetrix. As a result, enhanced expression of EZH2 was detected in breast and lung cancer tissues. In the analyzed normal tissues, expression was below the detection limit [determined as Absent by GeneChip analysis software (Affymetrix)] except for the rectum, cerebellum and testis (Tables 1 and 2).

表中の遺伝子発現量比は、以下の式で算出された数値を表す。
遺伝子発現量比＝（がん組織由来cDNA 1μl当たりのEZH2コピー数）／（周辺正常組織由来cDNA 1μl当たりのEZH2コピー数） Example 2
(1) The EZH2 full-length gene was cloned.
Using two primers (SEQ ID NO: 4 and SEQ ID NO: 5), PCR was performed using Marathon ready cDNA library (Clontech) as a template and Pfu polymerase (Straragene). The PCR was performed in a 20 μl reaction solution containing 10 μl of 2xGC buffer I (manufactured by Takara), 2.5 μm each dNTP mixture 1.6 μl, each of the above two primers prepared at 20 μM, 0.4 μl, template cDNA solution 0.5 μl, and 0.4 μl of Pfu polymerase. After a pretreatment at 96 ° C. for 2 minutes, 35 cycles of a reaction at 94 ° C. for 10 seconds, 61 ° C. for 15 seconds, and 72 ° C. for 5 minutes were performed. After the PCR reaction, the product was separated by agarose gel electrophoresis, the target band was cut out, and the reaction product was purified using a GelExtraction kit (manufactured by Qiagen). Thereafter, A was added to both ends of the reaction product based on the protocol attached to pcDNA3.1 / V5 His TA Expression vector (manufactured by Invitrogen) and cloned into the vector. The nucleotide sequence of the inserted fragment was confirmed, and it was confirmed that there was no error in the sequence. Thus, an EZH2 expression vector (vector 1) was obtained.
(2) It was decided to confirm the enhanced expression of EZH2 in various cancer types using a quantitative PCR method.
Using two primers (SEQ ID NO: 6 and SEQ ID NO: 7) and a TaqMan probe (SEQ ID NO: 8), quantitative PCR was performed using Matched cDNA Pairs (Clontech) as a template (Table 3). Using TaqMan Universal PCR Master Mix (manufactured by Applied Biosystems), prepare a 15 μl reaction solution to a final primer concentration of 500 nM, a final probe concentration of 100 nM, and 1 μl of template cDNA, and prepare an ABI PRISM ^™ 7900HT Sequence Detector (Applied Biosystems). Default reaction conditions). Further, the vector 1 obtained in the above (1) was subjected to a constant copy number PCR, and the copy number of EZH2 per 1 μl of the matched cDNA pair was determined. The ratio of the expression amount of EZH2 per 1 μl of cDNA derived from cancer tissue and the ratio of the expression amount of EZH2 per 1 μl of cDNA derived from surrounding normal tissues forming a pair were determined, and the results are shown in Table 3.
In 3 out of 4 breast cancers, 2 out of 3 colorectal cancers, and 4 out of 4 ovarian cancers, EZH2 expression was found to be higher than in surrounding normal tissues.

The gene expression ratio in the table represents a numerical value calculated by the following equation.
Gene expression ratio = (number of EZH2 copies per μl of cDNA derived from cancer tissue) / (number of EZH2 copies per 1 μl of cDNA derived from surrounding normal tissue)

Example 3
EZH2 antisense oligonucleotide transfection experiments were performed to analyze the effect of EZH2 gene, which was upregulated in various cancer types, on apoptosis.
First, after designing an antisense (SEQ ID NO: 9) to the base sequence represented by SEQ ID NO: 3, a phosphorothioated oligonucleotide was synthesized, purified by HPLC, and used for an introduction experiment (Amersham Pharmacia Biotech) (hereinafter, referred to as “A”). Abbreviated as antisense oligonucleotide). As a control oligonucleotide, the reverse sequence (SEQ ID NO: 10) of the nucleotide sequence represented by SEQ ID NO: 9 was similarly phosphorothioated, purified by HPLC, and used (Amersham Pharmacia Biotech).
As a test cell, a breast cancer cell line MDA-MB-231 (In Vitro, vol. 14 (11), 911-915, purchased from ATCC, 1978) was used, and 1 × 10 ⁵ cells were transferred 24 days before the introduction of the oligonucleotide. The cells were seeded in a well plate (Falcon). Oligonucleotides were introduced using Lipofectamine2000 (manufactured by Invitrogen) and the protocol was followed. 15 hours after introduction, RNA was extracted according to the protocol of RNeasy mini kit (manufactured by Qiagen), and cDNA was prepared using TaqMan Reverse Transcription Reagents (manufactured by Applied Biosystems). Quantitative PCR was carried out in the same manner as in Example 2 using ABI PRISM ^™ 7900HT Sequence Detector (Applied Biosystems) using two types of primers (SEQ ID NO: 6 and SEQ ID NO: 7) and a probe (SEQ ID NO: 8). I went.
On the other hand, the effect on apoptosis was determined by seeding 7 × 10 ³ cells on the day before oligonucleotide introduction into a 96-well plate (Falcon) and using Lipofectamine2000 (Invitrogen) in the same manner as described above. On day 3 after the introduction of the sense oligonucleotide, a comparison was made with the control oligonucleotide-introduced sample using Cell death detection ELISA (Roche).
As a result, at 7 hours after introduction of the antisense oligonucleotide, the expression (RNA) of EZH2 was reduced to 63% as compared with that at the time of introduction of the control oligonucleotide (100%). (174%) compared to 100%).
Thus, EZH2 is not only upregulated in colon cancer, breast cancer, lung cancer, pancreatic cancer, and ovarian cancer, but also involved in the growth and apoptosis of cancer cells such as MDA-MB-231. It became clear that.

表中の遺伝子発現量比は、以下の式で算出された数値を記載した。
（がん細胞由来cDNA 1μl当たりのEZH2コピー数）／（がん細胞由来由来cDNA 1μl当たりのβActinコピー数） Example 4
The expression levels of EZH2 in various cancer cell lines were compared. The cell lines shown in Table 4 were used. After culturing at 37 ° C., RNA was extracted according to the protocol of RNeasy mini kit (Qiagen), and cDNA was prepared using TaqMan Reverse Transcription Reagents (Applied Biosystems). did. Quantitative PCR was performed in the same manner as in Example 2 using ABI PRISM ^™ 7900HT Sequence Detector (Applied Biosystems) using two types of primers (SEQ ID NO: 6 and SEQ ID NO: 7) and a probe (SEQ ID NO: 8). I went. To correct the expression level between cells, the amount of β-Actin RNA was quantified using a Human ACTB TaqMan MGB probe (manufactured by Applied Biosystems).
As a result, the expression of EZH in normal mammary epithelial cells HMEC and MCF10A, which is relatively close to normal cells, is low, and hormone-independent breast cancer strain MDA-MB-231 and various lung cancers (eg, NCI-H1299 etc.) ), High expression in colorectal cancer (eg, RKO).
When the results of Example 3 are combined, EZH2 is widely expressed in hormone-independent cancers, and it is expected that there are many types of cancer cells that cause apoptosis by suppressing their functions.

In the table, the gene expression ratio is a numerical value calculated by the following equation.
(Number of EZH2 copies per μl of cancer cell-derived cDNA) / (number of βActin copies per μl of cancer cell-derived cDNA)

Example 5
Example 4 also revealed that EZH2 expression was enhanced in colorectal cancer and lung cancer. Therefore, it was examined whether suppression of the function of EZH2 in these cell lines would induce apoptosis.
After designing a double-stranded RNA (hereinafter abbreviated as siRNA) for the base sequence represented by SEQ ID NO: 3 (SEQ ID NO: 11 and SEQ ID NO: 12), a synthetic product (Qiagen) was used for an introduction experiment. Provided. As the control siRNA, a commercially available Control (non-silencing) siRNA (SEQ ID NO: 13 and SEQ ID NO: 14) (manufactured by QIAGEN) was used.
As test cells used below, colon cancer cell line HCT116 (purchased from ATCC) and lung cancer cell line NCI-H1299 (purchased from ATCC) were used. 48 hours before the introduction of the siRNA, 5 × 10 ⁵ cells were seeded on a 24-well plate (Falcon). When introducing these siRNAs, Lipofectamine2000 (manufactured by Invitrogen) was used and the protocol was followed. Final siRNA concentration was performed at 45 nM. 36 hours after introduction, RNA was extracted according to the protocol of RNeasy mini kit (manufactured by Qiagen), and cDNA was prepared using TaqMan Reverse Transcription Reagents (manufactured by Applied Biosystems). Quantitative PCR was performed using ABI PRISM ^™ 7900HT Sequence Detector (Applied Biosystems) using two types of primers (SEQ ID NO: 6 and SEQ ID NO: 7) and a probe (SEQ ID NO: 8) in the same manner as in Example 4. I went to.
On the other hand, in order to see the effect on apoptosis, 1 × 10 ³ cells were seeded in a 96-well plate (manufactured by Falcon), and 48 hours later, Lipofectamine2000 (manufactured by Invitrogen) was used, and siRNA or EZH2 was used. Control siRNA was introduced. On the third day after the introduction of the siRNA, the Caspase 3/7 activity, which is an indicator of apoptosis, was compared using the Caspase Glo-3 / 7 Assay (Promega).
As a result, the expression (RNA) of EZH2 was reduced to 13% for NCI-H1299 and to 50% for HCT116 at 36 hours after the introduction of siRNA to EZH2, compared to the control siRNA (100%). On the third day after siRNA introduction into EZH2, apoptosis was increased to 143% in NCI-H1299 and 185% in HCT116, as compared to the control (100%).
Thus, it is clear that EZH2 is not only upregulated in colon cancer, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, etc., but also involved in the proliferation and apoptosis of various cancer cells. became.

Example 6
(1) Preparation of recombinant EZH2 protein
A recombinant EZH2 protein is prepared to obtain an enzyme inhibitory compound of the EZH2 protein.
PCR is performed using the full-length EZH2 gene obtained in Example 2 as a template, or an appropriate restriction enzyme is selected to cut out the EZH2 gene and insert it into a vector having an appropriate tag. When the expression cell is a mammal, for example, pFLAGCMV4 (SIGMA) having a FLAG tag is used as the vector. Expression is performed by transfecting a cell line such as COS7 using Fugene6 (Roche) or the like, and protein purification is performed 2 to 3 days later. A nuclear fraction is obtained using an NE-PER kit (Pierce) and purified using M2-Agarose and FLAG peptide (both from SIGMA) to obtain a FLAG-fused EZH2 protein.
(2) Enzyme reaction 50 μl of a reaction buffer containing the recombinant EZH2 protein obtained in the above (1) [50 mM Tris-HCl (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 are mixed on a streptavidin-coated 96-well plate (manufactured by Perkin-Elmer) and left at 37 ° C. for 10 minutes. Next, the reaction is started by adding and mixing 1 ng of biotin-labeled histone H3 peptide (manufactured by Upstate) and [ ³ H] S-adenosylmethionine (manufactured by Amersham) (0.5 μCi). After 3 hours of reaction at 37 ° C., the reaction solution was discarded, washed three times with a washing buffer [phosphate buffer containing 0.05% Tween-20], and 100 μl of a liquid scintillator (manufactured by Wako Pure Chemical Industries, Ltd.) was added. The radioactivity is measured by a liquid scintillation counter (Wallac). The activity when a DMF solution containing no test compound is added is defined as 100, and the 50% inhibitory concentration (IC ₅₀ value) is determined. The compound providing lower an IC ₅₀ value, is selected as a compound that inhibits stronger activity of EZH2 protein.

Claims (27)

Prevention and prevention of cancer comprising a compound or a salt thereof which inhibits the activity of a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Therapeutic agent. Cancer containing a compound or a salt thereof that inhibits the expression of a protein or a partial peptide thereof or a gene thereof which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 Prophylactic and therapeutic agents. A nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of a polynucleotide 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, or one thereof Antisense polynucleotide containing a part. An agent for preventing or treating cancer comprising the antisense polynucleotide according to claim 3. A prophylactic / therapeutic agent for cancer comprising 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. The prophylactic / therapeutic agent according to claim 1, 2, 4, or 5, wherein the cancer is colon cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer. A diagnostic agent for 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. A diagnostic agent for cancer, comprising a polynucleotide 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 diagnostic agent according to claim 7, wherein the cancer is colorectal cancer, breast cancer, lung cancer, pancreatic cancer or ovarian cancer. A prophylactic / therapeutic agent for cancer comprising a compound having an enzymatic activity inhibitory effect of transferring a methyl group to the ninth and / or 27th lysine residue of histone H3 or a salt thereof. An apoptosis-inducing agent comprising a compound having an inhibitory activity on an enzyme activity for transferring a methyl group to the ninth and / or 27th lysine residue of histone H3 or a salt thereof. A prophylactic / therapeutic agent for cancer comprising a compound having an inhibitory effect on the expression of an enzyme that transfers a methyl group to the 9th and / or 27th lysine residue of histone H3, or a salt thereof. An apoptosis-inducing agent comprising a compound having an enzyme-inhibiting effect of transferring a methyl group to the ninth and / or 27th lysine residue of histone H3 or a salt thereof. A method for screening for a prophylactic / therapeutic agent for cancer, which comprises using a protein having 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. A kit for screening for a prophylactic / therapeutic agent for cancer, comprising 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. . A method for screening a prophylactic / therapeutic agent for cancer, comprising using a polynucleotide 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. For screening a prophylactic / therapeutic agent for cancer, comprising a polynucleotide 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. kit. An apoptosis-inducing agent comprising a compound having 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 compound inhibiting the activity of a salt thereof, or a salt thereof. An apoptosis-inducing agent comprising a compound having 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 compound inhibiting the gene expression of a salt thereof, or a salt thereof . A method for screening an apoptosis-inducing agent, which comprises using a protein having 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. A method for screening an apoptosis-inducing agent, comprising 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. For mammals, (i) a compound or a salt thereof that inhibits the activity of 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, ii) a compound or a salt thereof that inhibits the expression of the gene of the protein or its partial peptide or its salt, (iii) an antibody against the protein or its partial peptide or its salt, or (iv) an encoding of the protein or its partial peptide. A method for preventing or treating cancer, which comprises administering an effective amount of an antisense polynucleotide containing a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of a polynucleotide or a part thereof. For mammals, (i) a compound or a salt thereof that inhibits the activity of 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, ii) a compound or a salt thereof that inhibits the expression of the gene of the protein or its partial peptide or its salt, (iii) an antibody against the protein or its partial peptide or its salt, or (iv) an encoding of the protein or its partial peptide. A method for inducing apoptosis, which comprises administering an effective amount of an antisense polynucleotide containing a base sequence complementary or substantially complementary to a base sequence of a polynucleotide to be treated or a part thereof. Inhibits the activity of 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, or expresses a gene of the protein or a partial peptide thereof or a salt thereof A method for preventing and treating cancer characterized by inhibiting cancer. Inhibits the activity of 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, or expresses a gene of the protein or a partial peptide thereof or a salt thereof A method for inducing apoptosis, characterized by inhibiting the following. (I) inhibits the activity of a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof for producing a prophylactic / therapeutic agent for cancer (Ii) a compound or a salt thereof that inhibits the gene expression of the protein or its partial peptide or its salt, (iii) an antibody against the protein or its partial peptide or its salt, or (iv) an antibody against the protein or its partial peptide or its salt. Use of an antisense polynucleotide containing a base sequence complementary to or substantially complementary to a base sequence of a polynucleotide encoding a protein or a partial peptide thereof, or a part thereof. (I) a compound that inhibits the activity of 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, for producing an apoptosis inducer; A salt, (ii) a compound or a salt thereof that inhibits expression of the gene of the protein or its partial peptide or a salt thereof, (iii) an antibody against the protein or its partial peptide or a salt thereof, or (iv) an antibody to the protein or its portion Use of an antisense polynucleotide containing a nucleotide sequence complementary or substantially complementary to a nucleotide sequence of a polynucleotide encoding a peptide or a part thereof.