JP2004298175A - New protein and its dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an excellent preventive/treating agent for cancers and provide a method for screening the same. <P>SOLUTION: A protein or a polynucleotide is useful as a diagnosis marker and the like for diagnosing cancers, neurodisgenerative disorders, autoimmune diseases and the like. A compound inhibiting activity of the protein, or the protein obtained by the method for screening the cancer using the polynucleotide or a compound inhibiting gene expression of the protein is used as a preventive/curing agent for cancers and an apotosis promoter. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for screening a novel protein, its DNA, a compound that regulates the expression of the protein gene, a compound obtained by the screening method, a medicament containing the compound (eg, an agent for preventing or treating cancer, an agent for promoting apoptosis) Etc.).

  Recent research has demonstrated that cancer is a genetic disease in which multiple genetic mutations accumulate in somatic cells, resulting in the inability to control cell growth. Of these, p53 is the most frequently detected tumor suppressor gene in human cancer, and has various physiological functions such as induction of G1 arrest, apoptosis, and checkpoint mechanism when DNA is damaged. It has been revealed that it has. It is believed that these functions are exerted by the p53 protein acting as a transcription factor and controlling the expression of its target gene. Therefore, when an abnormality occurs in this p53 (mutant p53), the above-mentioned action is not exhibited, and the cell is considered to progress to cancer. On the other hand, when mutant p53 is introduced into cancer cells lacking p53, the cancer cells will form colonies even in soft agar, or will form tumors in a bile cancer nude mouse model (Nature Genetics, 4 , P. 42-46, 1993), mutant p53 not only loses the function of normal p53 (wild-type p53), but mutant p53 itself actively promotes canceration. It is suggested that they are involved. Mutant p53 is classified into several types depending on the location of the mutation. It is reported that cancer with mutant p53 in which arginine (R) at position 175 is replaced with histidine (H) has the worst prognosis. (Cancer Research, Vol. 55, pp. 5217-5221, 1995). Furthermore, it has been reported that this mutant p53 induces downstream genes as well as wild-type p53 (Oncogene, 12, 1941-1952, 1996). Among genes induced by mutant p53, genes related to the control of cell proliferation, drug resistance, and apoptosis have been reported (Gene, 277, pp. 15-30, 2001). It seems that they are closely involved. In addition, p53 mutations are reported not only in cancer but also in rheumatoid arthritis (Semin Arthritis Rheum April, 31 (5), 299-310, 2002).

The cells of most organisms, including humans, have an apoptotic mechanism, which is involved in various life phenomena such as morphogenesis during embryonic development, maintenance of tissue homeostasis, and removal of harmful and unnecessary cells. I have. On the other hand, abnormalities in apoptosis also cause the onset of many diseases such as cancer and neurodegenerative diseases, and therefore, studies on apoptosis have attracted attention in terms of disease treatment. Apoptosis signals are controlled by the balance of molecules that promote apoptosis and those that suppress it. Furthermore, it is considered that the survival of the cell is determined by the involvement of the cell proliferation signal in this signal. Many cell growth or apoptosis control molecules have been reported to date, but unknown control molecules may be present.
In order to comprehensively analyze gene expression, a microarray method in which cDNAs and 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 being widely used for diagnosis of various diseases and discovery of target genes for drug discovery. However, many GeneChip probes are prepared based on EST sequences, and the protein encoded by cDNA containing the EST sequence is often unknown.
Human-derived TSLRP (testis specific leucine rich repeat protein) is a gene reported in Genbank Accesiion No. NM_012472 (Non-Patent Document 1). No. 97/26001 (Patent Document 1).
The human-derived TSLRP gene has high homology with the mouse LRTP gene. This mouse LRTP gene is specifically expressed in testis and its protein has a leucine rich repeat region at the N-terminal. This region is thought to be involved in protein-protein interaction, but its detailed function has not been clarified (Non-Patent Document 2 Biology of Reproduction, 62, 1278-1284, 2000 Year).
WO 97/26001 Genbank Accesiion No. NM_012472 Biology of Reproduction, 62, 1278-1284, 2000

  New genes related to the regulation of cell proliferation and apoptosis enable the development of pharmaceuticals useful for the prevention and treatment of various diseases caused by up- and down-regulation of the genes, such as cancer, neurodegenerative diseases and autoimmune diseases I do. Accordingly, it has been desired to find a novel human-derived gene relating to control of cell proliferation and apoptosis.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, succeeded in cloning cDNA having a novel nucleotide sequence from a cancer cell line, and the protein encoded thereby Was found to be relevant. The present inventors have conducted further studies based on these findings, and as a result, have completed the present invention.

That is, the present invention
(1) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof;
(2) a protein comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof;
(3) a partial peptide of the protein according to (1) or a salt thereof;
(4) a polynucleotide containing a polynucleotide encoding the protein of (1) or the partial peptide of (3),
(5) the polynucleotide according to (4), which is DNA;
(6) a polynucleotide having a base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4,
(7) a recombinant vector containing the polynucleotide according to (4),
(8) a transformant transformed with the recombinant vector according to (7),
(9) The transformant according to (1), wherein the transformant according to (8) is cultured to produce and accumulate the protein according to (1) or the partial peptide according to (3), and collect the protein. )) Or a method for producing the partial peptide according to (3) or a salt thereof,
(10) a diagnostic agent comprising the polynucleotide according to the above (4),
(11) an antibody against the protein according to (1) or the partial peptide according to (3) or a salt thereof,
(12) a medicine comprising the antibody according to the above (11),
(13) A diagnostic agent comprising the antibody according to (11) above,
(14) a polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide according to (4) or a part thereof;
(15) a medicine comprising the polynucleotide according to the above (14),
(16) A method for screening a compound or a salt thereof that inhibits the expression of the protein gene according to (1), which comprises using the polynucleotide according to (4).
(17) A kit for screening a compound or a salt thereof that inhibits the expression of the protein gene according to (1), comprising the polynucleotide according to (4).
(17a) a compound or a salt thereof, which inhibits expression of the protein gene according to (1), obtained by using the screening method according to (16) or the screening kit according to (17);
(17b) a medicine comprising the compound of the above (17a) or a salt thereof,
(17c) The medicament according to the above (17b), which is an agent for preventing or treating cancer.
(17d) the medicament according to the above (17b), which is an apoptosis promoting agent;
(18) The method for quantifying a protein according to (1), comprising using the antibody according to (11);
(19) The medicament according to the above (12) or (15), which is an agent for preventing or treating cancer.
(20) The diagnostic agent according to (10) or (13) above, which is a diagnostic agent for cancer.
(21) the medicament according to the above (12) or (15), which is an apoptosis promoting agent;
(22) a method for screening a prophylactic / therapeutic agent for cancer, which comprises using a polynucleotide containing a polynucleotide encoding a protein containing the amino acid sequence represented by SEQ ID NO: 15 or a partial peptide thereof;
(22a) a compound having a prophylactic / therapeutic activity for cancer, or a polynucleotide thereof, which comprises using a polynucleotide containing a protein containing the amino acid sequence represented by SEQ ID NO: 15 or a polynucleotide encoding a partial peptide thereof Salt screening method,
(22b) a method for screening for an apoptosis-promoting agent, comprising using a polynucleotide containing a protein containing the amino acid sequence represented by SEQ ID NO: 15 or a polynucleotide encoding a partial peptide thereof,
(22c) Screening for a compound having a pro-apoptotic activity or a salt thereof, comprising using a polynucleotide containing a protein containing the amino acid sequence represented by SEQ ID NO: 15 or a polynucleotide encoding a partial peptide thereof. Method,
(22d) A kit for screening for a prophylactic / therapeutic agent for cancer, comprising a polynucleotide containing a protein containing the amino acid sequence represented by SEQ ID NO: 15 or a polynucleotide encoding a partial peptide thereof. ,
(22e) a kit for screening an apoptosis-promoting agent, comprising: a polynucleotide containing a protein containing the amino acid sequence represented by SEQ ID NO: 15 or a polynucleotide encoding a partial peptide thereof;
(23) a polynucleotide containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of a polynucleotide encoding the protein or the partial peptide thereof containing the amino acid sequence represented by SEQ ID NO: 15, or a part thereof A prophylactic / therapeutic agent for cancer comprising
(23a) a polynucleotide containing a nucleotide sequence complementary to or substantially complementary to a nucleotide sequence of a polynucleotide encoding a protein containing the amino acid sequence represented by SEQ ID NO: 15 or a partial peptide thereof, or a part thereof An apoptosis-promoting agent, comprising
(24) a method for preventing or treating cancer, which comprises administering to a mammal an effective amount of a compound or a salt thereof that inhibits the expression of the gene for the protein or salt thereof according to (1) above;
(24a) A method for promoting apoptosis, which comprises administering to a mammal an effective amount of a compound or a salt thereof that inhibits the expression of the gene for the protein or salt thereof according to (1) above,
(25) a method for preventing or treating cancer, which comprises inhibiting the expression of the gene for the protein or salt thereof according to (1);
(25a) A method for promoting apoptosis, which comprises inhibiting the expression of the gene for the protein or salt thereof according to (1) above,
(26) Use of a compound or a salt thereof that inhibits the expression of a gene of the protein or a salt thereof according to (1) for producing a prophylactic or therapeutic agent for cancer;
(26a) It is intended to provide a use of the compound or the salt thereof for inhibiting the expression of the gene of the protein or the salt thereof according to the above (1) for producing an apoptosis-promoting agent.

  The protein of the present invention, the polynucleotide of the present invention, the antibody of the present invention, the antisense polynucleotide of the present invention, a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein of the present invention, or the gene of the protein of the present invention Compounds or salts thereof that regulate (preferably inhibit) the expression of, for example, cancers (eg, colon cancer, breast cancer, ovarian 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, brain tumor, melanoma or hematological tumors), neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, Glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., kidney disease (eg, sugar Prophylactic / therapeutic agent / diagnostic agent for pathological nephropathy, chronic renal failure, nephritis, renal edema, interstitial renal disease, etc., preferably a prophylactic / therapeutic agent / diagnostic agent for cancer; ) It can be used safely as an agent.

A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 15 (hereinafter 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, adipocytes, immune cells (eg, macrophages, T cells) Cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, bone Cells, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells, stem cells, or cancer cells of these cells) or any tissue in which those cells are present, for example, the brain, various parts of the brain (eg, Olfactory bulb, amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonads, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle Proteins from the lungs, gastrointestinal tract (eg, large and small intestines), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovaries, placenta, uterus, bones, joints, skeletal muscle, etc. Or a synthetic protein.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 has 99.5% or more homology with the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. Amino acid sequence, etc.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 15 is, for example, about 60% or more, preferably about 70% or more, and preferably about 80% or more as the amino acid sequence represented by SEQ ID NO: 15. And preferably about 90% or more, and more preferably about 95% or more amino acid sequences having homology.
Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 15 include, for example, the aforementioned SEQ ID NO: 1, SEQ ID NO: 2 or It contains an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 15, and is substantially the same as a protein containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 15. Are preferred.
Substantially homogenous indicates that the properties are homogenous (eg, physiologically or pharmacologically). For example, the activities are preferably equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). And other quantitative factors may be different.

Examples of the protein of the present invention include, for example, (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 (eg, 1 to 3, preferably 1 to (Ii) the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2; or (1) two or more amino acids (e.g., about 1 to 200, preferably 1 to 100) (Preferably about 1 to 30, preferably about 1 to 10, and more preferably number (1 to 5) amino acids), (iii) SEQ ID NO: 1 or SEQ ID NO: 2. One or more (for example, about 1 to 200, preferably about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10 5) pcs of amino (Iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 (eg, about 1 to 200, preferably about 1 to 100, preferably Contains an amino acid sequence in which about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids have been substituted with another amino acid, or (v) an amino acid sequence obtained by combining them. (2) (i) 1 or 2 or more (for example, about 1 to 200, preferably about 1 to 100, and preferably 1 to 100) in the amino acid sequence represented by SEQ ID NO: 15. About 30 amino acids, preferably about 1 to 10, more preferably about 1 to 5 amino acids, and (ii) one amino acid sequence represented by SEQ ID NO: 15. Has two or more (for example, about 1 to 200, preferably about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids. (Iii) 1 or 2 or more amino acids (for example, about 1 to 200, preferably about 1 to 100, preferably about 1 to 30, preferably about 1 to 30 in the amino acid sequence represented by SEQ ID NO: 15) An amino acid sequence in which about 1 to 10, more preferably a number (1 to 5) amino acids have been inserted, and (iv) one or more (for example, 1 to 2) in the amino acid sequence represented by SEQ ID NO: 15 Amino acid in which about 200, preferably about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) of amino acids have been substituted with other amino acids Array, Others also include so-called muteins such as proteins containing the amino acid sequence comprising a combination thereof (v).
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 of 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 ⁻ ), an amide (—CONH ₂ ) or an ester at the C-terminus. (-COOR).
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 of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a carboxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Furthermore, in the protein of the present invention, the amino group of the N-terminal amino acid residue (eg, a methionine residue) may have a protecting group (eg, a C _1-6 acyl such as a C _1-6 alkanoyl such as a formyl group or an acetyl group). 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. 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 a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
Examples of the protein of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, a protein containing the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence represented by SEQ ID NO: 15. And the like.

The partial peptide of the protein of the present invention is the partial peptide of the protein of the present invention described above, and preferably any peptide having the same properties as the above-described protein of the present invention.
For example, a peptide having an amino acid sequence of 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 of the constituent amino acid sequences of the protein of the present invention. Is used.
In the partial peptide of the present invention, one or more (preferably, about 1 to 10, more preferably, about 1 to 5) amino acids in the amino acid sequence are deleted, or 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 acids are added to the amino acid sequence, or the amino acid sequence is added to 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 inserted, or one or more amino acids in the amino acid sequence are inserted. Two or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids may be substituted with another amino acid.

In the partial peptide, the C-terminus carboxyl group of the present invention (-COOH), carboxylate (-COO ^-), may be any of an amide (-CONH ₂₎ or an ester (-COOR).
Furthermore, the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, an N-terminal amino acid residue (eg, a methionine residue), similarly to the above-described protein of the present invention. ) Wherein the amino group is protected with a protecting group, glutamine residues generated by cleavage of the N-terminal side in vivo are pyroglutamine-oxidized, and the substituent on the side chain of the amino acid in the molecule is an appropriate protecting group. Or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.
The partial peptide of the present invention can also be used as an antigen for producing an antibody.

As the salt of the protein or partial peptide of the present invention, a salt with a physiologically acceptable acid (eg, an inorganic acid or an organic acid) or a base (eg, an alkali metal salt) is used. Preferred acid addition salts are: 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) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
The protein of the present invention or a partial peptide thereof or a salt thereof can be produced from the above-mentioned method for purifying a protein from human or warm-blooded animal cells or tissues, or can be transformed using a DNA encoding the protein. It can also be produced by culturing the body. 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 of the present invention, or a salt thereof, or an amide thereof, 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. Examples thereof include 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. 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 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 carbonyl hydrazide, trityl hydrazide 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 to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, meta-cresol, para-cresol, dimethyl sulfide, 1,4- It is effective to add a cation scavenger such as butanedithiol or 1,2-ethanedithiol. 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 C-terminal carboxyl group 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 of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein of 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 target peptide can be produced by condensing a partial peptide or amino acid that can constitute the partial peptide of the present invention with the remaining portion, and removing the protective group when the product has a protective group. 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, Vol. 14, Peptide Synthesis, Hirokawa Shoten After the reaction, normal purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. Can be used to purify and isolate the partial peptide of 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 of the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein of 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 preparation of a total RNA or mRNA fraction from the cells and tissues described above.
Examples of the DNA encoding the protein of the present invention include (1) a DNA containing the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or a base represented by SEQ ID NO: 3 or SEQ ID NO: 4 A protein having a base sequence that hybridizes under high stringent conditions to a sequence and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 described above. An encoding DNA, (2) a DNA containing the nucleotide sequence represented by SEQ ID NO: 14 or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 14; D encoding a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 15 It may be of any if A.

Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 and 99. DNA containing a base sequence having a homology of 5% or more is used.
Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 14 under high stringent conditions include, for example, about 60% or more, more preferably about 70% or more, with the nucleotide sequence represented by SEQ ID NO: 14 More preferably, a DNA containing a nucleotide sequence having a homology of about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more is used.
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: 3 or the like is represented by SEQ ID NO: 2. Examples of the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 4 include the DNA containing the base sequence represented by SEQ ID NO: 4 and the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 15 For example, a DNA containing the base sequence represented by SEQ ID NO: 14 is used.

The polynucleotide (eg, DNA) encoding the partial peptide of the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the aforementioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
As the DNA encoding the partial peptide of the present invention, for example, a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 14, or SEQ ID NO: 3, DNA containing a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 14 under high stringent conditions, and encoding a protein having substantially the same activity as the protein of the present invention DNA containing a part of the above is used.
DNAs capable of hybridizing with the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 14 have 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 the protein or partial peptide of the present invention (hereinafter, in the description of the cloning and expression of the DNA encoding these may be simply referred to as the protein of the present invention) include: Amplifying by a PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or incorporating a DNA incorporated in an appropriate vector into a DNA encoding a part or the whole region of the protein of the present invention. Selection can be performed by hybridization with fragments or those labeled with 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 is, 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 a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. 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 roth moth (Spodoptera frugiperda cell; Sf cell), MG1 cells derived from the midgut of Trichoplusia ni, and High Five ^™ derived from eggs 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).
To transform yeast, for example, the method described in Methods in Enzymology, vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) can be used. 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, a Burkholder minimal 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 to 35 ° C. for about 24 to 72 hours, and aeration and / or agitation are added as necessary.
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 used as appropriate. 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 whose animal 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 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.
Purification of the protein contained in the culture supernatant or extract thus obtained can be carried out 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. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and use of difference in hydrophobicity such as reverse 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 the protein obtained is 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 applying 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 of the present invention or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide of the present invention or a salt thereof.
An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these may be simply abbreviated as the protein of the present invention) is a known antibody using the protein of the present invention as an antigen. Alternatively, it can be produced according to a method for producing an antiserum.
[Preparation of monoclonal antibody]
(A) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal 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, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal 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 a complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of the carrier protein and the mixing ratio of the carrier protein and the hapten are determined by the antibody against the hapten immunized by crosslinking the carrier protein. Any material may be crosslinked at any ratio as long as it can be efficiently prepared.For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 20, preferably 1 to hapten 1 based on hapten. A method of coupling at a rate of about 1 to 5 is used.
In addition, various condensing agents can be used for coupling the hapten and the carrier protein, and glutaraldehyde, carbodiimide, 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, or the like, 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.

Complementary to the nucleotide sequence of a polynucleotide encoding the protein or partial peptide of the present invention (eg, DNA (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of the antisense polynucleotide)) An antisense polynucleotide having a base sequence substantially or complementary to a base sequence or a part thereof includes bases complementary to or substantially complementary to the base sequence of the polynucleotide (eg, DNA) of the present invention. Any antisense polynucleotide may be used as long as it has a sequence or a part thereof and has an action capable 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: 3 or SEQ ID NO: 4, or a part thereof Preferably, 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 SEQ ID NO: 4, or a part thereof (more preferably, SEQ ID NO: 3 or A base sequence complementary to the base sequence of the DNA containing the base sequence represented by SEQ ID NO: 4, or an antisense polynucleotide having a portion 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, for example, 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 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, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the cloned or determined nucleotide sequence information of the DNA encoding the protein. And can be synthesized. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention and inhibit the synthesis or function of the RNA, or through the interaction with the protein-related RNA of the present invention. Thus, 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. "Corresponding" between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. . 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation termination codon, 3' end untranslated region, 3 ' The terminal palindrome region and the 3'-end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as a target.
The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region can be said to be "antisense" if the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target is. 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, or Other polymers having non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymer is a base such as found in DNA or RNA And nucleotides having a configuration permitting the attachment of a base). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides), or even known. Such as labeled, capped, methylated, one or more naturally-occurring nucleotides replaced by analogs, intramolecular nucleotides Modified, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), Such as proteins (nucleases, nuclease inhibitors, toxic , Antibodies, signal peptides, poly-L-lysine, etc.), sugars (eg, monosaccharides), etc., having side chain groups, interacting compounds (eg, acridine, psoralen, etc.), chelates Those containing compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) It may be. 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 by halogens, aliphatic groups, etc., or by functional groups such as ethers, amines, etc. It may have been converted.
The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids.
Thus, many modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed. , Antisense Research and Applications, CRC Press, 1993.
The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It could 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 ( For example, hydrophobic substances such as phospholipid and cholesterol can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. 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 nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various methods known per se.

  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 may be abbreviated as the antibody of the present invention), and DNA of the present invention. The use of the antisense polynucleotide (hereinafter, may be abbreviated as the antisense polynucleotide of the present invention) will be described.

Since the protein of the present invention regulates (preferably inhibits / suppresses) the apoptotic effect, the protein of the present invention, the polynucleotide of the present invention, the antibody of the present invention, etc. may be used, for example, for cancer (eg, colon cancer, breast cancer, ovarian cancer) , Lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testis cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood tumor), neurodegenerative disease (Eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), kidney Disease markers (eg, diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial renal disease, etc.), preferably cancer, etc .; Te is useful. Furthermore, the antisense polynucleotide of the present invention, a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein of the present invention, a compound or a salt thereof that regulates (preferably inhibits) the expression of the gene of the protein of the present invention, etc. For example, cancer (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer , Brain tumors, melanoma or hematological tumors), neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistance) Diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), kidney diseases (eg, diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial kidney disease, etc.) Prophylactic or therapeutic agent, preferably useful as an agent for preventing or treating cancer.
In addition, since the gene of the protein of the present invention has low expression in normal tissues, screening for a prophylactic / therapeutic agent for cancer with few side effects becomes possible. Further, it is known that cancer retaining mutant p53 has a high degree of malignancy, such as poor efficacy of an anticancer drug in clinical practice and poor prognosis. The protein gene of the present invention acts on the downstream of mutant p53, and thus acts as an antisense polynucleotide of a polynucleotide encoding the protein of the present invention, a compound inhibiting the activity of the protein of the present invention or a salt thereof, and a protein of the present invention. A compound or a salt thereof that inhibits the expression of the above-mentioned gene, or an antibody against the protein of the present invention is also effective for highly malignant cancer.

(1) Screening of drug candidate compounds for diseases Inhibiting the activity or expression of the protein of the present invention causes apoptosis of cancer cells. Accordingly, compounds or salts thereof that regulate (enhance or inhibit, preferably inhibit) the activity of the protein of the present invention include, for example, cancers (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver) Cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood tumor, etc., neurodegenerative disease (eg, Alzheimer's disease, Parkinson's syndrome, etc.), self Immune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), kidney diseases (eg, diabetic nephropathy, chronic renal failure) , Nephritis, renal edema, interstitial renal disease, etc.). Preferably cancer (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer , Brain tumors, melanomas or hematological tumors).
Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates the activity of the protein of the present invention.
That is, the present invention provides a method for screening a compound or a salt thereof that regulates the activity of the protein of the present invention, which comprises using the protein of the present invention.
More specifically, for example, a comparison between (i) the activity of a cell capable of producing the protein of the present invention and (ii) the activity of a mixture of a cell capable of producing the protein of the present invention and a test compound. A method for screening a compound or a salt thereof that regulates the activity of the protein of the present invention, which is characterized in that
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 in which the protein of the present invention is expressed on a cell membrane and in a cell by culturing by the above-described method is preferably used. The method for culturing cells capable of expressing the protein of the present invention is the same as the method for culturing the transformant of the present invention described above.
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 promotes the 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), by using the protein of the present invention A test compound which inhibits the activity in the case (ii) above by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case (i) as a compound which promotes the activity of Can be selected as compounds that inhibit the activity 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, compounds or salts thereof that regulate (enhance or inhibit, preferably inhibit) the expression of the gene of the protein of the present invention include, for example, cancers (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer) , Liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood tumor, etc., neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.) , Autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), kidney diseases (eg, diabetic nephropathy, chronic Renal failure, nephritis, renal edema, interstitial renal disease, etc.). Preferably, cancer (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreas) Cancer, brain tumor, melanoma or blood tumor).
Therefore, the polynucleotide (eg, DNA) of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates the expression of the gene of the protein of the present invention.
The screening methods include (iii) culturing cells capable of producing the protein of the present invention, and (iv) culturing cells capable of producing the protein of the present invention in the presence of the test compound. And a screening method characterized by performing a comparison.
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 the same cells as 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 is measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 3 or SEQ ID NO: 4 or a part thereof as a probe, or SEQ ID NO: 3 or SEQ ID NO: 4 as a primer or It can be measured according to a PCR method using a nucleic acid containing a part thereof or a method analogous thereto.
For example, a test compound which promotes the expression of a gene 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) can As a compound that promotes the expression of the protein gene, the gene expression in the case of the above (iv) is about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). % Of the test compound can be selected as compounds that inhibit the expression of the gene of the protein of the present invention.

The screening kit of the present invention contains the protein or partial peptide of the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide of 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, or the like, or a salt thereof, a compound or a salt thereof that regulates the activity of the protein of the present invention, or a compound or a salt thereof that regulates gene expression of the protein of the present invention. .
As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
The compound or its salt that regulates (preferably inhibits) the activity of the protein of the present invention and the compound or its salt that regulates (preferably inhibits) the expression of the gene of the protein of the present invention are, for example, cancer (eg, colon cancer) , Breast, ovarian, lung, prostate, esophageal, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid, pancreas, brain, melanoma or blood tumor ), Neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic Prophylactic / therapeutic agents for renal diseases (eg, diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial renal disease, etc.), preferably cancer (eg, lupus erythematosus) Colorectal cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood It is useful as a safe medicine such as a prophylactic / therapeutic agent for tumors.
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 formulation. 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. 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. 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 composition is conveniently prepared in a unit dosage 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 compound.
Each of the above-mentioned compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the compound.
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, the activity of the protein of the present invention or the expression of the protein gene is inhibited 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, the target disease, and the like. For example, the activity of the protein of the present invention or the expression of the protein gene for the purpose of treating breast cancer. When a compound or a salt thereof is administered to an adult (with a body weight of 60 kg) usually in the form of an injection, the compound or a salt thereof is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day. Conveniently, more preferably about 0.1 to 10 mg is administered 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.

(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 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, a competitive method, an immunometric method, and a sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance 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, 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 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 the 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 site to which the protein of the present invention binds 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 precipitates generated 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 immunoassays 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” (3rd Edition), Ishikawa, 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 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, ovarian cancer, lung cancer) , Prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testis cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood tumor, etc., neurodegenerative disease (example) , Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), kidney diseases ( E.g., diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial kidney disease, etc.) (preferably cancer), etc., or can be diagnosed as likely to be affected 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) Genetic Diagnostic Agent 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, such as 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 USA, 86, 2766-2770 (1989)).
For example, when overexpression is detected by Northern hybridization or DNA mutation is detected by PCR-SSCP method, for example, cancer (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer) , Gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood tumor, etc., neurodegenerative disease (eg, Alzheimer's disease, Parkinson's syndrome) Autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), kidney diseases (eg, diabetic nephropathy) , Chronic renal failure, nephritis, renal edema, interstitial renal disease, etc.) and preferably 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 low toxicity and exhibits the present invention in vivo. Can inhibit the function of the protein of the present invention or the DNA of the present invention. Cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood tumor, etc., neurodegenerative disease (eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune disease (eg, myasthenia gravis) , Glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), kidney diseases (eg, diabetic kidney Disease, chronic renal failure, nephritis, renal edema, interstitial kidney disease, etc.). Preferably, cancer (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreas) Cancer, brain tumor, melanoma or blood tumor).
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, they can be aerosolized and administered locally into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection) and administered intravenously, subcutaneously, etc. May be.
The dose 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 suppress the expression of the protein and the function of the protein of the present invention or the DNA used in the present invention in a living body. Thus, for example, cancer (eg, colon cancer, breast cancer, ovarian 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, brain tumor, melanoma or blood tumor, etc., neurodegenerative disease (eg, Alzheimer's disease) , Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, chronic joint Rheumatic, such as systemic lupus erythematosus), kidney disease (e.g., diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial renal disease, etc.) can be used as a preventive or therapeutic agent for such. Preferably, cancer (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreas) Cancer, brain tumor, melanoma or blood tumor).
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) Medicine containing the antibody of the present invention The antibody of the present invention can be used, for example, in cancer (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer) Cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood tumor, etc., neurodegenerative disease (eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune disease (eg, myasthenia gravis) , Glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistance diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc., renal diseases (eg, diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitium) The present invention can be used as a prophylactic / therapeutic agent (eg, vaccine, antibody drug, etc.) for sexual renal disease. Preferably, cancer (eg, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreas) Cancer, brain tumor, melanoma or blood tumor).
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity, and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form in humans or mammals (eg, rats, rabbits, sheep, pigs, It can be administered orally or parenterally (eg, intravascular, subcutaneous, etc.) to cattle, cats, dogs, monkeys, etc.). Preferably, it can be administered as a vaccine according to a standard method.
The antibodies of the present invention may be administered per se or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the antibody of the present invention and a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
As compositions for parenteral administration, for example, injections, suppositories, vaccines and the like are used. Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. Dosage forms may be included. Such an injection can be prepared according to a known method. The injection can be prepared, for example, by dissolving, suspending, or emulsifying the antibody of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents 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 and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository for rectal administration may be prepared by mixing the antibody or a salt thereof with a conventional suppository base.
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), syrups Agents, emulsions, suspensions and the like. Such compositions are prepared by known methods and may contain carriers, diluents or excipients commonly used in the field of formulation. As carriers and excipients for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.
The above-mentioned parenteral or oral pharmaceutical composition is conveniently prepared in dosage unit form so as to be compatible with the dosage of the active ingredient. Such dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The content of the antibody is usually about 5 to 500 mg per dosage unit dosage form, particularly preferably about 5 to 100 mg for injection, and about 10 to 250 mg for other dosage forms.
The dose of the prophylactic / therapeutic agent containing the antibody of the present invention varies depending on the administration subject, target disease, symptom, administration route, and the like.For example, when used for the treatment / prevention of adult breast cancer, As a single dose of the antibody of the present invention, usually 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 per day It is convenient to administer by intravenous injection about 1 to 5 times, preferably about 1 to 3 times a day. 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, intravascular injection, subcutaneous injection, etc.).
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the antibody.

(6) DNA-transferred animal The present invention relates to a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (hereinafter 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 preparation of disease animal model systems, and are easy to breed, especially 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, or the like, 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 is 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 type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophy Int, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally 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 acti , 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 above vector preferably has a sequence that terminates transcription of a messenger RNA of interest in a DNA-transferred mammal (generally called a terminator). 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 between 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 transposed animal by a conventional DNA engineering technique in which it is ligated downstream of the above-mentioned 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 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 eventually promotes the hyperactivity of the protein of the present invention by promoting the function of endogenous normal DNA. 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 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 the mammal into 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 (eg, Colorectal cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood Tumors), neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistance diabetes, rheumatoid arthritis, Screening test for prophylactic / therapeutic agents such as systemic lupus erythematosus) and renal diseases (eg, diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial renal disease, etc.) It is possible to use.
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the target foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is 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 progeny 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 so that all progeny 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 inactive refractory disease of the protein of the present invention. A model to elucidate.
In addition, since a mammal to 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, such as cancer ( For example, colon cancer, breast cancer, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma Or blood tumors), neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, chronic joints) Screening for prophylactic / therapeutic agents for rheumatism, systemic lupus erythematosus, etc.), kidney diseases (eg, diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial kidney disease, etc.) Also to experience it is available.
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.

(7) 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 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 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 cells are knocked out of normal cells (for example, chromosomes in mice). It is desirable to clone again into cells (number 2n = 40).
The embryonic stem cell line obtained in this manner 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 subculture is usually performed 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, Proc. Natl. Acad. Sci. USA 78, 7634, 1981; TC Doetschman et al. Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], and the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention can Is useful in cell biology studies of proteins.

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 mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector 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 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 non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. 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 a mutated DNA locus of the present invention, all tissues are artificially mutated from a 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. 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 breeding 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.

(7a) Method of screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention The non-human mammal deficient in DNA expression of the present invention is deficient or damaged of the DNA of the present invention Can be used for screening for a compound having a therapeutic / preventive effect on a disease 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 having a therapeutic or preventive effect on a disease, for example, cancer, or a salt thereof.
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 an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.
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, ovarian cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, Brain tumors, melanoma or hematological tumors), neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes) Compounds that have a prophylactic / therapeutic effect on rheumatoid arthritis, systemic lupus erythematosus, etc.), renal diseases (eg, diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial renal disease, etc.) When screening, a test compound is administered to a non-human mammal deficient in expression of the DNA of the present invention, and the difference in the degree of onset of cancer and the degree of healing of cancer in the non-test compound non-administered group are determined over time in the tissue. Observation.
In the screening method, when the test compound is administered to a test animal, if 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, the test compound is administered. 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 preventive / therapeutic 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.). And the like, 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.
Since the preparation thus obtained is safe and low toxic, it 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. 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 as an injection to an adult (with a body weight of 60 kg) breast cancer patient. In that 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 the weight per 60 kg.

(7b) Screening method for compound that promotes or inhibits the activity of the 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. And a method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for the DNA of the present invention.
In the above-described screening method, the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing 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). Of the protein in an animal body can be observed. 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 of the present invention or a salt thereof that inhibits the promoter activity for the DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein. , Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, melanoma or blood tumor, etc., neurodegenerative disease (example) , Alzheimer's disease, Parkinson's syndrome, etc.), autoimmune diseases (eg, myasthenia gravis, glomerulonephritis, multiple sclerosis, Sjogren's syndrome, insulin resistant diabetes, rheumatoid arthritis, systemic lupus erythematosus, etc.), kidney diseases ( Eg, diabetic nephropathy, chronic renal failure, nephritis, renal edema, interstitial renal disease, 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.
Since the preparation thus obtained is safe and low toxic, it 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 example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally the adult (body weight) is used. For breast cancer patients (as 60 kg), the compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, a compound that inhibits the promoter activity of the DNA of the present invention is usually administered to adults ( 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, 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: thyronine : 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]
1 shows the amino acid sequence of the MTp53TSLRPH (Ile) protein obtained in Example 1.
[SEQ ID NO: 2]
1 shows the amino acid sequence of the MTp53TSLRPH (Thr) protein obtained in Example 1.
[SEQ ID NO: 3]
The nucleotide sequence of the DNA encoding the amino acid sequence represented by SEQ ID NO: 1 is shown.
[SEQ ID NO: 4]
This shows the base sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 2.
[SEQ ID NO: 5]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 6]
1 shows the nucleotide sequence of a primer used in Example 1.
[SEQ ID NO: 7]
2 shows the base sequences of primers used in Examples 1 and 2.
[SEQ ID NO: 8]
2 shows the base sequences of primers used in Examples 1 and 2.
[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]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 12]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 13]
The nucleotide sequence of GenBank Accession No. NM_012472 is shown.
[SEQ ID NO: 14]
This shows the base sequence of the ORF of GenBank Accession No. NM_012472.
[SEQ ID NO: 15]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 14 is shown.
The transformant Escherichia coli JM109 / MTp53TSLRPH (Ile) obtained in Example 1 described below was obtained from March 1, 2003 at 1-1-1, Higashi, Chuo-dai, Tsukuba City, Ibaraki Prefecture, Japan (zip code 305-8566). Deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the deposit number FERM BP-8329.
The transformant Escherichia coli JM109 / MTp53TSLRPH (Thr) obtained in Example 1 described below was obtained from March 1, 2003 at 1-1-1, Higashi 1-chome, Chuo No. 6 (Postal code 305-8566), Higashi 1-1-1 Tsukuba, Ibaraki Prefecture, Japan Deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the deposit number FERM BP-8330.

Hereinafter, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto.
Example 1
(1) Construction of Mutant p53 Vector Forward and reverse primers in which the 524th base counted from A of translation initiation codon ATG of wild-type p53 (GenBank Accession No. AF307851) has been changed from G to A (SEQ ID NO: : 5 and SEQ ID NO: 6) were prepared. Using the pcDNA3.1-WTp53 vector in which wild-type p53 (WTp53) was incorporated into pcDNA3.1 (Invitrogen) at the Bam HI-Eco RI site as a template, using the above two primers, QuikChange ^™ Site-Directed Mutagenesis Kit ( Mutant p53 (MTp53) gene was prepared using STRATAGENE). The method followed the attached protocol. The MTp53 gene was treated with Bam HI and Eco RI with restriction enzymes, and incorporated into a pIND vector (attached to the Ecdysone-Inducible Mammalian Expression System (Invitrogen)) treated with the same enzymes to obtain a pIND-MTp53 vector. In this mutant p53, the amino acid sequence at position 175 replaces arginine (R) with histidine (H). As a control, a pIND-WTp53 vector in which a wild-type p53 gene was incorporated into a pIND vector was also prepared. As another control, a pIND-CAT vector (Invitrogen) incorporating CAT (chloramphenicol acetyltransferase) was also used.

(2) Construction of mutant p53 gene expression control cells
A p53-deficient lung cancer-derived cell line H1299 (ATCC) was seeded at 4 × 10 ⁵ cells in a 6-cm Petri dish, cultured overnight, and obtained using Fugene 6 (Roche) according to the attached protocol according to (1) above. The pIND-MTp53 vector, pIND-WTp53 vector or pIND-CAT vector, and pVgRXR vector (attached to the Ecdysone-Inducible Mammalian Expression System (invitrogen)) were co-transfected, respectively. Geneticin (GIBCO BRL) and Zeosin (Invitrogen) were used to select cell lines (H1299-MTp53, H1299-WTp53, H1299-CAT) into which the respective genes had been introduced. When 5 μM Muristerone A (attached to the above kit) was added to these three stable expression strains, H1299-MTp53 expressed MTp53, H1299-WTp53 expressed WTp53, and H1299-CAT expressed CAT. Each was induced. Furthermore, it was confirmed by a Western blotting method using an anti-p53 antibody (Oncogene) that the expression of MTp53 was induced in H1299-MTp53 and the expression of WTp53 was induced in H1299-WTp53.

(3) GeneChip analysis 5 μM Muristerone A was added to H1299-MTp53, H1299-WTp53 and H1299-CAT after 0, 12, 24 and 48 hours, respectively, in order to clarify the genes induced by mutant p53 Gene expression analysis was performed using GeneChip (Human Genome U95A, U95B, U95C, U95D, U95E; Affymetrix) with total RNA prepared from these three cell lines as materials. The experimental method was in accordance with the experimental manual (Expression analysis technical manual) of Affymetrix.
As a result, a gene (Affymetrix No. 36738_at; a partial sequence of GenBank Accession No. NM_012472) whose expression was increased only by mutant p53 and not induced by wild-type p53 was found.

[Table 1]
Cell line (culture time) Affymetrix number 36738_at relative expression level
H1299-MTp53 (0 hours) 0.15
H1299-MTp53 (12 hours) 0.25
H1299-MTp53 (24 hours) 0.33
H1299-MTp53 (48 hours) 0.21
H1299-WTp53 (0 hours) 0.00
H1299-WTp53 (12 hours) 0.00
H1299-WTp53 (24 hours) 0.02
H1299-WTp53 (48 hours) 0.00

(4) Isolation of novel gene Based on the base sequence of GenBank Accession No. NM_012472 obtained in (3) above, primer 3 having different restriction enzyme recognition sites (Hind III and Xba I) (SEQ ID NO: 7) and primer 4 (SEQ ID NO: 8) were prepared, and cDNA prepared from Marathon-ready human testis cDNA (CLONTECH) or MTp53-derived H1299 cells (H1299-MTp53) constructed in (2) above was used as a template. , A PCR reaction was performed. The reaction was carried out at 94 ° C. for 1 minute, followed by 30 cycles of 98 ° C. for 5 seconds—65 ° C. for 10 seconds—72 ° C. for 3 minutes 30 times. The obtained cDNA fragment was digested with Hind III and Xba I, and the plasmid p3 × FLAG-CMV-14 Expression Vector (SIGMA) was inserted into the site digested with the above restriction enzymes, followed by a sequencing reaction. As a result of the sequencing, insertion of 9 bp was detected in frame between the 723rd base and the 724th base, counting from the start codon A of the ORF (SEQ ID NO: 14) of GenBank Accession No. NM_012472 sequence. The obtained base sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4.
The plasmid containing the DNA having the nucleotide sequence represented by SEQ ID NO: 3 was p3 × FLAG-CMV-14-MTp53TSLRPH (Ile), and the plasmid containing the DNA having the nucleotide sequence represented by SEQ ID NO: 4 was p3 × It was named FLAG-CMV-14-MTp53TSLRPH (Thr), respectively. These plasmids were introduced into Escherichia coli JM109 to obtain transformants Escherichia coli JM109 / MTp53TSLRPH (Ile) and Escherichia coli JM109 / MTp53TSLRPH (Thr), respectively.
The 695th base in the base sequence represented by SEQ ID NO: 3 is T as in the corresponding GenBank Accession No. NM_012472 sequence, but the 695th base in the base sequence represented by SEQ ID NO: 4 is C It is. This base substitution involves an amino acid substitution.
A protein containing the amino acid sequence (SEQ ID NO: 1) encoded by the nucleotide sequence represented by SEQ ID NO: 3 is referred to as an MTp53TSLRPH (Ile) protein and an amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 4 (sequence) The protein containing No. 2) is named MTp53TSLRPH (Thr) protein, respectively.
Position 232 of the amino acid sequence represented by SEQ ID NO: 1 is Ile, and position 232 of the amino acid sequence represented by SEQ ID NO: 2 is Thr.

Example 2
Examination of MTp53TSLRPH gene expression level in human breast cancer matched pair Hereinafter, the genes of the MTp53TSLRPH (Ile) protein, MTp53TSLRPH (Thr) protein and the protein having the amino acid sequence represented by SEQ ID NO: 15 are collectively referred to as MTp53TSLRPH gene.
Using the Human Breast Matched cDNA Pair Panel (Clontech) derived from cancer tissues and normal tissues of human breast cancer patients as a template, the expression level of the MTp53TSLRPH gene was examined. By performing a PCR reaction using the primers 3 and 4 used in Example 1, the expression level of the TSLRP gene was compared between cancer tissues and normal tissues. The reaction was performed at 94 ° C. for 1 minute, and then a reaction cycle of 98 ° C. for 5 seconds-65 ° C. for 10 seconds-72 ° C. for 3 minutes was performed 40 times.
As a result, it was confirmed that the expression of the MTp53TSLRPH gene in cancer tissues was more remarkable than in normal tissues.

Example 3
Cell death by antisense oligonucleotides
In order to analyze the effect of the MTp53TSLRPH gene on apoptosis, an MTp53TSLRPH antisense oligonucleotide introduction experiment was performed.
First, after designing an antisense (SEQ ID NO: 9) to the base sequence represented by SEQ ID NO: 13, a phosphorothioated oligonucleotide was synthesized, purified by HPLC (Amersham Pharmacia Biotech), and used for an introduction experiment. As a control oligonucleotide, the reverse sequence (SEQ ID NO: 10) of the base sequence represented by SEQ ID NO: 9 was similarly phosphorothioated and purified by HPLC (Amersham Pharmacia Biotech) for use.
Lung cancer cell line A549 (Dainippon Pharmaceutical) was used as test cells, and 3.3 × 10 ³ cells were seeded on a 96-well plate (Falcon) the day before oligonucleotide introduction. OligofectAMINE (Invitrogen) was used for oligonucleotide introduction, and the protocol was followed. Twenty hours after the introduction, RNA was extracted according to the protocol of RNeasy mini kit (Qiagen), and cDNA was prepared using SuperScript Preamplification System for First Strand cDNA Synthesis (GIBCO BRL). The expression level of the MTp53TSLRPH gene was measured using two primers (SEQ ID NO: 11 and SEQ ID NO: 12), and the β-actin expression level was corrected using TaqMan human β-actin control reagent (Applied Biosystem) for correction. PCR was performed according to the ABI7700 (Applied Biosystem) manual. The composition of the reaction solution in the above PCR reaction when measuring the expression level of the MTp53TSLRPH gene was 7.5 μl of SYBR PCR Master Mix (Applied Biosystem), and the cDNA template prepared above diluted 3 times with sterile distilled water. Was added to each of 5 μl and two kinds of primers (SEQ ID NO: 11 and SEQ ID NO: 12) so as to have a concentration of 0.5 μM each to give a liquid volume of 15 μl. In the PCR reaction, a cycle of 50 ° C. for 2 minutes, 95 ° C. for 10 minutes, 95 ° C. for 15 seconds and 60 ° C. for 1 minute was repeated 40 times. The expression level of the MTp53TSLRPH gene was corrected for β-actin expression level for comparison.
On the other hand, the effect on apoptosis was compared with control oligonucleotide-transfected cells using Cell death detection ELISA (Roche) three days after introduction of the antisense oligonucleotide.
As a result, 20 hours after the introduction of the antisense oligonucleotide, the expression (RNA) of MTp53TSLRPH was reduced to 37% as compared to the time when the control (reverse) oligonucleotide was introduced (100%).
On the 3rd day after the introduction, the apoptosis when the antisense oligonucleotide was introduced was increased to 204% as compared with the case where the control oligonucleotide was introduced (100%).
Thus, it was confirmed that suppressing the expression of the MTp53TSLRPH gene causes cancer cells to undergo apoptosis and cell death.

Claims (26)

A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof. A protein comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or a salt thereof. A partial peptide of the protein according to claim 1 or a salt thereof. A polynucleotide comprising a polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 3. The polynucleotide according to claim 4, which is DNA. A polynucleotide consisting of the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. A recombinant vector containing the polynucleotide according to claim 4. A transformant transformed with the recombinant vector according to claim 7. The protein according to claim 1, wherein the transformant according to claim 8 is cultured to produce and accumulate the protein according to claim 1 or the partial peptide according to claim 3, and the collected protein is collected. 3. The method for producing the partial peptide or a salt thereof according to item 3. A diagnostic agent comprising the polynucleotide according to claim 4. An antibody against the protein according to claim 1, the partial peptide according to claim 3, or a salt thereof. A medicament comprising the antibody according to claim 11. A diagnostic agent comprising the antibody according to claim 11. A polynucleotide comprising a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide according to claim 4, or a part thereof. A medicament comprising the polynucleotide according to claim 14. A method for screening a compound or a salt thereof that inhibits expression of the protein gene according to claim 1, wherein the polynucleotide according to claim 4 is used. A kit for screening a compound or its salt that inhibits expression of the protein gene according to claim 1, comprising the polynucleotide according to claim 4. A method for quantifying a protein according to claim 1, wherein the antibody according to claim 11 is used. The medicament according to claim 12 or 15, which is a prophylactic / therapeutic agent for cancer. 14. The diagnostic agent according to claim 10 or 13, which is a diagnostic agent for cancer. The medicament according to claim 12 or 15, which is an apoptosis promoting agent. A method for screening for a prophylactic / therapeutic agent for cancer, comprising using a polynucleotide containing a protein encoding the amino acid sequence represented by SEQ ID NO: 15 or a partial peptide thereof. It contains a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide encoding the protein or the partial peptide thereof containing the amino acid sequence represented by SEQ ID NO: 15, or a polynucleotide containing a part thereof Prevention and treatment of cancer. A method for preventing or treating cancer, comprising administering to a mammal an effective amount of a compound or a salt thereof that inhibits the expression of the gene for the protein or a salt thereof according to claim 1. A method for preventing or treating cancer, comprising inhibiting the expression of the gene for the protein according to claim 1 or a salt thereof. Use of the compound or its salt for inhibiting the expression of the gene of the protein or its salt according to claim 1, for producing a prophylactic / therapeutic agent for cancer.