JP2003093079A - New disease-associated gene and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening a medicine by using a disease- associated gene product and to obtain an antibody against the disease-associated gene product and an antisense DNA inhibiting the expression of the disease- associated gene. SOLUTION: The protein containing the same or substantially the same amino acid sequence as that of a specific amino acid sequence, its partial peptide or their salts are obtained. The method for screening a medicine by using the disease-associated gene product is provided. The antisense polynucleotide to the disease-associated gene is obtained. A medicine containing the antisense polynucleotide is obtained. The antibody against the disease-associated gene product is obtained. A medicine containing the antibody is obtained. A diagnostic containing the antibody is obtained. The method for screening a compound controlling the activity of the disease-associated gene product is provided. A compound is obtained by the screening method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a disease-related gene,
It relates to the use of genes associated with diseases such as heart disease, cancer or the retina. More specifically, a method for screening a drug using the disease-related gene product, an antisense polynucleotide against a disease-related gene useful as a diagnostic marker for heart diseases such as cardiomyopathy, myocardial infarction, heart failure, and angina, and the disease-related gene The present invention relates to antibodies to products.

[0002]

2. Description of the Related Art Heart failure is considered to be myocardial contraction failure. The mechanism of onset may be as follows. Myocardial disorders, mechanical and functional abnormalities of the heart pump,
Due to a pressure load due to hypertension and pulmonary hypertension, anemia, and a volume load such as acute nephritis, a state in which the heart cannot pump the blood volume according to the demand of the living body occurs. On the other hand, compensatory mechanisms such as the sympathetic nervous system and the nerve-fluid-endocrine system operate to maintain homeostasis of the living body. The compensatory mechanism of heart failure is as follows. 1) The above-mentioned load increases, the contractile force of the heart increases, and cardiac expansion occurs with the increase in the length of sarcomere. 2) The contractile unit of myocardium increases. 3) Neuromuscular factors are activated to compensate for the condition in which the necessary blood cannot be ejected throughout the body, which leads to myocardial fibrosis locally. Basically, it is a mechanism that tries to adapt to a given load, but in some cases, insufficient operation may worsen heart failure, but on the contrary, excessive operation may cause myocardial injury, resulting in insufficient operation. As in some cases, heart failure may be exacerbated. As a result of the activation of the compensatory mechanism,
Myocardial cells are enlarged and cardiac hypertrophy occurs. However, if the aforementioned obstacle or load is chronically continued, the compensatory mechanism fails. In other words, hypertrophic cardiomyocytes do not supply a sufficient amount of blood and enter into ischemia, which causes myocardial disorders such as myocardial contraction failure, decreased cardiac output, impaired organ circulation, venous congestion, and body fluids. This will result in heart failure syndrome with retention. Treatment for this requires improvement of cardiomyocyte damage, enhancement of cardioprotection, recovery of cardiac function decline due to myocardial contraction failure, suppression of compensatory failure of the body which is the cause, or improvement of excessive compensation mechanism. . Currently, for the treatment of the heart failure syndrome, clinically, as cardiotonics, 1) cardiac glycosides such as digoxin, 2) sympathomimetics such as dobutamine, 3) phosphodiesterase inhibitors such as amrinone, and vasodilation. As the drug, hydralazine, a calcium antagonist, an angiotensin I converting enzyme inhibitor, an angiotensin II receptor antagonist and the like are used. In addition, for the treatment of other dilated cardiomyopathy,
Beta blockers are used. However, there is no therapeutic drug that can promptly activate the compensatory mechanism, suppress the excessive compensatory mechanism or suppress the compensatory failure (including suppression of apoptosis). On the other hand, the function of G protein-coupled receptor phosphatase (GRK) desensitizes the receptor by directly phosphorylating the G protein-coupled receptor (GPCR) (Annu Rev Pharmacol Toxicol 3
8,289-319, 1998). Photor for GPCRs in the retina
The eceptor (rhodopsin) is well known, and desensitization by GRK1 has been reported (Physiol Rev 81,117-15).
1, 2002). Furthermore, it has been reported that squirrel GRK7 is involved in this desensitization (Mol. Vis., 4, 27, 1998).

[0003]

DISCLOSURE OF THE INVENTION As described above, GRK
Since 7 is considered to have an action of reducing sensitivity to light, a substance having an action of suppressing its function is considered to be promising as a prophylactic / therapeutic agent for eye diseases accompanied by reduced visual acuity and the like. In retinoblastoma, various GPCRs
Are thought to be involved in cell proliferation. When GRK7 is involved in the GPCR signal enhanced in this cancer cell, a malignant tumor can be regressed by a function promoter.
GRK7 is localized in the retina in normal people, but
When expression is observed in various cancer tissues, the GRK7 function inhibitor or promoter can be expected to act as a preventive or therapeutic agent for cancer. The present invention aims to develop a preventive / therapeutic agent for eye diseases and cancer, discovers a novel GRK7 homolog gene, and screens a compound that regulates the activity of a protein that is a gene product thereof, and a compound obtained by the screening method. And so on.

[0004]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found an mRNA whose expression is increased when heart failure develops in a myocardial infarction model rat by coronary artery ligation. . As a result of examining homology with a known gene or protein using the nucleotide sequence, a gene showing high homology with the mouse G protein signal regulator (hereinafter abbreviated as RGS. Gene Bank Accession No. U67188) from the nucleotide sequence. It turned out to be Furthermore, a group of genes forming a family of RGS genes has been reported (Chips,
20, 376-382 (1999)), it is known that these genes have a highly conserved RGS domain. Therefore, the existence of a very highly rigid motif was found from an alignment using amino acid sequences deduced from these RGS domains (Figs. 1 to 3). Therefore, an amino acid sequence deduced from human GRK4, which is one of the known RGS family genes having this RGS motif, was used to perform a search based on the amino acid sequence deduced from a novel DNA sequence database, and as a result, 48%
The amino acid sequence represented by SEQ ID NO: 1 having the homology of SEQ ID NO: 1 was found (human RGS No. 9). Found the base sequence represented by SEQ ID NO: 2 encoding the amino acid sequence,
The present invention has been completed.

That is, the present invention provides (1) SEQ ID NO: 1.
A protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by
(2) The protein according to the above (1) or a salt thereof, which contains the amino acid sequence represented by SEQ ID NO: 1, (3) a partial peptide of the protein according to the above (1) or a salt thereof,
(4) A polynucleotide containing a polynucleotide encoding the protein according to (1) above or a partial peptide thereof, (5) a polynucleotide according to (4) above which is DNA, and (6) represented by SEQ ID NO: 2. And (7) a recombinant vector containing the polynucleotide described in (4) above, (8) a recombinant vector containing the nucleotide sequence containing the nucleotide sequence (9) A transformant, (9) The transformant according to (8) above is cultured to produce the protein according to (1) above or a partial peptide thereof, (1) above.
(10) A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof, Of a compound or a salt thereof that regulates the activity of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Screening method, (11) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof is used as the amino acid sequence represented by SEQ ID NO: 1 Proteins containing identical or substantially identical amino acid sequences DN encoding the partial peptide
The screening method according to the above (10), which is expressed in the cytoplasm of a transformant transformed with the DNA containing A, and (12) the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 1. To a protein containing the same amino acid sequence or a partial peptide thereof or a salt thereof, 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: obtained by using a screening kit for a compound or a salt thereof that regulates the activity of a partial peptide or a salt thereof, (13) the screening method according to (10) above or the screening kit according to (12) above A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by 1. Click proteins or compounds or salts thereof that regulate its partial peptide or activity of their salts,
(14) A medicament comprising the compound according to (13) or a salt thereof, (15) a medicament according to (14), which is a prophylactic / therapeutic agent for heart disease, cancer or retinal disease, (16) SEQ ID NO: An antisense polynucleotide having a base sequence complementary to or substantially complementary to a DNA encoding a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 1: or a part thereof (17) A medicament comprising the antisense polynucleotide according to (16) above, (1
8) An antibody against a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, (19) the amino acid sequence represented by SEQ ID NO: 1 The antibody according to (18) above, which is a neutralizing antibody that neutralizes the activity of a protein containing the same or substantially the same amino acid sequence as described above, a partial peptide thereof, or a salt thereof.
(20) A medicine comprising the antibody according to (18) above,
(21) The diagnostic agent comprising the antibody according to (18) above, (22) the antibody according to (18) above, wherein the antibody or the partial peptide thereof or a salt thereof is used. (23) A method for diagnosing a disease associated with cardiac function, which comprises using the quantification method described in (22) above, (24) The compound described in (13) above or a salt thereof for mammals. (13) A method for preventing / treating a heart disease, cancer or retinal disease, which comprises administering an effective amount of And the use of the compound or salt thereof.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter also referred to as the protein of the present invention or the protein used in the present invention. ) Is a cell of a human or non-human warm-blooded animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) (eg, hepatocyte, splenocyte, nerve cell, glial cell, pancreas). 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) , B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, Bone cells, mammary cells, hepatocytes or stromal cells, or their precursor cells, stem cells or cancer cells) or any tissue in which these cells are present, for example, the brain, various parts of the brain (eg, olfactory bulb, tonsils) Nucleus, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, lung,
Digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen,
Submandibular gland, peripheral blood, prostate, testicles, ovaries, placenta, uterus,
It may be a protein derived from bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

The amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 6% of the amino acid sequence represented by SEQ ID NO: 1.
An amino acid sequence having a homology of 0% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 above. , A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable. Examples of substantially the same activity include cardiac function lowering activity and cell stimulating activity. The term “substantially the same” means that those properties are qualitatively (eg, physiologically or pharmacologically) the same. Therefore, the activity to lower cardiac function is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times).
However, quantitative factors such as the degree of activity and the molecular weight of the protein may be different. The activity such as cardiac function lowering activity and cell stimulating activity can be measured by a known method or a method analogous thereto. Cardiac function-decreasing activity can be measured, for example, by an echocardiography device (Cell, Vol. 97, pages 189-198, 1999) or cardiac catheter (circulation research, No. 1).
69, 370-377, 1991). Furthermore, for example, angiotensin I converting enzyme (ACE)
The renin angiotensin system (RAS) enhancement such as is measured using a commercially available measurement kit (eg, Peninsular, Phoenix, etc.), or blood catecholamine increasing activity (Tosoh, fully automatic catecholamine analyzer) ) Etc. can be used as an index to measure the activity. The cell stimulating activity is obtained by contacting the protein of the present invention with a substance (eg, ligand) that activates the protein of the present invention, and activating the protein of the protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ^{2 +} Release, intracellular c
AMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation,
c-fos activation, pH decrease, protein kinase A
Activity, protein kinase C activity, map kinase activity-promoting activity, etc.) are measured according to known methods. Examples of the ligand include angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, PACAP (eg, PACAP).
27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, G
HRH, CRF, ACTH, GRP, PTH, VIP
(Vasoactive intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily ( Example, IL-8, GROα, GROβ, GROγ, NA
P-2, ENA-78, GCP-2, PF4, IP-1
0, Mig, PBSF / SDF-1 and other CXC chemokine subfamilies; MCAF / MCP-1, MCP-
2, MCP-3, MCP-4, eotaxin, RAN
TES, MIP-1α, MIP-1β, HCC-1, M
IP-3α / LARC, MIP-3β / ELC, I-3
09, TARC, MIPF-1, MIPF-2 / eot
axin-2, MDC, DC-CK1 / PARC, SL
CC chemokine subfamily such as C; lympho
C chemokine subfamily such as tactin; fr
CX3C chemokine subfamily such as actalkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) or sphingosine 1-phosphate, etc. are used.

The protein used in the present invention is, for example, 1 or 2 or more (preferably about 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1.
Preferably about 1 to 10, more preferably a number (1 to
5) The amino acid sequence in which the amino acid is deleted, or 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably about 1 to 2 in the amino acid sequence represented by SEQ ID NO: 1. 1 or 2 or more (preferably 1 to 30 or so, preferably 1 to 10 or so) in the amino acid sequence represented by SEQ ID NO: 1, in which an amino acid number (1 to 5) is added. More preferably, an amino acid sequence in which several (1 to 5) amino acids are inserted,
1 or 2 in the amino acid sequence represented by SEQ ID NO: 1
1 or more (preferably about 1 to 30 and preferably 1 to 30)
A so-called mutein such as a protein containing an amino acid sequence in which about 10 amino acids, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence combining these is also included.

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

The partial peptide of the protein used in the present invention is a partial peptide of the above-mentioned protein used in the present invention, and preferably has the same properties as the above-mentioned protein used in the present invention. Any one will do. For example, at least 20 of the constituent amino acid sequences of the protein used in the present invention
Or more, preferably 50 or more, more preferably 70
Peptides containing an amino acid sequence of 1 or more, more preferably 100 or more, and most preferably 200 or more are used. The partial peptide used in the present invention has 1 or 2 or more amino acids in its amino acid sequence (preferably about 1 to 10 and more preferably about 1 to 5).
1 or 2 or more (preferably 1 to 20 or so, more preferably 1 to 10 or so, still more preferably a number (1 to 2)
5) amino acids are added, or 1 or 2 or more (preferably about 1 to 20 amino acids) is added to the amino acid sequence,
More preferably about 1 to 10, more preferably a number (1 to 5) amino acid is inserted, or 1 or 2 or more (preferably 1 to 1) in the amino acid sequence is inserted.
About 0, more preferably several, and further preferably 1 to
(About 5 amino acids) may be substituted with other amino acids, or a combination thereof may be used.

The partial peptide used in the present invention has a C-terminal carboxyl group (-COOH) or carboxylate (-C) as in the above-mentioned protein of the present invention.
OO ^-), amide (-CONH ₂₎ or ester (-C
OOR). Furthermore, in the partial peptide used in the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group, as in the above-mentioned protein used in the present invention, Glutamine residue produced by cleavage at the N-terminal side in vivo is pyroglutamine-oxidized, a substituent on the side chain of an amino acid in the molecule is protected by an appropriate protecting group, or a so-called sugar chain-bonded Complex peptides such as glycopeptides are also included. The partial peptide used in the present invention can also be used as an antigen for antibody production.

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

For the synthesis of the protein or partial peptide used in the present invention, a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl resin. Phenylacetamide methyl resin, polyacrylamide resin,
4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl)
Phenoxy resin, 4- (2 ', 4'-dimethoxyphenyl-Fm
oc aminoethyl) phenoxy resin and the like. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein. At the end of the reaction, the protein is cleaved from the resin, at the same time various protecting groups are removed, and an intramolecular disulfide bond-forming reaction is carried out in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. Regarding the condensation of the above-mentioned protected amino acids, various activation reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid was added directly to the resin together with a racemization-suppressing additive (eg, HOBt, HOOBt), or the protected amino acid was previously activated as the target acid anhydride or HOBt ester or HOOBt ester. It can later be added to the resin.

The solvent used for activation of the protected amino acid and condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol,
Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from the range known to be applicable to protein bond-forming reactions, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained by repeating the reaction, it is possible to acetylate the unreacted amino acid with acetic anhydride or acetylimidazole so that the subsequent reaction is not affected.

Examples of the protecting group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2
-Nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc and the like are used. The carboxyl group is
For example, alkyl esterification (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl,
Cyclohexyl, cycloheptyl, cyclooctyl, 2
-Straight-chain, branched or cyclic alkyl esterification such as adamantyl), aralkyl esterification (eg benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification) , Phenacyl esterification, benzyloxycarbonyl hydrazide formation, t-butoxycarbonyl hydrazide formation, trityl hydrazide formation and the like. The hydroxyl group of serine is
For example, it can be protected by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group. Used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, a t-butyl group and the like. Examples of the protective group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-
Nitrobenzyl, Br-Z, t-butyl and the like are used.
Examples of the protecting group for imidazole of histidine include:
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc
Are used.

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

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

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

The polynucleotide encoding the protein used in the present invention includes a nucleotide sequence (DNA or R) encoding the above-mentioned protein used in the present invention.
It may be any as long as it contains NA, preferably DNA). The polynucleotide may be DN that encodes the protein used in the present invention.
A, RNA such as mRNA, which may be double-stranded or single-stranded. In the case of double-stranded, double-stranded DNA,
It may be double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). Using the polynucleotide encoding the protein used in the present invention, the method described in the publicly known experimental medicine special number "New PCR and its application" 15 (7), 1997 or a method similar thereto, for example, TaqMan PCR or the like. The mRNA of the protein used in the present invention can be quantified. The DNA encoding the protein used in the present invention may be any DNA as long as it contains the nucleotide sequence encoding the protein used in the present invention. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the cells or tissues described above, cDNA library derived from the cells or tissues described above, or synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse RNA can be directly analyzed by using a total RNA or mRNA fraction prepared from the cells or tissues described above.
Transcriptase Polymerase Chain Reaction (hereinafter R
It can also be amplified by the T-PCR method). DNA encoding the protein used in the present invention
As the DNA containing, for example, DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 2
Any DNA can be used as long as it has a nucleotide sequence that hybridizes with the nucleotide sequence represented by the following under high stringent conditions and encodes a protein having substantially the same properties as the protein used in the present invention. Good.

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

The polynucleotide containing the DNA encoding the partial peptide used in the present invention may be any polynucleotide containing the base sequence encoding the partial peptide used in the present invention. .. In addition, genomic DNA, genomic DNA library,
It may be any of the above-mentioned cells or tissues-derived cDNA, the above-mentioned cells or tissues-derived cDNA library, or synthetic DNA. The polynucleotide containing the DNA encoding the partial peptide used in the present invention includes:
For example, D containing the base sequence represented by SEQ ID NO: 2
It has a DNA having a part of NA or a base sequence which hybridizes with the base sequence represented by SEQ ID NO: 2 under high stringent conditions and has substantially the same activity as the protein used in the present invention. For example, DNA containing a part of DNA encoding a protein is used. The DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 has the same meaning as described above. The same hybridization method and high stringent conditions as described above are used.

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

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

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. Examples of selection markers include dihydrofolate reductase (hereinafter, dhfr).
The gene [methotrexate (M
TX) resistance], ampicillin resistance gene (hereinafter referred to as Amp
^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r , G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells lacking the dhfr gene, the target gene can also be selected by a thymidine-free medium. In addition, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention, if necessary. When the host is a bacterium of the genus Escherichia, the PhoA signal sequence, OmpA signal sequence, etc., and when the host is a bacterium of the genus Bacillus, the α-amylase signal sequence, the subtilisin signal sequence, etc., the host is yeast. In some cases, the MFα signal sequence,
When the host is an animal cell such as SUC2 / signal sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used respectively. A transformant can be produced using the vector containing the polynucleotide 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 Escherichia bacteria include:
For example, Escherichia coli K1
2. DH1 [Proc. Natl. Acad. Sci. USA], 60
Vol. 160 (1968)], JM103 [Nukuirekku.
Acids Research, (Nucleic Acids Research), 9
Vol., 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecul
ar Biology)], 120, 517 (1978)], HB
101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like are used. Examples of the genus Bacillus include, for example, Bacillus subtilis MI114.
[Gene, 24, 255 (1983)], 207-21
[Journal of Biochemistry
Biochemistry), Vol. 95, 87 (1984)] and the like. Examples of yeast include Saccharomyces
Saccharomyces cerevisiae AH22, A
^{H22R -, NA87-11A, DKD-5D} , 20B
-12, Schizosaccharomyces pombe (Schizosaccha
romyces pombe) NCYC1913, NCYC203
6. Pichia pastoris KM71
Are used.

Examples of insect cells include virus A
In the case of cNPV, the cell line derived from the larva of Spodoptera frugiperda (Spod
optera frugiperda cell; Sf cell), Trichoplusia n
MG1 cells from the midgut of i, from the eggs of Trichoplusia ni
High Five ^TM cells, cells from Mamestra brassicae or
Cells derived from Estigmena acrea are used. When the virus is BmNPV, the silkworm-derived cell line (Bombyx
mori N cells; BmN cells) and the like are used. The Sf
Examples of the cells include Sf9 cells (ATCC CRL1711),
Sf21 cells (above Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used.
As the insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592.
(1985)]. Examples of animal cells include monkey cells C
OS-7, Vero, Chinese hamster cell CHO
(Hereinafter, abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dhf
r ^-) cell), mouse L cells, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells, H9c2 cells and the like are used. Transformation of Escherichia can be performed, for example, by Proc. Natl. Acad. Sci.
USA), Volume 69, 2110 (1972) and Gene (Gen)
e), Vol. 17, 107 (1982) and the like.

For transforming Bacillus, for example, Molecular & General Genetics, 168,
111 (1979) and the like. For transforming yeast, for example, Methods in Enzymolog
y), 194, 182-187 (1991), Procedures of the National Academy of Sciences of the USA (Proc. Na).
tl. Acad. Sci. USA), 75, 1929 (197).
It can be performed according to the method described in 8). Transformation of insect cells or insects can be performed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988). To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 263-267 (1995)
(Published by Shujunsha), Virology, Volume 52,
456 (1973). In this way, the DNA encoding the protein
It is possible to obtain a transformant transformed with an expression vector containing When the host is culturing a transformant that is a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source necessary for the growth of the transformant, A nitrogen source, an inorganic substance, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal and potato extract. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York1
972] is preferable. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter so as to work efficiently. When the host is a bacterium belonging to the genus Escherichia, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and stirring can be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder's minimum medium [Bostian, KL
Et al., Proc. Natl. Acad. Sci. USA, 77, 4505.
(1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Procedures of the National Academy of Sciences of
The USA (Proc. Natl. Acad. Sci. USA), 8
1, 5330 (1984)]. P of medium
H is preferably adjusted to about 5-8. Culture is usually about 2
It is carried out at 0 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added if necessary. When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Inse
ct Medium (Grace, TCC, Nature, 195,
788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added is used. The pH of the medium is about 6.
It is preferably adjusted to 2 to 6.4. Culture is usually about 27
It is performed for about 3 to 5 days at 0 ° C., and if necessary, aeration and stirring are added. When culturing a transformant whose host is an animal cell,
Examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum [Science, Volume 122, 5].
01 (1952)], DMEM medium [Virology (Viro
logy), Volume 8, 396 (1959)], RPMI 164
0 Medium [The Journal of the American Medical Association (The Journal of the American
Medical Association) 199, 519 (196
7)], 199 medium [Proceeding of the Society for the Biological Medi
cine), 73, 1 (1950)] and the like. p
H is preferably about 6-8. Culture is usually about 30 ℃
Perform at about -40 ° C for about 15-60 hours, adding aeration and agitation as needed. As described above, the transformant can be used to produce the protein of the present invention.

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

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

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

Examples of myeloma cells include NS-1,
Examples include myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000-PEG6000)
Is added at a concentration of about 10 to 80%, and 20 to 40 ° C.,
Cell fusion can be efficiently performed by preferably incubating at 30 to 37 ° C. for 1 to 10 minutes. Although various methods can be used for screening a monoclonal antibody-producing hybridoma, for example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed with a carrier, and then a radioactive substance or Anti-immunoglobulin antibody labeled with an enzyme (when the cells used for cell fusion are mice, anti-mouse immunoglobulin antibody is used) or protein A
And detecting the monoclonal antibody bound to the solid phase, adding the hybridoma culture supernatant to the solid phase adsorbing the anti-immunoglobulin antibody or protein A, adding the protein labeled with a radioactive substance or enzyme, Examples include a method of detecting a monoclonal antibody bound to a phase. Selection of the monoclonal antibody can be performed according to a known method or a method similar thereto. Normal HAT
(Hypoxanthine, aminopterin, thymidine) can be added to the medium for animal cells. As the medium for selection and breeding, any medium may be used as long as the hybridoma can grow. For example, 1-2
RP containing 0%, preferably 10-20% fetal bovine serum
MI 1640 medium, G containing 1-10% fetal bovine serum
IT medium (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of Monoclonal Antibody Monoclonal antibody can be separated and purified by a known method, for example, a method for separating and purifying immunoglobulin [eg salting out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, Adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen binding solid phase or active adsorbent such as protein A or protein G to collect only antibody and dissociate the binding Specific Purification Method to Obtain]].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, an immune antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-mentioned method for producing a monoclonal antibody. It can be produced by collecting the substance and separating and purifying the antibody. Regarding the complex of the immunizing antigen and the carrier protein used for immunizing warm-blooded animals, the type of carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is effective against the hapten immunized by crosslinking with the carrier. If possible, any kind may be cross-linked in any ratio, but, for example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. in a weight ratio of about 0.1 to 20, preferably about 1 to 1 hapten. ~ 5
A method of coupling at a ratio of is used. Although various condensing agents can be used for coupling the hapten and carrier, glutaraldehyde, carbodiimide, maleimide active ester, active ester reagent containing thiol group, dithiopyridyl group, etc. are used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent.
Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from the blood, ascites, etc., of the warm-blooded animal immunized by the above method, preferably from the blood. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the antiserum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

[0035] Complementary or substantially complementary to DNA encoding the protein or partial peptide used in the present invention (hereinafter, these DNAs are abbreviated as the DNA of the present invention in the description of antisense polynucleotides). As an antisense polynucleotide having a specific base sequence, it has a base sequence complementary to or substantially complementary to the DNA of the present invention or a part thereof, and has an action of suppressing the expression of the DNA. Any antisense polynucleotide may be used, but antisense DNA is preferred. The term “substantially complementary nucleotide sequence to the DNA of the present invention” refers to, for example, a total nucleotide sequence or a partial nucleotide sequence of a nucleotide sequence complementary to the DNA of the present invention (that is, a complementary strand of the DNA of the present invention) and about 70 nucleotides. % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homologous base sequences. In particular, of the total nucleotide sequence of the complementary strand of the DNA of the present invention, about 70% or more with the complementary strand of the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the nucleotide sequence near the start codon), Preferably about 80% or more, more preferably about 90% or more, most preferably about 9%.
Antisense polynucleotides with 5% or greater homology are preferred. The antisense polynucleotide is usually composed of about 10 to 40 bases, 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 changed to, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, or phosphorodithionate. It may be substituted. These antisense polynucleotides can be produced using a known DNA synthesizer or the like. According to the present invention, an antisense gene capable of inhibiting the replication or expression of the protein gene of the present invention.
A polynucleotide (nucleic acid) can be designed and synthesized based on the nucleotide sequence information of a DNA encoding a cloned or determined protein. Such a polynucleotide (nucleic acid) can hybridize with RNA of the protein gene of the present invention, can inhibit the synthesis or function of the RNA, or can interact with the protein-related RNA of the present invention. The expression of the protein gene of the present invention can be regulated / controlled. A polynucleotide complementary to a selected sequence of a protein-related RNA of the invention, and a protein-related RN of the invention
The polynucleotide capable of specifically hybridizing with A is useful for regulating and controlling the expression of the protein gene of the present invention in vivo and in vitro, 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, a nucleotide sequence or a nucleic acid and a peptide (protein) usually refers to an amino acid of a peptide (protein) in a command derived from the nucleotide (nucleic acid) sequence or its complement. . 5'end hairpin loop of protein gene, 5'end 6-base pair repeat, 5'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3'end untranslated region,
The 3'-end palindromic region and the 3'-end hairpin loop can be selected as the preferred region of interest, but any region within the protein gene can be selected. It can be said that the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, and the relationship between the polynucleotide capable of hybridizing with the target region are “antisense”. Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Alternatively, other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that such polymers are found in DNA or RNA. Containing a nucleotide having a configuration that allows pairing of bases and attachment of bases) and the like. They are double-stranded DNA, single-stranded DNA, double-stranded R
NA, single-stranded RNA, DNA: RNA hybrid, unmodified polynucleotide (or unmodified oligonucleotide), and known modification can be added, for example, a label known in the art. , Capped, methylated, substituted with one or more natural nucleotides by analogs, modified intramolecularly with nucleotides such as uncharged bonds (eg methylphosphonate, With phosphotriesters, phosphoramidates, carbamates, etc., charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.)
Having a side chain group such as a protein (for example, nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or sugar (for example, monosaccharide), Interactive compounds (eg, acridine,
Those having psoralen, etc., chelate compounds (eg,
Metal, radioactive metal, boron, oxidizing metal, etc.), alkylating agent, modified bond (eg, α-anomer type nucleic acid, etc.) Good. As used herein, the term "nucleoside", "nucleotide" and "nucleic acid" may include those having not only purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications include methylated purines and pyrimidines,
It may include acylated purines and pyrimidines, or other heterocycles. The modified nucleotide and modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen, an aliphatic group, or a functional group such as ether or amine. It may have been converted. The antisense polynucleotide (nucleic acid) of the present invention has R
NA, DNA, or modified nucleic acid (RNA, DN
A). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, it makes the antisense nucleic acid more stable in the cell, increases the cell permeability of the antisense nucleic acid, increases the affinity for the target sense strand, and Make sense nucleic acid less toxic. Many such modifications are known in the art, e.g.
Kawakami et al., Pharm Tech Japan, Vol. 8, pp.247,
1992; Vol. 8, pp. 395, 1992; ST Crooke et al. E
d., Antisense Research and Applications, CRC Pres
s, 1993 etc. The antisense nucleic acid of the present invention may contain altered or modified sugars, bases, linkages, provided in a special form such as liposomes and microspheres, or applied by gene therapy, It can be provided in added form.
Thus, as an additive form, polycationic substances such as polylysine that act to neutralize the charge of the phosphate group, lipids that enhance the interaction with cell membranes and increase the uptake of nucleic acids ( Examples thereof include hydrophobic ones such as phospholipids and cholesterol). Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These things are
Can be attached to the 3'or 5'end of the nucleic acid,
It may be attached via a base, a sugar or an intramolecular nucleoside bond. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of nucleic acid,
Examples thereof include those for preventing decomposition by nucleases such as exonuclease and RNase. Examples of the group for such a cap include protective groups for hydroxyl groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol.
It is not limited to that. The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various known methods.

The proteins or partial peptides or salts thereof used in the present invention (hereinafter sometimes abbreviated as the protein of the present invention), DNAs encoding the proteins or partial peptides of the present invention (hereinafter, the present invention) May be abbreviated as "DNA" of the present invention), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as "antibody of the present invention"), and an antisense polynucleotide of the DNA of the present invention (hereinafter , May be abbreviated as the antisense polynucleotide of the present invention).

Since the expression of the protein of the present invention varies in ophthalmic diseases associated with reduced visual acuity, retinocytoma, and various cancers, it can be used as a disease marker. The protein of the present invention is, for example, a heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer,
Non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vascular occlusion) ,
Ischemic retinal disease, retinal vitreous disease, eye tumor, cataract,
For early diagnosis of glaucoma, retinal detachment, retinal vitreous hyperplasia, retinitis, retinolipidemia, branch retinal vein occlusion, retinoblastoma, etc., determination of severity of symptoms, prediction of disease progression Is useful as a marker. In addition, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, by administration of an antisense polynucleotide of the gene encoding the protein of the present invention,
Angina), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, Thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vascular occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retinal vitreous hyperplasia, retinitis, retina Since lipemia, branch retinal vein occlusion, retinoblastoma, etc. are suppressed, a compound or a salt thereof that regulates the activity of the protein of the present invention or an antibody against the protein of the present invention also exhibits the same action. it is conceivable that. Therefore, a drug containing an antisense polynucleotide of a gene encoding the protein of the present invention, a compound or a salt thereof that regulates the activity of the protein of the present invention, or an antibody against the protein of the present invention is, for example, a heart disease (eg, myocardium Disease, myocardial infarction, heart failure, angina, etc., cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer) , Rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vessel occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retinal vitreous) It can be used as a therapeutic / preventive agent for proliferative diseases, retinitis, retinolipidemia, branch retinal vein occlusion, retinoblastoma, etc.).

[1] Screening for Drug Candidate Compounds against Diseases Since the expression of the protein of the present invention varies in ophthalmic diseases accompanied by reduced visual acuity, retinal cell tumor, and various cancers, a compound or its compound that regulates the activity of the protein of the present invention Salt is, for example, a heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.),
Cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc.), retina Diseases (eg, diabetic retinopathy, retinal vascular occlusion, ischemic retinal disease, retinal vitreous disease, ocular tumor, cataract, glaucoma, retinal detachment, vitreoretinal hyperplasia, retinitis, retinal lipemia, retinal vein) It can be used as a therapeutic / preventive drug for branched occlusion, retinoblastoma, etc.). Therefore, the protein of the present invention is useful as a reagent for screening a compound or its salt that regulates the activity of the protein of the present invention.

That is, the present invention provides (1) a compound which regulates the activity of the protein of the present invention, which comprises using the protein of the present invention, such as a compound which regulates cell stimulating activity, cardiac function lowering activity or the like. Provided is a method for screening a salt thereof (hereinafter, sometimes abbreviated as a modulator). Specifically, for example, (2) (i) a cell having the ability to produce the protein of the present invention when stretch stimulus is added under hypoxic conditions, and (ii) a cell having the ability to produce the protein of the present invention And a mixture of a test compound and a compound to which a stretch stimulus is applied under hypoxic conditions, to provide a method for screening a compound or a salt thereof that regulates the activity of the protein of the present invention. More specifically, in the above screening method, for example,
In the cases (i) and (ii), the expression level of the gene of the protein of the present invention is measured and compared. Here, the low oxygen condition means, for example, an oxygen concentration of 20% O ₂ or less and 2% (Nature, Vol. 394, pages 485-490, 1998). The stretch stimulus is a stimulus in which a cardiomyocyte is cultured on a stretchable silicon film and a mechanical load is applied by pulling the silicon film (JBC, Vol. 271, 335).
92-33597, 1996, Circulation, Vol. 89, 22
04-2211, 1994, JBC, Vol 271, 3221-3228, 1
996). The gene expression level of the present invention can be determined by a known method, for example, Northern blotting or Reverse transcription-
polymerase chain reaction (RT-PCR) and real-time PCR analysis system (ABI, TaqMan polymerase
It can be measured according to a method such as chain reaction) or a method similar thereto. For example, above (i
A test compound which inhibits or promotes the gene expression in the case of i) by about 20% or more, preferably 30% or more, more preferably about 50% or more compared with the case of the above (i) is used as a test compound of the protein of the present invention. It can be selected as a compound that inhibits or promotes the activity.

Further, (3) (iii) when cells having the ability to produce the protein of the present invention or cells into which cDNA encoding the protein of the present invention has been introduced are cultured under lethal conditions, And (iv) cells that have the ability to produce the protein of the present invention or cDNA that encodes the protein of the present invention, when cultured in the absence of serum or in the addition of an anticancer agent such as adriamycin to cardiomyocytes When the cultured cells and a mixture of test compounds were cultured under lethal conditions, specifically, comparison with the case of culturing under serum removal or addition of an anticancer agent such as adriamycin, which has relatively high toxicity, to cardiomyocytes A method for screening a regulatory drug is used which is characterized in that The screening method is characterized in that, for example, in the cases of (iii) and (iv), the cytoprotective effect and the expression level of the gene of the protein of the present invention are measured and compared. The expression level of the gene of the present invention can be determined by a known method, for example, Northern blotting or Reverse transcription-polymerase chain reaction.
(RT-PCR) and a real-time PCR analysis system (ABI, TaqMan polymerase chain reaction) or the like or a method similar thereto can be used for the measurement. The cytoprotective effect can be shown by the activation or survival rate of cardiomyocytes. Specifically, MTT (3- (4,
5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazoliu
m) Assay method, trypan blue staining method, TUNNEL staining method (Terminal deoxytransferase-mediated dUTP-X ni
ck end labeling, Cell, Vol. 97, pp. 189-198, 1999)
It can be measured with. For example, a test compound that inhibits or promotes the gene expression in the case of the above (iv) by about 20% or more, preferably by 30% or more, more preferably by about 50% or more, as compared with the case of the above (iii), is used. It can be selected as a compound that inhibits or promotes the activity of the protein of the present invention.

The function of the protein of the present invention is similar to that of the RGS protein because of its structural similarity.
That is, GT of G protein possessed by RGS protein
Activity to activate Pase (JBC, Vol. 272, 8679-8)
685 pages, 1997). It is known that GTPase activity of G protein promotes dissociation of G protein subunits and suppresses signal transduction mediated by G protein. In heart failure, it is considered that the pathology of heart failure is exacerbated by abnormality of G protein signal transduction mechanism such as desensitization of β receptor. Therefore, it is considered that the enhancement of expression of RGS-like gene found this time is involved in exacerbation of heart failure. . Therefore, in the present invention, a signal transducing substance (eg, cAMP, calcium) derived from a G protein by ligand stimulation is used by using a cell having the ability to produce the protein of the present invention or a cell into which a cDNA encoding the protein of the present invention is introduced. Etc.) is used as an index to provide a screening system.

Therefore, according to the present invention, for example, (4) (v)
When the cell containing the protein of the present invention is contacted with a substance that activates the protein of the present invention (eg, a ligand), (vi) the cell of the present invention is added to the cell containing the protein of the present invention. Cell-stimulating activity of the protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP) when brought into contact with a substance to be activated (eg, ligand) and a test compound
Production, intracellular cGMP production, inositol phosphate production,
Cell membrane potential fluctuation, intracellular protein phosphorylation, cf
os activation, pH reduction, protein kinase A activity,
Also provided is a method for screening a modulator, which comprises comparing protein kinase C activity, map kinase activity-promoting activity or suppressing activity). Examples of the ligand include angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxytocin, P
ACAP (eg, PACAP27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH,
GRP, PTH, VIP (vasoactive intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatatin, prostaglandin, thromboxane, adenosine, adrenaline. , Chemokine superfamily (eg, IL-8, GROα, GRO
β, GROγ, NAP-2, ENA-78, GCP-
2, PF4, IP-10, Mig, PBSF / SDF-
CXC chemokine subfamily such as 1; MCAF /
MCP-1, MCP-2, MCP-3, MCP-4, e
Otaxin, RANTES, MIP-1α, MIP-
1β, HCC-1, MIP-3α / LARC, MIP-
3β / ELC, I-309, TARC, MIPF-1,
MIPF-2 / eotaxin-2, MDC, DC-C
CC chemokine subfamily such as K1 / PARC and SLC; C chemokine subfamily such as lymphoactin; CX3 such as fractalkine
C chemokine subfamily, etc.), endothelin, enterogastrin, histamine, neurotensin, TR
H, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) or sphingosine 1
-Phosphoric acid or the like is used. The cell stimulating activity can be measured using a known method or a commercially available measurement kit.

As a cell containing the protein of the present invention
For culturing a transformant containing the DNA of the present invention.
The receptor protein of the present invention expressed on the cell membrane by
The protein, the cell membrane fraction of the protein of the present invention, etc.
Be done. When using cells containing the protein of the present invention
Cells, fix the cells with glutaraldehyde, formalin, etc.
May be standardized. The immobilization method is performed according to a known method.
be able to. A cell containing the protein of the present invention
For example, a host cell expressing the protein may be used.
The host cells include Escherichia coli, Bacillus subtilis, yeast, and insects.
Cells, animal cells and the like are preferred. Preferably H9c2 fine
Cells. As the cell membrane fraction, the cells were disrupted
Then, a fraction containing a large amount of cell membrane obtained by a known method
Say that. As a cell disruption method, Potter-Elvehj
How to crush cells with em type homogenizer, waring
Break by blender or polytron (Kinematica)
Do not crush, crush with ultrasonic waves, press with a French press, etc.
Do not crush the waste cells by ejecting them from a thin nozzle.
Which can be mentioned. Fractionation centrifugation or
Fractionation method by centrifugal force such as density gradient centrifugation method is mainly
Used. For example, the cell lysate should be slow (500 rp).
m to 3000 rpm) for a short time (usually about 1 minute to 10 minutes)
Centrifuge, and the supernatant is spun at a higher speed (15000 rpm to 3
It is usually obtained by centrifugation at 0000 rpm for 30 minutes to 2 hours.
The precipitate is used as the membrane fraction. In the membrane fraction, the expressed
Phospholipids and membrane proteins derived from cell-derived proteins
It contains a lot of film components such as quality. Contains the protein
The amount of protein in cells or membrane fraction is 10
³-10⁸Preferably it is a molecule, 10^Five-10⁷In the molecule
Preferably. The higher the expression level, the more the membrane fraction
Higher sensitivity due to higher ligand binding activity (specific activity)
Not only is it possible to construct a sophisticated screening system,
It becomes possible to measure a large number of samples in the same lot. test
Examples of the compound include peptides, proteins and non-peptides
Compounds, synthetic compounds, fermentation products, cell extracts, plants
Extracts, animal tissue extracts, etc. are used and these compounds
May be a novel compound or a known compound.
May be. To carry out the above screening method,
The cells capable of producing the protein of the present invention are screened.
Culture using a medium suitable for leaning. The medium is a book
That does not affect the gene expression of the protein of the invention
Any one will do. Produces a protein of the invention
A cell having the ability is, for example, the protein of the present invention.
Primary cardiomyocytes with or without the ability to produce protein
Contains the DNA encoding the protein of the present invention described above
Used by a host (transformant) transformed with the vector
To be The host may be, for example, an animal such as H9c2 cell.
Product cells are preferably used. The screening includes
For example, by culturing by the method described above,
A transformant expressing the protein intracellularly is preferable.
Used.

For example, the cell stimulating activity in the case of (vi) above is about 20% or more as compared with the case of (v) above.
A test compound that inhibits or promotes preferably 30% or more, more preferably about 50% or more can be selected as a compound that inhibits or promotes the activity of the protein of the present invention. The compound or salt thereof obtained by the above-mentioned screening is, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer,
Liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retina) Vascular occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retinal vitreous hyperplasia, retinitis, retinolipidemia, branch retinal vein occlusion, retinoblastoma Etc.) and can be used as a medicine such as a safe and therapeutic drug with low toxicity. The screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell having the ability to produce the protein or partial peptide used in the present invention.

The compound or its salt obtained by using the screening method or the screening kit of the present invention is a test compound described above, for example, a peptide, a protein, a non-peptidic compound of biological origin (eg, carbohydrate, lipid, etc.), A compound or a salt thereof selected from a synthetic compound, a microbial culture, a fermentation product, a cell extract, a plant extract, an animal tissue extract, plasma, etc., and the activity of the protein of the present invention (cardiac function lowering activity, cell A compound or a salt thereof that regulates (promotes or inhibits) stimulatory activity and the like. As the salt of the compound, those similar to the salts of the protein of the present invention described above are used. The compound or its salt that regulates (promotes or inhibits) the activity of the protein of the present invention is, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer, Liver cancer, non-small cell lung cancer,
Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer,
Colon cancer, rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vessel occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retina Treatment for vitreous hyperplasia, retinitis, retinal lipemia, branch retinal vein occlusion, retinoblastoma, etc.
It is useful as a medicine such as a preventive agent.

When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic / prophylactic agent, it can be formulated according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be used. Since the preparation thus obtained is safe and has low toxicity, for example, humans or warm-blooded animals (for example, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Orally or parenterally. The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route, etc., but for example, a compound or a salt thereof that modulates the activity of the protein of the present invention is orally administered for the purpose of treating heart failure. When administered, it is generally adult (with a body weight of 60 kg)
In the above, the amount of the compound or salt thereof is about 0.
1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg is administered. When administered parenterally, the single dose of the compound or its salt varies depending on the administration subject, target disease, etc., but for example, a compound or its compound that regulates the activity of the protein of the present invention for the purpose of treating heart failure. Adult salt (60 kg) in the form of salt
As a compound), about 0.01 to 30 mg, preferably about 0.1 to 30 mg of the compound or a salt thereof per day is administered.
It is convenient to administer about -20 mg, more preferably about 0.1-10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

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

In addition to quantifying the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like can also be performed. . For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used. The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may include an antibody, an antigen or an antibody-antigen complex corresponding to the amount of antigen (for example, the amount of protein) in the liquid to be measured. Any measuring method may be used as long as it is a method of detecting the amount by chemical or physical means and calculating it from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used, but from the viewpoint of sensitivity and specificity, the sandwich method described later is particularly preferably used. As the labeling agent used in the measuring method using the labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, etc. are used. Examples of radioisotopes include, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴
C] or the like is used. As the above-mentioned enzyme, those having a stable and large specific activity are preferable, and for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. Luminol, a luminol derivative, luciferin, lucigenin, etc. are used as a luminescent substance, for example. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing or immobilizing proteins or enzymes. Examples of the carrier include insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicon, and glass. In the sandwich method, a test solution is reacted with the insolubilized monoclonal antibody of the present invention (first reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. The amount of the protein of the present invention in the test liquid can be quantified by measuring the activity of the labeling agent. The primary reaction and the secondary reaction may be carried out in the reverse order, may be carried out simultaneously, or may be carried out at different times. The labeling agent and the method of insolubilization can be the same as those described above. In addition, in the immunoassay method by the sandwich method,
The antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies may be used for the purpose of improving the measurement sensitivity. In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibodies of the present invention used in the primary reaction and the secondary reaction are preferably antibodies having different binding sites for the protein of the present invention. That is, when the antibody used in the secondary reaction recognizes the C-terminal portion of the protein of the present invention, the antibody used in the primary reaction and the secondary reaction is preferably an antibody used in the primary reaction. An antibody that recognizes an N-terminal other than the C-terminal is used.

The monoclonal antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competitive method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen is reacted.
(F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), the labeled amount of either B or F is measured, and the amount of antigen in the test liquid is quantified. In this reaction method, a soluble antibody is used as the antibody, a liquid phase method using polyethylene glycol, a second antibody against the antibody is used for B / F separation, and a solid phase antibody is used as the first antibody, or The first antibody is soluble and the second antibody is a solid-phased antibody. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are competitively reacted with a fixed amount of the labeled antibody and then the solid phase and the liquid phase are separated, or the antigen in the test liquid is separated. After reacting with an excess amount of the labeled antibody and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated.
Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the quantification method of the present invention, it is not necessary to set special conditions, operations and the like. The protein measurement system of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like. For example, Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. "Enzyme Immunoassay" (Medical Shoin, Showa Showa) 1993), Eiji Ishikawa et al., "Enzyme Immunoassay" (second edition) (Medical Shoin, published in 1982), Eiji Ishikawa, et al. "Enzyme Immunoassay" (3rd edition) (Medicine Shoin, Showa) 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunochem
ical Techniques (Part A)), ibid Vol. 73 (Immunochem
ical Techniques (Part B)), ibid Vol. 74 (Immunochem
ical Techniques (Part C)), ibid Vol. 84 (Immunochem
ical Techniques (Part D: Selected Immunoassays)),
Ibid Vol. 92 (Immunochemical Techniques (Part E: Mono
clonal Antibodies and General Immunoassay Method
s)), ibid Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodie
s)) (above, published by Academic Press). As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, by quantifying the concentration of the protein of the present invention using the antibody of the present invention, (1)
When an increase in the concentration of the protein of the present invention is detected, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell) Lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vascular occlusion, ischemic) Diseases such as retinal disease, retinal vitreous disease, ocular tumor, cataract, glaucoma, retinal detachment, retinal vitreous hyperplasia, retinitis, retinolipidemia, branch retinal vein occlusion, retinoblastoma) is there,
Alternatively, it 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 body fluid or tissue. In addition, for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction during purification, analysis of the behavior of the protein of the present invention in a test cell, etc. Can be used.

[3] Gene Diagnostic Agent The DNA of the present invention can be used as a probe, for example, to prepare a human or non-human warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, It is possible to detect an abnormality (gene abnormality) of DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in a horse, cat, dog, monkey, chimpanzee, etc.). It is useful as a gene diagnostic agent for mutation or decreased expression, increased DNA or mRNA, or excessive expression. The above-mentioned gene diagnosis using the DNA of the present invention is carried out, for example, by known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989).
,), Proceedings of the National Academy of Sciences o
f the United States of America), Vol. 86, 276
6 to 2770 (1989)) and the like. For example, when overexpression is detected by Northern hybridization or when a DNA mutation is detected by PCR-SSCP method, for example,
Heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer,
Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer,
Colon cancer, rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vessel occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retina It can be diagnosed as being likely to be a disease such as vitreous hyperplasia, retinitis, retinolipidemia, branch retinal vein occlusion, and retinoblastoma).

[4] Medicament Containing Antisense Polynucleotide The antisense polynucleotide of the present invention capable of binding to the DNA of the present invention in a complementary manner and suppressing the expression of the DNA has low toxicity, Since it is possible to regulate (inhibit) the function of the protein of the present invention or the DNA of the present invention (such as cardiac function lowering activity and cell stimulating activity) in
For example, diseases associated with organ dysfunction, heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer,
Kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retina) Disease, retinal vascular occlusion, ischemic retinal disease, retinal vitreous disease, ocular tumor, cataract, glaucoma, retinal detachment, retinal vitreous hyperplasia, retinitis, retinolipidemia, branch retinal vein occlusion, retina Blastoma, etc.) and the like. The above antisense polynucleotide is treated according to the above
When used as a prophylactic agent, it can be formulated and administered according to a known method. For example, when using the antisense polynucleotide, after inserting the antisense polynucleotide alone or in a suitable vector such as retrovirus vector, adenovirus vector, adenovirus associated virus vector,
It can be orally or parenterally administered to a human or non-human mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) according to a conventional method. The antisense polynucleotide can be administered as it is or in the form of a preparation with a physiologically acceptable carrier such as an auxiliary agent for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. The dose of the antisense polynucleotide varies depending on the target disease, administration subject, administration route, etc.,
For example, when the antisense polynucleotide of the present invention is orally administered for the purpose of treating heart failure, about 0.1 to 100 mg of the antisense polynucleotide is generally administered to an adult (body weight 60 kg) per day. Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA of the present invention or the expression state thereof in tissues or cells.

[5] Pharmaceutical containing the antibody of the present invention The antibody of the present invention having an action of neutralizing the activity of the protein of the present invention is, for example, a heart disease (eg, cardiomyopathy, myocardial infarction,
Heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer,
Breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vascular occlusion,
Ischemic retinal disease, retinal vitreous disease, eye tumor, cataract,
It can be used as a prophylactic / therapeutic agent for glaucoma, retinal detachment, retinal vitreous hyperplasia, retinitis, retinolipidemia, retinal vein occlusion, retinoblastoma, etc.). The prophylactic / therapeutic agent for the above diseases containing the antibody of the present invention has low toxicity, and as a liquid formulation as it is or as a pharmaceutical composition in a suitable dosage form, a human or non-human mammal (eg, rat,
Rabbit, sheep, pig, cow, cat, dog, monkey, etc.)
Can be administered orally or parenterally. The dose varies depending on the administration subject, target disease, symptom, administration route, etc., but when it is used for the treatment / prevention of heart failure in adults, for example, the antibody of the present invention is usually used as a single dose. 0.01 to 20 mg / kg body weight, preferably 0.1 to 10 mg / kg body weight, more preferably 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 to 3 times a day. It is convenient to administer by intravenous injection once or twice. In the case of other parenteral administration and oral administration, an amount similar to this can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a composition is provided as a dosage form suitable for oral or parenteral administration. That is, for example, as a composition for oral administration, a solid or liquid dosage form, specifically, tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (soft capsules Including), syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, etc. Dosage forms of Such an injection is prepared according to a known method, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like are used. Suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg polysorbate 80,
HCO-50 (polyoxyethylene (50mol) adduct of
hydrogenatedcastor oil)] and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is usually filled in a suitable ampoule. The suppository used for rectal administration is prepared by mixing the above antibody or a salt thereof with an ordinary suppository base. The oral or parenteral pharmaceutical compositions described above are conveniently prepared in dosage unit form for administration of the active ingredient.
Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., usually 5 to 500 mg per dosage unit dosage form, especially 5 to 100 mg for injections, 10-2 for other dosage forms
It is preferable to contain 50 mg of the above antibody.
Each composition described above may contain other active ingredients as long as the composition does not cause an unfavorable interaction with the antibody.

[6] DNA-Introduced Animal The present invention provides D which encodes an exogenous protein of the present invention.
Provided is a non-human mammal having NA (hereinafter abbreviated as exogenous DNA of the present invention) or a mutant DNA thereof (may be abbreviated as exogenous mutant DNA of the present invention). That is, the present invention provides (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, (2) an animal according to (1), wherein the non-human mammal is a rodent, (3) (2) An animal according to (2), wherein the rodent is a mouse or rat; and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof, which can be expressed in a mammal. . A non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof (hereinafter, abbreviated as the DNA-introduced animal of the present invention) is used for embryo cells such as unfertilized eggs, fertilized eggs, sperms and their progenitor cells. Preferably, at the stage of embryogenesis in development of non-human mammals (more preferably at the stage of single cells or fertilized egg cells and generally before the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, agglutination method, Micro injection method, particle gun method, DEA
It can be produced by introducing the target DNA by the E-dextran method or the like. Also, the DNA
The foreign DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells and the like by the introduction method and used for cell culture, tissue culture and the like. It is also possible to produce the DNA-introduced animal of the present invention by fusing it with a known cell fusion method.

Non-human mammals include, for example, bovine,
Pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Among them, rodents, particularly mice (for example, C57B as a pure line), which have relatively short ontogeny and biological cycle from the viewpoint of creating a pathological animal model system and are easy to reproduce.
B6C3 as a hybrid system such as L / 6 line, DBA2 line, etc.
F1 system, BDF1 system, B6D2F1 system, BALB
/ C strain, ICR strain, etc.) or rat (eg, Wi
(star, SD, etc.) and the like are preferable. Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.
The exogenous DNA of the present invention does not mean the DNA of the present invention originally possessed by the non-human mammal, but the DNA of the present invention once isolated and extracted from the mammal. The mutant DNA of the present invention is a DNA having a mutation (eg, mutation) in the base sequence of the original DNA of the present invention, specifically, addition of base, deletion, substitution with other base, etc. The generated DNA or the like is used, and abnormal DNA is also included. The abnormal DN
As A, D which expresses the abnormal protein of the present invention
It means NA, and for example, DNA or the like that expresses a polypeptide that suppresses the normal function of the protein of the present invention is used. The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. When the DNA of the present invention is introduced into a target animal, it is generally advantageous to use the DNA as a DNA construct in which the DNA is linked downstream of a promoter that can be expressed in animal cells. For example, when the human DNA of the present invention is introduced, it expresses DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology with the human DNA. D of the present invention is obtained by microinjecting a DNA construct (eg, a vector, etc.) in which the human DNA of the present invention is bound to the downstream of various promoters that can be allowed, into a fertilized egg of a target mammal, for example, a mouse fertilized egg.
A DNA-introduced mammal that highly expresses NA can be produced.

Examples of the expression vector for the protein of the present invention include Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage such as λ phage, retrovirus such as Moloney leukemia virus, vaccinia virus or baculovirus. The animal viruses and the like are used. Among them, Escherichia coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, and the like are preferably used. Examples of the above promoter for controlling DNA expression include promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), various mammals (human , Rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase. , Platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, tartaric acid Anti alkaline phosphatase, atrial natriuretic factor (abbreviated as generally Tie2) endothelial receptor tyrosine kinases, sodium potassium adenosine triphosphatase (Na, K-ATPase), neurofilament light chain, metallothioneins I and IIA,
Metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TP
O), polypeptide chain elongation factor 1α (EF-1α), β
Actin, α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, T
Promoters such as hy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A and vasopressin are used. Among them, cytomegalovirus promoter, human polypeptide chain elongation factor 1α, which can be highly expressed systemically
(EF-1α) promoter, human and chicken β
The actin promoter and the like are preferable.

It is preferable that the vector has a sequence (generally called terminator) that terminates the transcription of the target mRNA in a DNA-transferred mammal. Sequences can be used, and preferably the SV40 terminator of simian virus and the like are used.
In addition, the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. 5'upstream of the promoter region, between the promoter region and the translation region or in the translation region for the purpose of further highly expressing the desired foreign DNA. Depending on the purpose, it is possible to connect the 3'downstream thereof. The translation region can be prepared as a DNA construct that can be expressed in a DNA-introduced animal by a conventional DNA engineering method in which it is linked to the downstream of the promoter and, if desired, the upstream of the transcription termination site. The introduction of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of DNA means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of its germinal cells and somatic cells. means. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germ cells and somatic cells.

It is confirmed that the non-human mammal introduced with the exogenous normal DNA of the present invention stably retains the exogenous DNA by mating, and subcultured in a normal breeding environment as the DNA-carrying animal. Can be done. The introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured so as to be present in excess in all the germinal cells and somatic cells of the target mammal. The excessive presence of the exogenous DNA of the present invention in the germinal cells of the producing animal after the introduction of the DNA means that all the progeny of the producing animal have the exogenous DNA of the present invention in all of their germinal cells and somatic cells. Means The offspring of this type of animal that inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in excess in all of their germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all offspring can be passaged so that the DNA has an excess. The non-human mammal having the normal DNA of the present invention is highly expressed with the normal DNA of the present invention, and by promoting the function of the endogenous normal DNA, finally, the hyperactivity of the protein of the present invention is suppressed. It may develop, and can be used as a model animal for its pathological condition. For example, using the normal DNA-introduced animal of the present invention, it is possible to elucidate the pathological mechanism of hyperfunction of the protein of the present invention and diseases related to the protein of the present invention, and to investigate treatment methods for these diseases. It is possible.
In addition, since the mammal into which the exogenous normal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used for screening tests for therapeutic agents for diseases related to the protein of the present invention. .

On the other hand, the non-human mammal having an exogenous abnormal DNA of the present invention should be subcultured as a DNA-carrying animal in a normal breeding environment after confirming that the exogenous DNA is stably retained by mating. Can be done. Furthermore, the foreign DNA of interest can be incorporated into the above-mentioned plasmid and used as a protozoa. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. Abnormality D of the present invention at the fertilized egg cell stage
The introduction of NA is ensured to be present in all germinal and somatic cells of the subject mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the producing animal after DNA transfer means that all the progeny of the producing animal have the abnormal DNA of the present invention in all of its germinal cells and somatic cells. The offspring of this type of animal that inherits the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the progeny can be passaged so that they have the DNA. The non-human mammal having the abnormal DNA of the present invention is highly expressed with the abnormal DNA of the present invention, and finally inhibits the function of the endogenous normal DNA, thereby finally deactivating the protein of the present invention. It may become refractory and can be used as a model animal for its pathological condition. For example, using the abnormal DNA-introduced animal of the present invention, it is possible to elucidate the pathological mechanism of the functionally inactive refractory disease of the protein of the present invention and to examine the treatment method for this disease. Further, as a specific applicability, the animal with high expression of abnormal DNA of the present invention is characterized by inhibition of normal protein function (dominant negat) by the abnormal protein of the present invention in functionally inactive refractory disease of the protein of the present invention.
ive action) becomes a model to elucidate. In addition, since the mammal into which the foreign abnormal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used in a therapeutic drug screening test for functionally inactive refractory disease of the protein of the present invention. is there.

Further, other uses of the above-mentioned two kinds of the DNA-introduced animal of the present invention include, for example, use as a cell source for tissue culture, DNA or R in tissue of the DNA-introduced animal of the present invention.
Whether NA is compared with that of non-introduced animals (control group)
Alternatively, analysis of genes or proteins specifically expressed, activated or inactivated by the protein of the present invention by comparative analysis of protein composition, cells of a tissue having DNA are cultured by a standard tissue culture technique, and It is generally used to study the function of cells from tissues that are difficult to culture, to screen for drugs that enhance cell function by using the cells described above, and to isolate and purify the mutant protein of the present invention and to produce its antibody. Etc. are possible. Furthermore, using the DNA-introduced animal of the present invention, it is possible to investigate the clinical symptoms of diseases related to the protein of the present invention, including functionally inactive refractory diseases of the protein of the present invention,
Further, more detailed pathological findings in each organ of the disease model related to the protein of the present invention can be obtained, which contributes to the development of new therapeutic methods, and further to the research and treatment of secondary diseases caused by the diseases. You can Further, each organ is taken out from the DNA-introduced animal of the present invention, and after being minced, it is possible to obtain free DNA-introduced cells, culture the cells or systematize the cultured cells with a protein (protein) degrading enzyme such as trypsin. It is possible. Furthermore, it is possible to investigate the specification of the protein-producing cell of the present invention, its relation to apoptosis, differentiation or proliferation, or the signal transduction mechanism in them, and to investigate their abnormalities. It becomes an effective research material for 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-introduced animal of the present invention, the above-mentioned test method and quantification It is possible to provide an effective and rapid screening method for the therapeutic drug for the disease using the method. Further, using the DNA-introduced animal of the present invention or the foreign DNA expression vector of the present invention,
It is possible to investigate and develop a DNA therapeutic 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 DNA expression-deficient non-human mammal of the present invention. That is, the present invention provides (1) a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated,
(2) The embryonic stem cell according to item (1), wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), (3) neomycin-resistant item (1) The embryonic stem cell according to item (4), the embryonic stem cell according to item (1), wherein the non-human mammal is a rodent, (5) the embryonic stem cell according to item (4), wherein the rodent is a mouse, ( 6) The DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated, (7) The DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from Escherichia coli), The non-human mammal according to item (6), wherein the reporter gene can be expressed under the control of a promoter for the DNA of the present invention,
(8) The non-human mammal according to item (6), wherein the non-human mammal is a rodent, (9) the non-human mammal according to item (8), wherein the rodent is a mouse, and (10) ) D of the present invention, which comprises administering a test compound to the animal according to item (7) and detecting the expression of a reporter gene.
Provided is a screening method for a compound or its salt that promotes or inhibits the promoter activity against NA.

The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated means that the expression ability of the DNA is suppressed by artificially mutating the DNA of the present invention possessed by the non-human mammal. Alternatively, the DNA does not substantially have the ability to express the protein of the present invention by substantially losing the activity of the protein of the present invention encoded by the DNA (hereinafter, referred to as the knockout DNA of the present invention. Sometimes non-human mammalian embryonic stem cells (hereinafter ES
Abbreviated as cell). As the non-human mammal, the same ones as described above are used. As a method for artificially mutating the DNA of the present invention, for example, a part or all of the DNA sequence is deleted by a genetic engineering method, or another DN is used.
This can be done by inserting or substituting A. Due to these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the reading frame of the codon or disrupting the function of the promoter or exon. Non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter referred to as the DNA inactivated ES of the present invention
(Abbreviated as cell or knockout ES cell of the present invention)
Specific examples include, for example, the DNA of the present invention contained in the target non-human mammal is isolated, and the exon portion thereof has a neomycin resistance gene, a drug resistance gene represented by a hygromycin resistance gene, or lacZ (β-
Galactosidase gene), a DNA sequence that terminates transcription of the gene by inserting a reporter gene typified by cat (chloramphenicol acetyltransferase gene) or the like, or by disrupting the function of the exon. For example, po
DN having a DNA sequence constructed so as to disrupt the gene by inserting a lyA addition signal or the like) to thereby make it impossible to synthesize a complete messenger RNA.
A chain (hereinafter abbreviated as targeting vector)
Is introduced into the chromosome of the animal by, for example, a homologous recombination method, and the obtained ES cells are targeted to a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe. It can be obtained by selecting the knockout ES cells of the present invention by analysis by the PCR method using the DNA sequence of the neighboring region other than the DNA of the present invention used for vector preparation as a primer.

Further, the DN of the present invention can be prepared by the homologous recombination method or the like.
As the original ES cells for inactivating A, those already established as described above may be used, or those newly established according to the known method of Evans and Kaufman. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used. However, since the immunological background is not clear, an alternative pure line immunological genetic background is used. For the purpose of obtaining ES cells with clear evidence, for example, C57BL / 6 mouse and C57BL /
BDF1 mice (C57BL / 6 and DBA / 2 F with improved egg collection number of 6 improved by crossing with DBA / 2)
Those established using 1) can also be used favorably. BDF
1 mouse has the advantage that the number of eggs collected is large and the eggs are strong, and since it has C57BL / 6 mice as a background, the ES cells obtained using this, when the pathological model mouse was produced, By backcrossing (backcrossing) with C57BL / 6 mice, the genetic background was changed to C57BL /
It can be advantageously used because it can be replaced with 6 mice. In addition, when ES cells are established, blastocysts on the 3.5th day after fertilization are generally used, but other than this, 8-cell stage embryos are ovulated and cultured up to the blastocysts for efficient and large numbers. The early embryos of can be obtained. Although either male or female ES cells may be used, male ES cells are usually more convenient for producing a germ line chimera. Also,
It is desirable to distinguish males and females as soon as possible in order to reduce troublesome culturing. As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in the sex determining region on the Y chromosome by the PCR method can be mentioned. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells to the karyotyping, ES cell number of about 1 colony (about 50
Therefore, the primary selection of ES cells in the early stage of culture can be performed by discriminating males and females, and the early selection of male cells can significantly reduce the labor required in the early stage of culture.

The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of ES cells obtained is preferably 100% of the normal number, but if it is difficult due to physical manipulations during establishment, after knocking out the gene of ES cells, normal cells (for example, in mice, The number is 2n
= 40 cells). The embryonic stem cell line thus obtained usually has a very good proliferative property, but since it is easy to lose the ability to develop ontogeny, it is necessary to carefully subculture. For example, in a carbon dioxide incubator in the presence of LIF (1-10000 U / ml) on a suitable feeder cell such as STO fibroblast (preferably about 5% carbon dioxide, about 95% air or about 5% oxygen). , About 5% carbon dioxide, about 90% air) about 3
It is cultivated by a method such as culturing at 7 ° C., and at the time of subculture, for example, trypsin / EDTA solution (usually about 0.001-0.
5% trypsin / about 0.1-5 mM EDTA, preferably about 0.1% trypsin / about 1 mM EDTA) to form single cells, and seeding on newly prepared feeder cells. Such passages are usually 1-3
Although it is performed every day, it is desirable to observe the cells at this time and to discard the cultured cells when morphologically abnormal cells are found. ES cells are differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by performing monolayer culture until reaching a high density or suspension culture until forming a cell aggregate under appropriate conditions. [MJ Evans and MH Kaufman, Nature (Na
ture) 292, 154, 1981; GR Martin Proceedings of National Academy of
Science USA (Proc. Natl. Acad. Sci.
USA) 78, 7634, 1981; TC Doetschman
Et al., Journal of Embiology and Experimental Morphology, Vol. 87, p. 27, 19
1985], the DNA expression-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is useful in the cell biological study of the protein of the present invention in vitro. The non-human mammal deficient in DNA expression of the present invention is the mR of the animal.
It is possible to distinguish from a normal animal by measuring the NA amount using a known method and indirectly comparing the expression amount. As the non-human mammal, the same ones as described above are used.

The non-human mammal deficient in expression of the DNA of the present invention can be obtained by, for example, introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells, and introducing the targeting vector D of the present invention.
It is possible to knock out the DNA of the present invention by performing homologous recombination in which the DNA sequence in which NA is inactivated replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. it can. The cells in which the DNA of the present invention has been knocked out are the DNA sequences on the Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the mouse-derived cells of the present invention used for the targeting vector. It can be determined by an analysis by the PCR method using a DNA sequence in a neighboring region other than DNA as a primer. When a non-human mammal embryonic stem cell is used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cell is cloned at an appropriate time, for example, at the 8-cell stage. It is injected into an animal embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has the mutated DNA locus of the present invention, all tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. D of the present invention added
It can be obtained by selecting individuals composed of cells having the NA locus, for example, by determining the coat color. The individual thus obtained is usually an individual deficient in hetero expression of the protein of the present invention, and heterologous individuals deficient in hetero expression of the protein of the present invention are crossed with each other to homo-express the protein of the present invention from their offspring. Defective individuals can be obtained. When using egg cells, for example, a transgenic non-human mammal having a targeting vector introduced into its chromosome by injecting a DNA solution into the egg cell nucleus by a microinjection method can be obtained,
Compared to these transgenic non-human mammals,
It can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.

As described above, for the individual in which the DNA of the present invention is knocked out, the animal individual obtained by mating is confirmed to have the DNA knocked out, and then the animal is subcultured in a normal breeding environment. You can further,
The acquisition and maintenance of the germ line may be carried out according to the usual method. That is, by mating male and female animals carrying the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The homozygous animals obtained were normal individuals 1,
It can be efficiently obtained by breeding in a state in which a plurality of homozygotes are present. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred. The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated is very useful in producing the non-human mammal deficient in expression of the DNA of the present invention. Further, the DN of the present invention
Since a non-human mammal deficient in A expression lacks various biological activities that can be induced by the protein of the present invention, it can be a model of diseases caused by inactivation of the biological activity of the protein of the present invention. It is useful for investigating the cause of disease and studying treatment methods.

(7a) Method for Screening Compound Having Curative / Preventive Effect Against Disease Caused by Defect or Damage of DNA of the Present Invention The non-human mammal deficient in DNA expression of the present invention is D
Diseases caused by NA deficiency or damage, such as heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer) ,
Prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer,
Rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vascular occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retinal vitreous proliferation) , Retinitis, retinal lipidemia, branch retinal vein occlusion,
It can be used for screening of compounds having therapeutic / preventive effects on retinoblastoma, etc.). That is, the present invention results from a deficiency or damage of the DNA of the present invention, which comprises administering a test compound to the non-human mammal deficient in DNA expression of the present invention and observing and measuring the change in the animal. Provided is a method for screening a compound having a therapeutic / preventive effect against a disease or a salt thereof. Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include the same ones as described above. Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products,
Examples thereof include cell extracts, plant extracts, animal tissue extracts, plasma and the like, and these compounds may be novel compounds or known compounds. Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound and compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / prophylactic effect of a compound can be tested. As a method for treating the test animal with the test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected depending on the symptoms of the test animal, the properties of the test compound and the like. Further, the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

The compound obtained by using the screening method of the present invention is a compound selected from the above-mentioned test compounds, and is a disease caused by a deficiency or damage of the protein of the present invention, such as heart disease (eg, cardiomyopathy). , Myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, Rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vascular occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retinal vitreous proliferation) , Retinitis, retinal lipemia, branch retinal vein occlusion, retinoblastoma, etc.), and therefore a safe and low-toxic therapeutic or prophylactic agent for the disease. Can be used as Furthermore, compounds derived from the compounds obtained by the above screening can be used as well. The compound obtained by the screening method may form a salt, and as the salt of the compound, a physiologically acceptable acid (eg, inorganic acid, organic acid), base (eg, alkali metal), or the like is used. Are used, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include 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). Salts with acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
The drug containing the compound or its salt obtained by the screening method can be manufactured in the same manner as the drug containing the protein of the present invention. Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in mammals (for example, humans, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered. The dose of the compound or its salt varies depending on the target disease, administration subject, administration route and the like. However, for example, when the compound is orally administered for the purpose of treating cancer, it is generally an adult (body weight 60 k
(as g), the compound is about 0.1 per day.
~ 100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but for example, the compound is usually administered to an adult (as 60 kg) in the form of an injection for the purpose of treating cancer. When administered, about 0.01 to 30 mg of the compound per day
Conveniently, about 0.1 to 20 mg, more preferably about 0.1 to 10 mg, is administered by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(7b) Method for Screening Compound Promoting or Inhibiting Promoter Activity for DNA of the Present Invention The present invention provides a non-human mammal deficient in expression of DNA of the present invention,
Provided is a method for screening a compound or its salt that promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene. In the above screening method, the non-human mammal deficient in DNA expression of the present invention, among the non-human mammal deficient in DNA expression of the present invention, the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used. Examples of the test compound include the same ones as described above. As the reporter gene, the same ones as described above are used, and the β-galactosidase gene (lacZ), the soluble alkaline phosphatase gene, the luciferase gene and the like are preferable. DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene
In a non-human mammal deficient in expression, since the reporter gene is under the control of the promoter for the DNA of the present invention, the activity of the promoter can be detected by tracing the expression of the substance encoded by the reporter gene.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with the β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue of the present invention is originally expressed in the tissue of the present invention. Β-galactosidase is expressed instead of protein. Thus, for example, 5-bromo-4-chloro-3-indolyl-β-
By staining with a reagent that serves as a substrate for β-galactosidase such as D-galactopyranoside (X-gal), the expression state of the protein of the present invention in an animal body can be easily observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, and a phosphate buffered saline (PB
After washing with S), it is reacted with a staining solution containing X-gal at room temperature or around 37 ° C. for about 30 minutes to 1 hour, and then the tissue specimen is washed with 1 mM EDTA / PBS solution to give β-galactosidase. The reaction may be stopped and the color may be observed. In addition, mRNA encoding lacZ may be detected according to a conventional method.

The compound or its salt obtained by the above screening method is a compound selected from the above-mentioned test compounds, and is a compound which promotes or inhibits the promoter activity for the DNA of the present invention. The compound obtained by the screening method may form a salt,
The salt of the compound includes a physiologically acceptable acid (eg,
A salt with an inorganic acid) or a base (eg, an organic acid) is used, and a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include 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). Salts with acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. The compound or a salt thereof that promotes the promoter activity for the DNA of the present invention can promote the expression of the protein of the present invention and the function of the protein, and therefore, for example, heart disease (eg, cardiomyopathy, myocardial infarction). , Heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer,
Liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retina) Vascular occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retinal vitreous hyperplasia, retinitis, retinolipidemia, branch retinal vein occlusion, retinoblastoma It is useful as a medicine such as a safe and low toxic therapeutic / prophylactic agent against diseases such as). Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.

The drug containing the compound or its salt obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or its salt. Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in mammals (for example, rat, human, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered. The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, etc.,
For example, when a compound that promotes the promoter activity against the DNA of the present invention is orally administered for the purpose of treating cancer, generally, in an adult (body weight is 60 kg), the compound is about 0.1 to 100 mg, preferably about 100 mg per day. Is about 1.
The dosage is 0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease and the like, but for example, a compound that promotes the promoter activity against the DNA of the present invention for the purpose of treating cancer can be administered by injection. Adult in the form (60
(as kg), about 0.01 to 30 mg of the compound per day, preferably about 0.1 to 20 m
It is convenient to administer about g, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

On the other hand, for example, when a compound which inhibits the promoter activity for the DNA of the present invention is orally administered,
Generally, in an adult (as a body weight of 60 kg) cancer patient, the compound is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to about 100 mg per day.
Administer 20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but the compound that inhibits the promoter activity against the DNA of the present invention in the form of an injection is usually used for adults (60 kg). )
When administered to patients with cancer of about 0.
It is convenient to administer about 01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. For other animals,
The amount converted per 60 kg can be administered.

In the present specification and the drawings, when an abbreviation is used for a base or amino acid, IUPAC-IUB is used.
It is based on the abbreviations by the Commission on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri 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: Cysteine Met: Cysteine Met : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine T r: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid

Further, substituents, protecting groups and reagents commonly used in the present specification are represented by the following symbols. Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine-4 (R) -carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc : N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene -2,3-
Dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification show the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of human RGS No9 gene product. [SEQ ID NO: 2] This shows the base sequence of DNA encoding human RGS No9 containing the amino acid sequence represented by SEQ ID NO: 1. [SEQ ID NO: 3] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 4] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 5] This shows the base sequence of T7 primer. [SEQ ID NO: 6] This shows the base sequence of U-19 primer. [SEQ ID NO: 7] This shows the base sequence of 5'untranslated region of human GRK. [SEQ ID NO: 8] This shows the base sequence of 3'untranslated region of human GRK. [SEQ ID NO: 9] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 10] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 11] This shows the base sequence of the PCR product obtained in Example 3. [SEQ ID NO: 12] This shows the amino acid sequence encoded by the base sequence represented by SEQ ID NO: 11.

Transformant Esch obtained in Example 3 below
Since June 7, 2002, erichia coli DH5α / pTB2271 has been located at Central 1-1, Higashi 1-1-chome, Tsukuba, Ibaraki, Japan.
Deposit number FER at the Japan Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Postal code 3058566)
Deposited as MBP-8067.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

Example 1 A group of RGS genes forming a family has been reported (Chips, Vol. 20, p. 376-382 (199).
9)), these genes are known to have a highly conserved RGS domain. So these RG
The existence of the motif was examined from the alignment using the amino acid sequence deduced from the S domain. The RGS genes used were mouse RGS1, human RGS2, mouse RGS3, human RGS4, mouse RGS5, rat RGS.
GS-like gene (Japanese Patent Application No. 2000-350183), human RGS
6, human RGS9, human RGS10, human RGS11,
Human RGS16, human RGS19 and human RGS20. When an alignment of the amino acid sequences of these RGS domains was created (FIGS. 1 to 3), all RGSs were found.
It was revealed that there is an amino acid common to the.
Using the amino acid sequence deduced from human GRK4, which is one of the known RGS family genes having this RGS motif, homology search was performed based on the amino acid sequence deduced from the novel DNA sequence database. Blast method was used for the search. As a result, the amino acid sequence represented by SEQ ID NO: 1 having 48% homology (human R
GSNo9), and the base sequence represented by SEQ ID NO: 2 encoded by the amino acid sequence were found.

Example 2 Expression Analysis Two types of primers (SEQ ID NO: 3 and SEQ ID NO: 4) were synthesized as PCR primers from the nucleotide sequence represented by SEQ ID NO: 2, and a commercially available human cDNA library was synthesized. The expression organs were examined by the PCR method using the above as a template. Marathon ready human heart cDNA library, marathon ready human skeletal muscle cDNA library, marathon ready human lung cDNA library, marathon ready human brain cDNA library, marathon ready human kidney cDNA library,
Marathon ready human liver cDNA library, Marathon ready human pancreas cDNA library, Marathon ready human small intestine cDNA library, Marathon ready human retina cd
NA library, marathon ready human testis cDNA library, marathon ready human placenta cDNA library,
PCR was carried out using a marathon ready human spleen cDNA library and a marathon ready human thymus cDNA library (manufactured by Clontech) as templates. The reaction is TaKaRa La Taq w
Thermal cycler gene amp PCR system 9700 (Perkin Elmer) using ith GC buffer (Takara Shuzo), 95 ° C for 30 seconds, 62 ° C for 30 seconds, 72 ° C
Then, 33 cycles were repeated with 3 minutes as one cycle.
As a result, a band specific to the retina was recognized. The obtained gene fragment was cloned into pT7-T vector (Takara Shuzo Co., Ltd.), and then using known synthetic primers [T7 primer (SEQ ID NO: 5) and U-19 primer (SEQ ID NO: 6)], The reaction was carried out with a cycle sequence kit from PE Applied Biosystems, and analyzed with a fluorescent DNA sequencer (ABI PRISM 377, manufactured by Perkin Elmer) to decipher the base sequence, and this gene fragment was
It was confirmed to have the sequence contained in the base sequence represented by SEQ ID NO: 2. Furthermore, as a result of conducting a blast search with MCBI (Gene Bank), which is a public database, using the sequence thus obtained as a query, it was revealed that it has 85% or more homology with squirrel GRK7 (AF063016) at the DNA level. Became. From this, the obtained human RGS No9 gene was considered to be GRK7 human ortholog.

Example 3 Cloning of human RGS No9 cDNA The protein coding region of squirrel GRK7 (AF063016) was searched in the Celera human genome database, and human GRK 5'untranslated region (SEQ ID NO: 7) which is a non-coding region.
And 3'untranslated region (SEQ ID NO: 8) were obtained. From the obtained nucleotide sequence (SEQ ID NO: 7 and SEQ ID NO: 8) to PC
SEQ ID NO: 9 and SEQ ID NO: 10 as R primer
Primers each having the nucleotide sequence represented by were synthesized, and PCR was performed using a marathon ready rat retina cDNA library (manufactured by Clontech) as a template. The reaction is TaK
aRa La Taq with GC buffer (Takara Shuzo Co., Ltd.) is used in a thermal cycler gene amp PCR system 9700 (Perkin Elmer Co., Ltd.) at 95 ° C. for 30 seconds and 62 ° C. for 30 seconds.
33 cycles were repeated with one second consisting of 3 minutes at 72 ° C. for 2 seconds. The obtained gene fragment was cloned into a pT7-T vector (Takara Shuzo), and then using known synthetic primers [T7 primer (SEQ ID NO: 5) and U-19 primer (SEQ ID NO: 6)], PE was used. The reaction was performed using a cycle sequence kit from Applied Biosystems, and a fluorescent DNA sequencer (ABI PRISM 37
7, manufactured by Perkin Elmer Co., Ltd.), and the nucleotide sequence was decoded to obtain the nucleotide sequence represented by SEQ ID NO: 11. The protein encoded by the base sequence represented by SEQ ID NO: 11 is shown in SEQ ID NO: 12. The base sequence represented by SEQ ID NO: 11 contained the base sequence represented by SEQ ID NO: 2. Next, from the pT7-T vector, the restriction enzymes EcoR1 and
A gene fragment containing RGSNo9 cDNA was obtained by digesting with BamH1, and then cloned into EcoR1 and BamH1 sites of pBluescript II (SK-) (Takara Shuzo). The plasmid containing the RGS No9 protein coding region thus prepared was named pTB2271, this plasmid was introduced into E. coli, and the transformant was transformed into Escherichia coli DH5α / p.
It was named TB2271.

[0083]

EFFECT OF THE INVENTION A compound or a salt thereof that regulates the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, and a neutralizing antibody that regulates the activity of the protein are , For example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina, etc.), cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer,
Prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer,
Rectal cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vascular occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retinal vitreous proliferation) , Retinitis, retinal lipidemia, branch retinal vein occlusion,
It can be used as a prophylactic / therapeutic agent for retinoblastoma, etc.). In addition, the antisense DNA can suppress the expression of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, and for example, heart disease (eg, cardiomyopathy, Myocardial infarction, heart failure, angina, etc., cancer (eg, lung cancer, kidney cancer, liver cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectum) Cancer, pancreatic cancer, thymoma, etc., retinal disease (eg, diabetic retinopathy, retinal vascular occlusion, ischemic retinal disease, retinal vitreous disease, eye tumor, cataract, glaucoma, retinal detachment, retinal vitreous hyperplasia) , Retinitis, retinal lipemia, branch retinal vein occlusion, retinoblastoma, etc.) and the like.

[0084]

[Sequence list]                             SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> A novel gene relating to disease and use thereof <130> P02-0071 <140> <141> <150> JP 2001-182654 <151> 2001-06-15 <160> 12 <170> PatentIn Ver. 2.1 <210> 1 <211> 397 <212> PRT <213> Human <400> 1 Met Val Asp Met Gly Ala Leu Asp Asn Leu Ile Ala Asn Thr Ala Tyr   1 5 10 15 Leu Gln Ala Arg Lys Pro Ser Asp Cys Asp Ser Lys Glu Leu Gln Arg              20 25 30 Arg Arg Arg Ser Leu Ala Leu Pro Gly Leu Gln Gly Cys Ala Glu Leu          35 40 45 Arg Gln Lys Leu Ser Leu Asn Phe His Ser Leu Cys Glu Gln Gln Pro      50 55 60 Ile Gly Arg Arg Leu Phe Arg Asp Phe Leu Ala Thr Val Pro Thr Phe  65 70 75 80 Arg Lys Ala Ala Thr Phe Leu Glu Asp Val Gln Asn Trp Glu Leu Ala                  85 90 95 Glu Glu Gly Pro Thr Lys Asp Ser Ala Leu Gln Gly Leu Val Ala Thr             100 105 110 Cys Ala Ser Ala Pro Ala Pro Gly Asn Pro Gln Pro Phe Leu Ser Gln         115 120 125 Ala Val Ala Thr Lys Cys Gln Ala Ala Thr Thr Glu Glu Glu Arg Val     130 135 140 Ala Ala Val Thr Leu Ala Lys Ala Glu Ala Met Ala Phe Leu Gln Glu 145 150 155 160 Gln Pro Phe Lys Asp Phe Val Thr Ser Ala Phe Tyr Asp Lys Phe Leu                 165 170 175 Gln Trp Lys Leu Phe Glu Met Gln Pro Val Ser Asp Lys Tyr Phe Thr             180 185 190 Glu Phe Arg Val Leu Gly Lys Gly Gly Phe Gly Glu Val Cys Ala Val         195 200 205 Gln Val Lys Asn Thr Gly Lys Met Tyr Ala Cys Lys Lys Leu Asp Lys     210 215 220 Lys Arg Leu Lys Lys Lys Gly Gly Glu Lys Met Ala Leu Leu Glu Lys 225 230 235 240 Glu Ile Leu Glu Lys Val Ser Ser Pro Phe Ile Val Ser Leu Ala Tyr                 245 250 255 Ala Phe Glu Ser Lys Thr His Leu Cys Leu Val Met Ser Leu Met Asn             260 265 270 Gly Gly Asp Leu Lys Phe His Ile Tyr Asn Val Gly Thr Arg Gly Leu         275 280 285 Asp Met Ser Arg Val Ile Phe Tyr Ser Ala Gln Ile Ala Cys Gly Met     290 295 300 Leu His Leu His Glu Leu Gly Ile Val Tyr Arg Asp Met Lys Pro Glu 305 310 315 320 Asn Val Leu Leu Asp Asp Leu Gly Asn Cys Arg Leu Ser Asp Leu Gly                 325 330 335 Leu Ala Val Glu Met Lys Gly Gly Lys Pro Ile Thr Gln Arg Ala Gly             340 345 350 Thr Asn Gly Tyr Met Ala Pro Glu Ile Leu Met Glu Lys Val Ser Tyr         355 360 365 Ser Tyr Pro Val Asp Trp Phe Ala Met Gly Cys Ser Ile Tyr Glu Met     370 375 380 Val Ala Gly Arg Thr Pro Phe Lys Asp Tyr Lys Glu Lys 385 390 395 <210> 2 <211> 1191 <212> DNA <213> Human <400> 2 atggtggaca tgggggccct ggacaacctg atcgccaaca ccgcctacct gcaggcccgg 60 aagccctcgg actgcgacag caaagagctg cagcggcggc ggcgtagcct ggccctgccc 120 gggctgcagg gctgcgcgga gctccgccag aagctgtccc tgaacttcca cagcctgtgt 180 gagcagcagc ccatcggtcg ccgcctcttc cgtgacttcc tagccacagt gcccacgttc 240 cgcaaggcgg caaccttcct agaggacgtg cagaactggg agctggccga ggagggaccc 300 accaaagaca gcgcgctgca ggggctggtg gccacttgtg cgagtgcccc tgccccgggg 360 aacccgcaac ccttcctcag ccaggccgtg gccaccaagt gccaagcagc caccactgag 420 gaagagcgag tggctgcagt gacgctggcc aaggctgagg ccatggcttt cttgcaagag 480 cagcccttta aggatttcgt gaccagcgcc ttctacgaca agtttctgca gtggaaactc 540 ttcgagatgc aaccagtgtc agacaagtac ttcactgagt tcagagtgct ggggaaaggt 600 ggttttgggg aggtatgtgc cgtccaggtg aaaaacactg ggaagatgta tgcctgtaag 660 aaactggaca agaagcggct gaagaagaaa ggtggcgaga agatggctct cttggaaaag 720 gaaatcttgg agaaggtcag cagccctttc attgtctctc tggcctatgc ctttgagagc 780 aagacccatc tctgccttgt catgagcctg atgaatgggg gagacctcaa gttccacatc 840 tacaacgtgg gcacgcgtgg cctggacatg agccgggtga tcttttactc ggcccagata 900 gcctgtggga tgctgcacct ccatgaactc ggcatcgtct atcgggacat gaagcctgag 960 aatgtgcttc tggatgacct cggcaactgc aggttatctg acctggggct ggccgtggag 1020 atgaagggtg gcaagcccat cacccagagg gctggaacca atggttacat ggctcctgag 1080 atcctaatgg aaaaggtaag ttattcctat cctgtggact ggtttgccat gggatgcagc 1140 atttatgaaa tggttgctgg acgaacacca ttcaaagatt acaaggaaaa g 1191 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 agccacagtg cccacgttcc gc 22 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gagacaatga aagggctgct gacc 24 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 taatacgact cactata 17 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 ggttttccca gtcacgacg 19 <210> 7 <211> 120 <212> DNA <213> Human <400> 7 acaggccaca ggactcactg taaatccctt ggacgttgtc tcacccggga agggaaagca 60 gccagcagcc ctccagccct cttgtgcttt ccctgggagt gcgccccgtg ctcagccatg 120 <210> 8 <211> 120 <212> DNA <213> Human <400> 8 attgctctct ttaccagaca ggcagcagga gtctcggctg acataatcct cgaatgttcc 60 acacgtggaa atctgtggaa tgagggctaa tcagttagga gggacatcac aaccacaaaa 120 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 ctgggagtgc gccccgtgct cagcc 25 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 agatttccac gtgtggaaca ttcgag 26 <210> 11 <211> 1761 <212> DNA <213> Human <400> 11 ctgggagtgc gccccgtgct cagccatggt ggacatgggg gccctggaca acctgatcgc 60 caacaccgcc tacctgcagg cccggaagcc ctcggactgc gacagcaaag agctgcagcg 120 gcggcggcgt agcctggccc tgcccgggct gcagggctgc gcggagctcc gccagaagct 180 gtccctgaac ttccacagcc tgtgtgagca gcagcccatc ggtcgccgcc tcttccgtga 240 cttcctagcc acagtgccca cgttccgcaa ggcggcaacc ttcctagagg acgtgcagaa 300 ctgggagctg gccgaggagg gacccaccaa agacagcgcg ctgcaggggc tggtggccac 360 ttgtgcgagt gcccctgccc cggggaaccc gcaacccttc ctcagccagg ccgtggccac 420 caagtgccaa gcagccacca ctgaggaaga gcgagtggct gcagtgacgc tggccaaggc 480 tgaggccatg gctttcttgc aagagcagcc ctttaaggat ttcgtgacca gcgccttcta 540 cgacaagttt ctgcagtgga aactcttcga gatgcaacca gtgtcagaca agtacttcac 600 tgagttcaga gtgctgggga aaggtggttt tggggaggta tgtgccgtcc aggtgaaaaa 660 cactgggaag atgtatgcct gtaagaaact ggacaagaag cggctgaaga agaaaggtgg 720 cgagaagatg gctctcttgg aaaaggaaat cttggagaag gtcagcagcc ctttcattgt 780 ctctctggcc tatgcctttg agagcaagac ccatctctgc cttgtcatga gcctgatgaa 840 tgggggagac ctcaagttcc acatctacaa cgtgggcacg cgtggcctgg acatgagccg 900 ggtgatcttt tactcggccc agatagcctg tgggatgctg cacctccatg aactcggcat 960 cgtctatcgg gacatgaagc ctgagaatgt gcttctggat gacctcggca actgcaggtt 1020 atctgacctg gggctggccg tggagatgaa gggtggcaag cccatcaccc agagggctgg 1080 aaccaatggt tacatggctc ctgagatcct aatggaaaag gtaagttatt cctatcctgt 1140 ggactggttt gccatgggat gcagcattta tgaaatggtt gctggacgaa caccattcaa 1200 agattacaag gaaaaggtca gtaaagagga tctgaagcaa agaactctgc aagacgaggt 1260 caaattccag catgataact tcacagagga agcaaaagat atttgcaggc tcttcttggc 1320 taagaaacca gagcaacgct taggaagcag agaaaagtct gatgatccca ggaaacatca 1380 tttctttaaa acgatcaact ttcctcgcct ggaagctggc ctaattgaac ccccatttgt 1440 gccagaccct tcagtggttt atgccaaaga catcgctgaa attgatgatt tctctgaggt 1500 tcggggggtg gaatttgatg acaaagataa gcagttcttc aaaaactttg cgacaggtgc 1560 tgttcctata gcatggcagg aagaaattat agaaacggga ctgtttgagg aactgaatga 1620 ccccaacaga cctacgggtt gtgaggaggg taattcatcc aagtctggcg tgtgtttgtt 1680 attgtaaatt gctctcttta ccagacaggc agcaggagtc tcggctgaca taatcctcga 1740 atgttccaca cgtggaaatc t 1761 <210> 12 <211> 553 <212> PRT <213> Human <400> 12 Met Val Asp Met Gly Ala Leu Asp Asn Leu Ile Ala Asn Thr Ala Tyr                  5 10 15 Leu Gln Ala Arg Lys Pro Ser Asp Cys Asp Ser Lys Glu Leu Gln Arg              20 25 30 Arg Arg Arg Ser Leu Ala Leu Pro Gly Leu Gln Gly Cys Ala Glu Leu          35 40 45 Arg Gln Lys Leu Ser Leu Asn Phe His Ser Leu Cys Glu Gln Gln Pro      50 55 60 Ile Gly Arg Arg Leu Phe Arg Asp Phe Leu Ala Thr Val Pro Thr Phe  65 70 75 80 Arg Lys Ala Ala Thr Phe Leu Glu Asp Val Gln Asn Trp Glu Leu Ala                  85 90 95 Glu Glu Gly Pro Thr Lys Asp Ser Ala Leu Gln Gly Leu Val Ala Thr             100 105 110 Cys Ala Ser Ala Pro Ala Pro Gly Asn Pro Gln Pro Phe Leu Ser Gln         115 120 125 Ala Val Ala Thr Lys Cys Gln Ala Ala Thr Thr Glu Glu Glu Arg Val     130 135 140 Ala Ala Val Thr Leu Ala Lys Ala Glu Ala Met Ala Phe Leu Gln Glu 145 150 155 160 Gln Pro Phe Lys Asp Phe Val Thr Ser Ala Phe Tyr Asp Lys Phe Leu                 165 170 175 Gln Trp Lys Leu Phe Glu Met Gln Pro Val Ser Asp Lys Tyr Phe Thr             180 185 190 Glu Phe Arg Val Leu Gly Lys Gly Gly Phe Gly Glu Val Cys Ala Val         195 200 205 Gln Val Lys Asn Thr Gly Lys Met Tyr Ala Cys Lys Lys Leu Asp Lys     210 215 220 Lys Arg Leu Lys Lys Lys Gly Gly Glu Lys Met Ala Leu Leu Glu Lys 225 230 235 240 Glu Ile Leu Glu Lys Val Ser Ser Pro Phe Ile Val Ser Leu Ala Tyr                 245 250 255 Ala Phe Glu Ser Lys Thr His Leu Cys Leu Val Met Ser Leu Met Asn             260 265 270 Gly Gly Asp Leu Lys Phe His Ile Tyr Asn Val Gly Thr Arg Gly Leu         275 280 285 Asp Met Ser Arg Val Ile Phe Tyr Ser Ala Gln Ile Ala Cys Gly Met     290 295 300 Leu His Leu His Glu Leu Gly Ile Val Tyr Arg Asp Met Lys Pro Glu 305 310 315 320 Asn Val Leu Leu Asp Asp Leu Gly Asn Cys Arg Leu Ser Asp Leu Gly                 325 330 335 Leu Ala Val Glu Met Lys Gly Gly Lys Pro Ile Thr Gln Arg Ala Gly             340 345 350 Thr Asn Gly Tyr Met Ala Pro Glu Ile Leu Met Glu Lys Val Ser Tyr         355 360 365 Ser Tyr Pro Val Asp Trp Phe Ala Met Gly Cys Ser Ile Tyr Glu Met     370 375 380 Val Ala Gly Arg Thr Pro Phe Lys Asp Tyr Lys Glu Lys Val Ser Lys 385 390 395 400 Glu Asp Leu Lys Gln Arg Thr Leu Gln Asp Glu Val Lys Phe Gln His                 405 410 415 Asp Asn Phe Thr Glu Glu Ala Lys Asp Ile Cys Arg Leu Phe Leu Ala             420 425 430 Lys Lys Pro Glu Gln Arg Leu Gly Ser Arg Glu Lys Ser Asp Asp Pro         435 440 445 Arg Lys His His Phe Phe Lys Thr Ile Asn Phe Pro Arg Leu Glu Ala     450 455 460 Gly Leu Ile Glu Pro Pro Phe Val Pro Asp Pro Ser Val Val Tyr Ala 465 470 475 480 Lys Asp Ile Ala Glu Ile Asp Asp Phe Ser Glu Val Arg Gly Val Glu                 485 490 495 Phe Asp Asp Lys Asp Lys Gln Phe Phe Lys Asn Phe Ala Thr Gly Ala             500 505 510 Val Pro Ile Ala Trp Gln Glu Glu Ile Ile Glu Thr Gly Leu Phe Glu         515 520 525 Glu Leu Asn Asp Pro Asn Arg Pro Thr Gly Cys Glu Glu Gly Asn Ser     530 535 540 Ser Lys Ser Gly Val Cys Leu Leu Leu 545 550

[Brief description of drawings]

FIG. 1 shows the results of motif search between RGS domains common to the RGS family. In the figure, mRGS1D.pro is mouse RGS1, hRGS2D.pro is human RGS2, mRGS3D.pro is mouse RGS3, hRGS4D.pro is human RGS4, mRGS5D.pro is mouse RGS5, ratRGShomoD. Is rat RGS-like gene. To h
RGS6D.pro is human RGS6, hRGS9D.pro is human RGS9, hRG
S10D.pro is human RGS10, hRGS11D.pro is human RGS11, h
RGS16D.pro is human RGS16, hRGS19D.pro is human RGS19
And hRGS20D.pro shows human RGS20, respectively. In addition, when all the amino acids match, the amino acid is displayed in the consensus, and otherwise, "." Is displayed.

FIG. 2 shows the results of motif search between RGS domains common to the RGS family (continuation of FIG. 1).

FIG. 3 shows the results of motif search between RGS domains common to the RGS family (continuation of FIG. 2).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 9/00 4C084 A61P 9/00 9/04 4C085 9/04 9/10 4C086 9/10 27/02 4H045 27/02 27/06 27/06 27/12 27/12 35/00 35/00 43/00 105 43/00 105 C07K 14/705 C07K 14/705 16/28 16/28 C12N 1 / 15 C12N 1/15 1/19 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/02 C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z G01N 33/15 33/53 D 33/50 C12N 15/00 ZNAA 33/53 5/00 A (72) Inventor Koji Yamamoto 9 7-1, Kasuga, Tsukuba, Ibaraki Prefecture Takeda Kasuga Heights 1202 F-term (reference) 2G045 AA34 AA35 DA13 DA36 FB02 FB03 4B024 AA01 AA11 BA63 CA04 EA04 GA11 HA01 HA19 4B063 QA01 QA05 QA18 QQ08 QQ67 QR10 QR32 QR58 QR75 QR77 QS35 QS36 QX02 4B064 AG20 AG26 CA19 CC24 DA01 DA13 4B065 AA26X AA93Y AB01 BA02 CA24 CA25 CA44 CA46 NA86 MA86A86 BB16A61 A16 A4 A11 A14 A61A16 A4 A11 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A3 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A1 A4 A4 A4 A0 A4 A4 A1 A4 A4 A4 A3 A of A of A of A of A of Z of 4 ZA40 ZB21 ZB26 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA75 EA20 FA74

Claims (25)

[Claims] 1. A protein containing an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof. 2. The protein according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof. 3. A partial peptide of the protein according to claim 1 or a salt thereof. 4. A polynucleotide containing a polynucleotide encoding the protein according to claim 1 or a partial peptide thereof. 5. The polynucleotide according to claim 4, which is DNA. 6. The polynucleotide according to claim 5, which contains the base sequence represented by SEQ ID NO: 2. 7. A recombinant vector containing the polynucleotide according to claim 4. 8. A transformant transformed with the recombinant vector according to claim 7. 9. The transformant according to claim 8 is cultured to produce the protein according to claim 1 or a partial peptide thereof, wherein the protein according to claim 1 or a partial peptide thereof or a salt thereof is used. Manufacturing method. 10. SEQ ID NO: 1, characterized in that a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof is used. A method for screening a compound or a salt thereof, which regulates the activity of a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the represented amino acid sequence. 11. A protein, a partial peptide thereof, or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1,
DN containing DNA encoding a protein or its partial peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1
The screening method according to claim 10, which is expressed in the cytoplasm of the transformant transformed with A. 12. SEQ ID NO: 1, characterized in that it contains a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof. A kit for screening a compound or a salt thereof that regulates the activity of a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the represented amino acid sequence. 13. An amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1, which is obtained by using the screening method according to claim 10 or the screening kit according to claim 12. A compound or a salt thereof that regulates the activity of a protein or a partial peptide thereof or a salt thereof. 14. A medicine comprising the compound according to claim 13 or a salt thereof. 15. The medicine according to claim 14, which is a prophylactic / therapeutic agent for heart disease, cancer or retinal disease. 16. A DNA encoding a protein or its partial peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1.
An antisense polynucleotide having a nucleotide sequence complementary to or substantially complementary to, or a part thereof. 17. A medicine comprising the antisense polynucleotide according to claim 16. 18. An antibody against a protein or a partial peptide thereof or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 19. A neutralizing antibody which neutralizes the activity of a protein containing the amino acid sequence represented by SEQ ID NO: 1 or a substantially identical amino acid sequence, or a partial peptide thereof or a salt thereof. 18. The antibody according to 18. 20. A medicine comprising the antibody according to claim 18. 21. A diagnostic agent comprising the antibody according to claim 18. 22. The method for quantifying the protein according to claim 1 or a partial peptide thereof or a salt thereof, wherein the antibody according to claim 18 is used. 23. A method for diagnosing a disease associated with cardiac function, which comprises using the quantification method according to claim 22. 24. A method for preventing or treating heart disease, cancer or retinal disease, which comprises administering an effective amount of the compound according to claim 13 or a salt thereof to a mammal. 25. Use of the compound according to claim 13 or a salt thereof for producing a prophylactic / therapeutic agent for heart disease, cancer or retinal disease.