WO2002036772A1

WO2002036772A1 - Novel disease-associated gene and use thereof

Info

Publication number: WO2002036772A1
Application number: PCT/JP2001/009477
Authority: WO
Inventors: Nobuyuki Koyama; Seiichi Tanida; Toshifumi Watanabe
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2000-10-30
Filing date: 2001-10-29
Publication date: 2002-05-10
Also published as: AU2002210960A1

Abstract

It is intended to provide a novel protein, its DNA, etc. A compound controlling the activity of the above-described protein or its salt and a neutralizing antibody controlling the activity of this protein are useful as, for example, preventives and remedies for heart diseases.

Description

New disease-related genes and their uses

The present invention relates to uses of disease-related genes. More specifically, a method for screening a drug using the disease-related gene product, an antisense nucleotide against a disease-related gene useful as a marker for diagnosis of heart diseases such as cardiomyopathy, myocardial infarction, heart failure, and angina; It relates to an antibody against a related gene product and the like. Background art

Heart failure is considered to be inadequate contraction of the heart muscle. The mechanism of the onset may be as follows. Myocardial damage, mechanical and functional abnormalities of the heart pump, pressure overload due to hypertension and pulmonary hypertension, volume overload such as anemia and acute nephritis can cause the heart to pump out blood volume according to the demands of the organism A state of disappearance occurs. On the other hand, the sympathetic nervous system, the nervous system, the endocrine system and other compensatory mechanisms operate to maintain the homeostasis of the body. The compensatory mechanisms of heart failure include: 1) the above-mentioned load increases and the contractility of the heart increases, and the heart expands as the length of the sarcomer increases.2) The contraction unit of the myocardium increases, resulting in Myocardial hypertrophy occurs as a result. 3) Nerve f nocturnal factors are activated to compensate for the inability to excrete the blood necessary for the whole body, and myocardial fibrosis progresses locally. Basically, the mechanism is to adapt to a given load.However, insufficient operation may promote heart failure, while excessive operation may cause myocardial injury and exacerbate heart failure There is also. As a result of activation of the compensatory mechanism, myocardial cells become enlarged and cardiac hypertrophy occurs. However, if the aforementioned obstacles or loads are chronically sustained, the compensatory mechanism will fail. In other words, ischemia occurs without a sufficient amount of blood being supplied to the enlarged cardiomyocytes, resulting in myocardial damage such as myocardial contractile dysfunction, decreased cardiac output, impaired organ circulation, venous congestion, This results in heart failure syndrome with fluid retention. Treatment for this requires improvement of myocardial cell damage, enhancement of cardioprotective action, recovery of cardiac dysfunction due to myocardial contractile dysfunction, and suppression of decompensation of the living organism, which is the cause, or improvement of excessive compensation mechanisms . Currently, the heart For the treatment of insufficiency syndrome, clinically cardiotonic drugs include: 1. cardiac glycosides such as digoxin, 2. sympathomimetics such as dobuyumin, and 3. phosphogesterase inhibitors such as amrinone. As vasodilators, hydralazine, calcium antagonists, angiotensin I converting enzyme inhibitors, angiotensin II receptor antagonists and the like have been used. For treatment of other dilated cardiomyopathy, a / 3 blocker is used.

However, there are no therapeutic agents that can activate the compensatory mechanism promptly, suppress excessive compensatory mechanisms or suppress compensatory failure (including suppression of apoptosis). There is a need for the development of therapeutic agents from the viewpoint of prompt activation of the compensation mechanism and suppression of excessive compensation mechanisms or failure. Disclosure of the invention

The present inventors have conducted intensive studies in order to solve the above-described problems, and as a result, have found a gene whose expression increases when heart failure occurs in a myocardial infarction model rat due to coronary artery ligation. As a result of detailed examination of the expression profile of mRNA having the nucleotide sequence represented by SEQ ID NO: 2, this mRNA was significantly reduced one week after the operation, and decreased from 8 weeks after the operation to 20 weeks. Weeks showed an increasing trend, and at week 30 it was evident that the expression port file returned to the same level as the sham-operated group. This gene is believed to be the chimney mammalian counterpart reported in Drosophila, Nkx2.5 (also known as Csx, Processings of the National Academy of Sciences'). Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 8145-8149, 1993; Development, Vol. 119, pp. 419-431, 1993; History of Medicine 185, pp. 1-7, 1998) and cloned from a rat heart cDNA library. Functional analysis of Nkx2.5 as a transcription factor that plays an important role in the developmental process of the heart is ongoing, but its relationship to heart failure pathology has not been clarified. On the other hand, the Nkx2.5 similar gene cloned this time increased at the onset of heart failure. As a result of further studies based on these findings, the present invention was completed.

That is, the present invention (1) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by arrangement J number: 1, or a salt thereof;

(2) a protein containing the amino acid sequence represented by SEQ ID NO: 1 or a protein thereof;

'

(3) a partial peptide of the protein according to (1) or a salt thereof,

(4) a polynucleotide comprising a polynucleotide encoding the protein of (1) or the partial peptide of (3),

(5) the polynucleotide according to (4), which is a DNA,

(6) a DNA according to the above (5) containing the nucleotide sequence represented by SEQ ID NO: 2, (7) a recombinant vector containing the polynucleotide according to the above (4),

(8) a transformant transformed with the recombinant vector according to (7),

(9) The transformant according to (8) is cultured, and the protein or salt thereof according to (1) or the partial peptide or salt thereof according to (3) is produced, accumulated, and collected. A method for producing the protein according to the above (1) or a salt thereof, or the partial peptide according to the above (3) or a salt thereof,

(10) a pharmaceutical comprising the protein or salt thereof according to (1) or the partial peptide or salt thereof according to (3);

(11) an antibody against the protein or salt thereof according to (1) or the partial peptide or salt thereof according to (3),

(12) a diagnostic agent comprising the antibody according to (11),

(13) The protein or salt thereof according to (1), or the salt thereof, wherein the protein or salt thereof according to (1) or the partial peptide or salt thereof according to (3) is used. A method for screening a compound or a salt thereof that regulates the activity of the partial peptide or a salt thereof according to the above,

(14) The protein or salt thereof according to (1) or the partial peptide or salt thereof according to (3) contains a DNA encoding the protein according to (1) or the partial peptide according to (3). The screening method according to the above (13), which is expressed in the cytoplasm of a transformant transformed with the DNA; (15) The protein or salt thereof according to (1), which comprises the protein or salt thereof according to (1) or the partial peptide or salt thereof according to (3). A) a kit for screening a compound or a salt thereof that regulates the activity of the partial peptide or a salt thereof described above;

(16) The protein or salt thereof according to (1) or the partial peptide or salt thereof according to (3), which is obtained by using the screening method according to (13) or the screening kit according to (15). A compound or a salt thereof that regulates the activity of

(17) a medicament comprising the compound according to (16) or a salt thereof, (18) a medicament according to (17), which is a prophylactic or therapeutic agent for heart disease.

(19) an antisense nucleotide having a base sequence complementary or substantially complementary to DNA encoding the protein according to (1) or the partial peptide according to (3),

(20) a medicine comprising the antisense nucleotide according to (19), (21) a medicine comprising the polynucleotide according to (4),

(22) a diagnostic agent comprising the polynucleotide according to (4),

(23) a method for preventing or treating heart disease, which comprises administering to a mammal an effective amount of the compound or a salt thereof according to (16);

(24) Use of the compound or a salt thereof according to the above (16) for producing an agent for preventing or treating heart disease.

Further, the present invention provides

(25) a DNA encoding the protein according to the above (1) or a non-human DNA transgenic animal having a mutant DNA thereof,

(26) the animal according to (25), wherein the non-human animal is a rodent;

(27) The animal according to (26), wherein the rodent is a mouse or a rat.

(28) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,

(29) The embryonic stem cell according to the above (28), wherein the DNA is inactivated by introducing a reporter gene,

(30) The embryonic stem cell according to (28), which is neomycin-resistant, (31) the embryonic stem cell according to (28), wherein the non-human mammal is a rodent; (32) the embryonic stem cell according to (31), wherein the rodent is a mouse;

(33) a non-human mammal deficient in expression of the DNA in which the DNA of the present invention has been inactivated, (34) the DNA has been inactivated by introducing a repo overnight gene, and the repo overnight gene is The non-human mammal according to the above (33), which can be expressed under the control of a promoter for the DNA of the present invention,

(35) the non-human mammal according to the above (34), wherein the non-human mammal is a rodent;

(36) the non-human mammal according to the above (35), wherein the rodent is a mouse; and

(37) A compound or a salt thereof, which comprises administering a test compound to the animal according to (36) and detecting the expression of a reporter gene β, which promotes or inhibits the activity of a promoter against DNA of the present invention. Also provided are screening methods and the like. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of DNA (NolOrCsx full) and AF006664 (AF006664Csx. SE) encoding the novel Nkx2.5 analogous protein of the present invention shown in Example 1 (continued from FIG. 2).

FIG. 2 shows an alignment of DNA (NolOrCsx full) and AF006664 (AF006664Csx.SE) encoding the novel Nkx2.5-like protein of the present invention shown in Example 1 (continued from FIG. 1 and FIG. Continue) .

FIG. 3 shows an alignment of DNA (NolOrCsx full) and AF006664 (AF006664Csx. SE) encoding the novel Nkx2.5-like protein of the present invention shown in Example 1 (continued from FIG. 2 and FIG. Continue) .

FIG. 4 shows the alignment of DNA (NolOrCsx full) and AF006664 (AF006664Csx.SE) encoding the novel Nkx2.5-like protein of the present invention shown in Example 1 (continued from FIG. 3, FIG. Continue) .

FIG. 5 shows the alignment of DNA (NolOrcsx full) and AF006664 (AF006664Csx. SE) encoding the novel Nkx2.5 analogous protein of the present invention shown in Example 1 (FIG. (Continued from Figure 4 and continued from Figure 6).

FIG. 6 shows an alignment between DNA (NolO rCsx ful) encoding the novel Nkx2.5 analogous protein of the present invention shown in Example 1 and AF006664 (AF006664Csx.SE) (continuation of FIG. 5 and FIG. 7). Continue) .

FIG. 7 shows an alignment between DNA (NolO rCsx ful) encoding the novel Nkx2.5 analogous protein of the present invention and 删 6664 (AF006664Csx.SE) shown in Example 1 (continuation of FIG. 6 and FIG. Continue) .

FIG. 8 shows an alignment between DNA (NolO rCsx ful) encoding the novel Nkx2.5 analogous protein of the present invention and AF006664 (AF006664Csx. SE) shown in Example 1 (continuation of FIG. 7, FIG. followed by) .

FIG. 9 shows an alignment of DNA (NolO rCsx ful) encoding the novel Nkx2.5 analogous protein of the present invention shown in Example 1 and AF006664 (AF006664Csx. SE) (continuation of FIG. 8).

FIG. 10 shows the time course of DNA encoding the novel Nkx2.5-like protein of the present invention in a myocardial infarction model rat. 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 of the present invention (hereinafter, also referred to as the protein of the present invention or the protein used in the present invention. The protein of the present invention and the protein used in the present invention may be used in the sense that their amides and esters are also included.) Are warm-blooded animals (for example, humans, guinea pigs, rats, mice, chickens, Egret, bush, sheep, wedge, monkey, etc.) cells (eg, hepatocytes, spleen cells, nerve cells, glial cells, knees | 3 cells, bone marrow cells, mesangial cells, Langer's cells, epidermal cells, Epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages) , T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, ruptures Osteocytes, breast cells, hepatocytes or stromal cells, or their precursors, stem cells or cancer cells Vesicles) or any tissue where these cells are present, such as the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal nucleus, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, Pituitary, stomach, knee, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, It may be a protein derived from peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 97% or more, preferably about 98% or more, more preferably the amino acid sequence represented by SEQ ID NO: 1. Examples include amino acid sequences having about 99% or more homology.

Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described above. However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 1 is preferable. Examples of the activity of substantially the same quality include, for example, activity for promoting cardiac function decline. Substantially identical indicates that the activity is qualitatively (eg, physiologically or pharmacologically) equivalent. Therefore, it is preferable that the activity to promote cardiac function decline 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 this activity and the molecular weight of the protein may be different.

Measurement of activity such as cardiac dysfunction promoting activity can be measured using an echocardiograph (Cell, Vol. 97, pp. 189-199, 1989) or cardiac function measurement using a cardiac catheter (circulation) (Research, Vol. 69, p. 370—p. 377, 1991). Further, for example, the enhancement of renin-angiotensin system (RAS) such as angiotensin I converting enzyme (ACE) is measured using a commercially available assay kit (eg, manufactured by Peninsula Inc., Phoenix Inc.). The activity can be measured by using, as an index, the activity of increasing catecholamine in blood (a full-automatic catecholamine analyzer manufactured by Tosoh Corporation).

Examples of the protein of the present invention include: (1) an amino acid represented by SEQ ID NO: 1; An amino acid sequence in which one or two or more (preferably about 1 to 10 and more preferably (1 to 5)) amino acids have been deleted from the amino acid sequence; 2) the amino acid represented by SEQ ID NO: 1 An amino acid sequence obtained by adding one or more (preferably about 1 to 10, more preferably a number of (1 to 5)) amino acids to the sequence; ③ the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence into which 1 or 2 or more (preferably about 1 to 10, more preferably a number (1 to 5)) amino acids have been inserted; ④ in the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence in which one or more (preferably preferably about 1 to 10 and more preferably 1 to 5) amino acids have been substituted with another amino acid, or an amino acid sequence combining them So-called muti, such as contained protein It is also included.

In the protein in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling. The proteins used in the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, include C-terminal lipoxyl group (one COOH), carboxylate (one COO—), and amide It may be either (—CONH ₂ ) or ester (one COOR).

Here, as R in the ester, e.g., methyl, Echiru, n _ propyl, isopropyl, ₆ alkyl group, such as n- heptyl, for example, C _3, such as cyclohexyl cyclopentyl, cyclohexylene - ₈ cycloalkyl group, for example, phenyl, α- naphthyl, such as C ₆ - _{1 2} Ariru group, e.g., benzyl, C ₇ _ ₄ Ararukiru groups such as flying one Nafuchiru § alkyl group such as a phenyl primary alkyl group or flying one naphthylmethyl such phenethyl, Bibaroiruo A xymethyl group or the like is used.

When the protein used in the present invention has a carboxyl group (or propyloxylate) other than the C-terminal, the protein used in the present invention includes amidated or esterified lipoxyl group. . As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Further, in the protein used in the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) has a protecting group (eg, formyl group, acetyl group, etc.). It is protected with a protecting such Ashiru group such bets ₆ Arukanoiru), which dull evening Min residues of N-terminal region is cleaved in vivo to form pin hole dull evening Min oxide, the side chains of amino acids in the molecular The above substituents (eg, 1 OH, 1 SH, amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (eg, alkenyl groups such as alkanoyl groups such as formyl group, acetyl group, etc.) Protected proteins, or multiproteins such as so-called glycoproteins to which sugar chains are bound are also included.

Specific examples of the protein of the present invention include, for example, the rat-derived protein represented by SEQ ID NO: 1.

Partial peptide of the protein of the present invention (hereinafter sometimes referred to as the partial peptide of the present invention. Further, the partial peptide of the present invention may be used in the sense of including its amide form and ester form.) Is a partial peptide of the above-mentioned protein of the present invention, and preferably any peptide having the same activity as the above-mentioned protein of the present invention.

For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 150 or more of the constituent amino acid sequences of the protein of the present invention. Peptides having the amino acid sequence of

In the partial peptide of the present invention, one or more (preferably 1 to 5) amino acids in the amino acid sequence are deleted, or one or two or more ( Preferably, about 1 to 20 amino acids, more preferably about 1 to 10 amino acids, and even more preferably about 1 to 5 amino acids are added, or 1 or 2 or more (preferably , 1 to 5) amino acids may be inserted, or one or more (preferably 1 to 5) amino acids in the amino acid sequence may be substituted with another amino acid.

More specifically, as the partial peptide of the present invention, in the amino acid sequence represented by SEQ ID NO: 1, (1) the 53rd (G1u) to 55th (Tyr) )) A partial amino acid sequence consisting of amino acid residues, ② a partial amino acid sequence consisting of the 131st (Arg) to 134th (Ala) amino acid residues from the N-terminal, ③ And the partial amino acid sequence consisting of the 164th (Ala) to 165th (Pro) amino acid residues, and ④ the 266th (A1a) to 266th from the N-terminal. A partial amino acid sequence consisting of the (A1a) amino acid residue and a partial amino acid sequence consisting of the 277th (Gin) to 282nd (Ala) amino acid residue from the N-terminal. The above-mentioned partial peptides of the protein of the present invention having any or a plurality (2 to 5) of the selected partial amino acid sequences are exemplified.

In the partial peptide, the C-terminus of the present invention the force Rupokishiru group (one CO OH), Cal Pokishireto (one COO-), amide (- CO NH ₂₎ or an ester (one COOR) (R is as defined above ).

Further, similar to the protein of the present invention, the partial peptide of the present invention has an amino terminal residue at the N-terminal (eg, methionine residue) in which the amino group is protected by a protecting group, and the N-terminal side has a native amino acid residue. Glutamine residues generated by cleavage in the body and pyroglutamine oxidized, those in which the substituents on the side chains of the amino acids in the molecule are protected with appropriate protecting groups, and so-called glycopeptides to which sugar chains are bonded Also included are composite peptides.

The partial peptide of the present invention can also be used as an antigen for producing an antibody. As the salt of the protein or partial peptide of the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used. Preferred are acid addition salts that are chemically acceptable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The protein of the present invention or a partial peptide thereof or a salt thereof may be produced by a known protein purification method from human or warm-blooded animal cells or tissues as described above, or by culturing a transformant containing DNA encoding the protein. It can be manufactured by Further, it can be produced according to the peptide synthesis method described later.

When manufactured from human or warm-blooded animal tissues or cells, human or warm-blooded animal After homogenizing the tissue or cells, the extract can be extracted with an acid or the like, and the extract can be purified and isolated by a combination of chromatography such as reverse phase chromatography and ion exchange chromatography.

For the synthesis of the protein or partial peptide of the present invention or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be usually used. Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethyl resin Methyl phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4, dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4-(2', 4 'dimethoxyphenyl-Fmocaminoethyl) phenoxy resin And so on. Using such a resin, an amino acid having a suitably protected amino group and side chain functional group is condensed on the resin according to the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or partial peptide or an amide thereof.

For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of the carpoimides include DCC, N, N'-diisopropylcarpo- imide, N-ethyl N '-(3-dimethylaminoprolyl) carbopimide. For these activations, the protected amino acid may be added directly to the resin along with a racemization inhibitor additive (eg, H〇Bt, HOOBt) or as a symmetrical anhydride or HOBtester or HOOBtester. After the protected amino acid is activated in advance, it can be added to the resin.

The solvent used for activating the protective amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpiperidone, and halogenated carbons such as methylene chloride and chloroform. Hydrogens, alcohols such as trifluoroethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; methyl acetate and ethyl acetate; Esters or an appropriate mixture thereof are used. The reaction temperature is appropriately selected from a range that is known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about 120 ° 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 a sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isopolnyoxycarbonyl, 4-methoxybenzyloxycarbonyl, C11Z, Br-Z, and adaman. Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

The carboxyl group may be, for example, alkyl-esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.). Alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl It can be protected by hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of a group suitable for the esterification include a lower group such as an acetyl group ((: an aroyl group such as an alkanoyl group and a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group). It is. Examples of groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.

The protecting group of the phenolic hydroxyl group of tyrosine, for example, Bz and C 1 ₂ - B z K 2_ nitrobenzyl, B r- Z, such as t one-heptyl is used.

As the protecting group for imidazole of histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the activated form of the carboxylic group of the raw material include, for example, corresponding acid anhydrides, azides, and active esters [alcohols (eg, phenol, 2,4,5-trichlorophenol, 2,4 —Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOB t)]. As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.

Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a stream of hydrogen in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane. Acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia Also used. The elimination reaction by the above-mentioned acid treatment is generally performed at a temperature of about 120 ° C to 40 ° C. In the acid treatment, for example, anisol, phenol, thioanisole, methacresol, paracresol, dimethylsulfur It is effective to add a force-thione scavenger such as FID, 1,4-butanedithiol, 1,2-ethanedithiol, etc. In addition, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-1. In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc. The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.

As another method for obtaining an amide form of a protein or partial peptide, for example, first, after amidating and protecting a lipoxyl group of a carboxy terminal amino acid, a peptide (protein) chain having a desired chain length on the amino group side is obtained. And a protein or partial peptide from which only the protecting group at the N-terminal α-amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group at the C-terminal carboxyl group has been removed. Are produced, and these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein or peptide can be obtained. The crude protein or peptide is purified by using various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein or peptide.

To obtain an ester of a protein or peptide, for example, after condensing the carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein can be obtained in the same manner as the amide of a protein or peptide. Alternatively, an ester of a peptide can be obtained.

The protein or partial peptide of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the desired peptide can be produced by condensing a partial peptide or amino acid capable of constituting the partial peptide of the present invention with the remaining portion, and if the product has a protective group, removing the protective group to produce the desired peptide. Can be. Known condensation methods and elimination of protecting groups include, for example, the methods described in the following ① to ⑤.

ΦΜ. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publ ishers, New York (1966) ② Sdiroeder and Luebke, The Peptide, Academic Press, New York (1965)

(3) Nobuo Izumiya et al. Basics and experiments on peptide synthesis, Maruzen Co., Ltd. (1975)

治 Haruaki Yajima and Shunpei Sakakibara, Biochemistry Laboratory Lecture 1, Protein Chemistry IV, 205, (1977)

治 Supervised by Haruaki Yajima, Development of Continuing Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten

After the reaction, the partial peptide used in the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the protein or partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the protein or the partial peptide is obtained by a salt, it is known. It can be converted to the free form or other salts by the method of or a method analogous thereto.

The polynucleotide encoding the protein of the present invention may be any polynucleotide containing a base sequence (DNA or RNA, preferably DNA) encoding the protein of the present invention. . The polynucleotide is RNA such as DNA or mRNA encoding the protein of the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. If single-stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).

Using a polynucleotide encoding the protein of the present invention, the method described in a known method, for example, “Experimental Medicine Special Edition,“ New PCR and Its Application ”15 (7), 1997, or a method analogous thereto, is used. The mRNA of the protein of the invention can be quantified.

The DNA encoding the protein of the present invention may be any DNA as long as it contains the above-described nucleotide sequence encoding the protein of the present invention. In addition, any of genomic DNA, genomic DNA library, the above-mentioned cells, tissue-derived cDNA, the above-mentioned cell 組織 tissue-derived cDNA library, and synthetic DNA May be.

The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be directly performed by reverse transcription polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells and tissues described above.

Examples of the DNA encoding the protein of the present invention include a DNA having the nucleotide sequence represented by SEQ ID NO: 2 or a DNA having the nucleotide sequence represented by SEQ ID NO: 2 and a high stringency. Any DNA may be used as long as it contains DNA that hybridizes under the conditions and encodes a protein having substantially the same properties as the protein of the present invention.

Examples of a DNA that can hybridize under high stringent conditions with a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 include, for example, about 97% or more of the nucleotide sequence represented by SEQ ID NO: 2, Preferably, a DNA containing a base sequence having a homology of about 98% or more, more preferably about 99% or more is used.

Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can be. 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 performed under high stringent conditions.

High stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° (: The conditions are from 0 to 65. Particularly preferred is a sodium concentration of about 19 mM and a temperature of about 65 ° C.

More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, a DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used.

Examples of the DNA encoding the partial peptide of the present invention include the aforementioned partial peptide of the present invention. Any DNA containing a nucleotide sequence encoding the peptide may be used. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.

Examples of the DNA encoding the partial peptide of the present invention include a DNA having a partial base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, or a base sequence represented by SEQ ID NO: 2 A partial nucleotide sequence of a DNA encoding a protein having a DNA that hybridizes with DNA under high stringent conditions and having substantially the same activity as a protein containing the amino acid sequence represented by SEQ ID NO: 1 DNA or the like is used.

Examples of the DNA that can hybridize with the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 97% or more, preferably about 98%, of the nucleotide sequence represented by SEQ ID NO: 2 %, More preferably a DNA containing a base sequence having a homology of about 99% or more is used.

More specifically, assuming that the DNA encodes the partial peptide of the present invention, in the amino acid sequence represented by SEQ ID NO: 1, (1) 53rd (G1u) to 55th (Ty r) the base sequence encoding the partial amino acid sequence consisting of the amino acid residues (eg, in the base sequence represented by SEQ ID NO: 2, the partial base sequence from the 157th to the 165th from the 5 'end (sequence No .: 18)), ② Nucleotide sequence encoding the partial amino acid sequence consisting of the 131st (Arg) to 134th (Ala) amino acid residues from the N-terminus (eg, SEQ ID NO: 2 391st to 402th partial nucleotide sequence from the 5 'end (SEQ ID NO: 19)), ③ 164th (A1a) to 165th (Pro) from the N-terminal A nucleotide sequence encoding an amino acid sequence (eg, 5 ′ in the nucleotide sequence represented by SEQ ID NO: 2) 490th to 495th partial base sequence from the end (SEQ ID NO: 20)), 部分 partial amino acid consisting of 266th (A1a) to 268th (A1a) amino acid residues from the N-terminus A nucleotide sequence encoding the sequence (eg, in the nucleotide sequence represented by SEQ ID NO: 2, a partial nucleotide sequence from 796th to 804th from the 5 'end (SEQ ID NO: 21)); 277th (G1n) to 282nd A nucleotide sequence encoding a partial amino acid sequence consisting of the amino acid residue at position (A la) (eg, in the nucleotide sequence represented by SEQ ID NO: 2, the 829th to 846th partial nucleotide sequence from the 5 'end) (SEQ ID NO: 22)) and a DNA encoding a partial peptide of the protein of the present invention having any one or a plurality (2 to 5) of base sequences selected from the group consisting of:

Hybridization methods and high stringency conditions are the same as described above.

DNA that completely encodes the protein of the present invention or the partial peptide of the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding them, these may be simply abbreviated as the protein of the present invention). Cloning means may be amplified by the PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA incorporated into an appropriate vector may be used as the DNA of the present invention. Selection can be performed by hybridization with a DNA fragment encoding a part or the whole region of the protein or labeled with a synthetic DNA. Hybridization can be carried out, for example, according to the method described in Molecular-Cloning (Molecular Cloning) 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted using PCR or a known kit, for example, Mutan ^™ -suer Expression Km (Takara Shuzo), Mutan ™ -K (Takara Shuzo), etc. The method can be carried out according to a known method such as the Gap ed dulex method or the Knuke 1 method or a method analogous thereto.

The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used by digesting with a restriction enzyme or adding a linker if desired. The DNA may have ATG as a translation initiation codon on its 5, terminal side, and may have TAA, TGA or TAG as a translation termination codon on its 3, terminal side. These translation start codons and translation stop codons are It can also be added using one.

The expression vector for the protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by

Examples of the vector include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, TP5, pC194), a plasmid derived from yeast (eg, pSH 19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, XT1, ρRc / CMV, pRc / RSV, pcD NA I ZN eo or the like is used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as host, SRa promoter, SV40 promoter, LTR motor, CMV promoter, HSV-TK promoter, etc. are mentioned. Among them, CMV (cytomegalovirus) promoter, SRa promoter It is preferable to use one or the like. When the host is Eshierihia genus bacteria, tr [rho promoter, lac promoter, re cA promoter, AP _L promoter, if 1 pp promoter, T 7 including promoter Isseki one is, host Ru der Bacillus, When the host is yeast, such as SPOL promoter, SPO2 promoter, penP promoter, etc., PH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. In addition to the above, the expression vector may further include an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter, sometimes abbreviated as SV40 ori), and the like, if desired. Can be used. Selection markers include, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene (methotrexate (MTX) resistance), ampicillin Phosphorus resistant gene (hereinafter sometimes abbreviated as Amp ¹ "), neomycin resistance gene (hereinafter sometimes abbreviated as Ne o ^r, G41 8 resistance). In particular, dh fr genetic defects When the dhfr gene is used as a selection method using Chinese hamster cells, the target gene can be selected even on a thymidine-free medium.

If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. If the host is Escherichia, Pho A signal sequence, OmpA signal sequence, etc., if the host is Bacillus, 《— amylase signal sequence, subtilisin signal sequence, etc. In the case of yeast, MFa signal sequence, SUC2 signal sequence, etc. In the case of host animal cells, use of insulin signal sequence, one interferon, signal sequence, antibody molecule, signal sequence, etc. it can.

Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant can be produced.

As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.

Specific examples of the genus Escherichia include, for example, Esdieric hia coli Kl 2 · DH1 [Procedures of the national academy 'ob' (Proc. Natl. Acad. ScI. USA), Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research], (Nucleic Acids Research), Vol. 9, 309 (1981)], JA2 21 [Journal of Molecular Biology, 120, 517 (1978)], HB 101 [Journal of Molecular Biology, Biology, 41, 459 (1969)] ], C600 [Genetics, 39, 440 (1954)] and the like.

Examples of Bacillus bacteria include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95] , 87 (1 984)]. Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD- 5D, 20B-12, Shizosaccharomyces' bomb (Sdiizosaccharomyces pombe) NCYC 1913, NCYC 2036, Pichia 'Pichia pastoris KM71 etc. is used.

Insect cells include, for example, when the virus is Ac NPV, a cell line derived from the larvae of night moth (Spodoptera frugiperda cell; S f cell); High Five ™ cells, cells derived from Mamestra b rassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a cell line derived from silkworm (Bombyx mori N cell; BmN cell) is used. Examples of the Sf cell include Sf9 cell (ATCC CRL1711), Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like. Used.

As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cells COS-7, Vero, Chinese Hamster cells CHO (hereinafter abbreviated as CHO cells), dh fr gene-deficient Chinese Hamster Yuichi cells CHO (hereinafter CHO (dhfr_) cells) Abbreviations), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, H9c2 cells, etc. are used.

Transformation of Escherichia sp. Is described, for example, in Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. , 2110 (1972) and Gene, 17, 107 (1982). Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).

To transform yeast, see, for example, Methods in Enzymology, 194, 182-187 (1991), According to the method described in, for example, Goods-of-the-National Academy of Things-of-the-Sciences-of-the-Us. (Proc. Natl. Acad. Sci. USA), Vol. 75, 1929 (1978) Can do it.

Insect cells or insects can be transformed, for example, according to the method described in Bio Z Technology (Bio / Technology), 6, 47-55 (1988). In order to transform animal cells, for example, Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol. 263—267 (1995) (published by Shujunsha), Virology, 52, 456 (1973) Can be performed according to the method described in (1).

Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The H of the medium is preferably about 5-8.

As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Exp. lar Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. Here, if necessary, a drug such as 3-indolylacrylic acid can be added to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.

When the host is a bacterium belonging to the genus Bacillus, culturing is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring may be added. When culturing a transformant in which the host is yeast, for example, Burkholder's minimal medium [Bostian, KL, et al., Processings * of the National Academy of Sciences] Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and SD medium containing 0.5% casamino acid, CBiUer, GA et al. -The National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The cultivation is usually performed at about 20 t to 35 ° C for about 24 to 72 hours, and aeration and agitation are added as necessary.

When culturing an insect cell or a transformant in which the host is an insect, the culture medium used was a 10% strain immobilized in Grace's Insect Medium (Grace, TC, Nature, 195, 788 (1962)). Those to which additives such as serum are appropriately added are used. It is preferable to adjust the ρ 培 of the culture medium to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPM I 1640 medium [The Journal of the American Medical Association at ion, 199, 519 (1967) )], And 199 medium [Proceding of the Society for the Biological Medicine, Vol. 73, 1 (1950)]. Preferably, the pH is about 6-8. Cultivation is usually carried out at about 30 to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.

As described above, the protein of the present invention can be produced in the cytoplasm or extracellularly of the transformant.

The protein of the present invention can be separated and purified from the culture by, for example, the following method.

When extracting the protein of the present invention from cultured cells or cells, The cells or cells are collected by a known method, suspended in an appropriate buffer, and

After the cells or cells are disrupted by lysozyme, Z or freeze-thawing, a method of obtaining a crude protein extract by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.

Purification of the protein contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting-out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Method using difference in charge, such as ion exchange chromatography, method using specific affinity such as affinity chromatography, hydrophobicity such as reversed-phase high-performance liquid chromatography, etc. A method using a difference in gender, a method using a difference in isoelectric point such as isoelectric focusing, and the like are used.

When the protein thus obtained is obtained as a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained as a salt, a known method or analogous method Depending on the method, it can be converted into a free form or another salt.

The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The presence of the protein of the present invention thus produced is determined using a specific antibody.

Antibodies against the protein or partial peptide of the present invention or a salt thereof are described in Any polyclonal antibody or monoclonal antibody may be used as long as it can recognize the protein or partial peptide or its salt.

An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these may be simply abbreviated to the protein of the present invention in the description of the antibody) is obtained by using the protein of the present invention as an antigen. The antibody or antiserum can be produced according to the following method.

[Preparation of monoclonal antibody]

(a) Preparation of monoclonal antibody-producing cells

The protein of the present invention is administered to a warm-blooded animal at a site where the antibody can be produced by administration to itself or together with a carrier or a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of the warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them. By fusing the antibody-producing cells obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kellar and Milstein (Nature, 256, 495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG6000) Is added at a concentration of about 10 to 80%, and incubating at 20 to 40 ° (preferably at 30 to 37 for 1 to 10 minutes) enables efficient cell fusion. Various methods can be used to screen for produced hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which protein antigens are directly or adsorbed together with a carrier, and then radioactive substances are added. Method to detect monoclonal antibody bound to solid phase by adding anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is mouse) or protein A labeled with proteins or enzymes Then, add the hybridoma culture supernatant to the solid phase to which the anti-immunoglobulin antibody or protein A is adsorbed, and use radioactive substances, enzymes, etc. A method of detecting a monoclonal antibody bound to a solid phase by adding a labeled protein may be used.

The selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) Alternatively, a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually from 20 to 40 ° C, preferably about 37 ° C. The culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(b) Purification of monoclonal antibody

Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods [eg, salting out, alcohol precipitation, isoelectric point precipitation, electrophoresis, ion exchangers (eg, DEAE) ), Ultracentrifugation, gel filtration, antigen-binding solid phase or specific antibody purification by collecting only the antibody with an active adsorbent such as protein A or protein G, and dissociating the bond to obtain the antibody). It can be carried out (Preparation of polyclonal antibody)

The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as the protein of the present invention) and a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting an antibody-containing substance against the antibody and separating and purifying the antibody.

Regarding the complex of immunizing antigen and carrier protein used for immunizing warm-blooded animals, the type of carrier protein and the mixing ratio of carrier and hapten are determined by the antibody against hapten immunized by cross-linking with the carrier. If it can be efficiently performed, any kind may be crosslinked at any ratio.For example, serum serum albumin, thyroglobulin, hemocyanin, etc. may be used in a weight ratio of about 0 to 1 for hapten. A method of condensing at a rate of 1 to 20, preferably about 1 to 5 is used. Further, various condensing agents can be used for force coupling between the hapten and the carrier. For example, an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.

The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.

The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be carried out according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.

DNAs encoding the protein or partial peptide of the present invention (hereinafter, these DNAs are abbreviated as DNAs of the present invention in the description of antisense nucleotides) or are substantially complementary to the DNAs. Antisense nucleotides having a base sequence or a part thereof are complementary or substantially complementary to the DNA of the present invention. Any antisense nucleotide may be used as long as it has a complementary base sequence or a part thereof and has an action capable of suppressing the expression of the DNA, but antisense DNA is preferable.

The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, the entire nucleotide sequence or a partial base of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). A nucleotide sequence having a homology of about 97% or more, preferably about 98% or more, and more preferably about 99% or more with the sequence is exemplified. In particular, of the entire base sequence of the complementary chain of the DNA of the present invention, the complementary sequence of the base sequence (for example, the base sequence near the start codon) of the portion encoding the N-terminal site of the protein of the present invention is about 9%. Antisense nucleotides having a homology of 7% or more, preferably about 98% or more, more preferably about 99% or more are suitable.

The antisense nucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.

In order to prevent degradation by hydrolases such as nucleases, the phosphate residues (phosphates) of each nucleotide constituting the antisense nucleotide are, for example, chemically modified phosphates such as phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted with a residue. These antisense nucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is cloned or a DNA encoding a determined protein is obtained. It can be designed and synthesized on the basis of the base sequence information. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or inhibit the interaction with the protein-related RNA of the present invention. Thus, the expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of a protein-associated RNA of the present invention, and polynucleotides that can specifically hybridize with a protein-associated RNA of the present invention can be used in vivo and in vitro. It is useful for regulating and controlling the expression of a protein gene in a plant, and is also useful for treating or diagnosing a disease or the like. Term "corresponding The term "is" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. The “correspondence” between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) under instructions derived from the nucleotide (nucleic acid) sequence or its complement. ing. 5 'end hairpin loop of protein gene, 5' end 6-base spare repeat, 5 'end untranslated region, polypeptide translation start codon, protein code region, ORF translation stop codon, 3' end untranslated The region, the 3 'end parindrome region, and the 3' end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected.

The relationship between the nucleic acid of interest and a polynucleotide complementary to at least a part of the target region can be said to be "antisense" with the polynucleotide capable of hybridizing with the target. Antisense polynucleotides include polynucleotides containing 2-dexoxy D-reports, polynucleotides containing D-reports, other types of polynucleotides that are N-daricosides of purine or pyrimidine bases, Alternatively, other polymers having non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (伹, such polymers may be found in DNA or RNA) (Including a nucleotide having a configuration that allows base pairing and base attachment as found)). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified polynucleotides). (Nucleotides), and also those with known modifications, such as those with labels, caps, methylated, and one or more natural nucleotides known in the art. Substituted, or modified with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or Those having a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (nucleases, nucleases / inhibitors) , Toxins, antibodies, signal peptides, poly -L one lysine, etc.) or a sugar (e.g., Mo No.3, etc., which have side chain groups, etc.

(E.g., acridine, psoralen, etc.), chelate compounds (e.g., metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, modification (Eg, a nucleic acid of the type I). Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functionalities such as ethers, amines, etc. It may be converted to a group.

The antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, 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, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Make sense nucleic acid less toxic.

Thus, many modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. ed., Ant isense Research and Applicat ions, CRC Press, 1993.

The antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form. In this way, the charge in the phosphate skeleton is used in the additional form. Polycationic substances such as polylysine, which acts to neutralize, and crude water-based substances, such as lipids (eg, phospholipids, cholesterol, etc.), which enhance the interaction with cell membranes and increase the uptake of nucleic acids. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include cap groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonucleases and RNases. . Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as dalicol such as polyethylene glycol and tetraethylene dalicol.

The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various known methods.

Hereinafter, the protein or partial peptide used in the present invention or a salt thereof (hereinafter, sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or the partial peptide of the present invention (hereinafter, the DNA of the present invention) ), An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the antibody of the present invention), and an antisense nucleotide of the DNA of the present invention (hereinafter, the present invention) May be abbreviated as “antisense nucleotide”).

Since the expression of the protein of the present invention increases in the heart at the stage of heart failure transition (heart failure decompensation / heart failure decompensation) after myocardial infarction, it can be used as a disease marker. That is, early diagnosis of diseases characterized by decreased cardiac function (eg, heart failure after myocardial infarction; angina pectoris; cardiomyopathy; heart diseases such as heart failure resulting from diseases such as angina pectoris and cardiomyopathy). It is useful as a marker for determining the severity of symptoms and predicting disease progression.

Antisense nucleotide of a gene encoding the protein of the present invention; Pharmaceuticals containing a compound that regulates the activity of a protein or a salt thereof or an antibody against the protein of the present invention include, for example, diseases characterized by decreased cardiac function (eg, heart failure after myocardial infarction; angina pectoris; cardiomyopathy) Useful in the treatment and prevention of heart diseases such as heart failure due to diseases such as angina pectoris and cardiomyopathy).

[1] Screening of drug candidate compounds for disease

Since the expression of the protein of the present invention increases with a decrease in cardiac function after myocardial infarction (heart failure decompensation Z cardiac decompensation), compounds or salts thereof that regulate the activity of the protein of the present invention include, for example, Hearts such as diseases characterized by reduced cardiac function (eg, heart failure after myocardial infarction; angina pectoris; cardiomyopathy; heart diseases such as heart failure resulting from diseases such as angina pectoris and cardiomyopathy) It can be used as a treatment and prevention drug for diseases.

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

(1) A method for screening a compound or a salt thereof that regulates the activity of the protein of the present invention, characterized by using the protein of the present invention, is provided.

Specifically, for example,

(2) (i) A cell capable of producing the protein of the present invention is preferably tested under the condition of low oxygen conditions when stimulated with extension, and (ii) A cell capable of producing the protein of the present invention is tested. It is intended to provide a method for screening a compound or a salt thereof that regulates the activity of the protein of the present invention, which is characterized by comparing a mixture of compounds with a case where a stretching stimulus is applied, preferably under hypoxic conditions.

More specifically, the above-mentioned screening method is characterized in that, for example, in (i) and (ii), the gene expression levels of the protein of the present invention are measured and compared.

The activity of the protein of the present invention includes, for example, an activity of promoting cardiac function decline accompanying decompensation failure, an effect of suppressing an excessive compensation mechanism, and the like.

Here, the low-oxygen conditions e.g. 2 0% 0 ₂ or less of oxygen concentration, for example, 2% (Neichiya primary, third 9 Volume 4, 4 8 5 page one 4 9 0 p., 1 9 9 8 years) Means the following conditions. Stretch stimulation means that cardiomyocytes are cultured on a stretchable silicon membrane, It is a stimulus that applies a mechanical load by pulling the membrane (JBC, '271, 33 592-33597, 1996, Saccuration, 89, 2204-2211, 1994) , JBC, 271: 3221-3228, 1996). Moreover,

(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 (as specific examples, (When serum is removed or cardiomyocytes are cultured with an anticancer agent such as adriamycin which is relatively toxic) and (iv) cells having the ability to produce the protein of the present invention or the protein of the present invention. When a mixture of cells into which the cDNA has been introduced and the test compound are cultured under lethal conditions (for example, adding anticancer drugs such as adriamicin, which is relatively toxic to serum cells or cardiomyocytes, is relatively toxic to cardiomyocytes). And a method for screening a compound or a salt thereof that regulates the activity of the protein of the present invention.

In the above-mentioned screening method, for example, the cytoprotective action in the cases (iii) and (iv) and the expression level of the gene encoding the protein of the present invention are measured by a known method and compared.

Cytoprotective action can be shown by cardiomyocyte activation or viability. Specifically, the MTT (3- (4,5-Dimethyl-2-t iazolyl) -2,5-di henyl -2H-tetrazol iuni) method, which can measure commonly used respiratory activity, and trypan-bleed staining It can be measured by the TUNNEL staining method (Terminal deoxytransferase-mediated d UTP-X nick end labeling, Cell, Vol. 97, pp. 189-198, 1999).

Appropriate expression enhancement at the time of cell death or cell damage can be expected to have a cytoprotective effect. In addition, overexpression of the gene encoding the protein of the present invention is considered to cause excessive activation of cells and accelerate cell exhaustion. Therefore, it is considered important to appropriately control the expression level of the gene encoding the protein of the present invention. It is thought that cell damage is caused by promotion of expression of the gene encoding the protein of the present invention, for example, during the heart failure decompensation period and the heart failure decompensation period. If administered, an inhibitor (a compound that inhibits the activity of the protein of the present invention or a salt thereof) is administered to reduce the expression of a gene encoding the protein of the present invention, for example, in the acute phase of heart failure. If cell damage is considered to have occurred, an enhancer (a compound that promotes the activity of the protein of the present invention or a salt thereof) is administered.

Since the DNA (gene) encoding the protein of the present invention has a function of positively regulating the expression of a heart-specific gene, extreme decrease in the expression of the gene encoding the protein of the present invention impairs cell function. Injury and, conversely, overexpression may cause more activation of cells than necessary, resulting in cell damage.

Therefore, when an excessive decrease in the expression of the protein of the present invention or the DNA of the present invention is found, the function of the protein of the present invention, the DNA of the present invention, or the function of the protein of the present invention obtained by the above-described screening method is considered. By administering a compound or a salt thereof (which will be specifically described later), which promotes cell growth, the disorder of the cell function can be prevented or treated, and conversely, an excess of the protein of the present invention or the DNA of the present invention can be obtained. If any expression is found, administration of a compound that inhibits the function of the protein of the present invention obtained by the above-described screening method or a salt thereof (specifically, described below) will increase the cell function. Disability can be prevented and treated.

Further, the present invention provides

(4) a gene whose expression is positively regulated by the protein of the present invention (e.g., atrial sodium diuretic peptide, cardiac adoriamycinresvonshib 'protein (CARP), myosin light chain 2V (MLC 2v), Nkx 2.5, GAT A-4, MEF2 C, N-myc, HAND-1, Ventricular natriuretic peptide (BBR, Vol. 270, pp. 1074-1079, 2000; Development, Vol. 126, 1269-1280 , 1999)), a compound that regulates the activity of the protein of the present invention, characterized by measuring the enzymatic activity of a reporter gene in a reporter gene using a promoter such as It also provides a method for screening the salt. This utilizes the fact that the enzymatic activity of the repo overnight gene is increased depending on the expression level and activation of the protein of the present invention. Specifically, primary cardiomyocytes, H9c2 cell line (ATCC No. CRL-1446), Alternatively, the primary cardiomyocyte or the H9c2 cell line into which the Nkx2.5 gene has been introduced is used as a host cell to perform repo overnight gene access. The reporter gene assay is, for example, a promoter region of a gene whose expression is positively regulated by the protein of the present invention [eg, the promoter region of the atrial natriuretic peptide gene (JBC, Vol. 272, 22800). -22808, 1997; Development, Vol. 124, pp. 793-804, 1997)) and a repo overnight gene (eg, 8 galactosidase, chloramphenicol acetyltransferase, luciferase). Is constructed by using a cell in which the plasmid has been introduced into cardiomyocytes or the like by a known method. Construction of the plasmid, introduction of the plasmid, and the like can be performed by known methods, for example, a method for producing a recombinant vector containing a DNA encoding the protein of the present invention, and a method for producing a transformant transformed with the recombinant vector. It can be performed according to a similar method.

As a specific example,

(v) Promoter of a gene whose expression is positively regulated by the protein of the present invention In a cell into which a plasmid having a reporter gene ligated downstream of the promoter region is introduced.

And (vi) introducing a plasmid into which a reporter gene has been ligated downstream of the promoter region of a gene whose expression is positively regulated by the protein of the present invention. The activity of the protein of the present invention is compared with the case where the protein of the present invention and a test compound are mixed or brought into contact with the isolated cells, and the enzymatic activity of the repo overnight gene is compared. A method for screening a compound or a salt thereof is provided. The enzyme activity of the reporter gene is measured according to a known method.

The compound or salt thereof obtained by the screening method is a substance that affects the expression level and activation of DNA (gene) encoding the protein of the present invention. Any of a compound that promotes the activity of the protein and a compound that inhibits the activity of the protein of the present invention can be selected.

Test compounds include, for example, peptides, proteins, non-peptidic compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds, microbial cultures, cell extracts, and plant extracts Liquid, animal tissue extract and the like. These compounds may be novel compounds or known compounds.

To carry out the above-described screening method, cells having the ability to produce the protein of the present invention are cultured in a medium suitable for screening. The medium may be any as long as it does not affect the gene expression of the protein of the present invention. Examples of cells having the ability to produce the protein of the present invention include, for example, primary cardiomyocytes having the ability to produce the protein of the present invention or vectors containing DNA encoding the protein of the present invention described above. A host (transformant) transformed with is used. As the host, for example, animal cells such as H9c2 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed in a cell by culturing by the method described above is preferably used.

The expression level of the gene of the present invention can be determined by a known method, for example, Northern blotting, Reverse transcript ion-polymerase chain react ion (RT-PCR), or a real-time PCR analysis system (Ad, TadMan polymerase chain react ion). The measurement can be performed according to any method or a method according to the method.

For example, the gene expression level in the case (ii) is inhibited or promoted by about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case (i). The test compound to be tested can be selected as a compound that inhibits or promotes the activity of the protein of the present invention.

In addition, in the cases (iii) and (iv) above, the cytoprotective effect and the expression level of the gene encoding the protein of the present invention were measured, and the gene expression level was reduced, resulting in cell damage. A compound that increases the expression level under the conditions is used as a compound that promotes the activity of the protein of the present invention. In an Atsushi system in which expression is enhanced and cell damage has occurred, a compound that reduces the expression level is used as a compound that inhibits the activity of the protein of the present invention, and can be used as a compound that has a cytoprotective action.

Furthermore, the enzyme activity of the reporter gene in the case of the above (V) is about 20% or more, preferably 30% or more, more preferably about 30% or more of that in the case of the above (iV). A test compound that inhibits or promotes 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 a salt thereof that inhibits the activity of the protein of the present invention selected by the above screening method is used during the heart failure decompensation phase and the decompensation phase in which the expression of the DNA (gene) encoding the protein of the present invention is enhanced. The administration can be expected to have a cardiac function recovery effect. In addition, the compound or salt thereof which promotes the activity of the protein of the present invention selected by the above screening method enhances the compensatory mechanism by being administered in the acute phase of heart failure where expression is reduced, thereby protecting cardiomyocytes. A heart protective effect can be expected.

The screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide used in the present invention.

The compound (a compound that promotes or inhibits the activity of the protein of the present invention) or a salt thereof obtained by using the screening method or the screening kit of the present invention may be a test compound as described above, for example, a peptide, a protein, a non-peptide derived from a living body. Compounds (eg, carbohydrates, lipids, etc.), synthetic compounds, compounds or salts thereof selected from microbial cultures, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. A compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention (eg, activity to lower cardiac function).

As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.

Compounds or salts thereof that regulate (promote or inhibit) the activity of the protein of the present invention include, for example, diseases characterized by reduced cardiac function (eg, heart failure after myocardial infarction; angina pectoris; cardiomyopathy; angina pectoris) And heart diseases such as heart failure caused by diseases such as cardiomyopathy).

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned therapeutic or prophylactic agent, it can be formulated according to a conventional method. For example, tablets, capsules, elixirs, micron capsules, sterile solutions, suspensions and the like can be used. The preparations obtained in this way are safe and have low toxicity, for example, warm-blooded animals (for example, humans, mice, rats, puppies, sheep, bush, puppies, puppies, birds, cats, dogs) , Monkeys, chimpanzees, etc.) orally or parenterally.

The dose of the compound or a salt thereof varies depending on its action, target disease, subject of administration, route of administration, and the like.For example, a compound or a salt thereof that regulates the activity of the protein of the present invention for the purpose of treating heart failure When the compound is orally administered, generally in an adult (assuming a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 10 Omg, preferably about 1.0 to 50 mg, more preferably about 0.1 to 50 mg per day. 1. Administer 0 to 20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like.For example, a compound or a compound thereof that regulates the activity of the protein of the present invention for the purpose of treating heart failure When the salt is administered to an adult (as 60 kg) usually in the form of an injection, the compound or a salt thereof is used in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 2 Omg. It is convenient to administer about 0.1 to 1 Omg by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered.

[2] Quantification of the protein of the present invention, its partial peptide or its salt

An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention, and therefore, the quantification of the protein of the present invention in a test solution, particularly It can be used for quantification by sandwich immunoassay.

That is, the present invention

(i) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody. A method for quantifying the protein of the present invention in a test solution, and

(ii) After reacting the test solution with the antibody of the present invention insoluble on the carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insoluble carrier is measured. To provide a method for quantifying the protein of the present invention in a test solution, characterized in that: In the quantification method (ii) above, it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention. .

In addition, the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. . For these purposes, the antibody molecule itself may be used, or the F (ab ') ₂ , Fab', or Fab fraction of the antibody molecule may be used.

The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. . For example, nephrometry, a competition method, an immunometric method and a sandwich method are suitably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.

As a labeling agent used in a measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, ^{[1 2 5} I], ^{[1 3 1} I], ^[3 H], and ^{[1 4} C] used. As the above-mentioned enzyme, those which are stable and have a large specific activity are preferable. For example, i3-galactosidase,) 8-darcosidase, alkaline phosphatase, peroxidase, and lignoic acid dehydrogenase are used. Can be As the fluorescent substance, for example, fluorescein, fluorescein isothiosinate and the like are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Carriers include insoluble polysaccharides such as agarose, dextran, and cellulose; and synthetic resins such as polystyrene, polyacrylamide, and silicon. Or glass.

In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. Also, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.

In the method for measuring the protein of the present invention according to the San Deutsch method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is an antibody having a different site to which the protein of the present invention binds. It is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephelometry method, or the like.In a competition method, an antigen in a test solution and a labeled antigen are applied to the antibody. After the competitive reaction, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), and the labeling amount of either B or F is measured. The amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using a polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. Alternatively, an immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.

In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. Reaction with an excess amount of the labeled antibody, and then adding immobilized antigen to the unreacted labeled antibody. After binding the body to the solid phase, the solid and liquid phases are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.

In nephrometry, the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations to those skilled in the art to the ordinary conditions and procedures in each method. For details of these general technical means, reference can be made to reviews and written documents.

For example, edited by Hiro Irie "Radio Nonotsusei" (Kodansha, published in 1949), edited by Hiro Irie "Radio Imunoatsusei" (Kodansha, published in 1954), Eiji Ishikawa et al. "Measurement Method" (Medical Shoin, published in 1958), Eiishi Ishikawa et al., "Enzyme Immunoassay" (Second Edition) (Medical Publishing, published in 1977), Eiji Ishikawa, "Enzyme Immunoassay" (3rd edition) (Medical Shoin, published in 1962), "Methods in ENZYMOLOGYj Vol. 70 (Immunochemical Tec niues (Part A)) ^ Ibid. 73 (Immunochemical TechniQues (Part B)), Ibid. Vol. 74 (Immunochemical Techniaues (Part C)), ibid.Vol. 84 (Immunochemical Techniaues (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochefflical Techniaues (Part E: Monoclonal Ant ibodies and General Immunoassay Methods))> ibid. 121 (Immunochemical Techniaues (P art I '.Hybridoma Technology and Monoclonal Ant ibodies)) ( The above can be referred to, for example, published by Academic Press.

As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.

Furthermore, when an increase in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, a disease characterized by decreased cardiac function (Eg, heart failure after myocardial infarction; angina; cardiomyopathy; heart disease such as heart failure from diseases such as angina and cardiomyopathy) or will be affected in the future Can be diagnosed as having a high possibility.

Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. In addition, for the preparation of an antibody column used for purifying the protein of the present invention, the detection of the protein of the present invention in each fraction during purification, and the analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.

(3) Gene diagnostic agent

The DNA of the present invention can be used, for example, as a probe to produce warm-blooded animals (eg, humans, rats, mice, guinea pigs, egrets, birds, higgies, bush, horses, dogs, cats, dogs, DNA or mRNA encoding the protein of the present invention or its partial peptide (monkey, chimpanzee, etc.) can be detected (gene abnormality). For example, the DNA or mRNA is damaged, mutated or expressed. It is useful as a gene diagnostic agent for a decrease, an increase in the DNA or mRNA, or an overexpression.

The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, p. 874-879 (1989). Proceedings of the National Academy of Sciences of the United States of America; Vol. 86, Vol. 27, Proceedings of the National Academy of Sciences of the United States of America; 66-27770 (11989)), etc. For example, when overexpression is detected by Northern hybridization or DNA mutation by PCR-SSCP method Is detected, it is possible to diagnose that the disease is highly likely to be a disease such as a heart disease accompanied by a decrease in cardiac function, etc. [4] A drug containing an antisense nucleotide

The antisense nucleotide of the present invention, which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, has low toxicity, and is characterized by having the protein of the present invention or the DNA of the present invention in vivo in vivo. It can regulate (inhibit) activities and functions (eg, cardiac dysfunction promoting activity), for example, diseases characterized by reduced cardiac function (eg, heart failure after myocardial infarction; angina pectoris; myocardium Sickness; derived from diseases such as angina pectoris and cardiomyopathy It can be used as a treatment and prevention agent for heart diseases such as heart failure. When the above-mentioned antisense nucleotide is used as the above-mentioned therapeutic or prophylactic agent, it can be formulated and administered according to a known method.

For example, when the antisense nucleotide is used, the antisense nucleotide is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. Oral or parenteral to warm-blooded animals (e.g., humans, rats, mice, guinea pigs, egrets, birds, higgins, pigs, pigs, dogs, cats, dogs, monkeys, chimpanzees, etc.) Can be administered. The antisense nucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter.

The dose of the antisense nucleotide varies depending on the disease to be treated, the subject to be administered, the administration route, and the like. For example, when the antisense nucleotide of the present invention is orally administered for the purpose of treating heart failure, generally the adult (body weight) is used. At 6 O kg), about 0.1 to 10 O mg of the antisense nucleotide is administered per day.

Furthermore, the antisense nucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of its expression.

The present invention further provides

(1) a double-stranded RNA containing a part of the RNA encoding the protein of the present invention,

(2) a medicament comprising the double-chain RNA,

(3) a lipozyme containing a part of the RNA encoding the protein of the present invention;

(4) To provide a medicine containing the lipozyme.

These double-stranded RNAs, lipozymes, and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention in the same manner as the above-mentioned antisense polynucleotide, and can inhibit the expression of the peptide of the present invention in vivo. It can regulate (inhibit) the activity or function of the polynucleotide (eg, DNA) of the present invention (eg, the activity of promoting cardiac function decline), for example, a disease (eg, a disease characterized by decreased cardiac function). Heart failure after myocardial infarction All; angina; cardiomyopathy; heart diseases such as heart failure derived from diseases such as angina and cardiomyopathy).

Double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).

The lipozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, 7, 221, 2001). For example, it can be produced by linking a known lipozyme to a part of the RNA encoding the peptide of the present invention. A part of the RNA encoding the peptide of the present invention includes a portion (RNA fragment) close to the cleavage site on the RNA of the present invention, which can be cleaved by a known lipozyme.

When the above double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.

[5] A drug containing the antibody of the present invention

The antibody of the present invention, which has the activity of neutralizing the activity of the protein of the present invention, is used for a disease characterized by a decrease in cardiac function (eg, heart failure after myocardial infarction; angina; cardiomyopathy; angina, cardiac muscle) It can be used as a prophylactic or therapeutic agent for diseases such as heart failure caused by diseases such as sickness.

The agent for preventing or treating the above-mentioned diseases, which contains the antibody of the present invention, has low toxicity, and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, as a warm-blooded animal (eg, human, rat, mouse, It can be administered orally or parenterally to guinea pigs, egrets, birds, sheep, pigs, puppies, pomas, cats, dogs, monkeys, chimpanzees, etc. The dosage varies depending on the administration subject, target disease, symptoms, administration route and the like.For example, when used for the treatment or prevention of heart failure in adults, the antibody of the present invention is usually administered as a single dose. 0.0 1-2 O mg / kg body weight, preferably 0.1-1 O mg / kg body weight, more preferably 0.1-5 mg / kg body weight, about 1-5 times a day, It is convenient to administer by intravenous injection preferably about once to three times a day. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. No.

The antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.

That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). ), Syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As compositions for parenteral administration, for example, injections, suppositories, etc. are used. Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Is included. Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used, and a suitable solubilizing agent, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), non-ionic surfactants (eg, polysorbate 80, HC-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)), etc. Good. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. A suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a usual suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form adapted to the dose of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories. Each dosage unit usually contains 5 to 500 mg, especially 5 to 100 mg for injections, and 10 to 25 mg for other dosage forms. preferable.

Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.

[6] A drug containing the protein of the present invention

(1) The protein of the present invention is used as a drug such as a vaccine for producing the antibody of the present invention. A vaccine for producing the antibody of the present invention can be produced by a known method using the protein of the present invention.

(2) Recombinant N obtained by linking a nuclear localization signal sequence (antenapedia: a nuclear localization peptide of Drosophi la, a report by Ito et al. Of Tokyo Medical and Dental University: 2nd Molecular Cardiovascular Conference) to the protein of the present invention. The reduced protein of the present invention can be supplemented by administering kx2.5 systemically or directly to the heart during a cardiac catheterization test.

(7) a drug containing the DNA of the present invention

The medicament containing DNA of the present invention is used for gene therapy of heart failure. One of the functions of the protein of the present invention is to enhance the expression of heart-specific genes. We believe that this will result in enhanced compensatory mechanisms following myocardial infarction. Therefore, during the period when the expression of the protein of the present invention is decreased, such as in the acute phase of heart failure, the DNA of the present invention can be administered so that the protein of the present invention maintains an appropriate expression level. Monkey

(8) Preparation of an animal having the DNA of the present invention.

Using the DNA of the present invention, transgenic animals expressing the protein of the present invention can be prepared. Examples of animals include mammals (for example, rats, mice, rabbits, rabbits, sheep, pigeons, pigs, cats, dogs, monkeys, etc.) (hereinafter sometimes abbreviated as animals). When introducing the DNA of the present invention into a target animal, the DNA is used as a gene construct linked downstream of a promoter capable of being expressed in animal cells. Is generally advantageous. For example, when a DNA of the present invention derived from a egret is introduced, a gene construct in which the DNA of the present invention derived from an animal having a high homology with the DNA is linked to a downstream of various promoters capable of expressing the same in animal cells is used, for example. By introducing microinjection into eggs, a DNA-introduced animal of the present invention that produces high levels of protein can be produced. As such a promoter, for example, a ubiquitous expression promoter such as a virus-derived promoter or meta-mouth thionein may be used, but preferably, an NGF gene promoter, a genolases gene promoter, etc., which are specifically expressed in the brain. Is used.

Introduction of the DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal. The presence of the protein of the present invention in the germinal cells of the animal after the introduction of DNA means that all the offspring of the animal have the protein of the present invention in all of the germinal and somatic cells. The progeny of such animals that have inherited the gene will have the protein of the invention in all of their germinal and somatic cells.

After confirming that the DNA-introduced animal of the present invention stably retains the gene by breeding, the animal having the DNA can be reared in an ordinary breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring will have the DNA Breeding to have Since the protein of the present invention is highly expressed in the animal into which the DNA of the present invention has been introduced, it is useful as an agonist or an animal for screening an gonist against the protein of the present invention.

The DNA-introduced animal of the present invention can also be used as a cell source for tissue culture. For example, by directly analyzing DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or by analyzing the tissue in which the receptor protein of the present invention expressed by a gene is present, the present invention can be used. Analyze protein. The cells of a tissue having the protein of the present invention can be cultured by standard tissue culture techniques, and these can be used to study the function of cells from generally difficult-to-cultivate tissues such as those derived from brain and peripheral tissues. . Also, use the cells Thus, for example, it is possible to select a medicine that enhances the function of various tissues. Further, if there is a high expression cell line, the protein of the present invention can be isolated and purified therefrom.

A test compound is administered to the DNA-introduced animal of the present invention, and the heart function, electrocardiogram, heart weight, and the like of the animal are measured. Heart weight is the parameter of hypertrophy. Specifically, the heart structure can be examined by calculating the heart weight per body weight, the left ventricular weight per body weight, and the left ventricular weight per right ventricle weight. Since the above parameters increase when cardiac hypertrophy occurs, test compounds can be evaluated using the suppression of this increase as an index. After administering a test compound to the DNA-introduced animal of the present invention, a myocardial infarction is performed, and the heart function, electrocardiogram, heart weight, and the like of the animal are measured. In addition, after performing cardiomyocardial infarction surgery, the infarction progress-inhibiting activity of the test compound can be examined by weighing the infarct layer. Administration of the test compound may be after infarction surgery. In addition, the animal can be crossed with a genetic hypertension model rat such as an SHR rat to create a new heart failure model. The compound is administered to the cardiac failure model thus prepared, and the animal is examined for cardiac function, electrocardiogram, cardiac weight, activity for suppressing infarction progression, and the like.

[9] Knockout animal

The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention.

That is, the present invention

(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,

(2) The embryonic stem cell according to the above (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a 3-galactosidase enzyme gene derived from Escherichia coli).

(3) The embryonic stem cell according to (1), which is neomycin-resistant,

(4) The embryonic stem cell according to (1), wherein the non-human mammal is a rodent,

(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,

(6) a DNA-deficient non-human mammal in which the DNA of the present invention has been inactivated,

(7) The DNA is inactivated by introducing a reporter gene (eg, a 3-galactosidase gene derived from Escherichia coli), and the reporter gene is transformed into the DN of the present invention. The non-human mammal according to the above (6), which can be expressed under the control of a promoter for A.

(8) The non-human mammal according to (6), wherein the non-human mammal is a rodent,

(9) The non-human mammal according to (8), wherein the rodent is a mouse; and

(10) Screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter of the DNA of the present invention, which comprises administering a test compound to the animal according to (7) and detecting the expression of a reporter gene. Provide a way.

The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are artificially mutated to the DNA of the present invention possessed by the non-human mammal to suppress the DNA expression ability, Alternatively, by substantially eliminating the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter referred to as the knockout DNA of the present invention). Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells).

As the non-human mammal, those similar to the above can be used.

The method of artificially mutating the DNA of the present invention can be performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. The knockout DNA of the present invention may be produced by, for example, shifting the codon reading frame or disrupting the function of the promoter or exon by these mutations.

Specific examples of the non-human mammalian embryonic stem cells of the present invention in which DNA is inactivated (hereinafter, abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The non-human mammal DNA of the present invention is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (13-galactosidase gene), A DNA sequence that disrupts exon function by inserting a reporter gene, such as cat (chloramphenicylacetyltransferase gene), or terminates gene transcription in the intron portion between exons (for example, po 1 gene, resulting in disruption of gene synthesis by preventing the synthesis of complete mRNA Have a DNA sequence that was constructed so that A DNA strand (hereinafter, abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, a homologous recombination method, and the obtained ES cell is treated with a DNA sequence on or near DN ^ A of the present invention as a probe. PCR method using the DNA sequence on the Southern hybridization analysis or evening getter vector and the DNA sequence of the neighboring region other than the DNA of the present invention used in the evening getter vector preparation as primers By analyzing and selecting the knockout ES cells of the present invention.

As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or the known methods of Evans and Kaufman may be used. It may be newly established according to. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, a pure line that substitutes them can be used for immunological inheritance. For example, for the purpose of obtaining ES cells with a clear background, BDFi mice (C57BLZ6 and DBAZ2 and C57BLZ6) BDFi mice can be used satisfactorily.Because BDFi mice have a high number of eggs collected and their eggs are durable, they have C57BLZ6 mice as their background. The obtained ES cells can be advantageously used when a pathological model mouse is created, since the genetic background can be replaced by C57BL / 6 mice by backcrossing with C57BLZ6 mice.

In addition, when establishing ES cells, blastocysts 3.5 days after fertilization are generally used. Early embryos can be obtained.

Although either male or female ES cells may be used, male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.

An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. Using this method, it has traditionally required about 10 ⁶ cell numbers to perform a karyotype analysis. On the other hand, the number of ES cells in one colony (approximately 50) is sufficient, so the primary selection of ES cells in the early stage of culture can be performed by gender discrimination. By making selection possible, labor in the initial stage of culture can be significantly reduced.

The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. It is desirable that the number of chromosomes in the obtained ES cells is 100% of the normal number. However, if it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, normal cells (for example, in mice) It is desirable to clone again into cells with 2 n = 40 chromosomes.

Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but they must be carefully subcultured because they tend to lose their ability to generate individuals. For example, on a suitable feeder cell, such as STO fibroblasts, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5%) in the presence of LIF (1-1000 OU / ml). Culture at about 37% oxygen, 5% carbon dioxide, 90% air) at the time of subculture. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0 1-5mM EDTA (preferably about 0.1% trypsin ZlmM EDTA) is used to convert the cells into single cells and seed them on freshly prepared feeder cells. Such subculture is usually performed every 11 to 13 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, discard the cultured cells.

ES cells can be cultured in monolayers at high densities or in suspension cultures to form cell clumps under appropriate conditions to produce various types of cells such as parietal, visceral, and cardiac muscles. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings 'Ob' National 'Academy of Science' Natl. Acad. Sci. USA, Vol. 78, p. 7634, 1981; TC Doetschman et al., Journal of Obelimborgologi 'and' Experimental Morphology, Vol. 87, 27. P., 1985], the DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are cells of the protein of the present invention in the mouth of the intestine Useful in biological studies.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level. It is possible.

As the non-human mammal, those similar to the aforementioned can be used.

The non-human mammal deficient in DNA expression of the present invention can be obtained, for example, by introducing the targeting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the DNA of the present invention into the evening targeting vector. The inactivated DNA sequence can be knocked out by homologous recombination of the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination. it can.

Cells in which the DNA of the present invention has been knocked out are combined with a DNA sequence on a Southern hybridization analysis or a targeting vector using a DNA sequence on or near the DNA of the present invention as a probe, and a targeting vector. The determination can be made by PCR analysis using the DNA sequence of the neighboring region other than the DNA of the present invention derived from the mouse used as the primer. When a non-human mammalian embryonic stem cell is used, the cell line in which the DNA of the present invention has been inactivated is cloned by homologous gene recombination, and the cell line is cloned at an appropriate time, for example, at the 8-cell stage. The chimeric embryo is injected into a human mammalian embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant 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 altered DNA locus of the present invention. When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individuals obtained in this manner are usually individuals with heterozygous expression of the protein of the present invention, which are mated with individuals with heterozygous expression of the protein of the present invention. It is possible to obtain an individual with poor homo-expression.

When using egg cells, for example, microinjection into the nucleus of the egg cell A transgenic non-human mammal in which the targeting vector has been introduced into the chromosome can be obtained by injecting a DNA solution into the chromosome of the present invention. It can be obtained by selecting those with mutations at the NA locus.

In the individual knocked out of the DNA of the present invention in this manner, it is possible to confirm that the DNA has been knocked out in the animal individual obtained by mating, and to carry out rearing in an ordinary rearing environment. it can.

Furthermore, the germline can be obtained and maintained according to a standard method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygous animals are obtained. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.

The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.

In addition, since the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a disease caused by inactivation of the biological activity of the protein of the present invention. It can be a model, so it is useful for studying the causes of these diseases and examining treatment methods.

(9a) A method for screening a compound having a therapeutic / preventive effect on diseases caused by DNA deficiency or damage of the present invention.

The non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by the deficiency or damage of the DNA of the present invention.

That is, the present invention is characterized in that a test compound is administered to a non-human mammal deficient in expression of the DNA of the present invention, and changes in the animal are observed and measured. Provided is a method for screening a compound or a salt thereof having a therapeutic or preventive effect on a disease caused by the disease. The non-human mammal deficient in DNA expression of the present invention used in the screening method includes the same ones as described above.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.These compounds are novel compounds. Or a known compound.

Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with an untreated control animal, and changes in the organs, tissues, disease symptoms, etc. of the animal are used as indices. The test compound can be tested for its therapeutic and prophylactic effects. .

As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

For example, to prevent diseases characterized by reduced cardiac function (eg, heart failure after myocardial infarction; angina pectoris; cardiomyopathy; heart diseases such as heart failure derived from diseases such as angina pectoris and cardiomyopathy). · When screening for a compound having a therapeutic effect, a test compound is administered to a non-human mammal deficient in the expression of DNA of the present invention, and the heart function, electrocardiogram, heart weight and the like of the animal are measured. Heart weight is a parameter of cardiac hypertrophy. Specifically, the heart structure can be examined by calculating the heart weight per body weight, the left ventricular weight per body weight, and the left ventricular weight per right ventricle weight. Since the above parameters increase when cardiac hypertrophy occurs, test compounds can be evaluated using the suppression of this increase as an index. After administering the test compound to the non-human mammal deficient in DNA expression of the present invention, a myocardial infarction is performed, and the heart function, electrocardiogram, heart weight, and the like of the animal are measured. After the myocardial infarction operation, the infarct growth inhibitory activity of the test compound can be examined by weighing the infarct layer. Administration of the test compound may be post-infarct surgery. In addition, a new heart failure model can be created by crossing the animal with a genetically high blood model rat such as an SHR rat. The compound is administered to the heart failure model thus prepared, and the animal is examined for cardiac function, electrocardiogram, heart weight, infarction progress inhibitory activity and the like. The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a prophylactic / therapeutic effect against a disease caused by deficiency or damage of the protein of the present invention. It can be used as a safe and low toxic prophylactic and therapeutic agent. Further, a compound derived from the compound obtained by the above-mentioned screening can also be used. The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Salts with acids, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.

A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention. The preparations obtained in this way are safe and low toxic, and can be used, for example, in humans or other mammals (eg, rats, mice, guinea pigs, egrets, higgs, bush, foxes, dogs, cats, Dogs, monkeys, etc.). The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered, it is generally used in adults (with a body weight of 6 kg). In a patient suffering from a disease, the compound is administered at about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound is usually administered in the form of an injection in an adult (as 60 kg) patient with heart disease. About 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 mg per day, by intravenous injection of the compound. is there. In the case of other animals, it can be administered in an amount converted per 60 kg.

(9b) a compound that promotes or inhibits the activity of promoter of DNA of the present invention. Screening method

The present invention provides a test compound administered to a non-human mammal deficient in expression of a DNA of the present invention, and detects or enhances the expression of a reporter gene. A method for screening a compound or a salt thereof.

In the above-described screening method, the non-human mammal deficient in expression of the DNA of the present invention may be one of the above-mentioned non-human mammals deficient in expression of the DNA of the present invention, in which the DNA of the present invention is not introduced by introducing a reporter gene. Those which are activated and which can express the reporter gene under the control of the promoter for the DNA of the present invention are used.

Examples of the test compound include the same compounds as described above.

As the repo overnight gene, the same gene as described above is used, and a) -galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is suitable.

In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention has been replaced with a reporter gene, the reporter gene is under the control of the promoter for the DNA of the present invention. By tracing the expression, the activity of the promoter can be detected.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with a [3-galactosidase gene (1 ac Z) derived from Escherichia coli, a tissue that originally expresses the protein of the present invention may be used. However, j3-galactosidase is expressed instead of the protein of the present invention. Thus, for example, by staining with a reagent that serves as a substrate for j8-galactosidase, such as 5-promote 4-monocloth-3- ^ f-indolyl-j8-galactovyranoside (X-gal) The state of expression 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 dartalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at 37 ° C. After reacting for about 30 minutes to 1 hour at about ° C, the tissue sample was washed with ImM EDTA / PBS solution to stop the 3-galactosidase reaction and to observe the coloration. I just think. In addition, mRNA encoding 1 ac Z may be detected according to a conventional method.

The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the DNA promoter activity of the present invention.

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). Salts are used, and physiologically acceptable acid addition salts are particularly preferred. ^ Such salts include, for example, inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) Salts or salts with organic acids (such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) Are used. Since the compound of the present invention or a salt thereof which promotes the promoter activity for DNA can promote the expression of the protein of the present invention and promote the function of the protein, for example, it is characterized by a decrease in cardiac function. It is useful as a drug for the prevention and treatment of diseases (eg, heart failure after myocardial infarction; angina pectoris; cardiomyopathy; heart diseases such as heart failure derived from diseases such as angina pectoris and cardiomyopathy).

In addition, the compound of the present invention or a salt thereof that inhibits the activity of a promoter for DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein. It is useful as a drug for the prevention and treatment of diseases (eg, heart failure after myocardial infarction; angina pectoris; cardiomyopathy; heart diseases such as heart failure derived from diseases such as angina pectoris and cardiomyopathy). .

Further, a compound derived from the compound obtained by the above screening can also be used.

A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.

The preparations obtained in this way are safe and low toxic, and thus can be used, for example, in humans or other mammals (eg, rats, mice, guinea pigs, egrets, sheep, Pigs, pests, pomas, cats, dogs, monkeys, etc.). 'The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the compound of the present invention that promotes the promoter overnight activity against DNA is orally administered, Generally, in an adult (assuming a body weight of 60 kg) patient with heart disease, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound per day is used. Administer. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, a compound that promotes the promoter activity for DNA of the present invention is usually administered in the form of an injection to an adult. When administered to a patient with a heart disease of about 60 kg (as 60 kg), the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 mg per day. It is convenient to administer about Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. On the other hand, for example, when the compound of the present invention that inhibits the promoter activity against DNA is orally administered, generally, in an adult (assuming a body weight of 6 O kg) heart disease patients, the compound is reduced to about 0 per day. 1 to 10 Omg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose of the compound varies depending on the subject of administration, the target disease, and the like. For example, the compound of the present invention that inhibits the promoter activity on DNA may be administered in the form of an injection. When administered to a normal adult (as 6 O kg) patient with a heart disease, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 2 Omg. It is convenient to administer about 0.1 to 1 Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention. Investigating or preventing the causes of various diseases caused by insufficient expression of DNA can greatly contribute to the development of therapeutic drugs.

In addition, using a DNA containing a promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene). Introduced animal) It is also possible to synthesize the polypeptide in an appropriate manner and to examine the action in the living body. Furthermore, by binding an appropriate repo overnight gene to a part of the above-mentioned promoter and establishing a cell line in which the gene is expressed, the action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body is achieved. It can be used as a search system for low molecular weight compounds with. In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations of the IU PAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When amino acids may have optical isomers, L-form shall be indicated unless otherwise specified.

DNA deoxylipo nucleic acid

c DNA complementary deoxylipo nucleic acid

A adenine

T thymine

G Guanin

C

RNA ribonucleic acid

mRNA-liponucleic acid

dATP 'Triphosphate

dTTP Deoxythymidine triphosphate

dGTP Deoxyguanosine triphosphate

dCTP Deoxycytidine triphosphate

ATP Adenosine triphosphate

EDTA ethylenediaminetetraacetic acid

SDS sodium dodecyl sulfate

G 1 y Glycine

A 1 a Alanin

Va 1 Valine L eu

I 1 e

S e r serine

Th r threonine

Cy s

Me t Methionin

G 1 u Glutamic acid

As p Aspartic acid

Lys lysine

A r g Arginine

H is histidine

P h e feniralanin

Ty r tyrosine

T r p Triple fan

Pro proline

A s n asparagine

G 1 n Glutamine

pG1u pyroglutamic acid Substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

Me methyl group

E tethyl group

Bu butyl group

P h phenyl group

TC thiazolidine-4 (R) one-pot lipoxamide group

T os p—toluenesulfonyl

CHO Holmill B z 1

_{C 1 2 -B z 1 2,} 6- dichloro base Njiru

Bom benzyloxymethyl

Z benzyloxycarponyl

C 1 -Z 2 -Brozen benzyloxycarbonyl

B r -Z 2 bromobenzyloxycarbonyl

B oc t-butoxycarbonyl

DNP dinitrophenyl

T r t Trittel

Bum t-butoxymethyl

Fmo c N— 9 _fluorenylmethoxycarbonyl

HOB t 1—Hydroxybenztriazole

HOOB t 3,4-Dihydro-3-hydroxy-1 4-oxo-1

1,2,3-benzotriazine

HONB 1-Hydroxy-5-norporene-2,3-dicarboxide, DCC SEQ ID NOs in the Sequence Listing of the present specification indicate the following sequences. [SEQ ID NO: 1]

2 shows the amino acid sequence of the protein of the present invention (derived from rat) obtained in Example 1.

[SEQ ID NO: 2]

This shows the base sequence of DNA (gene) encoding the amino acid sequence represented by SEQ ID NO: 1.

[SEQ ID NO: 3]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 4]

1 shows the nucleotide sequence of a primer used in Example 1. [SEQ ID NO: 5]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 6]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 7]

1 shows the nucleotide sequence of the gene fragment obtained in Example 1.

[SEQ ID NO: 8]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 9]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 10]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 11]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 12]

1 shows the entire nucleotide sequence including the non-coding region of DNA (gene) encoding the amino acid sequence represented by SEQ ID NO: 1 obtained in Example 1.

[SEQ ID NO: 13]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 14]

1 shows the nucleotide sequence of a primer used in Example 1.

[SEQ ID NO: 15]

3 shows the nucleotide sequence of a primer used in Example 2.

[SEQ ID NO: 16]

3 shows the nucleotide sequence of a primer used in Example 2.

[SEQ ID NO: 17]

4 shows the nucleotide sequence of a probe used in Example 2.

[SEQ ID NO: 18]

In the nucleotide sequence represented by SEQ ID NO: 2, from the 157th to the 165th from the 5 'end The third partial base sequence is shown.

[SEQ ID NO: 19]

In the nucleotide sequence represented by SEQ ID NO: 2, this shows the 391st to 402nd partial nucleotide sequence from the 5 'end.

[SEQ ID NO: 20]

In the nucleotide sequence represented by SEQ ID NO: 2, this shows the 490th to 495th partial nucleotide sequence from the 5 'end.

[SEQ ID NO: 21]

In the nucleotide sequence represented by SEQ ID NO: 2, this shows the 796th to 804th partial nucleotide sequence from the 5 'end.

[SEQ ID NO: 22]

In the nucleotide sequence represented by SEQ ID NO: 2, this shows the 829th to 846th partial nucleotide sequence from the 5 'end. The transformant Escherichia coli DH5 o; / pTB2165 obtained in Example 1 described below has been used since October 19, 2000 at 1-1-1 Tsukuba-Higashi, Ibaraki, Japan 1 6 (Postal Code 305-8566) at the National Institute of Advanced Industrial Science and Technology (AIST), the Patent Organism Depositary Center (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NI BH)) under the deposit number FERM BP-7327. From September 26, 2000, deposited with the Fermentation Research Institute (I FO) at 2-173, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (zip code 532-8686) as deposit number I FO 16481 Have been. Example

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. Genetic manipulation using Escherichia coli is performed according to the method described in Molecular cloning, 2nd, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989. Was. Example 1

(1) Preparation of myocardial infarction model rat

According to the report of Watanabe et al. (Circulation Research, Vol. 69, pp. 370-377, 1991), male Wistar rats (11 weeks old, weighing 300-400 g) were anesthetized with pentobarbital (50 mg / kg, ip). The chest was opened midline under artificial respiration. After incision of the pericardium, the heart was exposed. At the root of the left anterior descending branch of the coronary artery, the coronary artery and myocardium were tied with a suture needle with a thread (ELP, 5_0 silk) and the chest was closed. The sham surgery group closed the chest without tying the thread. After recovery from anesthesia, they were usually reared.

(2) Total RNA extraction

Rats 1 week, 8 weeks, 20 weeks, and 30 weeks after surgery were subjected to thoracotomy under pentobarbital anesthesia, the heart was removed, and the coronary artery was perfused retrograde from the aorta with saline. Blood was washed away. After removing tissues other than the left ventricle from the extracted heart with scissors, infarct formation was confirmed and then the infarct region (scar formation site) was removed, leaving only the non-infarcted region. This was cut into small pieces with scissors, and Tota1 RNA was extracted using ISOGEN (Wako Pure Chemical).

(3) Cloning of a novel rat Nkx2.5-like gene

Nucleotide sequence of mouse NkX2.5c DNA registered in GeneBank (Processing, Ob, Nachonar, Academic Science, USA, Vol. 90, pp. 8145-8149, 1993, Accession Nampa) : AF091351), a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 and SEQ ID NO: 4 was synthesized as a PCR primer, and PCR was performed using a marathon lady rat heart cDNA library (Clontech) as type II. . The reaction was performed using TaKaRa La Taa with GC buffer (Takara Shuzo) on a thermal cycler gene amp PGR syst em 9700 (manufactured by PerkinElmer), for 30 seconds at 95, 30 seconds at 62 ° C, and 3 at 72. 33 cycles were repeated with one minute as one cycle. After cloning the obtained DNA fragment into the pT7-T vector (Takara Shuzo), using known synthetic primers (Τ7 primer (SEQ ID NO: 5) and U-19 primer-1 (SEQ ID NO: 6)), The reaction was performed using the Applied Biosystems cycle sequence kit, and a fluorescent DNA sequencer (AB IPRISM 377 As a result of analyzing the nucleotide sequence, a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 7 was obtained. This sequence showed a high homology with the known mouse Nkx2.5 cDNA sequence. Therefore, the full-length sequence of the cDNA sequence was determined by the 5, RACE method and the 3 'RACE method. 5. Synthesizes a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 8 and SEQ ID NO: 9 as a primer for RACE, and 3, represented by SEQ ID NO: 10 and SEQ ID NO: 11 as primers for RACE A DNA consisting of the following base sequence was synthesized, and PCR was carried out using a marathon lady rat heart cDNA library 1 (Clontech) as type III. The nucleotide sequence of the obtained gene fragment was decoded to obtain a DNA comprising the nucleotide sequence represented by SEQ ID NO: 12 and containing the DNA encoding the novel Nkx2.5 analogous protein of the present invention. Next, two primers (SEQ ID NO: 13 and SEQ ID NO: 14) were synthesized using the marathon lady rat heart cDNA library (Clontech) as a type II and referring to the nucleotide sequence represented by SEQ ID NO: 12. PCR was performed to obtain the full-length DNA encoding the novel NkX2.5 analogous protein of the present invention. The thus-obtained DNA encoding the novel Nkx2.5 analogous protein of the present invention was cloned into pT71-vector (Takara Shuzo), and the vector (plasmid) was named ρ1652165. Further, using known synthetic primers (Τ7 primer (SEQ ID NO: 5) and U-19 primer (SEQ ID NO: 6)), the reaction was carried out using a cycle sequence kit of Applied Biosystems, Inc. to obtain a fluorescent DNA sequencer. (AB IPRISM 377, manufactured by PerkinElmer Inc.), and the nucleotide sequence was decoded. As a result, DNA (SEQ ID NO: 2) encoding the novel Nkx2.5 analogous protein of the present invention was obtained. The transformant in which the above-mentioned pTB2165 was introduced into Escherichia coli was named Escherichiacoli DH5α / ρTΒ2165.

Next, a homology search using B 1 ast N was performed using the public database Ge η eb 1 e database based on the revealed base sequence, and Ratt us norvegicus tin thigh homolog (rNkx-2.5) (AF006664), the DNA encoding the novel Nkx2.5-like protein of the present invention (SEQ ID NO: 2) was found to be a novel Nkx2.5-like gene (FIG. 1). ~ Figure 9). Example 2

Analysis of tissue distribution of DNA encoding the novel Nkx2.5 analogous protein of the present invention in normal rats

In order to obtain a probe for Northern blotting, the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 obtained in Example 1 was designated as type III, and the nucleotides represented by SEQ ID NO: 15 and SEQ ID NO: 16 Using the DNA consisting of the sequence as a primer, PCR was performed in the same manner as in Example 1- (3) to prepare a probe (SEQ ID NO: 17). Rat MTN Blot manufactured by Clontech was used as a membrane for northern blotting. Prehybridization was performed at 68 ° C using Express Hyb Hybridization solution (Clontech) as a hybridization solution. On the other hand, the Nkx2.5-like gene fragment prepared above was labeled as a probe using [α- ³² P] dCTP and BcaBEST Labeling Kit (Takara Shuzo). Hybridization was performed in Express Hyb Hybridization solution (Clontech) containing the labeled probe at 68 ° C for 1 hour. The membrane was finally washed at 50 ° C in 0.1 XSSC, 0.1% SDS solution, and BAS-2000 (Fujifilm) was used for detection. As a result, it was found that the heart was the main expression site of DNA encoding the novel Nkx2.5 analogous protein of the present invention. Example 3

Analysis of the time course of DNA encoding the novel Nkx2.5 analogous protein of the present invention in a myocardial infarction model rat

Example 1 Tota1 RNA derived from the non-infarcted region of the left ventricle of the rat after 1 week, 8 weeks, 20 weeks and 30 weeks after myocardial infarction described in (2) Eight weeks after the operation, cDNA was synthesized from the left ventricle-derived Tota 1 RNA using TadM an Reverse Transcription Reagents (manufactured by PE Applied Biosystems). Next, using a TadMan Rodent G3PDH control reagent VIC 'probe (manufactured by PE Applied Biosystems), the copy number of glycerol triphosphate dehydrogenase was determined by PCR using ABI Prism 7700 seauence Detectio. Performed by n System. In this reaction, a series of operations were performed using TadMan PCR Core Reagents kit (manufactured by PE Applied Biosystems) according to the attached instructions. After normalizing each cDNA library with the copy number of glycerol triphosphate dehydrogenase obtained as a result, the expression level of the DNA encoding the novel Nkx2.5 analogous protein of the present invention was measured by RT-PCR. Next, primers (SEQ ID NO: 16 and SEQ ID NO: 17) capable of specifically amplifying the DNA encoding the novel Nkx2.5 analogous protein of the present invention were synthesized, and PCR was performed by the following method. went. That is, the reaction was performed using TaKaRa La Ta with GC buffer (Takara Shuzo) in a samurai cycler gene amp PCR syst em 9700 (manufactured by PerkinElmer) at 95 ° C for 30 seconds and at 62 ° C. The cycle was performed at 72 ° C for 30 seconds and 3 minutes as one cycle. In addition, sampling was performed for each cycle from 30 to 40. The obtained DNA encoding the novel Nkx2.5 analogous protein of the present invention was subjected to electrophoresis with 2% agarose, and the band detected by ethidium bromide staining was analyzed with an image analyzer (Fluorlmager 595, manufactured by Molecular Dynamics). Quantified. On a semi-log plot, plastic! ^ The expression level was calculated from the measured value during a cycle that did not reach one. Next, the amount of expression over time when the measured value of the sham operation group was set to 1 is shown in FIG. In the figure, the vertical axis indicates the copy number (expression level) of the DNA encoding the novel Nkx2.5-like protein of the present invention in the left ventricle, which is the number of copies of the glycerol triphosphate dehydrogenase gene, which is a housekeeping gene. After correcting by dividing by, the numerical value obtained by dividing by the measurement value of the sham operation group was shown, and this was indicated as fold increase. The time (weeks) on the horizontal axis shows the progress of each sample of the heart failure model used. sham 8w for sham operation group, MI lw for 1 week after surgery, MI 8w for 8 weeks after surgery, MI 20w for 20 weeks after surgery, and Ml 30 w for heart after 30 weeks after surgery Indicates the sample name.

Thus, the DNA encoding the novel Nkx2.5 analogous protein of the present invention showed a marked decrease (0.29-fold) at 1 week after surgery and an increasing tendency (1.135-fold) at 8 weeks after surgery. However, it increased at 20 weeks after the operation (2 times) and returned to the sham operation group level at 30 weeks.

One week after the operation is considered to be the time when infarction is being formed. It is presumed that myocardial cells in the downstream region from the coronary artery died and died rapidly, and inflammation was caused by lymphocyte infiltration. Since 20 to 30 weeks after the operation is immediately before death is observed, 8 weeks after the operation is the time when the compensation mechanism is operating, and after 20 weeks after the operation, sufficient compensation is available. It is likely that the order is not working, or that there is a compensatory failure due to excessive compensation mechanisms. Therefore, one week after the operation was considered the acute phase (acute heart failure), eight weeks after the surgery was considered the chronic phase (heart failure decompensation), and 20 weeks after the surgery was considered the end stage (heart failure decompensation). The compensatory mechanism involved in the transition from myocardial infarction to heart failure is considered as follows. When the myocardial cells fall off (necrosis or apoptosis), the remaining myocardial cells become enlarged to compensate for the functions of the lost myocardial cells throughout the heart, and the heart is remodeled with diastolic and fibrotic changes (cardiac). Remodeling) occurs. This functionally compensates for cardiac function, but it is thought that this cardiac remodeling or excessive compensatory mechanism itself carries the risk of developing heart failure. Vol. 79, pp. 2-20, 1997). However, the molecules involved in compensatory failure itself have not yet been identified, and the mechanism has not been elucidated. As shown in the above Examples, using the rat myocardial infarction model described above, the novel N kx of the present invention, which is a gene highly homologous to N kX 2.5 which is a positive transcription regulator of the heart-specific gene group, is used. An expression profile for DNA encoding the 2.5 analogous protein was generated. This gene decreased remarkably in the acute phase, and increased from the chronic phase to the end phase. The detailed expression profile of the DNA encoding the novel N kx 2.5 analogous protein of the present invention, which has been clarified in the above example, can be considered as follows. Nkx2.5 induces the expression of atrial sodium diuretic peptide and ventricular sodium diuretic peptide, which are thought to protect the heart.Myosin, one of the cardiac contractile proteins It has been reported to positively regulate light chain 2V expression. Therefore, there is a possibility that the Nkx2.5-like gene has a similar function, and it is considered that the decreased expression in the acute phase is directly linked to the worsening of the disease state. On the other hand, from the chronic stage to the end stage, the expression is enhanced by the ability of diatsk-adriamycin-resbonsib 'protein (CARP), which is considered to be a cardiac-specific negative regulator (J.B, 272, 22800- 22808, 1997, Development, 124, 793-804, 1997, Development, 126, 4223- 4234, 1999), which is thought to directly lead to worsening of the disease state. As a therapeutic agent for heart disease prevention, it is important to actuate the compensatory mechanism promptly in the acute phase and to suppress compensatory failure. Appropriate control of the expression of DNA encoding the novel Nkx2.5 analogous protein of the present invention is important for solving this problem. Decreased expression suppresses the compensatory mechanism, and increased expression is thought to accelerate compensation failure. Therefore, the drug of the present invention that regulates the expression of DNA encoding the novel Nkx2.5-like protein and the function of the gene product is useful as a new agent for preventing and treating heart disease. Industrial applicability

The protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof is novel, and a compound or a salt thereof which regulates the activity of the protein or the salt thereof; An antibody that regulates the activity of the salt can be used, for example, as a prophylactic or therapeutic agent for heart disease and the like. An antisense nucleotide having a nucleotide sequence complementary or substantially complementary to DNA encoding the protein or a salt thereof can suppress the expression of the protein or a salt thereof. It can be used as a therapeutic agent.

Claims

The scope of the claims

1. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof.

2. A protein containing an amino acid sequence represented by SEQ ID NO: 1 or a salt thereof.

3. The partial protein of claim 1 or a salt thereof.

4. A polynucleotide containing the polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 3.

5. The polynucleotide according to claim 4, which is DNA.

6. The DNA according to claim 5, which comprises the nucleotide sequence represented by SEQ ID NO: 2.

7. A recombinant vector containing the polynucleotide according to claim 4.

8. A transformant transformed by the recombinant vector according to claim 7.

9. The method according to claim 8, wherein the transformant according to claim 8 is cultured to produce and accumulate the protein according to claim 1 or a salt thereof or the partial peptide according to claim 3 or a salt thereof, and collect the same. A method for producing the protein of claim 1 or a salt thereof or the partial peptide of claim 3 or a salt thereof.

10. A medicament comprising the protein of claim 1 or a salt thereof or the partial peptide of claim 3 or a salt thereof.

11. An antibody against the protein according to claim 1 or a salt thereof, or the partial peptide according to claim 3 or a salt thereof.

12. A diagnostic agent comprising the antibody according to claim 11.

13. The protein according to claim 1, a salt thereof, or the partial peptide according to claim 3 or a salt thereof, wherein the protein according to claim 1, a salt thereof, or the portion according to claim 3 is used. A method for screening a compound or a salt thereof that regulates the activity of the peptide or a salt thereof.

1 4. A protein or a salt thereof according to claim 1 or a partial peptide or a salt thereof according to claim 3 is characterized by a DNA containing a DNA encoding the protein according to claim 1 or the partial peptide according to claim 3. 14. The screening method according to claim 13, which is expressed in the cells of the transformed transformant.

15. The protein according to claim 1, or a salt thereof, or the partial peptide according to claim 3, or a salt thereof, wherein the protein according to claim 1, or a salt thereof, or the salt according to claim 3, is contained. A kit for screening a compound or a salt thereof that regulates the activity of a peptide or a salt thereof.

16. The protein according to claim 1, or a salt thereof, or the partial peptide or the partial peptide according to claim 3, obtained by using the screening method according to claim 13 or the screening kit according to claim 15. A compound or a salt thereof that regulates the activity of the salt.

17. A medicament comprising the compound according to claim 16 or a salt thereof.

18. The medicament according to claim 17, which is an agent for preventing or treating heart disease.

19. An antisense nucleotide having a nucleotide sequence complementary or substantially complementary to DNA encoding the protein according to claim 1 or the partial peptide according to claim 3.

20. A medicament comprising the antisense nucleotide according to claim 19.

21. A pharmaceutical comprising the polynucleotide according to claim 4.

22. A diagnostic agent comprising the polynucleotide according to claim 4.

23. A method for preventing and treating heart disease, comprising administering an effective amount of the compound according to claim 16 or a salt thereof to a mammal.

2 4. Use of the compound according to claim 16 or a salt thereof for producing an agent for preventing or treating heart disease.