WO2003097686A1 - Novel protein, its dna and use thereof - Google Patents

Abstract

A protein and a polynucleotide which are useful as diagnostic markers for heart diseases, central nerve diseases, genital diseases, etc. A compound obtained by screening with the use of the protein, the polynucleotide or an antibody and a neutralizing antibody inhibiting the activity of the protein are usable as preventives/remedies for heart diseases, central nerve diseases, genital diseases, etc.

Description

 Description Novel proteins, their DNA and their applications

 The present invention relates to a novel protein, DNA encoding the protein, a method for screening a compound that regulates the activity of the protein, a compound obtained by the screening method, and the like. More specifically, the present invention relates to a novel protein useful as a prophylactic / therapeutic agent or diagnostic agent for heart disease. Background art

Heart failure is considered to be contractile dysfunction of the myocardium, and the mechanism of its onset is as follows. Myocardial damage, mechanical abnormalities of the heart pump, abnormal function of the heart pump, pressure overload due to hypertension or pulmonary hypertension, and volumetric load such as anemia or acute nephritis. For these reasons, the heart can no longer pump the blood volume according to the demands of the living body, but the heart activates the sympathetic nervous system, the nerve-integrated fluid-endocrine system, and other compensatory mechanisms. However, try to maintain the homeostasis of the body. As a result, cardiac hypertrophy occurs due to the enlargement of cardiac muscle cells. However, if the aforementioned obstacles or loads are chronically sustained, the compensatory mechanism will fail. In other words, not enough blood is supplied to the enlarged cardiomyocytes, resulting in ischemia, which results in myocardial damage such as incomplete myocardial contraction, which results in decreased cardiac output, organ circulation disorders, venous congestion, This results in heart failure syndrome with fluid retention. Therapies for this need to improve myocardial dysfunction, enhance cardioprotection, restore cardiac dysfunction due to myocardial contractile dysfunction, and suppress the decompensation of the living body or improve the excessive compensation mechanism. Become. Currently, in treating the heart failure syndrome, clinically inotropic drugs include (1) cardiac glycosides such as digoxin, (2) sympathomimetics such as dobutamine, and (3) phosphodiesterase inhibitors such as amrinone. As vasodilators, hydralazine, potash antagonist, angiotensin exchange enzyme inhibitor, angiotensin receptor antagonist and the like are used. Also, for the treatment of other dilated cardiomyopathy, Which is used. On the other hand, there is no therapeutic agent from the viewpoint of suppression of excessive compensatory mechanisms or suppression of decompensation (including suppression of apo1 ^ -cis).

 There is an urgent need to develop an excellent therapeutic agent for heart failure with few side effects from the viewpoint of suppressing 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 further studies based on this finding, the present invention has been completed.

 That is, the present invention

 (1) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 13 or SEQ ID NO: 30, or a salt thereof;

 (2) a protein consisting of the amino acid sequence represented by SEQ ID NO: 25 or a salt thereof,

 (3) a protein consisting of the amino acid sequence represented by SEQ ID NO: 28 or a salt thereof;

 (4) a protein consisting of the amino acid sequence represented by SEQ ID NO: 13 or a salt thereof

 (5) a protein consisting of the amino acid sequence represented by SEQ ID NO: 30 or a salt thereof;

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

 (7) a polynucleotide containing a polynucleotide encoding the protein of (1) or the partial peptide of (6),

 (8) the polynucleotide according to the above (7), which is a DNA;

 (9) the polynucleotide according to the above (8), comprising the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31;

(10) a recombinant vector containing the polynucleotide according to the above (7), (11) a transformant transformed with the recombinant vector according to the above (10),

(12) culturing the transformant according to (11) above, producing and accumulating the protein according to (1) or the partial peptide according to (6), and collecting the protein; A method for producing the protein described in (1) or the partial peptide described in (6) or a salt thereof,

 (13) a medicament comprising the protein according to (1) or a partial peptide thereof or a salt thereof,

 (14) a medicament comprising the polynucleotide according to the above (7),

 (15) a diagnostic agent comprising the polynucleotide according to (7),

 (16) an antibody against the protein according to (1) or the partial peptide according to (6) or a salt thereof,

 (17) a diagnostic agent comprising the antibody according to the above (16),

 (18) a medicament comprising the antibody according to the above (16),

 (19) an antisense nucleotide containing a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the polynucleotide according to (7) or a part thereof,

(20) a medicine comprising the antisense nucleotide according to (19), (21) a protein according to (1) or a partial peptide according to (6) or a salt thereof, (1) a method for screening a compound or a salt thereof that regulates the activity of the protein or the partial peptide or the salt thereof according to (6);

 (22) The protein according to (1) or the partial peptide according to (6), which comprises the protein according to (1) or the partial peptide according to (6) or a salt thereof. A kit for screening a compound or a salt thereof that regulates the activity of the salt,

 (23) The protein according to (1) or the partial peptide according to (6) or a salt thereof, which can be obtained using the screening method according to (21) or the screening kit according to (22). A compound or a salt thereof that regulates the activity of

(24) a medicament comprising the compound according to (23) or a salt thereof, (25) A method for screening a compound or a salt thereof that regulates the expression of the protein gene according to (1), which comprises using the polynucleotide according to (7).

 (26) a kit for screening a compound or a salt thereof that regulates the expression of the protein gene according to (1), which comprises the polynucleotide according to (7);

 (27) A compound or a salt thereof that regulates the expression of the protein gene according to (1), which can be obtained using the screening method according to (25) or the screening kit according to (26).

 (28) a medicament comprising the compound according to (27) or a salt thereof,

(29) The method for quantifying a protein according to (1), which comprises using the antibody according to (16),

 (30) The method for diagnosing a disease associated with the function of evening protein as described in (1) above, which comprises using the quantification method as described in (29) above.

 (31) a method for screening a compound or a salt thereof that regulates the expression of the protein according to (1), which comprises using the antibody according to (16);

(32) a kit for screening a compound or a salt thereof that regulates the expression of the protein according to (1), which comprises the antibody according to (16);

 (33) A compound or a salt thereof that regulates the expression of the protein of (1), which is obtained by using the screening method of (31) or the screening kit of (32).

 (34) a medicament comprising the compound according to (33) or a salt thereof,

(35) The medicament according to (13), (14), (18), (20), (24), (28) or (34), which is a preventive or therapeutic agent for heart disease.

 (36) The medicament according to the above (24), (28) or (34), wherein the compound is a compound that suppresses excessive compensatory mechanism or compensatory failure.

 (37) The medicament according to the above (35) or (36), which is a preventive or therapeutic agent for cardiomyopathy, myocardial infarction, heart failure or angina pectoris.

(38) The diagnostic agent according to (15) or (17) above, which is a diagnostic agent for heart disease. (39) A method for preventing or treating heart disease, which comprises administering to a mammal an effective amount of the compound according to (23), (27) or (33) or a salt thereof,

(40) Use of the compound or a salt thereof according to (23), (27) or (33) for the manufacture of a prophylactic or therapeutic agent for heart disease.

 (41) A compound or a salt thereof that regulates the activity of the protein according to (1) or the partial peptide or the salt thereof according to (6) above,

Prevention of heart disease characterized by administering an effective amount of a compound or a salt thereof that regulates the expression of the protein gene described in (1) or a compound or a salt thereof that regulates the expression of the protein described in (1). ·Method of treatment,

 (42) A compound or a salt thereof that regulates the activity of the protein of (1) or the partial peptide of (6) or a salt thereof for producing a prophylactic or therapeutic agent for heart disease, The present invention also provides use of a compound or a salt thereof that regulates the expression of the protein gene described above, or a compound or a salt thereof that regulates the expression of the protein described in the above (1).

 Moreover,

(43) the protein is (i) an amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 13; (ii) an amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 13; (E.g., about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5 amino acids) (iii) SEQ ID NO: 25 or SEQ ID NO: 13 An amino acid sequence obtained by adding one or two or more amino acids (for example, about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) to the amino acid sequence to be obtained; (iv) SEQ ID NO: : 25 or 1 or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5) amino acids) in the amino acid sequence represented by SEQ ID NO: 13. An amino acid sequence into which an acid has been introduced; (V) SEQ ID NO: 25 or SEQ ID NO: 1 or 2 or more (eg, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) amino acids in the amino acid sequence represented by 13 are replaced with other amino acids (Vi) the protein according to the above (1), which is a protein containing an amino acid sequence obtained by combining (ii) to (V) above; (44) A polynucleotide or the like that hybridizes with the polynucleotide of the above (7) under high stringency conditions is also provided. BEST MODE FOR CARRYING OUT THE INVENTION

 A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 13 or SEQ ID NO: 30 (hereinafter, the present invention) Are sometimes called human non-human warm-blooded animals (eg, guinea pigs, rats, mice, chickens, egrets, bushes, sheep, horses, monkeys, etc.) (eg, hepatocytes, Spleen cells, nerve cells, glial cells, kidney cells] 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, soft Cells, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells) or any tissue in which these cells are present, such as the brain , Each part of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, stomach, kidney, 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, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, It may be a protein derived from a joint, skeletal muscle, or the like, or may be a synthetic protein.

 As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25, about 85% or more, preferably about 90% or more, more preferably the amino acid sequence represented by SEQ ID NO: 25 Is an amino acid sequence having about 95% or more, more preferably about 97% or more homology.

Examples of a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 25 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 25 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 25 is preferable. As the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 28, the amino acid sequence represented by SEQ ID NO: 28 is 75% or more, preferably about 80% or more, and about 85% or more. % Or more, preferably about 90% or more, more preferably about 95% or more, more preferably about 97% or more. Examples of a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 28 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 28 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 28 is preferable. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 13 includes about 80% or more, preferably about 85% or more, preferably Amino acid sequences having about 90% or more, more preferably about 95% or more, more preferably about 97% or more homology, and the like.

 Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 13 include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 13 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 13 is preferable. As the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 29, the amino acid sequence represented by SEQ ID NO: 29 is at least about 50%, preferably at least about 60%, preferably About 70% or more, about 80% or more, preferably about 85% or more, preferably about 90% or more, more preferably about 95% or more, and more preferably about 97% or more homology. And the like.

Examples of a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 29 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 29 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 29 is preferable. Examples of substantially equivalent activities include, for example, cardiac function promoting or inhibiting activity, cardiomyocyte functional activity or inactivating activity, and the like. Specific examples include cardiac function-promoting activity as one of the compensatory mechanisms of cardiac function, and cardiac function-suppressing activity due to compensatory failure caused by excessive activation of the compensatory mechanism. Cardiomyocyte function activating effects include cells Also includes protective action.

 The activity of the protein of the present invention for promoting or inhibiting cardiac function and the activity for inducing or inactivating cardiomyocyte function are measured by measuring cardiac function.

 The measurement of cardiac function can be measured according to a known method or a method analogous thereto. For example, the measurement is performed by an echocardiograph (Cell, Vol. 97, pp. 189-198, 1999) or cardiac function measurement using a cardiac catheter (circulation research, Vol. 69, pp. 370-377, 1991). Furthermore, for example, angiotensin I converting enzyme

(ACE) and other renin-angiotensin (RAS) enhancements can be measured using a commercially available assay kit (eg, Peninsula One, Phoenix, etc.), or blood catecholamine increasing activity ( The heart function is measured using an index such as a fully automatic catecholamine analyzer manufactured by Tosoh Corporation.

 When measuring the activation or inactivation of cardiomyocyte function using cells having the ability to produce the protein of the present invention, for example, measuring the respiratory activity of the cells, or a heart failure marker gene (e.g., ANP (atrial Changes in expression of BNP (brain sodium diuretic peptide, etc.) or production of heart failure marker gene product are measured according to a known method or a method similar thereto. For example, the MT T (3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium) method, trypable monostaining method, TUN NEL staining method (Terminal deoxytrans ferase-mediated dUTP-X nick end labeling, Cell, Vol. 97, pp. 189-198, 1999).

 Substantially identical indicates that the activity is qualitatively (eg, physiologically or pharmacologically) equivalent. Therefore, the activity of promoting or suppressing cardiac function and the activity of activating or inactivating cardiomyocyte function are equivalent (e.g., about 0.01 to 100 times, preferably about 0.1 to 10 times, (More preferably 0.5 to 2 times), but quantitative factors such as the degree of this activity and the molecular weight of the protein may be different.

Examples of the protein of the present invention include: (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 (for example, about 1 to 30 and preferably 1 to 10 About 1 amino acid, more preferably 1 to 5 amino acids are deleted. Amino acid sequence, (ii) 1 or 2 or more amino acid sequences represented by SEQ ID NO: 25 (eg, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) (Iii) 1 or 2 or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10 amino acids in the amino acid sequence represented by SEQ ID NO: 25) (Iv) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 (for example, about 1 to 30, preferably about 1 to 10, More preferably, a so-called mutein such as a protein containing an amino acid sequence in which a number (1 to 5) of amino acids are substituted with another amino acid, or (V) a protein containing an amino acid sequence combining them; No. 1 in the amino acid sequence represented by SEQ ID NO: 28 Is an amino acid sequence in which two or more (for example, about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5) amino acids have been deleted, and (ii) a sequence represented by SEQ ID NO: 28 An amino acid sequence in which one or more (eg, about 1 to 30, preferably about 1 to 10, and more preferably, about 1 to 5) amino acids are added to the amino acid sequence to be prepared; (iii) One or more amino acids (for example, about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5) amino acids are inserted into the amino acid sequence represented by SEQ ID NO: 28 (Iv) 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 28 (e.g., about 1 to 30, preferably about 1 to 10, more preferably about 1 to 10; Amino acid sequence in which 5) amino acids have been replaced with other amino acids, or (V) So-called mutin, such as proteins containing the combined amino acid sequence,

(3) (0 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 13 (for example, about 1 to 30, preferably about 1 to 10, more preferably number (1 to 5)) (Ii) 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 13 (for example, about 1 to 30, preferably about 1 to 10, more preferably (Iii) 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 13 (eg, about 1 to 30, preferably about 1 to 10, More preferably, an amino acid sequence having a number (1 to 5) of amino acids inserted therein; (iv) an amino acid represented by SEQ ID NO: 13 Amino acids in which one or more (for example, about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids in the acid sequence have been substituted with other amino acids Sequences or (V) so-called muteins such as proteins containing amino acid sequences combining them;

 (4) (i) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 30 (for example, about 1 to 30, preferably about 1 to 10, more preferably (Ii) an amino acid sequence having the amino acid sequence represented by SEQ ID NO: 30 or more (for example, about 1 to 30 amino acids, preferably 1 to 10 amino acids). (Iii) 1 or 2 or more amino acids (for example, about 1 to 30) to the amino acid sequence represented by SEQ ID NO: 30 Preferably about 1 to 10 amino acids, more preferably about 1 to 5 amino acids, and (iv) one or two amino acids in the amino acid sequence represented by SEQ ID NO: 30. More than one (for example, about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids So-called muteins, such as proteins containing amino acid sequences substituted with amino acids or (V) amino acid sequences combining them, are 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 (capilloxy terminus) according to the convention of peptide labeling. Proteins of the present invention, C-terminal, carboxyl groups (- C00H), carboxylate (- C00-), amide (-C0NH ₂₎ or ester - may be either (C00R).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, isopropyl, _alkyl groups such as n _-butyl, cyclopentyl, C _3, such as cyclohexyl - ₈ cycloalkyl group, for example, phenyl, α- naphth C ₆ _ _{1 2} Ariru group such le, for example, benzyl, phenylene Lou alkyl or α- naphthylmethyl etc. α- Nafuchiru C E such as phenethyl - ₂ C ₇ _ _{1 4} such as Al kill group An aralkyl group, a bivaloyloxymethyl group and the like are used.

 When the protein of the present invention has a C-terminal lipoxyl group (or carboxylate)

), The carbonyl group is amidated or esterified. Is also included in the protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Furthermore, in the protein of the present invention, the amino acid residues at the N-terminus (eg, Mechionin residues) Amino group protecting groups (e.g., formyl group, etc. Ashiru group such as C ₆ Al force Noiru such Asechiru group) Protected, Cleavage in vivo, N-terminal glutamine residue generated by pyroglutamine oxidation, Substituent on side chain of amino acid in molecule (eg, 1H, 1SH, amino) those protected like ₆ Ashiru group) - group, an imidazole group, I Ndoru group, etc. Guanijino group) appropriate protecting groups (e.g., formyl group, C such as C E one ₆ Arukanoiru group such Asechi Le group Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.

 Specific examples of the protein of the present invention include, for example, a protein containing an amino acid sequence represented by SEQ ID NO: 25, a protein containing an amino acid sequence represented by SEQ ID NO: 28, SEQ ID NO: 1 A protein containing the amino acid sequence represented by SEQ ID NO: 30; and a protein containing the amino acid sequence represented by SEQ ID NO: 30.

 The partial peptide of the protein of the present invention is the partial peptide of the protein of the present invention, and preferably any peptide having the same properties as the above-described protein of the present invention. For example, at least 20 or more, preferably 50 or more, preferably 70 or more, preferably 100 or more, preferably 200 or more of the amino acid sequences constituting the protein of the present invention. Peptides having an amino acid sequence are used.

Further, the partial peptide of the present invention lacks one or more (preferably about 1 to 10 and more preferably number (1 to 5)) amino acids in its amino acid sequence, Alternatively, one or more (preferably, about 1 to 20, more preferably, about 1 to 10, and more preferably, about 1 to 5) amino acids are added to the amino acid sequence. Or 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, about 1 to 5) amino acids are inserted into the amino acid sequence; Or 1 or 2 or more in the amino acid sequence (Preferably, about 1 to 10, more preferably, several, and still more preferably, about 1 to 5) amino acids may be substituted with another amino acid.

 More specifically, as the partial peptide of the present invention, for example, (1) in the amino acid sequence represented by SEQ ID NO: 25, the second to 57th or 58th to 24th from the N-terminus A partial peptide having a partial amino acid sequence having the ninth amino acid residue,

(2) a portion having a partial amino acid sequence having the 2nd to 239th or the 240th to 48th amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 28 Peptide,

 (3) having a partial amino acid sequence having the 2nd to 64th or the 65th to 256th amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: .13 Partial peptide,

 (4) a portion having a partial amino acid sequence having the 2nd to 239th or the 240th to 48th amino acid residues from the N-terminus in the amino acid sequence represented by SEQ ID NO: 30 Peptides and the like.

In the partial peptide of the present invention, the C-terminus may be any of a hydroxyl group (—CO OH), a carboxylate (—COO ″), an amide (one C〇NH ₂ ), and an ester (one COOR).

 Further, the partial peptides of the present invention include those having a carboxyl group (or lipoxylate) in addition to the C-terminus and N-terminal amino acid residues (eg, methionine), similarly to the above-mentioned protein of the present invention. Residue is protected with a protecting group, the N-terminal is cleaved in vivo, and the resulting daltamine residue is pyrodaltamic acid, and the substituent on the side chain of the amino acid in the molecule is appropriate. Also included are those protected with a protecting group, and complex peptides such as so-called glycopeptides to which sugar chains are bound.

The partial peptides of the present invention can also be used as antigens for preparing antibodies. 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 acid addition salts are: Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or salts with inorganic acids. Salts with mechanical acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

 The protein of the present invention or a partial peptide thereof or a salt thereof can be produced by a known protein purification method from cells or tissues of a human / non-human warm-blooded animal described above, or contains a DNA encoding the protein. Can also be produced by culturing the transformant. It can also be produced according to the peptide synthesis method described below.

 When producing from tissues or cells of a human / non-human mammal, the tissues or cells of a human / non-human mammal are homogenized and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography or ion exchange chromatography. Purification and isolation can be achieved by combining such chromatographs.

 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. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2,4, dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2 ', 4, dimethoxyphenyl Fmocaminoethyl) phenoxy Resin and the like can be mentioned. Using such a resin, amino acids having appropriately protected amino groups and side chain functional groups are condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the target protein or partial peptide or their amide Get the body.

For the condensation of the protected amino acids described above, various active reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of carpoimides include DCC, N, N'-diisopropylcarpoimide, N-ethyl Ru-N '-(3-dimethylaminoprolyl) carbodiimide is used. For these activations, the protected amino acid may be added directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt) or may be pre-formed as the corresponding acid anhydride or HOBT or HOOBt ester. It can be added to the resin after activation of the protected amino acid.

 The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylvinylidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol. , Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. Used. The reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually appropriately selected from the range of about -2θυ to 50. 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, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid 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, isopolnyloxycarponyl, 4-methoxybenzyloxycarponyl, C 1 _Z, Br—Z, and adaman Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

The lipoxyl group can be, for example, a linear, branched or cyclic alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl) A 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, etc.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Use Examples of groups appropriately used for the esterification, for example, low-grade (〇 DOO ₆₎ Arukanoiru groups such Asechiru group, Aroiru group such Benzoiru group, Benjiruokishi carbonyl group, and a group derived from carbonic acid such as ethoxycarbonyl group It is required. Examples of a group suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a t-butyl group.

The protecting group of the phenolic hydroxyl group of tyrosine, for example, Bz and C 1 ₂ - B zl, 2_ nitrobenzyl, B r- Z, such as t one-butyl 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 activated raw oxypoxyl groups include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol) Phenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOB tester) and the like. 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., reduction with sodium in liquid ammonia, etc. Used. The elimination reaction by the above acid treatment is generally about -2 The acid treatment is performed at a temperature of 0 ° (: up to 40 ° C, for example, anisol, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol, It is effective to add a thione scavenger such as 2-ethanedithiol, etc. Also, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and a tryptophan indole protecting group is used. The formyl group used as a catalyst is removed by acid treatment in the presence of 1,2-ethanedithiol or 1,4-butanedithiol as described above, or by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc. Is done.

 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 of a protein or partial peptide, for example, first, after amidating and protecting a carboxyl group of a carboxyl-terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After elongation, the protein or partial peptide from which only the protecting group for the α-amino group at the N-terminal of the peptide chain has been removed and the protein or partial peptide from which only the protecting group for the C-terminal lipoxyl group have been removed And condensing these proteins or peptides 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 freeze-dried to obtain an amide of the desired protein or peptide.

 In order to obtain an ester of a protein or peptide, for example, after condensing an α; -hydroxyl group of a carboxy-terminal amino acid with a desired alcohol to form an amino acid ester, the desired amino acid ester is prepared in the same manner as in the amide of a protein or peptide. An ester of a protein or a peptide can be obtained.

The partial peptide of the present invention or a salt thereof can be produced according to a known method for synthesizing a peptide. Alternatively, it can be produced by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can constitute the partial peptide of the present invention with the remaining portion, and if the product has a protective group, removing the protective group to produce the desired peptide. . Examples of the known condensation method and elimination of the protecting group include the methods described in the following [omega] to (V).

 (i) M. Bodanszky and M. A. Ondet t i, Peptide Synthes is;, Interscience Publ ishers, New York (1966)

 (ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

 (iii) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd. (1975)

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

 (V) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten After the reaction, normal purification methods, such as solvent extraction, distillation, column chromatography, liquid chromatography, and liquid chromatography The partial peptide of the present invention can be purified and isolated by combining crystals and the like. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto. It can be converted into a free form or another salt by an analogous method.

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 DNA, such as DNA or mRNA, encoding the protein of the present invention; NA, and may be double-stranded or single-stranded. In the case of a double-stranded DNA, it may be a double-stranded DNA, a double-stranded RNA or a hybrid of DNA: RNA. If single-stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand). Using the polynucleotide encoding the protein of the present invention, for example, the mRNA of the protein of the present invention can be obtained by the method described in the well-known experimental medicine special edition “New PCR and its Applications” 15 (7), 1997 or a method analogous thereto. Can be determined.

 The DNA encoding the protein of the present invention may be any DNA containing the above-described nucleotide sequence encoding the protein of the present invention. Further, it may be any of genomic DNA, genomic DNA library, the aforementioned cDNA derived from cells and tissues, the aforementioned cDNA library derived from cells and tissues, and synthetic DNA.

 The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse RNA was directly prepared using the whole RNA or mRNA fraction prepared from the cells and tissues described above.

It can also be amplified by Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method).

Examples of the DNA encoding the protein of the present invention include: (1) a DNA containing the base sequence represented by SEQ ID NO: 26, or a base sequence represented by SEQ ID NO: 26 and a highly stringent condition. A DNA encoding a protein having a property substantially identical to that of a protein having an amino acid sequence represented by SEQ ID NO: 25, which contains a base sequence hybridized with: (2) a base represented by SEQ ID NO: 29, DNA containing the sequence or a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 29, and substantially the same as the protein containing the amino acid sequence represented by SEQ ID NO: 28 DNA that encodes a protein having the same homologous properties, (3) a DNA having the nucleotide sequence represented by SEQ ID NO: 27, or a nucleotide sequence represented by SEQ ID NO: 27 A DNA encoding a protein containing a nucleotide sequence that hybridizes under high stringent conditions and having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 13, or (4) the sequence A DNA containing the nucleotide sequence represented by SEQ ID NO: 31 or a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 31; represented by SEQ ID NO: 30 A protein having substantially the same properties as the protein containing the amino acid sequence Any DNA can be used.

 Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 26 under high stringent conditions include, for example, about 85% or more, preferably about 90% or more of the nucleotide sequence represented by SEQ ID NO: 26 More preferably, a DNA containing a nucleotide sequence having a homology of about 95% or more, more preferably about 97% or more is used.

 Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 29 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 29 and 75% or more, preferably about 80% or more. DNA containing a nucleotide sequence having a homology of about 85% or more, preferably about 90% or more, more preferably about 95% or more, and more preferably about 97% or more is used.

 Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 27 under high stringent conditions include, for example, about 80% or more, and preferably about 85% or more of the nucleotide sequence represented by SEQ ID NO: 27 Preferably, a DNA containing a base sequence having a homology of about 90% or more, more preferably about 95% or more, more preferably about 97% or more is used.

 Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 31 under high stringent conditions include, for example, about 50% or more, preferably about 60% or more of the nucleotide sequence represented by SEQ ID NO: 31. A nucleotide sequence having a homology of preferably about 70% or more, about 80% or more, preferably about 85% or more, preferably about 90% or more, more preferably about 95% or more, and more preferably about 97% or more. And the like containing DNA.

 Hybridization is 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 done. 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 stringency conditions are, for example, when the sodium concentration is about 19-40 m M, preferably about 19-2 OmM, at a temperature of about 50-70 ° C, preferably about 60-65. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.

 More specifically, (1) the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 25 includes a DNA comprising the base sequence represented by SEQ ID NO: 26; Examples of the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 28 include a DNA containing the base sequence represented by SEQ ID NO: 29, and (3) a DNA represented by SEQ ID NO: 13. Examples of the DNA encoding the protein containing the amino acid sequence include a DNA containing the nucleotide sequence represented by SEQ ID NO: 27, and (4) a protein containing the amino acid sequence represented by SEQ ID NO: 30. As the DNA to be used, a DNA containing the base sequence represented by SEQ ID NO: 31 or the like is used.

 The DNA encoding the partial peptide of the present invention may be any DNA as long as it contains the above-described nucleotide sequence encoding the partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the 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 part of the DNA having the base sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31, or SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31, containing a nucleotide sequence that hybridizes under high stringent conditions with a nucleotide sequence represented by SEQ ID NO: 25, SEQ ID NO: 28, DNA containing a part of DNA encoding a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 30, and the like are used.

 The DNA hybridizable with the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31 has the same significance as described above.

The same hybridization method and high stringency conditions as described above are used. A DNA clone that completely encodes the protein or partial peptide of the present invention (hereinafter, these may be simply referred to as the protein of the present invention in the description of cloning and expression of the DNA encoding the same). As a means of mouth opening, DNA 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 DNA incorporated into an appropriate vector is used for the present invention. Can be selected by hybridization with a DNA fragment encoding a part or the entire region of the above-mentioned protein or labeled with a synthetic DNA. The hybridization method can be carried out, for example, according to the method described in Molecular 'Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted using PCR or a known kit, for example, Mutan ^™ _super Express Km (Takara Shuzo Co., Ltd.), Mutan ™ -K (Takara Shuzo Co., Ltd.), etc., using the 0DA-LA PCR method or the Gapped method. It can be performed according to a known method such as the duplex method or the Kunkel method, or a method analogous thereto.

 The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used by digesting with a restriction enzyme or adding a linker if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.

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

Examples of the vector include a plasmid derived from E. coli (eg, PBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pCl94), a plasmid derived from yeast (eg, P SH19, pSHl 5), In addition to bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus, pAl-11, pXTl, pRc / CMV, pRc / RSV, pcDNAIZNeo, and the like are used.

 The promoter of 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 hosts, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.

Of these, it is preferable to use CMV (Cytomegalovirus) Promo Overnight, SRo; Promo Overnight, and the like. When the host is Eshierihia genus bacterium, trp promoter, lac promoter, re cA promoter Isseki one, lambda P _L promoter evening one, l pp promoter evening one, T 7 including promoter Isseki one is, host Bacillus If the host is yeast, PH〇5 Promoter, PGK Promoter, GAP promoter, ADH Promoter One is preferred. When the host is an insect cell, a polyhedrin promoter, P10 promoter and the like are preferred. The expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired. Can be used. Examples of the selection marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene (methotrexate (MTX) resistance) and an ampicillin resistance gene (hereinafter abbreviated as Amp ¹ ) ), Neomycin resistance gene (hereinafter sometimes abbreviated as Ne ο ¹ ·, G418 resistance), etc. Particularly, using dh fr gene as a selectable marker using dh fr gene deficient Chinese hamster cells In this case, the target gene can also be selected by using a medium containing no thymidine.

If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is Escherichia, the PhoA 'signal sequence and OmpA signal sequence are used.When the host is Bacillus, the amylase and subtilisin signal sequences are used. In some cases, MF signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon 'signal sequence, antibody molecule, signal sequence, etc. can be used.

 A transformant can be produced using the thus constructed vector containing the DNA encoding the protein of the present invention. '

 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, Escherichia coli.

Escherichia coli) K12-DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA2 21 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like are used.

 Examples of Bacillus bacteria include, for example, Bacillus subtilis MI 114 ½61 ^, Vol. 24, 255 (1983)], 207-21 [Journal of

Biochemistry, 95, 87 (1984)].

 Examples of yeast include Saccharomyces cerevisiae AH22, AH22R-, 2287-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NC YC 1913, NCYC 2036, Pichia · Pastoris (Pichia pastoris) K # 71 or the like is used.

As insect cells, for example, when the virus is AcNPV, a cell line derived from a larva of Spodoptera (Spodoptera frugiperda cell; S f cell), an MG1 cell derived from the midgut of Trichoplusia ni, and a High Five ™ derived from an egg of Trichoplusia ni Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori cell; BmN cell) or the like is used. Examples of the Sf cell include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)). Used. As insects, for example, silkworm larvae are used [Nature, vol. 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 Hams Yuichi cells CHO (hereinafter CHO (dh fr-1) cells Abbreviations), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, H9c2 cells, etc. are used.

 To transform Escherichia sp., For example, Proc. Natl. Acad. Sci.

USA, 69, 2110 (1972) and Gene, 17, 107 (1982).

 To transform Bacillus, for example, use Molecular & General

Gene Ucs, Vol. 168, 111 (1979) and the like.

 The yeast can be transformed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978). Can be.

 Transformation of insect cells or insects can be performed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

 To transform animal cells, for example, a method described in Cell Engineering Annex 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). It can be carried out.

 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 a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn chipperica, peptone, power zein, meat extract, soybean meal, and potato extract. Inorganic or organic substances such as liquids, and inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. Pum and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

 As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferable. If necessary, a drug such as 3] 3-indolylacrylic acid can be added to make the promoter work efficiently.

 When the host is a bacterium belonging to the genus Escherichia, culturing is usually performed at about 15 to 43 T for about 3 to 24 hours, and if necessary, aeration and stirring may be added.

 When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours.

 When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Proc. Natl. Acad. Sci. USA, 77

, 4505 (1980)] and an SD medium containing 0.5% casamino acid [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. Culture is usually performed at about 20 to 35 for about 24 to 72 hours, and aeration and agitation are added as necessary.

 When culturing an insect cell or a transformant whose host is an insect, a medium such as Grace's Insect Medium (Nature, 195, 788 (1962)) was supplemented with an additive such as 10% inactivated serum. Things and the like are used. The pH of the medium is preferably adjusted 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, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [

Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)]. Preferably, the pH is about 6-8. Cultivation is usually performed 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 cells of the transformant, in the fine J5 envelope or extracellularly.

 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, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and Z or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is appropriately used. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X100 ^™ . 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. Methods that utilize differences in charge; methods that use differences in charge, such as ion exchange chromatography; methods that use specific affinity, such as affinity chromatography; hydrophobicity, such as reverse-phase high-performance liquid chromatography A method using a difference, a method using an isoelectric point difference such as an isoelectric focusing method, 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. Examples of protein modifying enzymes include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, and glycoprotein. Sidase and the like are used.

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

 The antibody against the protein or partial peptide of the present invention or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide of the present invention or a salt thereof.

 An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these may be simply abbreviated 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 itself or together with a carrier or diluent at a site capable of producing an antibody upon administration. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal 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 a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Köhler and Milstein [Nature, 256, 495 (1975)]. As a fusion promoter, for example, polyethylene glycol (PEG) And Sendai virus, but PEG is preferably used.

 Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is used at a concentration of about 10 to 80%. The cell fusion can be carried out efficiently by incubating at 20 to 40t: preferably at 30 to 37 ° C for 1 to 10 minutes. Various methods can be used to screen the monoclonal antibody-producing hybridoma.For example, the hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which the protein antigen is adsorbed directly or together with a carrier. Next, an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mouse) or protein A, and a monoclonal antibody bound to the solid phase A monoclonal antibody bound to a solid phase is prepared by adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A has been adsorbed, adding a protein labeled with a radioactive substance, an enzyme, or the like. And a method for detecting antibody.

 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 a medium for animal cells 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 1-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or hybridoma culture A serum-free medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culture temperature is usually 20 to 40, preferably about 37 ° C. The cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. 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 analysis. Separation and purification method (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, extraction solid phase Alternatively, a specific purification method for collecting an antibody alone using an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody]. (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 immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the method for producing a monoclonal antibody described above. It can be produced by collecting an antibody-containing substance against the protein of the present invention 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 crosslinking the carrier. If it is possible to efficiently crosslink hapten, any kind may be crosslinked at any ratio. For example, serum albumin ゃ ゥ thyroglobulin, hemocyanin, etc. may be used in a weight ratio of about 0 to 1 for hapten. A method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used. In addition, various condensing agents can be used for force coupling between the hapten and the carrier. For example, daltaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithiopyridyl group, or the like is used.

 The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant / 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. It is complementary to or substantially complementary to the base sequence of DNA encoding the protein or partial peptide of the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of antisense nucleotides). The antisense nucleotide having a base sequence that is partially complementary or a part thereof includes a base sequence that is complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof. Any antisense nucleotide may be used as long as it has an action of suppressing the expression of DNA, but antisense DNA is preferable.

 The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology. In particular, about 70% or more of the complementary nucleotide sequence of the nucleotide sequence encoding the N-terminal portion of the protein of the present invention (for example, the nucleotide sequence near the start codon) in the entire nucleotide sequence of the complementary strand of the DNA of the present invention. Antisense nucleotides having a homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more are suitable.

 Specifically, it is complementary to or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31. Nucleotide sequence or an antisense nucleotide containing a part thereof, preferably, for example, a base of DNA having a nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31 An antisense nucleotide having a base sequence complementary to the sequence or a part thereof (more preferably, a DNA having a base sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31) A base sequence complementary to the base sequence of SEQ ID NO: 1, or an antisense nucleotide having a part thereof).

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

To prevent degradation by hydrolases such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA should be It may be substituted with a chemically modified phosphoric acid residue such as mouth carbonate, methylphosphonate and phosphorodithionate. These antisense nucleotides can be produced using a known DNA synthesizer or the like.

 According to the present invention, an antisense nucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the nucleotide sequence information of the DNA encoding the cloned or determined protein. , Can be synthesized. Such a polynucleotide (nucleic acid) can hybridize to the RNA of the protein gene of the present invention and 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-related RNA of the present invention, and that can specifically hybridize with a protein-related RNA of the present invention, are in vivo and in vitro. It is useful for regulating and controlling the expression of the protein gene of the present invention, and is also useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids, including genes. The “correspondence” between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by 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 coding region, ORF translation stop codon, 3' end untranslated region The 3'-end palindrome region, and the 3'-end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as the region of interest.

The relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region or the relationship between the target nucleic acid and a polynucleotide that can hybridize with the target can be said to be “antisense”. The nucleic acid nucleotide may be a polynucleotide containing 2-dexoxy D-report, a polynucleotide containing D-report, another type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or Has a non-nucleotide backbone Other polymers (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymers are capable of pairing bases such as those found in DNA or RNA. Containing a nucleotide having a configuration that allows base attachment). 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 oligonucleotides). , And also with known modifications, e.g., labeled, capped, methylated, or substituted with one or more natural nucleotides as known in the art. Modified with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate) , Phosphorodithioate, etc., such as proteins (nucleases, nucleases, inhibitors, Has side-chain groups such as syn, antibody, signal peptide, poly-L-lysine, etc. and sugar (eg, monosaccharide), and has an inter-current compound (eg, acridine, psoralen, etc.) , Those containing chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, those with modified bonds (eg, α-anomer type 1) Nucleic acid). Here, "nucleoside", "nucleotide" and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.

The antisense nucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of modified nucleic acids include sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides. Examples include, but are not limited to, those that are resistant to nucleoside amide degradation. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and if toxic Reduce the toxicity of antisense nucleic acids.

 Thus, many modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; S. T.

Crooke et al. Ed., Ant isense Research and Appli cat 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. Such additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, enhance interactions with cell membranes, and increase nucleic acid uptake. Hydrophobic substances such as lipids (eg, phospholipids, cholesterol, etc.) can be mentioned. 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 capping groups specifically located at the 3 'or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNAse. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene 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 of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as the D NA), 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 the antisense nucleotide of the invention).

 The protein of the present invention can be used as a disease marker because its expression is increased in the heart at the stage of heart failure transition (heart failure decompensation / heart failure decompensation) after myocardial infarction. That is, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disease (eg, testicular hypersensitivity, ovary) It is useful as a marker for early diagnosis of dysfunction, infertility, etc.), judgment of symptom severity, and prediction of disease progression.

 Antisense nucleotide of a gene encoding the protein of the present invention (protein gene of the present invention), a compound or its salt that regulates the activity of the protein of the present invention, a compound or a salt thereof that regulates the expression of the protein gene of the present invention The pharmaceuticals containing the antibody against the protein according to the present invention include, for example, diseases characterized by decreased cardiac function, such as heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), It can be used as a prophylactic or therapeutic agent for neurological disorders (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disorders (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.), or as a contraceptive .

[1] Screening of drug candidate compounds for disease

 The protein of the present invention and the protein gene of the present invention

(Heart failure decompensation phase Heart failure decompensation) Since the expression increases, the compound or its salt that regulates the activity of the protein of the present invention, and the compound or its salt that regulates the expression of the protein gene of the present invention include, for example, Diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, It can be used as a prophylactic or therapeutic agent for genital disorders (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.), or as a medicament such as a contraceptive.

 Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates the activity of the protein of the present invention, and the polynucleotide encoding the protein is a protein gene of the present invention. It is useful as a reagent for screening a compound or a salt thereof that regulates the expression of chromosome.

 That is, the present invention provides (1) an activity of the protein of the present invention characterized by using the protein of the present invention (for example, activity for promoting or suppressing cardiac function, activating or inactivating cardiomyocyte function). A method of screening for a compound that regulates (promotes or inhibits) or a salt thereof; (2) regulates (promotes or inhibits) the expression of the protein gene of the present invention, which comprises using a polynucleotide encoding the protein of the present invention. And (3) a method for regulating (promoting or inhibiting) the expression of the protein of the present invention or a salt thereof, characterized by using the antibody of 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 using the protein of the present invention

 Specific examples of the screening method include (i) culturing a cell capable of producing the protein of the present invention and (ii) culturing a mixture of a cell capable of producing the protein of the present invention and a test compound. A method for screening for a modulator characterized by performing a comparison with the case is provided.

 In the above-mentioned screening method, for example, in the cases (i) and (ii), the activity of the protein of the present invention for promoting or suppressing cardiac function, the activity for activating or inactivating cardiomyocyte function, and the like are measured and compared.

 The activity of promoting or suppressing cardiac function and the activity of activating or inactivating cardiomyocyte function of the protein of the present invention are measured by measuring cardiac function.

The measurement of cardiac function can be measured according to a known method or a method analogous thereto. For example, the measurement is performed by an echocardiograph (Cell, Vol. 97, pp. 189-198, 1999) or cardiac function measurement using a cardiac catheter (circulation research, Vol. 69, pp. 370-377, 1991). Furthermore, for example, angiotensin I converting enzyme Increase in renin angiotensin system (RAS) such as (ACE) using a commercially available assay kit (eg, manufactured by Peninsula Inc., Phoenix Inc., etc.), or increased blood catecholamine activity (E. The heart function is measured using an index such as a fully automatic catecholamine analyzer manufactured by Soviet Union.

 When a cell having the ability to produce the protein of the present invention is used, for example, the respiratory activity of the cell is measured, or a heart failure marker gene [eg, ANP (atrial sodium diuretic peptide), BNP (brain natriuretic peptide, etc.) The respiratory activity of the cells can be measured according to a known method or a method analogous thereto. For example, MTT (3- (4, 5-Dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium) method, trypan blue staining method, TUNNEL staining method (Terminal deoxytransferase-mediated dUTP-X nick end labeling, cell, Vol. 97, 189) -P. 198, 1999) Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts. Etc., and these compounds may be novel compounds, it may be a known compound..

 To carry out the above screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any buffer such as 11 to 4 to 10 (preferably 11 to 6 to 8) phosphate buffer or borate buffer.

 As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing DNA encoding the protein of the present invention described above is used. As a 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 cells by culturing by the method described above is preferably used.

For example, a test compound that promotes the cardiomyocyte function activating effect in the case of the above (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (i) A compound or a compound that promotes the activity of the protein of the present invention. Can be selected as a salt. A test compound that inhibits the inactivation of cardiomyocyte function in case (ii) above by about 20% or more, preferably 30% or more, and more preferably about 50% or more, compared to the case (i). It can be selected as the compound or its salt that inhibits the activity of the protein of the present invention.

 (2) A method for screening a compound or a salt thereof that regulates the expression of the protein gene of the present invention using a polynucleotide encoding the protein of the present invention

 Specific examples of the screening method include (iii) culturing a cell capable of producing the protein of the present invention and (iv) culturing a mixture of a cell capable of producing the protein of the present invention and a test compound. In each case, the expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured and compared.

 For example, (iii ') a case where cells having the ability to produce the protein of the present invention are cultured under hypoxic conditions with extension stimulation, and (iv') a cell having the ability to produce the protein of the present invention is tested. The expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the case where the mixture of the compounds is cultured under hypoxic conditions with extension stimulation is measured. And compare.

The hypoxic condition means, for example, an oxygen concentration of 20% O ₂ or less and a condition of, for example, 2% (Nature, Vol. 394, pp. 485-490, 1998).

 The stretching stimulus is, for example, a stimulus in which a target cell is cultured on a stretchable silicon membrane and a mechanical load is applied by pulling the silicon membrane (JB, Vol. 271, 33592-33597, 1996 Circulation, 89, 2204-2221, 1994, JBC, 271, 3221-3228, 1996).

 For example, (iii '') a cell capable of producing the protein of the present invention when cultured under lethal conditions; and (iv '') a mixture of a cell capable of producing the protein of the present invention and a test compound. Are cultured under lethal conditions, and the expression level of the protein gene of the present invention (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) is measured and compared.

The culture under the above lethal conditions is, for example, culture under serum removal or an anticancer agent (eg, Cardiomyocytes can be cultured with relatively toxic adriamicin, etc.).

 The amount of the protein of the present invention can be measured by a known method, for example, by using an antibody recognizing the protein of the present invention to analyze the protein present in a cell extract or the like, by Western analysis, ELISA, etc. Alternatively, it can be measured according to an equivalent method.

 The expression level of the protein gene of the present invention can be determined by a known method, for example, Northern blotte ink, reverse transcript ion-polymerase chain reaction (RT-PCR), real-time PCR analysis system (ABI, TaqMan polymerase chain reaction). It can be measured according to such a method or a method equivalent thereto.

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

 To carry out the above screening method, cells having the ability to produce the protein of the present invention are prepared by suspending them in a buffer suitable for screening. The buffer may be any buffer that does not inhibit the activity of the protein of the present invention, such as a phosphate buffer or a borate buffer having a pH of about 4 to 10 (preferably, a pH of about 6 to 8).

 As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing DNA encoding the protein of the present invention described above is used. As a 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 cells by culturing by the method described above is preferably used.

For example, the expression level of the protein gene of the present invention in the case of the above (iv) is about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). A test compound that promotes the expression of the protein gene of the present invention Or a salt thereof. The expression level of the protein gene of the present invention in the case of the above (iv) is about 20% or more, preferably 30% or more, more preferably about 50% or more compared with the case of the above (iii). The test compound that inhibits can be selected as a compound that inhibits the expression of the protein gene of the present invention or a salt thereof.

 (3) A method for screening a compound or a salt thereof that regulates the expression of the protein of the present invention using the antibody of the present invention

 Specific examples of the screening method include: (a case where cells having the ability to produce the protein of the present invention are cultured; and (vi) a case where a mixture of cells having the ability to produce the protein of the present invention and a test compound are cultured). In the above screening method, the expression level of the protein of the present invention (specifically, the amount of the protein of the present invention) in cases (V) and (vi) is measured using the antibody of the present invention. (Eg, detection of expression, quantification of expression level, etc.) and compare.

 The amount of the protein of the present invention can be measured by a known method, for example, using an antibody recognizing the protein of the present invention to detect the protein present in a cell extract or the like, using a method such as Western analysis, ELISA, or the like. It can be measured in accordance with the corresponding method.

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

 As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing DNA encoding the protein of the present invention described above is used. As a 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 cells by culturing by the method described above is preferably used.

For example, the expression level of the protein of the present invention in the case of the above (vi) is about 20% or more, preferably 30% or more, more preferably about 5% or more as compared with the case of the above (V). A test compound that promotes 0% or more can be selected as a compound that promotes expression of the protein of the present invention or a salt thereof. In the case of the above (vi), the expression level of the protein of the present invention is inhibited by about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case of the above (V). The test compound to be tested can be selected as a compound that inhibits the expression of the protein of the present invention or a salt thereof.

 The screening kit of the present invention comprises a protein or partial peptide of the present invention or a salt thereof, a polypeptide encoding the protein or partial peptide of the present invention, an antibody of the present invention, or a protein or partial protein of the present invention. It contains cells having the ability to produce peptides.

 The compound 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-peptidic compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract. A compound selected from animal tissue extract, plasma, or the like, or a salt thereof, and the activity of the protein of the present invention (eg, cardiac function promoting or suppressing activity, cardiomyocyte function activating or inactivating effect, etc.), A compound or a salt thereof that regulates (promotes or inhibits) the expression of the protein gene of the present invention or the expression of the protein of the present invention.

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

It is thought that excessively enhanced expression of the gene encoding the protein of the present invention causes excessive activation of cells and accelerates cell exhaustion. On the other hand, moderate expression is thought to enhance the compensatory mechanism. Therefore, it is considered important to appropriately control the expression level of the gene encoding the protein of the present invention. For example, when it is considered that cell damage is caused by promotion of the expression of the gene encoding the protein of the present invention, such as in the acute phase of heart failure and the decompensated phase of heart failure, the activity of the protein of the present invention is inhibited. Administering a compound, a compound that inhibits the expression level of the protein gene of the present invention, a compound that inhibits the expression level of the protein of the present invention, or a salt (inhibitor) thereof, and conversely, the gene encoding the protein of the present invention. A compound that promotes the activity of the protein of the present invention or a compound that promotes the expression level of the protein gene of the present invention when the cell damage is considered to be caused by the decreased expression. Alternatively, the compound of the present invention that promotes the expression level of the protein or a salt thereof (promoter) is administered.

 Since the DNA (gene) encoding the protein of the present invention has a function of regulating cardiac function, extremely reduced expression of the gene encoding the protein of the present invention impairs cell function, and conversely, excessive expression may It is thought that it causes activation of cells more 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, administration of the protein of the present invention, the DNA of the present invention, or the promoter obtained by the above-described screening method is performed. Thus, the disorder of the cell function can be prevented and treated, and conversely, if overexpression of the protein of the present invention or the DNA of the present invention is found, the above-mentioned strain. By administering an inhibitor obtained by the above-mentioned method, the impairment of the cell function can be prevented and treated.

 A compound or a salt thereof that inhibits the activity of the protein of the present invention, the expression of the protein gene of the present invention, or the expression of the protein of the present invention, which is obtained by the above-described screening method, is used for the expression of DNA (gene) encoding the protein of the present invention. Administration in the acute phase or decompensated phase of heart failure where enhancement is observed can be expected to restore cardiac function. In addition, a compound or a salt thereof that promotes the activity of the protein of the present invention, the expression of the gene of the protein of the present invention, or the expression of the protein of the present invention decreases the expression of the DNA (gene) encoding the protein of the present invention. When administered, administration can enhance the compensatory mechanism, and can be expected to have a heart protective effect by protecting cardiomyocytes.

 The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention has excellent cardiac function promoting activity and cardiomyocyte function activating activity (cytoprotective activity etc.). Cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc., central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disorders (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.) It is useful as a prophylactic or therapeutic agent or a contraceptive drug.

Obtained by using the screening method or the screening kit of the present invention. The compound or a salt thereof can be safely administered by itself or as a suitable medicament. The medicament used for the above administration contains the above compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient, and is provided as a pharmaceutical composition suitable for oral or parenteral administration. You.

 For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). ), 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 liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like, suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol ( For example, propylene glycol, polyethylene glycol), and nonionic surfactants [eg, polysorbate 80, HCO-50 polyoxyethylene (5 O mol) adduct of hydrogenated cast oil) may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection 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), suppositories, etc., and usually 5 to 500 mg per dosage unit dosage form. 5 to 100 mg for the agent, Other dosage forms preferably contain 10-25 Omg of the above compound.

 The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (eg mice, rats, puppies, sheep, bush, pussi, puma, birds, cats, dogs, Monkeys, chimpanzees, etc.) orally or parenterally.

 The dose of the compound or its salt that regulates the activity of the protein of the present invention may vary depending on its action, target disease, subject to be administered, administration route and the like.For example, the activity of the protein of the present invention for the treatment of heart failure When a compound or a salt thereof is orally administered, generally in adults (assuming a body weight of 6 Okg), the compound or a salt thereof is added in an amount of about 0.1 to 10 Omg, preferably about 1.0 mg / day. 5050 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, etc., for example, a compound that regulates the activity of the protein of the present invention for the purpose of treating heart failure Or, when the salt thereof is administered to an adult (as 6 Okg) in the form of an injection, usually about 0.01 to 3 Omg, preferably about 0.1 to 20 mg of the compound or a salt thereof per day. More preferably, about 0.1 to 1 Omg is administered by intravenous injection. For other animals, the dose can be administered in terms of 60 kg.

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

 An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention, 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) Simultaneously or continuously reacting the test wave with the antibody of the present invention insolubilized on the carrier and another labeled antibody of the present invention, and measuring the activity of the labeling agent on the insolubilized carrier. The present invention provides 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 to quantification of the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like is also possible. it can. 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, antigen or 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, nephelometry, competition method, immunometric method and sandwich method are preferably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, / 3-galactosidase,; 3-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, 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 insolubilization of antigen or antibody, physical adsorption may be used. A method using a chemical bond used for insolubilizing or immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

 In the sandwich method, 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 by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds. . 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, or a nephrometry. In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) is separated from the labeled antigen (B) bound to the antibody. (BZF separation) Measure the amount of labeling of either B or F, and quantify the amount of antigen in the test solution. In this reaction method, a soluble antibody is used as an antibody, BZF separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, a solid phase antibody is used as the first antibody, or An immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used. '' In the immunometric method, a fixed amount of labeled Ability to separate solid phase and liquid phase after competitive reaction with the body or react the antigen in the test wave with an excess amount of labeled antibody, then add immobilized antigen and label unreacted After binding the antibody 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 wave.

 In nephelometry, the amount of insoluble sediment resulting from the 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 measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, documents, etc.

 For example, edited by Hiro Irie, "Radio Nonotsusei" (Kodansha, published in Showa 49), edited by Hiroshi Irie, "Continued Radio Immnoatsusy" (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, et al., "Enzyme Immunoassay" (3rd edition) (Medical Shoin, published in 1962), ("Methods in ENZYMOLOGYj Vol. 70 (Immunochemical Techniques (Part A)), ibid. Vol. 73 (Immunochemical Techniques (Part B)), ibid. 7 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)) Ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)), ibid. . 121 (Immunochemical

Techniques (Part I: Hybridoma Technology and Monoclonal Ant ibodies) ') (above, published by Academic Press) can be referred to.

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

Furthermore, when an increase in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, heart disease ( E.g., cardiomyopathy, myocardial infarction, heart failure, angina pectoris), central nervous system disorders (e.g., Alfheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disorders (e.g., testicular irritability, ovarian dysfunction, (Eg, infertility) or are more likely to be affected in the future.

 Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. 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, in humans or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, bushfishes, horsetails, dogs, cats, dogs, monkeys) by using them as probes. , Chimpanzee, etc.), it is possible to detect an abnormality (gene abnormality) in DNA or mRNA encoding the protein of the present invention or its partial peptide in, for example, DNA or mRNA damage, sudden mutation or It is useful as a diagnostic agent for genes such as decreased expression, increased DNA or mRNA or excessive expression.

 The above-described genetic diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Procaging). Proceedings of the National Academy of Sciences of the United States of America, Volume 86, pp. 2766-2770 (1989), etc. Can be implemented.

For example, when overexpression is detected by Northern hybridization or when DNA mutation is detected by PCR-SSCP method, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure) , Angina pectoris), central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), reproductive disorders (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.) Diagnosis is high Can be.

[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 functions of the protein of the present invention or the DNA of the present invention in vivo. (E.g., cardiac function inhibitory activity, cardiomyocyte function inactivating effect, etc.), for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system disease ( For example, it can be used as a prophylactic / therapeutic agent for congenital diseases such as Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc., genital disease (eg, testicular hypersensitivity, ovarian insufficiency, infertility), or as a contraceptive. .

 When the antisense nucleotide is used as the above-mentioned prophylactic / therapeutic 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 a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. It can be administered orally or parenterally to human or non-human mammals (eg, rats, puppies, sheep, higgs, bush, puppies, cats, dogs, monkeys, etc.). 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 can be administered with a gene gun or a catheter such as a hide mouth gel catheter. Alternatively, it can be made into an aerosol and administered topically into the trachea as an inhalant.

 The dosage of the antisense nucleotide varies depending on the target disease, the administration subject, 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, it is generally required for an adult ( At a body weight of 60 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

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

(i i) a medicine comprising the double-stranded RNA,

 (iii) a lipozyme containing a part of RNA encoding the protein of the present invention,

(iv) A pharmaceutical comprising the ribozyme is provided.

 These double-stranded RNAs (RNAi; RNA interference method), lipozymes, and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention similarly to the above-mentioned antisense nucleotides. It can inhibit the activity or function of the peptide of the present invention or the polynucleotide of the present invention (eg, DNA) in a living body (eg, cardiac function-suppressing activity, myocardial cell function-inactivating action, etc.). , Heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disease (eg, testicular hypersensitivity, ovarian function) It can be used as a prophylactic / therapeutic agent for insufficiency, infertility, etc.) or as a contraceptive.

 The double-stranded RNA can be designed and manufactured 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, Vol. 7, p. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of RNA encoding the peptide of the present invention. As a part of the RNA encoding the peptide of the present invention, a portion (RNA fragment) close to the cleavage site on the RNA of the present invention, which can be cleaved by a known lipozyme, can be mentioned.

 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 the antisense nucleotide.

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

The antibody of the present invention having the activity of neutralizing the activity of the protein of the present invention may be used for heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome) , Schizophrenia, etc.), genital disorders (eg, testicular hypersensitivity, It can be used as a medicine for diseases such as ovarian insufficiency and infertility.

 The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in human or non-human mammals (eg, rat, egret, sheep, etc.). Or parenteral, parenteral, parenteral, cat, dog, monkey, etc.). The dose varies depending on the administration subject, target disease, symptoms, administration route and the like.For example, when used for the treatment of heart failure in adults, the antibody of the present invention is usually used in a dose of 0.0. About 1 to 20 mg / kg body weight, preferably about 0.1 to 1 OmgZkg body weight, more preferably about 0.1 to 5 mgZkg body weight, about 1 to 5 times a day, preferably one day It is convenient to administer by intravenous injection about 1 to 3 times. 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 accordingly.

 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) ), 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 pharmaceutical field. 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, infusions, 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. Examples of aqueous liquids for injection include physiological saline Isotonic solutions containing water, glucose and other adjuvants are used, and suitable solubilizing agents, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non- It may be used in combination with an ionic surfactant (eg, polysorbate 80, HCO-50 (.polyoxy ethylene (50 mol) adduct of hydrogenated catalyst)) and the like. 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 dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg, especially The injection preferably contains 5 to 10 O mg of the above antibody, and other dosage forms contain 10 to 25 O mg of the above antibody.

 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 or DNA of the present invention

The protein of the present invention or a DNA encoding the protein of the present invention (hereinafter, sometimes abbreviated as the DNA of the present invention) may be a heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.) ), Central nervous system disorders (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disorders (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.) It is also useful. When the protein of the present invention is used as the above medicine, it can be formulated according to a conventional method. When the DNA of the present invention is used as the above-described prophylactic or therapeutic agent, the DNA of the present invention may be used alone or in an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. After insertion, it can be carried out according to conventional means. The DNA of the present invention is as it is It can be administered via a gene gun or a catheter such as a hydrogel catheter, together with adjuvants to promote uptake.

 For example, the protein of the present invention or the DNA of the present invention is orally acceptable as tablets, capsules, elixirs, microcapsules, etc., if necessary, or water or other pharmaceutically acceptable. It can be used parenterally in the form of sterile solutions with liquid or injectable forms such as suspensions. For example, the protein of the present invention or the DNA of the present invention is required for the generally accepted formulation of the preparation together with known carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc., which are physiologically recognized. It can be manufactured by mixing in a unit dosage form. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

 The dose of the protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. In the case of oral administration, for example, in a heart failure patient (with a body weight of 6 O kg), one dose is generally used. It is about 0.1 mg to 100 mg per day, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like. ), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day by intravenous injection. For other animals, the dose can be administered in terms of 60 kg.

The dosage of the DNA of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, in general, for example, in a heart failure patient (with a body weight of 6 O kg), It is about 0.1 mg to 10 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like. For example, in the case of an injection, it is usually, for example, a heart failure patient (weight of 6 O kg ), About 0.01 to 30 mg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day is administered by intravenous injection. It is convenient. In the case of other animals, the dose can be administered in terms of weight per 60 kg.

[7] Creation of an animal having the DNA of the present invention

 The present invention relates to a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). And a non-human mammal having the same.

 That is, the present invention

 (1) a non-human mammal having the exogenous DNA of the present invention or its mutant DNA, (2) the animal according to the above (1), wherein the non-human mammal is a rodent,

 (3) The animal according to (2), wherein the rodent is a mouse or a rat, and

(4) To provide a recombinant vector or the like containing the exogenous DNA of the present invention or its mutant DNA, which can be expressed in mammals.

 Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-introduced animal of the present invention) include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. Preferably, at the stage of embryonic development in the development of non-human mammals (more preferably, at the stage of a single cell or a fertilized egg cell and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, It can be produced by introducing the target DNA by a method such as a coagulation method, a coagulation method, a microinjection method, a particle gun method, or a DEAE-dextran method. In addition, the exogenous DNA of the present invention, which is intended for somatic cells, organs of living organisms, tissue cells, and the like, can be introduced by the DNA introduction method and used for cell culture, tissue culture, and the like. Can be fused with the above-mentioned germinal cells by a known cell fusion method to produce the DNA-introduced animal of the present invention.

As the non-human mammal, for example, red sea lions, bushes, higgins, goats, green egrets, dogs, cats, guinea pigs, hamsters, mice, rats, and the like are used. Above all, rodents with relatively short ontogeny and biological cycles in terms of the creation of disease animal model systems, and easily breeding rodents, especially mice (for example, C57B LZ6 strains, DBA2 strains as pure strains, etc.) B 6 C 3F, strain, BDF, strain, B 6D2Fi strain, BALB / c strain, ICR strain, etc.) or rat (for example,

Wistar, SD, etc.) are preferred.

 As the “mammal” in the recombinant vector that can be expressed in mammals, human and the like can be mentioned in addition to the above-mentioned non-human mammals.

 The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by a non-human mammal but to the DNA of the present invention once isolated and extracted from the mammal.

 As the mutant DNA of the present invention, DNA having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition, deletion, or substitution of another base to another base DNA that has been used is used, and also includes abnormal DNA.

 The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.

 The exogenous DNA of the present invention may be derived from a mammal of the same species or a different species as the target animal. In introducing the DNA of the present invention into a subject animal, a DNA linked to a downstream of a promoter capable of expressing the DNA in animal cells is used.

It is generally advantageous to use it as an A construct. For example, the human D of the present invention

When introducing NA, it is possible to express DNA derived from various mammals having the DNA of the present invention having a high homology with the DNA (eg, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.). The DNA construct of the present invention is microinjected downstream of various promoters into a fertilized egg of a target mammal, for example, a mouse fertilized egg, with a DNA construct (eg, vector 1) bound to the human DNA of the present invention. Can be created to highly express DNA.

 Examples of the expression vector for the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as λ phage, a retrovirus such as Moroni monoleukemia virus, a vaccinia virus or a baculovirus. For example, animal viruses such as E. coli are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.

Examples of promoters that regulate the expression of DN DN include: For example, the promotion of DNA derived from simian virus, cytomegalovirus, moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc., various mammals (human, egret, dog, cat, guinea pig, hamster, Rat, mouse, etc.), such as albumin, insulin II, perobrakin II, elasase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, daltathione S-transferase—, platelet derived Growth factor] 3, keratin K1, 10 and 14, collagen I and II, cyclic AMP-dependent protein kinase) 3 I-subunit, dystrophin, tartrate-resistant alkaline phosphatase, atrial sodium diuretic factor, Endothelium Putch mouth synthase (generally abbreviated as Tie 2), sodium potassium adenosine triphosphatase (Na, K-ATPase), double-mouth filament light chain, metamouth thionine I and IIA, metamouth proteinase 1 Tissue incipient, MHC class I antigen (H-2L), H-ras, renin, dopamine) 3-hydroxylase, thyroid peroxidase (TPO), polypeptide elongation factor la ( EF-1), β-actin, α and 3 myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy_l, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, mi Promoters such as odarobin, troponin C, smooth muscle actin, prebub enkephalin A, and vasopressin are used. Among them, a cytomegalovirus promoter capable of high expression throughout the whole body, a promoter of human polypeptide chain elongation factor 1a (EF-1), human and chicken, and 3 actin promoter are preferable.

 The vector preferably has a sequence that terminates transcription of the target messenger RNA in the DNA-transfected mammal (generally referred to as Yuichi Minei Yuichi). The sequence of each DNA derived can be used, and preferably, SV40 terminator of Simian virus or the like is used.

In addition, the splicing signal of each DNA, enhancer region, a part of the intron of eukaryotic DNA, etc. are used to further express the target exogenous DNA. It is also possible to ligate 5 ′ upstream of the promoter region, between the promoter overnight region and the translation region, or 3 ′ downstream of the translation region.

 The normal translation region of the protein of the present invention may be a liver, kidney, thyroid cell, fibroblast derived from a human or various mammals (eg, a heron, a dog, a cat, a guinea pig, a hamus, a rat, a mouse, etc.). All or part of genomic DNA from cell-derived DNA and various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cell, or fibroblast-derived RNA. It can be obtained as a raw material. In addition, a foreign abnormal DNA can produce a translation region obtained by mutating a normal polypeptide translation region obtained from the above-described cells or tissues by a point mutagenesis method.

 The translation region can be prepared as a DNA construct that can be expressed in a DNA-transduced animal by a conventional DNA engineering technique in which it is linked to the downstream of the promoter and, if desired, to the upstream of the transcription termination site.

 Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after the introduction of the DNA indicates that the progeny of the produced animal has the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to keep. The progeny of this type of animal that has inherited the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germinal and somatic cells.

 The non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and should be subcultured as an animal having the DNA in a normal breeding environment. Can be done.

Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of the DNA indicates that all the offspring of the produced animal have the exogenous DNA of the present invention in all of the germinal and somatic cells. To have. The offspring of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained. By crossing the animals, all the offspring can be bred and passaged so as to have the DNA in excess.

 In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately promoted by promoting the function of endogenous normal DNA. It may develop hypertension and can be used as a model animal for the disease. For example, using the normal DNA-introduced animal of the present invention to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. Is possible.

 In addition, since the mammal into which the exogenous normal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can also be used for screening tests for therapeutic drugs for diseases related to the protein of the present invention. It is.

 On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. I can do it. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material. The DNA construct with the promoter can be produced by a usual DNA engineering technique. Introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after the introduction of the DNA means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of the germ cells and somatic cells. The progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing the male and female animals, it is possible to breed and pass all the offspring so as to have the DNA.

The non-human mammal having the abnormal DNA of the present invention has a high level of expression of the abnormal DNA of the present invention, and finally inhibits the function of endogenous normal DNA, thereby finally obtaining the protein of the present invention. May become functionally inactive refractory, and can be used as a disease model animal. For example, using the abnormal DNA-introduced animal of the present invention to elucidate the pathological mechanism of function-inactive refractory of the protein of the present invention and to treat this disease A study of the method is possible.

 Further, as a specific possibility, the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of the normal protein by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention (dominant negative activity). Action). Further, since the mammal into which the foreign abnormal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, a therapeutic drug screening test for the protein of the present invention or its functionally inactive type refractory disease is performed. Is also available.

 Further, as other possible uses of the above two kinds of DNA-introduced animals of the present invention, for example,

 (0 use as cell source for tissue culture,

 (ii) by directly analyzing the DNA or RNA in the tissue of the DNA-introduced animal of the present invention, or by analyzing the polypeptide tissue expressed by the DNA, to specifically express or express the protein of the present invention. Analysis of the relationship to the activating protein,

 (iii) cells of tissues having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from generally difficult tissues,

 (iv) screening for a drug that enhances cell function by using the cell described in (iii) above, and

 (v) Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered. Furthermore, using the DNA-introduced animal of the present invention, it is possible to examine the clinical symptoms of diseases related to the protein of the present invention, including the inactive refractory type of the protein of the present invention. More detailed pathological findings in each organ of the disease model related to the protein of the present invention can be obtained, which will contribute to the development of new treatment methods and the research and treatment of secondary diseases caused by the disease. it can.

In addition, it is possible to remove each organ from the DNA-introduced animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain the released DNA-introduced cells, culture them, or systematize the cultured cells. It is possible. Furthermore, it is possible to examine the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and its abnormalities. It is an effective research material for elucidating the protein of the invention and its action.

 As a specific example, 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 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. When cardiac hypertrophy occurs, the above parameters increase all the time. Therefore, the test compound can be evaluated using the suppression of this increase as an index. After administering the 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. After the myocardial infarction operation, the infarct progress inhibition 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 breeding the animal with a genetically hypertensive model rat such as an SHR rat. The compound is administered to the heart failure model prepared as described above, and the animal is examined for cardiac function, electrocardiogram, heart weight, infarction progress inhibitory activity and the like.

[8] 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 the expression of DNA of the present invention.

 That is, the present invention

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

 (2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a repo overnight 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 monogalactosidase enzyme gene derived from Escherichia coli), and the reporter gene controls the promoter for the DNA of the present invention. The non-human mammal according to the above (6), which can be expressed under (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) Promoting or inhibiting (preferably, inhibiting) the activity of the promoter of the DNA of the present invention, which comprises administering a test compound to the animal described in (7) above and detecting the expression of a reporter gene. A method for screening a compound or a salt thereof.

 A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression of DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA has substantially no 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, the same one as described above is 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 by a genetic engineering technique, and inserting or substituting another DNA. With these mutations, for example, the knockout DNA of the present invention may be produced by shifting the codon reading frame or disrupting the function of promoters or exons.

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, DNA of the present invention possessed by the non-human mammal described above, and a neomycin resistance gene, a drug resistance gene represented by a hygromycin resistance gene, or a lacZ () 3-galactosidase gene), cat ( The exon function is destroyed by inserting a reporter gene or the like typified by the chloramphenic acid acetyltransferase gene), or the DNA sequence that terminates the transcription of the gene in the intron between exons (for example, po 1 (e.g., yA additional signal) to prevent synthesis of the complete messenger RNA DNA strand having a D NA sequence terminates gene (hereinafter, evening abbreviated as one Getting vector) and, For example, the ES cells obtained by introducing into the chromosome of the animal by the homologous recombination method and using the DNA sequence on or near the DNA of the present invention as a probe are analyzed by Southern hybridization analysis or on the evening getter vector. The DNA sequence of the present invention and the DNA sequence of the neighboring region other than the DNA of the present invention used in the preparation of the targeting vector are analyzed by PCR using the primers as primers, and are obtained by selecting the knockout ES cells of the present invention. be able to.

 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. It may be newly established according to the method of ma. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, an alternative pure immunological and genetic background is used. For example, to obtain ES cells in which C57BL / 6 mice and C57BL / 6 mice have a low number of eggs collected by crossing with DBAZ2, BDFi mice (C57BLZ6 and DBA / 2 BDF 1 mice can be used satisfactorily because they have a high number of eggs collected and their eggs are robust, and they have C 57 BL / 6 mice as their background. ES cells obtained using this method are advantageous in that, when a pathological model mouse is created, the genetic background can be replaced by C57BL / 6 mice by backcrossing with C57BLZ6 mice. Can be used.

 In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. Many 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.

As an example of a method for determining the sex of ES cells, a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR can be given as an example. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture First The primary selection of ES cells in the stage can be performed by gender discrimination, and the selection of male cells at an early stage greatly reduces the time and labor required for the initial culture.

 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, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5%) in the presence of LIF (1-1000 OUZml) on a suitable feeder cell such as STO fibroblasts. Incubate the cells at about 37 ° C with 5% CO 2, 5% CO 2, 90% air. At the time of subculture, for example, trypsin ZEDTA solution (usually 001 — 0.5% trypsin Z0.1. A single cell is treated by treatment with 5 mM EDTA (preferably about 0.1% trypsin ZlmM EDTA), and the cells are seeded on a freshly prepared feeder cell. Normally, such subculture is 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; G.R.

Martin Proceedings of National Academy of Sciences, Proc. Natl. Acad. Sci. USA, 78, 7634, 1981; TC Doetschman et al., Journal of Ob.ェ ペ リ ク ス ペ リ ク ス モ ル ク ス DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA. Useful in protein cell biology 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. .

 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 targeting vector into the DNA of the present invention. Knockout of the DNA of the present invention by homologous recombination of the DNA sequence in which NA has been inactivated by replacing the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination be able to.

 The cells in which the DNA of the present invention has been knocked out are subjected to Southern hybridization analysis using the DNA sequence on or near the DNA of the present invention as a probe or the DNA sequence on the getter vector, and It can be determined by analysis by PCR using, as a primer, the DNA sequence of a neighboring region other than the DNA of the present invention derived from the mouse used in the first vector. 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 the cells having the normal DNA locus of the present invention and the cells having the artificially changed 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 deficient in the hetero-expression of the protein of the present invention, and mated with individuals deficient in the hetero-expression of the protein of the present invention. A homozygous deficient individual can be obtained.

When using an egg cell, for example, a DNA solution is injected into the nucleus of the egg cell by a microinjection method to introduce the evening vector into the chromosome. The resulting transgenic non-human mammal can be obtained, and can be obtained by selecting a transgenic non-human mammal having a mutation in the DNA locus of the present invention by gene homologous recombination as compared to these transgenic non-human mammals.

 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 mating 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 the non-human mammal deficient in expression of 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. Since it can be a model, it is useful for investigating the causes of these diseases and examining treatment methods.

 C 8 a] A method for screening a compound having a therapeutic / preventive effect on diseases caused by DNA deficiency or damage according to the present invention

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

 That is, the present invention provides a method for administering a test compound to a non-human mammal deficient in expression of a DNA of the present invention, and observing and measuring a change in the animal. The present invention provides a method for screening a compound or a salt thereof, which has a therapeutic or preventive effect against a disease caused by the above.

Non-human mammal deficient in DNA expression of the present invention used in the screening method Examples of the animal include the same 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, compared with a non-treated control animal, and changes in the organs, tissues, disease symptoms, etc. of the animal are used as indicators. 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, diseases characterized by reduced cardiac function (eg, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia) ), Genital disorders (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.). The compound is administered, 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. When cardiac hypertrophy occurs, the above parameters increase all the time. Therefore, the test compound can be evaluated using the suppression of this increase as an index. After administration of the test compound to the non-human mammal deficient in DNA expression of the present invention, myocardial infarction is performed, and the heart function, electrocardiogram, cardiac weight, etc. of the animal are measured. After the myocardial infarction operation, the infarct progress-inhibitory activity of the test compound can be determined by weighing the infarct layer. Administration of the test compound may be post-infarction surgery. In addition, a new heart failure model can be created by mating the animal with a genetic hypertension model lad such as an SHR rat. The compound is administered to the heart failure model prepared as described above, and the animal is examined for cardiac function, electrocardiogram, heart weight, infarction progress inhibitory activity and the like. 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 physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and 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, etc.).

 A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the drug containing the protein of the present invention described above. The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or non-human mammals (e.g., Dogs, monkeys, etc.).

 The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, generally the heart failure of an adult (with a body weight of 6 O kg) In a patient, about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, and more preferably about 1.0 to 20 mg of the echium compound is administered per day. When administered parenterally, the single dose of the compound will vary depending on the subject of administration, the target disease, etc. For example, the compound is usually administered in the form of an injection to an adult (as 60 kg) heart failure patient. When administered, about 0.01 to 3 Omg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg of the compound per day is conveniently administered by intravenous injection. is there. In the case of other animals, the amount converted per 60 kg can be administered.

 [8b] A method for screening a compound that promotes or inhibits the activity of a promoter for DNA of the present invention

The present invention provides a method for administering a test compound to a non-human mammal deficient in expression of a DNA of the present invention and detecting the expression of a reporter gene, which promotes the activity of a promoter against DNA of the present invention. Provided is a method for screening a compound or a salt thereof that inhibits the inhibition. In the above screening method, the non-human mammal deficient in expression of DNA of the present invention may be the non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactive by introducing a reporter gene. The reporter gene which can be expressed under the control of the promoter for the DNA of the present invention is 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; 8-galactosidase gene (1 acZ), soluble alkaline phosphatase gene or luciferase gene and the like are preferable.

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

 For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase enzyme (1 ac Z) derived from Escherichia coli, a tissue that originally expresses the protein of the present invention will 3-galactosidase is expressed (instead of the protein of the invention). Therefore, for example, by staining with a reagent that serves as a substrate for j8-galactosidase, such as 5-bromo-4-monochloro-3-indolyl || 8-galactopyranoside (X-gal), In addition, the expression state of the protein of the present invention in an animal body can be 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, the 13-galactosidase reaction may be stopped by washing the tissue specimen with ImM EDTAZPBS solution, and the coloration may be observed. Further, mRNA encoding 1acZ may be detected according to a conventional method.

The compound or a salt thereof obtained by the above-mentioned screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter one activity with respect to the DNA 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). And the like, and especially preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, shinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used. In addition, the compound of the present invention or a salt thereof that inhibits the promoter activity for DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein. , Myocardial infarction, heart failure, angina, etc.) Prevention of central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital diseases (eg, testicular hypersensitivity, ovarian dysfunction, infertility, etc.) · It is useful as a therapeutic agent or a medicine such as a contraceptive.

 Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.

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

 The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or non-human mammals (eg, rats, mice, guinea pigs, egrets, sheep, higgs, bush, horses, cats, cats, Dogs, monkeys, etc.).

The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like.For example, when the compound of the present invention that promotes promoter overnight activity against DNA is orally administered, Generally, in an adult (assuming a body weight of 60 kg) heart failure patient, about 0.1 to 10 Omg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 10 mg of the compound per day is used. Administer 0-2 O mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, the compound of the present invention that promotes the activity of a promoter against DNA is usually administered in the form of an injection to an adult. If administered to heart failure patients weighing 6 O kg It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg of the compound by intravenous injection. In the case of other animals, the dose can be administered in terms of the weight per 6 O 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 Ok) heart failure patients, the conjugate can be used in an amount of about 0.1 to about 0.1 per day. Administer 10 Omg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, a compound that inhibits the promoter activity of DNA of the present invention is usually administered in the form of an injection to an adult. When administered to a patient with heart failure (assuming a body weight of 6 O kg), about 0.01 to 3 Omg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg of the compound per day is administered. It is conveniently administered by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.

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

In addition, using DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof, and the genes are injected into egg cells of an animal to produce a so-called transgene. If an animal (transgenic animal) is created, it will be possible to specifically synthesize the polypeptide and examine its action in the living body. Furthermore, by binding an appropriate repo allele to the above promoter portion and establishing a cell line in which this is expressed, the action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body can be achieved. It can be used as a search system for low molecular weight compounds. In the present specification, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations of the IUPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. In addition, optical differences When there is a sexual body, L body shall be indicated unless otherwise specified.

DNA deoxylipo nucleic acid

 cDNA complementary deoxylipo nucleic acid

 A adenine

 T thymine

 G Guanin

 C

 R adenine (A) or guanine (G)

 Y cytosine (C) or thymine (T)

 K guanine (G) or thymine (T)

 M adenine (A) or cytosine (C)

 S guanine (G) or cytosine (C)

 • W adenine (A) or thymine (cho)

 N Adenine (A), cytosine (C), guanine (G) or thymine (T)

 RNA ribonucleic acid

 mRNA messenger liponucleic acid

 dATP Deoxyadenosin triphosphate

 dTTP Deoxythymidine triphosphate

 dGTP Deoxyguanosine triphosphate

 dCTP Deoxycytidine triphosphate

 ATP Adenosine triphosphate

 EDTA ethylenediaminetetraacetic acid

 SD S Dodecyl sulfate:

 G 1 y

 A 1 a Alanin

 Va 1 Valine

 Le u

I 1 e Ser 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 Tribute fan

 Pro proline

 A s n asparagine

 G 1 n Dal Yu Min

 p G 1 u pyroglutamic acid

 The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

 Me methyl group

 E tethyl group

 B u butyl group

 Ph phenyl group

 TC thiazolidine-1 (R) One-pot lipoxamide group

 T os p—Toluenes refonyl

 CHO Holmill

 B z 1

Cl ₂ -Bzl 2, 6-dichlorobenzyl

 Bom benzyloxymethyl

Z benzyloxycarponyl C 1 -z 2 benzoyloxycarbonyl B r—Z 2 bromobenzyloxycarbonyl

 B oc t-butoxycarbonyl

 DNP dinitrophenyl

 T r t Trityl

 Bum t—butoxymethyl

 Fmo c N-9-Fluorenylmethoxycarbonyl

 HOB t 1—Hydroxybenztriazole

 H〇〇B t 3,4-dihydro-3-hydroxy-4oxo

 1,2,3_benzotriazine

 HONB 1-Hydroxy-5-norbornene-2,3-dicarpoxyimide DCC N, N'-dicyclohexylcarposimide The sequence numbers in the sequence listing in the present specification show the following sequences.

[SEQ ID NO: 1]

 1 shows the nucleotide sequence obtained in Example 1.

[SEQ ID NO: 2]

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

[SEQ ID NO: 5]

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

[SEQ ID NO: 8] 1 shows the nucleotide sequence obtained in Example 1. ^_

 [SEQ ID NO: 9]

 The base sequences of the primers used in Examples 1 to 5, Example 8, and Example 9 are shown.

 [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 obtained in Example 1.

 [SEQ ID NO: 12]

 1 shows the nucleotide sequence of DNA containing the r164-1h full-length gene obtained in Example 1. [SEQ ID NO: 13]

 1 shows the amino acid sequence of a novel rat-derived protein r164-1h.

 [SEQ ID NO: 14]

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

 [SEQ ID NO: 15]

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

 [SEQ ID NO: 16]

 1 shows the nucleotide sequence of DNA containing the r164-1h full-length gene obtained in Example 1. [SEQ ID NO: 17]

 The base sequences of the primers used in Examples 2 to 5 are shown.

 [SEQ ID NO: 18]

 3 shows the base sequence obtained in Example 2.

 [SEQ ID NO: 19]

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

 [SEQ ID NO: 20]

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

 [SEQ ID NO: 21]

 3 shows the base sequence obtained in Example 2.

[SEQ ID NO: 22] 3 shows the nucleotide sequence of a primer used in Example 2.

 [SEQ ID NO: 23]

 6 shows the nucleotide sequences of primers used in Examples 2 and 6.

 [SEQ ID NO: 24]

 3 shows the base sequence obtained in Example 2.

 [SEQ ID NO: 25]

 1 shows the amino acid sequence of a novel human-derived protein h164-1h.

 [SEQ ID NO: 26]

 The nucleotide sequence of DNA encoding human novel protein h164-1h is shown. [SEQ ID NO: 27]

 Shows the nucleotide sequence of DNA encoding r164-1h, a novel rat-derived protein.

 [SEQ ID NO: 28]

 3 shows the amino acid sequence of a novel human-derived protein h164-1b.

 [SEQ ID NO: 29]

 1 shows the nucleotide sequence of a DNA encoding a novel human-derived protein h164-1b. [SEQ ID NO: 30]

 2 shows the amino acid sequence of a novel rat-derived protein r164-1b.

 [SEQ ID NO: 31]

 1 shows the nucleotide sequence of DNA encoding a novel rat-derived protein r164-1b.

 [SEQ ID NO: 32]

 7 shows the nucleotide sequence of a primer used in Example 6.

 [SEQ ID NO: 33]

 7 shows the nucleotide sequence obtained in Example 6.

 [SEQ ID NO: 34]

 7 shows the nucleotide sequence of a primer used in Example 7.

 [SEQ ID NO: 35]

7 shows the nucleotide sequence of a primer used in Example 7. [SEQ ID NO: 36]

 7 shows the nucleotide sequence obtained in Example 7.

 [SEQ ID NO: 37]

 7 shows the nucleotide sequence of a primer used in Example 7.

 [SEQ ID NO: 38]

 7 shows the nucleotide sequence of a primer used in Example 7.

 [SEQ ID NO: 39]

 7 shows the nucleotide sequence obtained in Example 7.

 [SEQ ID NO: 40]

 7 shows the nucleotide sequence of a primer used in Example 7.

 [SEQ ID NO: 41]

 7 shows the nucleotide sequence of a primer used in Example 7.

 [SEQ ID NO: 42]

 7 shows the nucleotide sequence obtained in Example 7.

 [SEQ ID NO: 43]

 7 shows the nucleotide sequence obtained in Example 7.

 [SEQ ID NO: 44]

 10 shows the nucleotide sequence of a primer used in Examples 8 and 9. The transformant Escherichia coli JM109 / pTB2268 obtained in Example 1 described below has been obtained from June 12, 2002 at 1-1-1, Tsukuba-Higashi, Ibaraki, Japan, independent of Central No. 6 (zip code 305-8566). Deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the accession number FERM BP-8071.

 The transformant Escherichia coli JM109 / pTB2269 obtained in Example 2 described below was obtained from Jun. 12, 2002 at 1-1, Tsukuba-Higashi, Ibaraki, Japan 1 independent of Chuo No. 6 (zip code 305-8566). Deposited with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the accession number FERM BP-8072.

The transformant Escherichia coli JM109 / pTB2270 obtained in Example 6 described below has been used since June 12, 2002, Tsukuba East East, Ibaraki Pref., Japan IT No. 1 Central No. 6 (Postal code 305- 8566) at the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM BP-8073. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

Example 1

 r 164-1 h Cloning of gene

 (1) Preparation of myocardial infarction model rat

 According to the report of Watanabe et al. (Circulation Research, Vol. 69, pp. 370-377, 1991), male Wis rats (11 weeks old, weighing 300-400 g) were pentobarbi (50 ig / kg, ip). ) And anesthesia was performed. After incision of the pericardium, the heart was exposed. At the origin of the left anterior descending branch of the coronary artery, the coronary artery and the myocardium were tied together with a suture needle with a thread (5-0 silk) and the chest was closed. The sham-operated 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 opened under pentobarbital anesthesia, the heart was excised, and the coronary artery was perfused retrograde from the aorta with saline. And washed off the blood. After removing the tissue other than the left ventricle from the extracted heart with scissors, after confirming infarction formation, the infarct region (the site of the formation of the muscle force) was removed, leaving only the non-infarcted region. This was cut into small pieces with scissors, and total RNA was extracted using ISOGEN (Wako Pure Chemical Industries, Ltd.).

 (3) Cloning of r1644-1h gene and construction of expression vector

To remove genomic DNA from total RNA, DNA is digested using enzyme set for DD (Takara Shuzo), and then differentially displayed using Fluorescence Differential Display kit Fluorescein version (Takara Shuzo). DD). Total RNA from the left ventricle 8 weeks after sham operation was used as the target tissue. As a result, a band was found to increase in tissues 1 week, 20 weeks, and 30 weeks after the operation compared to the target tissues. The band is cut out of the acrylamide gel with a cutter, suspended in sterile distilled water, and heated for 95 or 10 minutes. The gene fragment was extracted from the gel. Next, after re-amplification by PCR, the DNA base sequence was decoded. A homology search using B 1 ast N was performed on the base sequence (SEQ ID NO: 1) identified using the public database EST database. AW435329 UI-R-BJOp-afc- c- 05- 0-UI.si UI-R-BJOp Rattus

norvegicus cDNA clone UI-R-BJ0p-afc-c-05-0-UI 3 ', found to be almost identical to the mRNA sequence. Therefore, two types of primers (SEQ ID NO: 2 and SEQ ID NO: 3) were designed based on this sequence, and rat heart was designed using these primers.

5, RACE PCR was performed on Marathon-Ready cDNA (CLONTECH) according to the protocol. As a result, two types of base sequences (SEQ ID NO: 4 and SEQ ID NO: 5) having the same 3 ′ sequence were obtained. In addition, a homology search was performed on the obtained base sequence represented by SEQ ID NO: 4 with B 1 ast N using a public database, nt database, to find that Homo sapiens hypothetical protein MGC14161 (MGC14161 ) It was found that the homology was high with mRNA (Genbank ID: XM_032377). Therefore, a forward primer (SEQ ID NO: 6) was prepared based on the base sequence represented by SEQ ID NO: 4, and a reverse primer (SEQ ID NO: 7) was prepared based on the base sequence represented by SEQ ID NO: 5. PCR was performed on rat heart Marathon-Ready cDNA (CLONTECH) using these primers to obtain the nucleotide sequence represented by SEQ ID NO: 8. In addition, two primers (SEQ ID NO: 9 and SEQ ID NO: 10) were designed based on the nucleotide sequence represented by SEQ ID NO: 4, and rat heart was designed using these primers.

5 'RACE PCR was again performed on Marathon-Ready cMA (CLONTECH) according to the protocol. As a result, the nucleotide sequence of SEQ ID NO: 11 was obtained.

 By combining the three nucleotide sequences of SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 11, the nucleotide sequence represented by SEQ ID NO: 12 is obtained, and this nucleotide sequence has 256 amino acid residues The nucleotide sequence represented by SEQ ID NO: 27 encoding a protein consisting of (SEQ ID NO: 13) was included. The protein containing the amino acid sequence represented by SEQ ID NO: 13 was named r164-1h.

Two kinds of primers (SEQ ID NO: 14 and SEQ ID NO: 15) were designed based on the nucleotide sequence represented by SEQ ID NO: 12, and using these primers, a rat heart Marathon-Ready cDNA (CLONTECH) was used. PCR was performed and the obtained SEQ ID NO: : The 1072 bp fragment having the nucleotide sequence represented by 16 was subcloned into a plasmid vector pTARGET vector (Promega) according to the prescription of pTARGET Mammalian Expression Vector system (Promega). The resulting expression vector was named rl641honTARGE.

 r164-1h (SEQ ID NO: 13) has 77% homology at the amino acid level with Homo sapiens hypothetical protein MGC14161.

 A transformant having the plasmid PTB2268 containing the nucleotide sequence represented by SEQ ID NO: 16 was designated as Escherichia coli JM109 / pTB2268. Example 2

 Cloning of h164-1h gene and construction of expression vector

 Using a primer designed based on the nucleotide sequence encoding r164—1h (SEQ ID NO: 17 and SEQ ID NO: 9), a human heart Marathon-Ready cDNA (

Respect CLONTECH Co.), the PCR, SEQ ID NO: 1 8 represented by nucleotide sequence obtained _c also SEQ ID NO: 2 types of primers based on the nucleotide sequence represented by 18 (SEQ ID NO: 1 9 and SEQ ID NO: 20) was designed, and 5 ′ RACE PCR was performed on human heart Marathon-Ready cA (CLONTECH) using these primers according to the protocol. As a result, the nucleotide sequence represented by SEQ ID NO: 21 was obtained. Using a primer (SEQ ID NO: 22) designed based on the nucleotide sequence of SEQ ID NO: 21 and a primer (SEQ ID NO: 23) designed based on the nucleotide sequence of MGC14161 (see Example 1), the human heart Marathon- PCR was performed on the Ready cDNA (CLONTECH) to obtain the nucleotide sequence represented by SEQ ID NO: 24. This nucleotide sequence contained the nucleotide sequence represented by SEQ ID NO: 26 which codes for a protein consisting of 249 amino acid residues (SEQ ID NO: 25). The protein containing the amino acid sequence represented by SEQ ID NO: 25 was designated as h164-1h.

The obtained fragment (SEQ ID NO: 24) is again amplified by PCR using two kinds of primers (SEQ ID NO: 22 and SEQ ID NO: 23), and the obtained fragment is PTARGET Mammalian Expression Vector system (Promega) The plasmid was subcloned into a plasmid vector pTARGET vector (Promega) according to the above procedure. Obtained expression level Kuta was named M641honTARGE.

 164-1 (SEQ ID NO: 25) had 82% homology at the amino acid level with Homo sapiens hypothetical protein MGC14161. In addition, hl64-lh (SEQ ID NO: 25) and r164-1h (SEQ ID NO: 13) obtained in Example 1 had 90% homology at the amino acid level.

 A transformant having the plasmid PTB2269 containing the nucleotide sequence represented by SEQ ID NO: 24 was designated as Escherichia coli JM109 / pTB2269. Example 3

 Analysis of time course of r164-1h gene in myocardial infarction model rat

 1 week, 8 weeks, 20 weeks, and 30 weeks after myocardial infarction described in Example 1 (2) Total RM derived from the non-infarcted region of the left ventricle of the rat and 1 week after sham operation as the subject CDNA was synthesized from total RNA derived from the left ventricle of rats at 8, 20 and 30 weeks old using TaqMan Reverse Transcription Reagents (PE Applied Biosystems). Next, using a primer having the nucleotide sequence represented by SEQ ID NO: 17 or SEQ ID NO: 9, which is a primer capable of specifically amplifying r164-1h, copying the r164-1h gene by PCR Numerical quantification was performed with the ABI Prism 7900 sequence Detection System. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. One week after myocardial infarction, total RNA from the non-infarcted region of the rat left ventricle was synthesized with cDNA using TaqMan Reverse Transcription Reagents (PE Applied Biosystems). Next, PCR was performed using the primers represented by SEQ ID NO: 17 and SEQ ID NO: 9, which are primers capable of specifically amplifying r164-1h, and the obtained r164-1h gene was partially subjected to PCR. The gene fragment having the sequence was used as the standard. Furthermore, the calculated copy number was used as an internal control in the same way as the copy number of r164-1h gene.

After correcting with the copy number of glycerol 3-phosphate dehydratase calculated using Rodent GAPDH Control regents VIC (manufactured by PE Applied Biosystems), the value was calculated. Was compared with the target tissue, and the change rate was converted to data.

 As a result, it was clarified that the expression of the r164-1h gene was increased 1.3-fold, 1.3-fold, and 1.4-fold at 1, 20, and 30 weeks after the operation, respectively. Example 4

 Analysis of tissue distribution of r164-1h gene in normal rat

 Using a primer having the nucleotide sequence represented by SEQ ID NO: 17 or SEQ ID NO: 9 as a primer capable of specifically amplifying r164-1h, the primer was used for the brain, heart, kidney, spleen, thymus, liver, stomach, PCR was performed using the Marathon-Ready cDNA library (CL0NTECH) derived from organs of the small intestine, muscle, lung, testis and skin, and the copy number of the r164-11h gene was quantified by the ABI Prism 7900 sequence Detection System. I did it. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. One week after myocardial infarction operation, cDNA was synthesized from TaqMan Reverse Transcription Reagents (manufactured by PE Applied Biosystems) using total RNA derived from the non-infarcted region of the left ventricle of the rat left ventricle. Next, PCR was performed using the primers represented by SEQ ID NO: 17 and SEQ ID NO: 9, which are primers capable of specifically amplifying r164-1h, and the resulting r164-1h gene was obtained. The gene fragment having the partial sequence of was used as the standard. Furthermore, the calculated copy number was used as an internal control, and the glycerol triphosphate dehydrogenase calculated using TaqMan Rodent GAPDH Control regents VIC (manufactured by PE Applied Biosystems) in the same manner as the copy number of the r16-1h gene. After correcting for the copy number, the value was converted to data as the expression level.

As a result, the expression of the r164-1h gene in the heart was as high as 0.0041, and the expression in the testis was 0.0005. In other organs, the expression of the r164-1h gene was less than 0.0002, indicating that the heart was the major expression site of the r164-1h gene. Example 5 Analysis of tissue distribution of h164-1h gene in human

 Brain, heart, kidney, spleen, thymus, liver, kidney, small intestine, skeletal muscle, lung, testis and aorta, using a primer having the nucleotide sequence represented by SEQ ID NO: 17 and SEQ ID NO: 9, respectively PCR was performed using the Marathon-Ready cDNA library (CLONTECH) derived from each organ of type III, and a copy of the h164—: L h gene was determined by PCR using the ABI Prism 7900 sequence Detection System. . The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. Using human heart Marathon-Ready cDNA (CLONTECH), PCR was performed with the above primers (SEQ ID NO: 17 and SEQ ID NO: 9), and the obtained gene fragment having the partial sequence of h164-1h gene was obtained. Standard. Further, the calculated copy number is used as an internal control, and corrected in the same manner as the h 164-1 h gene copy number by the glycerol triphosphate dehydratase copy number calculated using TaqMan GAPDH Control regents (manufactured by PE Applied Biosystems). After that, the value was converted into data as the expression level.

 As a result, the expression of the h164-1h gene in the heart was as high as 0.0021, and the expression in the testis was 0.0003. In other organs, the expression of the h164-1h gene was less than 0.0001, indicating that the heart was the major expression site of the h164-1h gene. Example 6

 Cloning of h164-1b gene and construction of expression vector

Using a primer designed based on the nucleotide sequence encoding MGC14161 (SEQ ID NO: 32 and SEQ ID NO: 23), PCR was performed on human heart Marathon-Ready cDNA (CLONTECH), and the results are shown in SEQ ID NO: 33. The base sequence to be obtained was obtained. This base sequence contained the base sequence represented by SEQ ID NO: 29, which codes for a protein consisting of 481 amino acid residues (SEQ ID NO: 28). The protein containing the amino acid sequence represented by SEQ ID NO: 28 was designated as h164-1b. hi64-1b (SEQ ID NO: 28) had 74% homology with Homo sapiens hypothetical protein MGC14161 at the amino acid level. After re-amplifying the nucleotide sequence represented by SEQ ID NO: 33, the obtained fragment was subcloned into the pTARGET Mammalian Expression Vector system (Promega), and the resulting plasmid was subcloned into the pTARGET vector-1 (Promega). did. The resulting expression vector was named hl641bonTARGE.

 A transformant having the plasmid PTB2270 containing the nucleotide sequence represented by SEQ ID NO: 33 was designated as Escherichia coli JM109 / pTB2270. Example 7

 Determination of the nucleotide sequence of the r164-1b gene

 Rat brain Marathon-Ready using a primer designed based on the nucleotide sequence encoding MGC14161 (SEQ ID NO: 34) and a primer designed based on the nucleotide sequence represented by SEQ ID NO: 12 (SEQ ID NO: 35) PCR was performed on the cDNA (CLONTECH) to obtain a nucleotide sequence represented by SEQ ID NO: 36. Using a primer designed based on the base sequence encoding MGC14161 (SEQ ID NO: 37) and a primer designed based on the base sequence represented by SEQ ID NO: 12 (SEQ ID NO: 38), rat brain Marathon PCR was performed on -Ready cDNA (CLONTECH) to obtain a nucleotide sequence represented by SEQ ID NO: 39. Based on the nucleotide sequence represented by SEQ ID NO: 39, two primers (SEQ ID NO: 40 and SEQ ID NO: 41) were designed and rat brain Marathon-Ready cDNA (CLONTECH), rat heart Marathon-Ready cDNA (CLONTECH), rat testis Marathon-Ready cDNA (CLONTECH)

5 'RACE PCR was performed on CLONTECH according to the protocol. As a result, the nucleotide sequence represented by SEQ ID NO: 42 was obtained. By combining the nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 39, and SEQ ID NO: 42, the nucleotide sequence of SEQ ID NO: 43 was obtained. This nucleotide sequence contained the nucleotide sequence of SEQ ID NO: 31 encoding a protein (SEQ ID NO: 30) consisting of 481 amino acid residues. The protein containing the amino acid sequence represented by SEQ ID NO: 30 was named r164-1b.

r164-1b (SEQ ID NO: 30) had 48% homology at the amino acid level with Homo sapiens hypothetical protein MGC14161. Also, r 164—lb ( SEQ ID NO: 30) and r164-1 (SEQ ID NO: 13) obtained in Example 1 had 78% homology at the amino acid level. Further, r164-1b (SEQ ID NO: 30) and h164-1b (SEQ ID NO: 28) obtained in Example 6 had 98% homology at the amino acid level. Example 8

 Analysis of temporal changes of r164-1b gene in myocardial infarction model rat

 1 week, 8 weeks, 20 weeks, and 30 weeks after myocardial infarction described in Example 1 (2) Total RNA from non-infarcted region of left ventricle of rat and 1 week after sham operation as target CDNA was synthesized from the total RNA derived from the left ventricle of the rat after 8 weeks, 20 weeks, and 30 weeks using TaqMan Reverse Transcription Reagents (manufactured by PE Affinity Systems). Next, as a primer for specifically amplifying r164-1b, primers having base sequences represented by SEQ ID NO: 44 and SEQ ID NO: 9, respectively, were used to copy r164-1b gene by PCR. One quantification was performed with the ABI Prism 7900 sequence Detection System. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. PCR was performed using Rat Brain Marathon-Ready cDNA (CLONTECH) and a primer having the nucleotide sequence represented by SEQ ID NO: 44 and SEQ ID NO: 9, respectively, and the obtained r164-1b was obtained. A gene fragment having a partial sequence of the gene was used as the standard. Glycerol triphosphate dehydrogenase calculated using TaqMan Rodent GAPDH Control regents VIC (manufactured by PE Applied Biosystems) in the same manner as the r164-1b gene copy number, using the calculated copy number as the internal control. After correcting for the copy number, the value was compared with the target tissue as the expression level, and the change rate was converted into data.

As a result, the r164-1b gene was expressed 2.2, 2.2, 2.7, and 3.3 times at 1, 8, 20, and 30 weeks after surgery, respectively. Was found to increase. Example 9

 Analysis of tissue distribution of r1644-1b gene in normal rats

 Using a primer having a nucleotide sequence represented by SEQ ID NO: 44 or SEQ ID NO: 9 as a primer for specifically amplifying r164-lb, brain, heart, kidney, spleen, thymus, liver PCR was performed on Marathon-Ready cDNA library (CL0NTECH) derived from stomach, small intestine, muscle, lung, testis, and skin organs to determine the copy number of r164-1b gene by ABI. Performed by Prism 7900 sequence Detection System. The reaction was performed using SYBR Green PCR Master Mix (manufactured by PE Applied Biosystems), and all the methods were performed according to the attached instructions. The method for preparing the standard for quantification is described below. PCR was performed using Rat Brain Marathon-Ready cDNA (CL0NTECH) and primers having the nucleotide sequences represented by SEQ ID NO: 44 and SEQ ID NO: 9, respectively, and the r164-1b gene obtained was obtained. The gene fragment having the partial sequence of was used as the standard. Glycerol triphosphate calculated using TaqMan Rodent GAPDH Control regents VIC (manufactured by PE Applied Biosystems) in the same manner as the r164-1—b gene copy number, using the calculated copy number as an internal control. After correcting for the copy number of acid dehydratase, the value was converted to data as the expression level.

 As a result, the expression of the r164-1b gene was maximal in the testis at 0.0039, and was expressed at 0.0005 in the brain. In the heart, the expression was 0.00002, and in other organs, the expression of the r164_1b gene was less than 0.0001. The testis and brain were found to be the major sites of expression of the r164-lb gene. Industrial applicability

The protein and polynucleotide of the present invention may be used for heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disease (Eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.), useful as a diagnostic marker, etc., and a compound or a salt thereof obtained by screening using the protein, polynucleotide or an antibody against the protein; Neutralizing antibodies that inhibit the activity of proteins Heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina), central nervous system disease (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital disease (eg, testicular hypersensitivity, ovary Dysfunction, infertility, etc.). It can be used as a therapeutic or contraceptive.

 The antisense nucleotide of the present invention can suppress the expression of the protein of the present invention. Examples thereof include heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), central nervous diseases ( It can be used as a prophylactic or therapeutic agent for, eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc., genital diseases (eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.), or as a contraceptive. Furthermore, the polynucleotide of the present invention can be used for, for example, heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina), central nervous system diseases (eg, Alzheimer's disease, Parkinson's syndrome, schizophrenia, etc.), genital diseases (eg, Eg, testicular hypersensitivity, ovarian insufficiency, infertility, etc.).

Claims

The scope of the claims 1. A protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 13 or SEQ ID NO: 30, or a salt thereof.  2. A protein comprising an amino acid sequence represented by SEQ ID NO: 25 or a salt thereof. 3. A protein comprising an amino acid sequence represented by SEQ ID NO: 28 or a salt thereof. 4. A protein comprising an amino acid sequence represented by SEQ ID NO: 13 or a salt thereof. 5. A protein consisting of the amino acid sequence represented by SEQ ID NO: 30 or a salt thereof. 6. A partial peptide of the protein according to claim 1, or a salt thereof.  7. A polynucleotide comprising a polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 6.  8. The polynucleotide according to claim 7, which is DNA.  9. The polynucleotide according to claim 8, comprising the nucleotide sequence represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 27 or SEQ ID NO: 31.  10. A recombinant vector containing the polynucleotide according to claim 7.  11. A transformant transformed with the recombinant vector according to claim 10.  12. The method according to claim 1, wherein the transformant according to claim 11 is cultured to produce and accumulate the protein according to claim 1 or the partial peptide according to claim 6, and the collected protein is collected. A method for producing the protein of claim 1 or the partial peptide of claim 6 or a salt thereof.  13. A pharmaceutical comprising the protein according to claim 1 or a partial peptide thereof or a salt thereof.  1 4. A pharmaceutical comprising the polynucleotide according to claim 7. 15. A diagnostic agent comprising the polynucleotide according to claim 7.  16. An antibody against the protein according to claim 1, the partial peptide according to claim 6, or a salt thereof.  17. A diagnostic agent comprising the antibody according to claim 16. 18. A pharmaceutical comprising the antibody according to claim 16. 19. An antisense nucleotide comprising a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the polynucleotide according to claim 7, or a part thereof. 20. A medicament comprising the antisense nucleotide according to claim 19. 21. The activity of the protein according to claim 1, the partial peptide according to claim 6, or a salt thereof, wherein the protein according to claim 1 or the partial peptide according to claim 6 or a salt thereof is used. A method for screening a compound or a salt thereof.  22. The activity of the protein of claim 1 or the partial peptide of claim 6, or a salt thereof, comprising the protein of claim 1 or the partial peptide of claim 6 or a salt thereof. For screening of a compound or a salt thereof that regulates the expression.  23. The protein or claim of claim 1, which can be obtained by using the screening method of claim 21 or the screening kit of claim 22. A compound or a salt thereof which regulates the activity of the partial peptide or a salt thereof according to 6. 24. A medicament comprising the compound according to claim 23 or a salt thereof.  25. A method for screening a compound or a salt thereof that regulates the expression of the protein gene according to claim 1, wherein the polynucleotide according to claim 7 is used.  26. A screening kit for a compound or a salt thereof that regulates the expression of the protein gene according to claim 1, which comprises the polynucleotide according to claim 7.  27. A compound or its salt that regulates the expression of the protein gene according to claim 1, which can be obtained by using the screening method according to claim 25 or the screening kit according to claim 26. 28. A medicament comprising the compound according to claim 27 or a salt thereof.  29. The method for quantifying a protein according to claim 1, wherein the antibody according to claim 16 is used. 30. The method for diagnosing a disease associated with protein function according to claim 1, wherein the quantification method according to claim 29 is used. 31. A method for screening a compound or a salt thereof that regulates the expression of the protein according to claim 1, characterized by using the antibody according to claim 16. 32. A screening kit for a compound or a salt thereof that regulates the expression of the protein according to claim 1, comprising the antibody according to claim 16. 33. A compound or a salt thereof, which regulates the expression of the protein according to claim 1, which is obtained by using the screening method according to claim 31 or the screening kit according to claim 32.  34. A medicament comprising the compound according to claim 33 or a salt thereof. 35. The medicament according to claim 13,14,18,20,24,28 or 34, which is an agent for preventing or treating heart disease.  36. The medicament according to claim 24, 28 or 34, wherein the compound is a compound that suppresses excessive compensatory mechanism or compensatory failure.  37. The medicament according to claim 35 or claim 36, which is an agent for preventing or treating cardiomyopathy, myocardial infarction, heart failure or angina. 38. The diagnostic agent according to claim 15 or 17, which is a diagnostic agent for heart disease.  39. A method for preventing and treating heart disease, which comprises administering to a mammal an effective amount of the compound according to claim 23, claim 27 or claim 33 or a salt thereof. 40. Use of the compound according to claim 23, claim 27 or claim 33 or a salt thereof for producing an agent for preventing or treating heart disease. 41. A compound that regulates the activity of the protein according to claim 1 or the partial peptide or the salt thereof according to claim 6, or a salt thereof, wherein the compound regulates the expression of the protein gene according to claim 1. Or a salt thereof, or a method for preventing or treating a heart disease, which comprises administering an effective amount of the compound or a salt thereof that regulates the expression of protein according to claim 1. 42. A compound or a salt thereof for regulating the activity of the protein of claim 1 or the partial peptide of claim 6 or a salt thereof for producing a prophylactic or therapeutic agent for heart disease. Use of a compound or a salt thereof that regulates the expression of the protein gene of claim 1, or a compound or a salt thereof that regulates the expression of the protein according to claim 1.