WO2004005512A1 - 新規タンパク質およびその用途 - Google Patents
新規タンパク質およびその用途 Download PDFInfo
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- WO2004005512A1 WO2004005512A1 PCT/JP2003/008666 JP0308666W WO2004005512A1 WO 2004005512 A1 WO2004005512 A1 WO 2004005512A1 JP 0308666 W JP0308666 W JP 0308666W WO 2004005512 A1 WO2004005512 A1 WO 2004005512A1
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- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to genes related to diseases such as heart diseases and uses thereof. More specifically, a method for screening a drug (eg, a prophylactic or therapeutic agent for heart disease, etc.) using the disease-related gene or gene product, an antisense polypeptide against a disease-related gene useful as a diagnostic marker for heart disease A nucleotide; an antibody against the disease-related gene product; Background art
- Heart failure is considered to be inadequate contraction of the heart muscle.
- the mechanism of the onset may be as follows. Myocardial damage, mechanical and functional abnormalities of the heart pump, pressure overload due to high blood pressure and pulmonary hypertension, volume overload such as anemia and acute nephritis can cause the heart to pump out blood volume according to the demands of the organism A state of disappearance occurs.
- compensatory mechanisms such as the sympathetic nervous system and the nervous system endocrine system operate to maintain the homeostasis of the living body.
- the compensatory mechanisms of heart failure include: 1) the above-mentioned load increases, the heart's contractile force increases, and the heart expands as the length of the sarcomer increases.2) The contraction unit of the myocardium increases, and as a result, Hypertrophy occurs. 3) Neurohumoral factors are activated to compensate for the inability to excrete the necessary blood throughout the body, and myocardial fibrosis progresses locally. Basically, the mechanism is to adapt to a given load.However, insufficient operation may promote heart failure, while excessive operation may lead to myocardial injury and exacerbate heart failure In some cases. As a result of activation of the compensatory mechanism, myocardial cells are enlarged and cardiac hypertrophy occurs.
- ischemia occurs when a sufficient amount of blood is not supplied to the enlarged cardiomyocytes, which results in myocardial damage such as myocardial contraction dysfunction, resulting in decreased cardiac output, organ circulatory disorders, venous congestion, Heart failure symptoms with fluid retention A group will come.
- Treatment for this requires improvement of myocardial cell damage, enhancement of cardioprotective effects, recovery of cardiac dysfunction due to myocardial systolic insufficiency, and suppression of the decompensation of the living body, which is the cause, or improvement of excessive compensation mechanisms. Become.
- inotropic drugs such as 1) cardiac glycosides such as digoxin, 2) sympathomimetics such as dobutamine, 3) phosphodiesterase inhibitors such as amrinone, and vascular Hydralazine, calcium antagonists, angiotensin I converting enzyme inhibitors, angiotensin II receptor antagonists, and the like have been used as extenders.
- cardiac glycosides such as digoxin
- sympathomimetics such as dobutamine
- phosphodiesterase inhibitors such as amrinone
- vascular Hydralazine calcium antagonists
- angiotensin I converting enzyme inhibitors angiotensin II receptor antagonists, and the like
- zero blockers and the like are used.
- the present inventors have conducted intensive studies to solve the above problems, and as a result, obtained a novel gene whose expression fluctuates at the onset of heart failure in a myocardial infarction model rat due to coronary artery ligation, and further studied. As a result, the present invention has been completed. That is, the present invention
- an antisense polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to DNA encoding the protein according to (1) or the partial peptide according to (5) or a part thereof,
- kits for screening a compound or a salt thereof that regulates the activity of the protein or its partial peptide or a salt thereof characterized by comprising:
- SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36 which can be obtained using the screening method described in (20) or the screening kit described in (21).
- SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36 which can be obtained using the screening method described in (24) or the screening kit described in (25).
- nucleotide sequence represented by SEQ ID NO: 17 or SEQ ID NO: 18 which can be obtained by using the screening method described in (36) or the screening kit described in (38).
- (40) a medicament comprising the compound according to (39) or a salt thereof, (41) a medicament according to (40), which is a prophylactic or therapeutic agent for heart disease,
- a heart disease characterized by administering to a mammal an effective amount of the compound or the salt thereof according to (22), (23), (26), (27) or (39).
- the present invention provides (44) DNA encoding the protein of (1) above or a mutant DN thereof
- a non-human DNA transgenic animal having A having A,
- test compound is administered to the non-human mammal according to (54), wherein the rodent is a mouse; and (56) the animal according to (53), wherein expression of the reporter gene is detected.
- a protein or partial peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 34 or SEQ ID NO: 36, or a salt thereof (protein B of the present invention) ) Is produced by culturing cells having the ability to produce the protein B of the present invention in the presence of the test compound, and (ii) culturing cells having the ability to produce the protein B of the present invention in the presence of the test compound.
- a method of screening for a compound or a salt thereof that promotes or inhibits the activity of protein B of the present invention which comprises measuring and comparing each of them.
- (61) (i) when cells having the ability to produce the protein A of the present invention were cultured, and (ii) when cells having the ability to produce the protein A of the present invention were cultured in the presence of the test compound.
- the protein A of the present invention is characterized in that the expression level of the gene of the protein A of the present invention when cells having the ability to produce Inhibition of evening protein A gene expression A method for screening a compound or a salt thereof,
- the present invention is characterized in that the expression level of the gene of the protein B of the present invention is measured and compared when cells having the ability to produce B are cultured under hypoxic conditions with extension stimulation. Screening method for a compound or a salt thereof that promotes or inhibits the expression of protein B gene
- the cells into which the DNA encoding the protein A of the present invention and the DNA encoding the protein that interacts with the protein A of the present invention have been introduced are subjected to extension stimulation under low oxygen conditions.
- the cells into which the DNA encoding the protein A of the present invention and the DNA encoding the protein interacting with the protein A of the present invention have been introduced are subjected to hypoxic conditions.
- the expression of the protein A gene of the present invention which is characterized by measuring and comparing the expression levels of the protein A gene of the present invention when cultured with extension stimulation under A method for screening a compound to be inhibited or a salt thereof,
- the cells into which the DNA encoding the protein B of the present invention and the DNA encoding the protein interacting with the protein B of the present invention have been introduced are subjected to extension stimulation under low oxygen conditions. And (ii) in the presence of the test compound, the cells into which the DNA encoding the protein B of the present invention and the DNA encoding the protein interacting with the protein B of the present invention have been introduced, and the cells are introduced under hypoxic conditions.
- a compound that promotes or inhibits the expression of the protein B gene of the present invention characterized by measuring and comparing the expression levels of the protein B gene of the present invention when cultured with a stretching stimulus in Or a method of screening for a salt thereof,
- a method for screening a compound or a salt thereof that inhibits the expression of the protein A gene of the present invention
- a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36 (hereinafter referred to as the protein of the present invention) May be) human or other Cells of warm-blooded animals (eg, guinea pigs, rats, mice, chicks, egrets, pigs, hidges, magpies, monkeys, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells, glands / 3 cells, Bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B Cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocyte
- amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 33 about 40% or more, preferably about 50% or more, preferably Amino acid sequences having a homology of about 60% or more, more preferably about 70% or more, more preferably about 80% or more, and particularly preferably about 90% or more.
- Examples of a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 33 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 33 However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 33 is preferred.
- the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 34 includes about 70% or more, preferably about 8% or more of the amino acid sequence represented by SEQ ID NO: 34.
- amino acid sequence having 0% or more, more preferably about 90% or more, and more preferably about 95% or more homology is exemplified.
- amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 34 For example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 34, and a protein having the amino acid sequence represented by SEQ ID NO: 34 substantially Proteins having the same activity are preferred.
- the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 35 includes about 60% or more, preferably about 70% or more, preferably Amino acid sequences having about 80% or more, more preferably about 90% or more, more preferably about 95% or more homology, and the like.
- Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 35 include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 35 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 35 is preferable.
- As the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 36 about 60% or more, preferably about 70% or more, preferably Amino acid sequences having about 80% or more, more preferably about 90% or more, more preferably about 95% 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: 36 include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 36 However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 36 is preferred.
- Amino acid sequence homology is calculated using the homology calculation algorithm NCBI BLAST (National
- 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 activation also includes cytoprotection. 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.
- 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).
- the enhancement of renin-angiotensin system (RAS) such as angiotensin I converting enzyme (ACE) can be measured using a commercially available measurement kit (eg, manufactured by Peninsula I, Phoenix, etc.).
- Cardiac function is measured using the index of increasing activity of catecholamine in blood (Tosoichi Co., Ltd., fully automatic catecholamine analyzer).
- cardiomyocyte function 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 (eg, ANP (atrial sodium Diuretic peptide), BNP (brain natriuretic peptide), etc.], or measure the production of heart failure marker gene product.
- a heart failure marker gene eg, ANP (atrial sodium Diuretic peptide), BNP (brain natriuretic peptide), etc.
- the respiratory activity of a cell can be measured according to a known method or a method analogous thereto.
- MTT (4,5-Dimethyl-2-thiazolyl) -2,5-di heny 1 -2H-tetrazolium
- trypan blue staining method TUN NEL staining method (Terminal deoxytransferase-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 cardiac myocyte function are equivalent (e.g., about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.1 to 10 times. (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: 33 (eg, about 1 to 100, preferably 1 to 50) Degree, preferably about 1 to 30, more preferably 1 to 10 (Ii) more than one or two (for example, about 1 to 100 amino acids) in the amino acid sequence represented by SEQ ID NO: 33; Preferably about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10, and more preferably about 1 to 5 amino acids; (iii) SEQ ID NO: : 1 or 2 or more in the amino acid sequence represented by 33 (for example, about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, more preferably 1 to 1 An amino acid sequence into which about 0, more preferably 1 to 5 amino acids have been inserted; (iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 33 (eg, 1 to 100 About, preferably about 1 to 50, preferably: about! To about 30, more preferably
- amino acids in the amino acid sequence represented by SEQ ID NO: 34 for example, about 1 to 100, preferably about 1 to 50, and preferably 1 to 30) (Preferably about 1 to 10, more preferably 1 to 5) amino acids, and (ii) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 34.
- amino acids (Iii) 1 or 2 or more amino acid sequences represented by SEQ ID NO: 34 (for example, about 1 to 100, preferably about 1 to 50, preferably 1 to 50) (Iv) about 30 amino acids, more preferably about 1 to 10 amino acids, and still more preferably 1 to 5 amino acids, No .: 1 or 2 or more in the amino acid sequence represented by 34 (for example, about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, more preferably 1 to 30) So-called muteins such as proteins containing an amino acid sequence in which about 10 (more preferably 1 to 5) amino acids have been substituted with other amino acids, or (V) an amino acid sequence combining them;
- amino acids in the amino acid sequence represented by SEQ ID NO: 35 eg, For example, about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10, and more preferably about 1 to 5 amino acids are deleted.
- amino acid sequences represented by SEQ ID NO: 35 for example, about 1 to 100, preferably about 1 to 50, and preferably 1 to 30) (Preferably about 1 to 10, more preferably 1 to 5) amino acid sequence; (iii) 1 or 2 amino acid sequences in the amino acid sequence represented by SEQ ID NO: 35
- the above amino acids for example, about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10, and more preferably about 1 to 5)
- amino acids for example, about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10, and more preferably about 1 to 5
- amino acids for example, 1 to 100
- muteins such as proteins containing a sequence or (V) an amino acid sequence combining them;
- amino acid sequence represented by SEQ ID NO: 36 for example, about 1 to 100, preferably about 1 to 50, and preferably 1 to 30
- Degree more preferably about 1 to 10 amino acids, and more preferably 1 to 5 amino acids
- 1 or 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 36 for example, about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, more preferably about 1 to 10, and more preferably about 1 to 5 amino acids.
- (Iii) 1 or 2 or more amino acid sequences represented by SEQ ID NO: 36 (for example, about 1 to 100, preferably about 1 to 50, and preferably 1 to 3) An amino acid sequence having about 0, more preferably about 1 to 10 and more preferably 1 to 5) amino acids, and (iv) a sequence.
- No .: 1 or 2 or more in the amino acid sequence represented by 36 (for example, about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, more preferably 1 to 30)
- (About 10 amino acids, more preferably 1 to 5 amino acids) are substituted with other amino acids, or
- the position of the insertion, deletion or substitution is not particularly limited.
- the protein has a N-terminus (amino terminus) at the left end and a C-terminus (carboxyl terminus) at the right end according to the convention of peptide labeling.
- Proteins of the present invention C-terminal, carboxyl group (-C00H), Karupokishireto (- C00-), amide (-C0NH 2) or ester - may be either (C00R).
- R in the ester e.g., methyl, Echiru, n- propyl, isopropyl, C, such as n- butyl, _ 6 alkyl groups, for example, C 3-8 cycloalkyl groups such as cyclohexyl cyclopentyl, to cyclo, for example, phenyl, alpha-naphthyl ⁇ / Les of any ⁇ 6 _ 12
- Ariru group e.g., benzyl, Fei one Nafuchiru C Bok 2 alkyl group such as phenylene Lou C doctor 2 ⁇ alkyl group or alpha-naphthylmethyl, such as phenethyl C 7 _ 14
- Ararukiru group such as, such as Viva Roy Ruo carboxymethyl group is used.
- the protein of the present invention When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention includes a carboxyl group amidated or esterified.
- the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
- amino acid residues e.g., Mechionin residues
- N-terminal Amino group protecting groups e.g., C, such as formyl group, Asechiru group, - 6 alkanol C, such as I-le
- a protecting _ like 6 Ashiru group which glutamine residue N-terminal region is cleaved in vivo to form pyroglutamic acid, a substituent on the side chain of an amino acid in the molecule
- a suitable protecting group e.g., formyl groups, C such Asechiru group is protected with a ⁇ like Bok 6 Ashiru group
- complex proteins such as so-called glycoproteins to which sugar chains are bound.
- protein of the present invention examples include, for example, a protein containing an amino acid sequence represented by SEQ ID NO: 33, a protein containing an amino acid sequence represented by SEQ ID NO: 34, SEQ ID NO: 3 A protein containing the amino acid sequence represented by 5 And a protein containing the amino acid sequence represented by SEQ ID NO: 36.
- the partial peptide of the protein of the present invention (hereinafter sometimes referred to as the partial peptide of the present invention) is the partial peptide of the protein of the present invention described above, and is preferably the same as the protein of the present invention described above. Any substance may be used as long as it has the following activities.
- the partial peptide of the present invention lacks one or more (preferably about 1 to 10, and more preferably a number (1 to 5)) of amino acids in its amino acid sequence, or One or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably a number (1 to 5)) amino acids are added to the amino acid sequence; or One or two or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are inserted into the amino acid sequence, or the amino acid sequence One or two or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids therein may be substituted with another amino acid.
- examples of the partial peptide of the present invention include: A partial peptide having an amino acid sequence having the Nth amino acid residue, (2) in the amino acid sequence represented by SEQ ID NO: 34, 1st to 20th, 21st to 40th, 41st to 60th, A partial peptide having an amino acid sequence having an amino acid residue at positions 61-80, 245-265, 355-375, or 514-527; (3) in the amino acid sequence represented by SEQ ID NO: 35 A partial peptide having an amino acid sequence having the 1st to 20th, the 112th to 133th or the 270th to 284th amino acid residues from the N-terminus; (4) SEQ ID NO: 36 In the amino acid sequence represented by 36, from the N-terminus, the 1st to 20th, the 21st to 40th, the 41st to 60th, the 61st to 80th, Partial peptides having an amino acid sequence having the 24th to 260th, the 32nd to 37th second or the 51s
- the C-terminus force Rupokishiru group (- C00H), carboxy Shireto (-C00-), or may be filed in any amide (_C0NH 2) or ester (-C00R).
- the partial peptide of the present invention has a N-terminal amino acid residue (eg, a methionine residue) in which the amino group is protected by a protecting group, and a N-terminal side having the same structure as the protein of the present invention described above.
- Glutamine residues generated by cleavage in the body are oxidized by pyroglutamine, substituents on the side chains of amino acids in the molecule are protected by appropriate protecting groups, or so-called glycopeptides with attached sugar chains Complex peptides and the like are also included.
- the partial peptides of the present invention can also be used as antigens for producing antibodies.
- a salt with a physiologically acceptable acid eg, an inorganic acid, an organic acid
- a base eg, an alkali metal salt
- Preferred acid addition salts are: Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.
- inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
- organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
- the protein of the present invention or its partial peptide or a salt thereof can be produced by the above-mentioned method for purifying a protein from human or other warm-blooded animal cells or tissues, or transformed with a DNA encoding the protein. It can be produced by culturing the body. It can also be produced according to the peptide synthesis method described below.
- human or other warm-blooded animal tissues or cells When producing from human or other warm-blooded animal tissues or cells, human or other warm-blooded animal tissues or cells are homogenized and then extracted with an acid or the like. The obtained extract can be purified and isolated by combining chromatography such as reverse phase chromatography and ion exchange chromatography.
- a commercially available resin for protein synthesis can be generally used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin,
- the condensation of the above protected amino acids various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
- the carpoimides DCC, N, ⁇ '-diisopropylcarpo- imide, and N-ethyl-N '-(3-dimethylaminoprolyl) carboimide are used.
- the protected amino acid may be added directly to the resin with a racemization inhibitor additive (eg, HOB t, HOOB t), or may be pre-protected as a symmetric anhydride or HOB t ester or H ⁇ B t ester
- the amino acid can be added to the resin after activation.
- 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.
- acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylvinylidone, and halogenated carbons such as methylene chloride and chloroform.
- Hydrogens alcohols such as trifluoroethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate.
- the reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about -2O: to 50.
- the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
- Examples of the protecting group for the starting amino group include Z, Boc, t_pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, CI—Z, Br—Z, and adaman.
- Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
- the lipoxyl group may be, for example, a linear, branched or cyclic alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl) Alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxy It can be protected by carbonylation, t-butoxycarbonylhydrazide, tritylhydrazide, etc.
- a linear, branched or cyclic alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cycl
- the hydroxyl group of serine can be protected, for example, by esterification or etherification.
- groups appropriately used for the esterification for example, low-grade, such as Asechiru group (C,. 6)
- Arukanoiru group, Aroiru group such Benzoiru group, Benjiruokishika Groups derived from carbonic acid such as a luponyl group and an ethoxycarbonyl group are used.
- Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
- the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz and C 1 2 - B zl, 2_ nitrobenzyl, B r- Z, such as t one-butyl is used.
- protecting group for imidazole of histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
- Examples of the activated carbonyl group of the raw material include, for example, corresponding acid anhydrides, azides, and active esters [alcohols (eg, phenol, 2,4,5-trichlorophenol, 2,4 —Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with H ⁇ Bt).
- active esters eg, phenol, 2,4,5-trichlorophenol, 2,4 —Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with H ⁇ Bt.
- 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 hydrogen stream in the presence of a catalyst such as Pd-black or Pd_carbon, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, or the like.
- the elimination reaction by the above acid treatment is generally carried out at a temperature of about 120: to 40.
- anisol for example, anisol, phenol, thioanisole, methacrylol, paracresol, dimethylsulfide, 1, It is effective to add a force-thione scavenger such as 4-butanedithiol or 1,2-ethanedithiol.
- the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment
- the formyl group used as an indole protecting group of tributophane is 1,2-ethanedithiol, 1,2 4 Desorption by acid treatment in the presence of 1-butanedithiol In addition to protection, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
- 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.
- an amide form of a protein or partial peptide for example, first, after amidating and protecting the ⁇ -hydroxyl group of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side with a desired chain length. After removing the peptide chain, the protein or partial peptide from which only the amino-terminal protecting group of the peptide chain is removed and the protein or partial peptide from which only the C-terminal carboxyl protecting group is 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.
- the crude protein or peptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain the desired protein or peptide amide.
- an ester of a protein or peptide for example, after condensing the ⁇ -carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein is prepared in the same manner as the amide of a protein or peptide. Alternatively, an ester of the peptide can be obtained.
- the partial peptide of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase.
- a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the desired peptide can be produced by condensing a partial peptide or amino acid capable of constituting the partial peptide of the present invention with the remaining portion, and if the product has a protective group, removing the protective group to produce the desired peptide. it can.
- Known condensation methods and elimination of protecting groups include, for example, those described in (i) to (V) below. Law.
- the polynucleotide encoding the protein of the present invention may be any polynucleotide containing a base sequence (DNA or RNA, preferably DNA) encoding the protein of the present invention.
- the polynucleotide is RNA such as DNA or mRNA encoding the protein of the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. If single-stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).
- the method of the present invention can be performed by a known method, for example, the method described in “Experimental Medicine Special Edition,“ New PCR and Its Applications ”15 (7), 1997, or a method analogous thereto.
- MRNA of the protein can be quantified.
- the DNA encoding the protein of the present invention includes the aforementioned protein of the present invention. Any substance may be used as long as it contains a nucleotide sequence encoding a quality. Further, it may be any of genomic DNA, genomic DNA library, cDNA derived from the cells and tissues described above, cDNA library derived from the cells and tissues described above, and synthetic DNA.
- the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
- RT-PCR method Transcriptase Polymerase Chain Reaction
- Examples of the DNA encoding the protein of the present invention include: (1) a DNA containing the nucleotide sequence represented by SEQ ID NO: 17 or the nucleotide sequence represented by SEQ ID NO: 17 which is highly stringent; A DNA containing a base sequence that hybridizes under the conditions, and encoding a protein having substantially the same properties as a protein containing the amino acid sequence represented by SEQ ID NO: 33;
- DNA containing the nucleotide sequence represented by SEQ ID NO: 17 and high stringency examples include, for example, about 40% or more, preferably about 50% or more, preferably about 60% or more, more preferably about 70% or more with the base sequence represented by SEQ ID NO: 17. More preferably, a DNA containing a nucleotide sequence having a homology of about 80% or more, particularly preferably about 90% or more is used.
- Examples of the DNA that can hybridize with the DNA containing the nucleotide sequence represented by SEQ ID NO: 18 under high stringency conditions include, for example, about 70% or more, preferably about 80%, of the nucleotide sequence represented by SEQ ID NO: 18. % Or more, more preferably about 90% or more, more preferably about 95% or more.
- Examples of the DNA that can hybridize with the DNA containing the nucleotide sequence represented by SEQ ID NO: 1 under high stringent conditions include, for example, about 60% or more, and preferably about 60% or more of the nucleotide sequence represented by SEQ ID NO: 1.
- Examples of the DNA that can hybridize with DNA containing the nucleotide sequence represented by SEQ ID NO: 32 under high stringent conditions include, for example, about 60% or more, preferably about 60% or more of the nucleotide sequence represented by SEQ ID NO: 32.
- DNA containing a base sequence having a homology of 70% or more, preferably about 80% or more, more preferably about 90% or more, and more preferably about 95% or more is used.
- Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using a commercially available library, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
- the high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 2 OmM, and the temperature is about 50 to 70, preferably about 60 to 65. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
- DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 33 a DNA containing the base sequence represented by SEQ ID NO: 17 is represented by SEQ ID NO: 34.
- the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 35 is a DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 35.
- A a DNA containing a base sequence represented by SEQ ID NO: 1 is used.
- a base represented by SEQ ID NO: 32 is used as a DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 32 is used. DNA containing a sequence is used.
- the DNA encoding the partial peptide of the present invention may be any DNA as long as it contains a base sequence encoding the partial peptide of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
- the DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention.
- Genomic DNA, genomic DNA library Any of the above-described cell-tissue-derived cDNA, the above-described cell-tissue-derived cDNA library, and synthetic DNA may be used.
- Examples of the DNA encoding the partial peptide of the present invention include, for example, a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1 or SEQ ID NO: 32, or SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1 or SEQ ID NO: 32, containing a nucleotide sequence that hybridizes under high stringency conditions with the nucleotide sequence represented by SEQ ID NO: 32; SEQ ID NO: 33, SEQ ID NO: 34, a DNA containing a part of a DNA encoding a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36, and the like are used.
- the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1 or SEQ ID NO: 32 has the same significance as described above.
- Hybridization methods and high stringency conditions are the same as described above.
- a DNA encoding a protein of the present invention or a partial peptide of the present invention (hereinafter, in the description of cloning and expression of DNAs encoding them, these may be simply abbreviated as the protein of the present invention).
- the DNA of the present invention is amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA of the present invention is incorporated into an appropriate vector.
- the method of hybridization is, for example,
- the DNA base sequence can be converted using PCR or a known kit, for example, Mutan TM -Super Express Km (Takara Shuzo Co., Ltd.), Mutan TM- K (Takara Shuzo Co., Ltd.), etc., using the 0DA-LA PCR method. It can be carried out according to a known method such as the gapped 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 digested with a restriction enzyme or added with a linker if desired.
- the DNA may have ATG as a translation initiation codon on its 5 'end and TAA, TGA or TAG as a translation stop codon on its 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
- the expression vector for the protein of the present invention can be prepared by, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) placing the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by linking.
- the vector examples include a plasmid derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast (eg, pSH19, pSH15), pacteriophages such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, pXTl, pRc / CMV, pRc / RSV, pc DNA IN eo is used.
- E. coli eg, pBR322, pBR325, pUC12, pUC13
- Bacillus subtilis eg, pUB110, pTP5, pC194
- yeast eg, pSH19, pSH15
- pacteriophages such
- the promoter used in the present invention may be any promoter that is suitable for the host used for gene expression.
- SRa promoter when animal cells are used as host, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. Of these, it is preferable to use the CMV (cytomegalovirus) promoter, SRa Promo overnight, and the like.
- the host is a bacterium belonging to the genus Escherichia
- the host is a bacterium belonging to the genus Bacillus, such as trp promoter, lac promoter, recA promoter, ⁇ ⁇ promoter, lpp promoter, T7 promoter, etc.
- the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a polyA addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired. Can be used.
- the selection marker examples include a dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance] and an ampicillin resistance gene (hereinafter abbreviated as Amp) ), Neomycin resistance Gene (hereinafter sometimes abbreviated as Ne o r, G418 resistance).
- dh fr dihydrofolate reductase
- Amp ampicillin resistance gene
- Ne o r, G418 resistance Neomycin resistance Gene
- a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention.
- the host is a bacterium belonging to the genus Escherichia
- the PhoA ′ signal sequence, the OmpA signal sequence, etc. is used.
- insulin 'signal sequence, ⁇ -interferon signal sequence, antibody molecule, signal sequence, etc. can be used, respectively. .
- a transformant can be produced.
- Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
- Escherichia include, for example, Escherichia coli.
- Bacillus subtilis MI114 Gene, 24, 255 (1983)
- 207-21 Journal of Biochemistry, 95, 87 (1984)] and the like are used. .
- yeast examples include Saccharomyces cerevisiae AH 22, AH22R—, ⁇ 87-11A, DKD—5D, 20 ⁇ 12, Schizosaccharomyces pombe NC YC 1913, NCYC 2036, Pichia pastoris K M71 or the like is used.
- insect cells for example, when the virus is Ac NPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, or egg derived from Trichoplusia ni egg High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
- SmNPV a silkworm-derived cell line (Bombyx mori cell; BmN cell) or the like is used.
- Sf cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, J.L., et al., In Vivo, 13, 213-217, (1977)) and the like are used.
- insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
- animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient chinini—zhamster cell CHO (hereinafter, CHO (dh fr ”) cell Abbreviations), mouse L cells, mouse At T_20, mouse myeloma cells, rat GH3, human FL cells, H9c2 cells, etc. are used.
- Transformation of the genus Escherichia can be performed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene., 17, 107 (1982). it can.
- Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
- Transformation of yeast is performed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). Can be.
- Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
- Transformation of animal cells can be performed, for example, by the method described in Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). It can be done according to the law.
- a liquid medium is suitable as a medium for culturing, and a carbon source necessary for growth of the transformant is contained therein.
- Nitrogen sources inorganic substances and others.
- carbon sources include glucose, dextrin, soluble starch, and sucrose.
- nitrogen sources include ammonium salts, nitrates, corn chipperica, peptone, power zein, meat extract, soybean meal, and potato extract.
- the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
- yeast extract, vitamins, growth promoting factors and the like may be added.
- the pH of the medium is preferably about 5-8.
- an M9 medium containing glucose and casamino acids As a medium for culturing a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Mi Her, Journal of Experiments in Molecular
- cultivation is usually performed at about 15 to 43 for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
- the cultivation is usually carried out at about 30 to 40 "for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
- a medium for example, Burkholder's minimal medium [Bostian, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980) )] And SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)].
- the pH of the medium is preferably adjusted to about 5-8. Culture is usually performed at about 20 to 35 t for about 24 to 72 hours, and aeration and agitation are added as necessary.
- the medium When culturing a transformant in which the host is an animal cell, the medium may be, for example, a MEM medium containing about 5 to 20% fetal bovine serum [3 616, 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
- the pH is about 6-8.
- the cultivation is usually performed at about 30 to 40 for about 15 to 60 hours, and if necessary, aeration and stirring are added.
- the protein of the present invention can be produced in the cytoplasm or extracellularly of the transformant.
- the protein of the present invention can be separated and purified from the above culture by, for example, the following method.
- the cells or cells are collected by a known method after culturing, 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 denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-1100 TM .
- the protein contained in the culture supernatant or extract obtained in this manner can be purified by appropriately combining known separation and purification methods.
- These known separation and purification methods include methods utilizing solubility such as salting-out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight.
- Method utilizing the difference in ion exchange chromatography -A method that uses a difference in charge such as, a method that uses a specific affinity such as affinity chromatography, a method that uses a difference in hydrophobicity such as reverse-phase high-performance liquid chromatography, and an isoelectric point.
- a method utilizing the difference between isoelectric points such as electrophoresis is used.
- the protein thus obtained when obtained in 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 in a salt, a known method or It can be converted to a free form or another salt by an analogous method.
- 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.
- an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
- the presence of the protein of the present invention thus produced can be measured by enzyme immunoassay western blotting using a specific antibody.
- the antibody against the protein or partial peptide or a salt thereof used in the present invention may be a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. .
- Antibodies against the protein or partial peptide of the present invention or a salt thereof can be obtained by using the protein of the present invention as an antigen.
- the antibody or antiserum can be produced according to the following method.
- the protein of the present invention is administered to a warm-blooded animal at a site capable of producing an antibody upon administration by itself or together with a carrier or diluent.
- Antibody production ability upon administration Complete Freund's adjuvant or incomplete Freund's adjuvant may be given to increase the level.
- Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
- the warm-blooded animals used include, for example, monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
- 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 included in them.
- 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 included in them.
- a monoclonal antibody-producing hybridoma can be prepared.
- the antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
- the fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)].
- the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
- PEG polyethylene glycol
- the myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and 'AP-1, but P3U1 is preferably used.
- the preferred ratio between the number of antibody-producing cells (spleen cells) 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 adding the mixture and incubating at 20 to 40 ° C., preferably 30 to 37, for 1 to 10 minutes.
- Various methods can be used to screen monoclonal antibody-producing hybridomas.
- 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.
- Anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mouse
- protein A protein A
- Detection method Add the hybridoma culture supernatant to a solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, and release A method of adding a protein labeled with a radioactive substance, an enzyme, or the like, and detecting a monoclonal antibody bound to a solid phase can be used.
- the selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
- HAT hyperxanthine, aminopterin, thymidine
- any medium can be used as long as it can grow a hybridoma.
- RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, and GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd. )
- serum-free medium for hybridoma culture SFM-101, Nissui Pharmaceutical Co., Ltd.
- the culturing temperature is usually 20 to 40: preferably about 37.
- the culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
- the culture can be usually performed under 5% carbon dioxide gas.
- the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
- Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)).
- immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
- Adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain antibody) Can be done.
- the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (antigen such as the protein of the present invention) and a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody.
- the antibody can be produced by collecting an antibody-containing substance against a protein and separating and purifying the antibody.
- the types of carrier-protein and the mixture of carrier-hapten may be any ratio as long as the antibody can be efficiently cross-linked to the hapten immunized by cross-linking with the carrier.
- ⁇ serum albumin ⁇ ⁇ thyroglobulin , Hemocyanin, etc. in a weight ratio of about 0.1 to 20 and preferably about 1 to 5 with respect to 1 hapten.
- various condensing agents can be used for force coupling between the hapten and the carrier.
- daltaraldehyde carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
- the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
- Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration.
- the dose is usually given about every 2 to 6 weeks, about 3 to 10 times in total.
- the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood, of the warm-blooded animal immunized by the above method.
- the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above.
- the separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.
- the antisense nucleotide containing a nucleotide sequence complementary to or a part thereof includes a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA of the present invention or a part thereof. Any antisense nucleotide may be used as long as it has an effect of suppressing the expression of DNA, but antisense DNA is preferable.
- the nucleotide sequence substantially complementary to the DNA of the present invention refers to, for example, 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). Approximately 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more nucleotide sequences having homology. It is. In particular, of the entire base sequence of the complementary strand of the DNA of the present invention, the complementary strand of the base sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the base sequence near the start codon) is about 0.70. Antisense nucleotides having homology of at least about 80%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% are suitable.
- nucleotide sequence of the DNA containing the nucleotide sequence represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1, or SEQ ID NO: 32 is complementary to or substantially complementary to the nucleotide sequence of the DNA containing the nucleotide sequence represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1, or SEQ ID NO: 32.
- the nucleotide sequence represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1 or SEQ ID NO: 32 is preferably a nucleotide sequence complementary to, or an antisense nucleotide containing a part thereof.
- a base sequence complementary to the base sequence of DNA containing, or an antisense nucleotide containing a part thereof is complementary to or substantially complementary to the nucleotide sequence of the DNA containing the nucleotide sequence represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1, or SEQ ID NO: 32.
- it contains a base sequence complementary to the base sequence of DNA having the base sequence represented by SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1 or SEQ ID NO: 32, or a part thereof.
- Antisense nucleotides and the like are particularly preferred.
- An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
- the phosphate residues (phosphates) of each nucleotide constituting the antisense polynucleotide are, for example, phosphorothioate, methylphosphonate, phosphorodithionate, etc. May be substituted with a chemically modified phosphate residue.
- These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
- an antisense polynucleotide capable of inhibiting the replication or expression of the protein gene of the present invention is cloned or the nucleotide sequence of a DNA encoding a protein whose amino acid sequence has been determined. Can be designed and synthesized based on information.
- a polynucleotide can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or bind to the protein-related RNA of the present invention.
- the expression of the protein gene of the present invention can be regulated and controlled through the interaction.
- Polynucleotides that are complementary to the selected sequence of the protein-related RNA of the present invention, and that can specifically hybridize with the protein-related RNA of the present invention, include the protein of the present invention in vivo and in vitro. It is useful for regulating and controlling gene expression, and is also useful for treating or diagnosing diseases.
- the term "corresponding" means having homology or being complementary to a particular 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. I have.
- 5'-end to 5'-end loop of protein gene 5'-end 6—base pair repeat, 5'-end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3'-end untranslated region
- 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 the polynucleotide complementary to at least a part of the target region, and the relationship between the target nucleic acid and the polynucleotide capable of hybridizing with the target can be said to be “antisense”.
- the antisense polynucleotide may be a polynucleotide containing 2-deoxy D-report, a polynucleotide containing D-report, another type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that such polymers are found in DNA or RNA) Base pairing and nucleotides having a configuration that allows base attachment).
- RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified oligonucleotides).
- Anoma first die nucleic acid may be used.
- “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.
- the modified nucleotide may have a sugar moiety modified, for example, one or more hydroxyl groups may be replaced with a halogen, an aliphatic group, or the like, or may be converted to a functional group such as an ether or an amine. .
- the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA).
- modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides, which are resistant to degradation of oligonucleoside amides.
- the antisense nucleic acid of the present invention can be preferably designed according to the following policy.
- the antisense nucleic acid is more stable in the cell, the cell permeability of the antisense nucleic acid is greater, the affinity for the target sense strand is greater, and if toxic, the Minimize the toxicity of sense nucleic acids.
- 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.
- additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes or increase nucleic acid uptake.
- polycations such as polylysine, which act to neutralize the charge on the phosphate backbone
- lipids which enhance interaction with cell membranes or increase nucleic acid uptake.
- hydrophobic substances such as phospholipid and cholesterol
- Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
- nucleic acids 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 that prevent degradation by nucleases such as exonucleases and RNases. . Examples of such a capping group include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene glycol.
- the inhibitory activity of the antisense polynucleotide can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention.
- the nucleic acid can be applied to cells by various known methods.
- a protein or partial peptide of the present invention or a salt thereof hereinafter, sometimes abbreviated as the protein of the present invention
- a DNA encoding the protein or partial peptide of the present invention hereinafter, a DNA of the present invention
- Antibodies against the protein or partial peptide of the present invention or a salt thereof hereinafter may be abbreviated as the antibody of the present invention
- antisense polynucleotides of the DNA of the present invention The use of the peptide (hereinafter sometimes abbreviated as the antisense polynucleotide of the present invention) will be described.
- Protein A of the present invention a protein or a partial peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 33 or SEQ ID NO: 35, or a salt thereof is designated as Protein A of the present invention.
- a protein or partial peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 34 or SEQ ID NO: 36, or a salt thereof may be abbreviated as protein B of the present 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 period and heart failure decompensation period) after myocardial infarction. That is, it is useful as a marker for early diagnosis of heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), determination of symptom severity, and prediction of disease progression.
- heart disease eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.
- 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
- a medicament containing an antibody against the protein of the present invention may be used for preventing diseases such as heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.). It can be used as a therapeutic agent and the like.
- the expression of the protein A of the present invention increases as the cardiac function decreases after myocardial infarction (heart failure decompensation phase heart failure decompensation).
- myocardial infarction heart failure decompensation phase heart failure decompensation
- the protein A gene sense strand of the present invention into animals, the rate of change in heart rate and left ventricular pressure is reduced. Therefore, the overexpression of the protein A gene in the stage of heart failure, and the accompanying enhancement of the function, leads to a decrease in cardiac function.
- Prognosis of diseases characterized by decline eg, heart failure after myocardial infarction; angina; cardiomyopathy; Can be used as an anti-therapeutic agent.
- a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention can be used as a preventive and therapeutic agent.
- 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.
- a method for screening eg, a compound or a salt thereof that regulates (promotes or inhibits) the expression of the protein of the present invention, which comprises using the antibody of the present invention.
- a method for screening a compound or a salt thereof that inhibits the expression of protein A of the present invention characterized by using (d) the activity of protein B of the present invention characterized by using protein B of the present invention (for example, a screen of a compound or a salt thereof that promotes or inhibits cardiac function promoting or inhibiting activity, cardiac muscle cell function activating or inactivating activity, etc.
- a method for screening a compound or a salt thereof that promotes or inhibits the expression of the protein B gene of the present invention which comprises using a polynucleotide encoding the protein B of the present invention; Promotes or inhibits the expression of protein B of the present invention, characterized by using the antibody of the present invention.
- a method for screening a compound or a salt thereof is provided.
- screening method examples include (i) culturing cells having the ability to produce the protein of the present invention, and ( ⁇ ) culturing cells having the ability to produce the protein of the present invention in the presence of the test compound.
- a screening method for a modulator (promoting or inhibiting) characterized by performing a comparison with the case of culturing may be mentioned.
- 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.
- 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).
- the enhancement of renin-angiotensin system (RAS) such as angiotensin I converting enzyme (ACE) can be measured using a commercially available measurement kit (eg, manufactured by Peninsula I, Phoenix, etc.).
- Cardiac function is measured using the index of increasing activity of catecholamine in blood (Tosoichi Co., Ltd., fully automatic catecholamine analyzer).
- the respiratory activity of the cells is measured, or a heart failure marker gene [eg, ANP (atrial sodium diuretic peptide), BNP (brain natriuretic peptide), etc. ] Or the production of a heart failure marker gene product is measured.
- ANP atrial sodium diuretic peptide
- BNP brain natriuretic peptide
- the respiratory activity of a cell can be measured according to a known method or a method analogous thereto.
- Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
- 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 of a phosphate buffer and a borate buffer having a pH of about 4 to 10 (preferably about 1 to about 6 to 8).
- the host (transformant) transformed with the vector containing the DNA encoding the protein of the present invention described above is used.
- a host for example, animal cells such as H9c2 cells are preferably used.
- a transformant in which the protein of the present invention is expressed in a cell by culturing by the method described above is preferably used.
- screening method include (iii) culturing cells having the ability to produce the protein of the present invention and (iv) cells having the ability to produce the protein of the present invention in the presence of the test compound.
- Expression level of the protein gene of the present invention in the case of culturing specifically, the amount of the protein of the present invention or Measure the amount of mRNA encoded and compare.
- the low oxygen conditions for example, 20% 0 2 or less of oxygen concentration, preferably 2% oxygen concentration (Neichiya, 394, pp. 485-490, 1998) Ru are like conditions.
- 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, pp. 33592-33597, 1996 , Circulation, Vol. 89, pp. 2204-2221, 1994, JB, Vol. 271, pp. 3221-3228, 1996)##
- (iii '') a cell capable of producing the protein of the present invention when cultured under lethal conditions
- the expression level of the protein gene of the present invention is measured and compared. .
- the culturing under the above lethal conditions includes, for example, culturing in the absence of serum or adding an anticancer agent (eg, adoriamycin which is relatively toxic to cardiomyocytes).
- an anticancer agent eg, adoriamycin which is relatively toxic to cardiomyocytes.
- 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, and analyzing the protein present in a cell extract or the like by Western analysis, ELISA, or the like. It can be measured according to a method according to it.
- the expression level of the protein gene of the present invention can be determined by a known method, for example, Northern blotte ink, reverse transcription-polymerase chain react ion (RT-PCR), It can be measured according to a method such as a real-time PCR analysis system (TaqMan polymerase chain reaction, manufactured by ABI) or a method analogous thereto.
- RT-PCR reverse transcription-polymerase chain react ion
- Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound.
- 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).
- a host transformed with a vector containing the DNA encoding the protein of the present invention described above is used.
- a host for example, animal cells such as H9c2 cells are preferably used.
- a transformant in which the protein of the present invention is expressed in a cell by culturing by the method described above is preferably used.
- the expression level of the protein gene of the present invention in the case of the above (iv), () ⁇ ') or (iv' ') is determined by the expression in the case of the above (iii), ( ⁇ ') or (iii '').
- a test compound that promotes about 20% or more, preferably 30% or more, more preferably about 50% or more can be selected as a compound that promotes expression of the protein gene of the present invention or a salt thereof.
- the expression level of the protein gene of the present invention is determined by the above (iii), (iii') or
- test compound that inhibits about 20% or more, preferably 30% or more, more preferably about 50% or more of the case of (iii "), is a compound that inhibits the expression of the protein gene of the present invention or It can be selected as its salt.
- a method for screening a compound or a salt thereof that regulates (promotes or inhibits) expression of the protein of the present invention using the antibody of the present invention includes (V) culturing cells having the ability to produce the protein of the present invention and (vi) culturing cells having the ability to produce the protein of the present invention in the presence of the test compound. Comparison with the case of culturing is performed.
- the expression level of the protein of the present invention (specifically, the protein level of the present invention) in the cases (V) and (vi) is measured using the antibody of the present invention (eg, expression level). Detection, 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, and the like. Or a known compound.
- a host transformed with a vector containing DNA encoding the protein of the present invention described above is used.
- a host for example, animal cells such as H9c2 cells are preferably used.
- a transformant in which the protein of the present invention is expressed in a cell by culturing by the method described above is preferably used.
- a test compound that promotes about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of (V) is selected as a compound or its salt that promotes expression of the protein of the present invention. can do.
- the compound can be selected as a compound that inhibits expression of the protein of the present invention or a salt thereof.
- the present invention relates to a method for measuring the enzymatic activity of a reporter gene in a reporter gene using a promoter of a gene encoding the protein of the present invention.
- the present invention also provides a method for screening a compound or a salt thereof, which regulates the expression of the gene of the protein of the present invention. This utilizes the fact that the enzyme activity of a reporter gene increases depending on the expression level and activation of the protein of the present invention.
- the promoter for example, a nucleotide sequence containing the nucleotide sequence represented by SEQ ID NO: 2 is used.
- the repo overnight gene Atsushi is, for example, a repo overnight gene downstream of the promoter region of the gene encoding the protein of the present invention (eg, ⁇ galactosidase, chloramphenicol acetyltransferase, luciferase ) Is constructed, and the plasmid is introduced into a host cell or the like by a known method.
- Construction of the plasmid, introduction of the plasmid, and the like can be performed by known methods, for example, a recombinant vector containing the DNA encoding the protein of the present invention, and a method for producing a transformant transformed with the recombinant vector.
- the host cell include primary cardiac myocytes, H9c2 cell line (ATCC No. CRL-1446), or primary cardiac myocytes or H9c2 cells into which a gene encoding the protein of the present invention has been introduced. Stocks are used.
- the present invention is characterized in that the enzyme activity of a reporter gene is compared with that in a case where a cell into which a plasmid in which a reporter gene is ligated downstream of a promoter region of a gene is introduced in the presence of a test compound and cultured. And a method for screening a compound or a salt thereof that regulates the expression of a protein gene. The enzyme activity of the reporter gene is measured according to a known method.
- the compound or a salt thereof obtained by the screening method may be a compound of the present invention.
- Test compounds include, for example, peptides, proteins, non-peptidic compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds, microorganism cultures, cell extracts, plant extracts, and animal tissue extracts.
- the compound may be a novel compound or a known compound.
- cells having the ability to produce the protein of the present invention are cultured in a medium suitable for screening.
- the medium may be any medium as long as it does not affect the gene expression of the protein of the present invention.
- the above cells the above host cells and the like are used.
- animal cells such as H9c2 cells (ATCC No. CRL-1446) are preferably used.
- a transformant in which the protein of the present invention is expressed in the cytoplasm by culturing by the method described above is preferably used.
- a method for screening a compound or a salt thereof that regulates (promotes or inhibits) the expression of the gene of the protein of the present invention using the expression of the gene that interacts with the protein of the present invention as an index.
- the enhanced expression of the protein of the present invention directly causes a decrease in cardiac function.
- the action of the protein A of the present invention (cardiac function exacerbating action) is achieved by suppressing the action of factors related to the maintenance of cardiac function homeostasis. Is estimated to occur.
- the factor include a cardiac-specific transcription factor (eg, NkX2.5 (heart-specific transcription factor; Journal of Biological Chemistry, Vol. 273, pp.
- GATA — 4 cell-specific transcription factors; The EMB0 Journal, 19, 2046-2055, 2000
- MLP cell-specific transcription factors
- FHL-2 cell-specific transcription factors
- MNF cell-specific transcription factors
- Sp-1 cell-specific transcription factors
- myocardial protective signals eg, gp130 signal, IGF-1 signal, etc.
- cell adhesion factors eg, Connexin 43 etc.
- a compound or a salt thereof that regulates the expression of the protein gene of the present invention can be obtained by the screening method using the binding activity of the protein of the present invention to a protein that interacts with the protein as an index.
- the screening method include (ix) culturing cells into which the DNA of the present invention and DNA encoding a protein interacting with the protein of the present invention have been introduced, and (X) the DN of the present invention.
- a and the expression level of the gene of the protein of the present invention when cells into which the DNA encoding the protein interacting with the protein of the present invention has been introduced in the presence of a test compound specifically, The amount of protein or the amount of mRNA encoding the protein is measured and compared.
- (ix ′) a case in which cells transfected with DNA of the present invention and a DNA encoding a protein interacting with the protein of the present invention are cultured under hypoxic conditions with extension stimulation
- ( ⁇ ′) test The method according to the present invention, wherein the cells into which the DNA of the present invention and the DNA encoding the protein interacting with the protein of the present invention have been introduced in the presence of a compound and cultured under low oxygen conditions with extension stimulation applied thereto.
- the expression level of the protein gene (specifically, the level of the protein of the present invention or the level of the mRNA encoding the protein) is measured and compared.
- Examples of the protein that interacts with the protein of the present invention include a cardiac-specific transcription factor (eg, Nkx2.5, GATA-4, MLP, MNF, Sp-1 and the like).
- a cardiac-specific transcription factor eg, Nkx2.5, GATA-4, MLP, MNF, Sp-1 and the like.
- the cells, the hypoxia condition, the stretching stimulation, the measurement of the amount of the protein of the present invention or the expression of the protein gene of the present invention, and the test compound are the same as those described in (2) above. Use the same one.
- the expression level of the protein gene of the present invention in the case of the above (X) or ( ⁇ ′) is about 20% or more, preferably 30% or more as compared with the case of the above (ix) or (ix ′). More preferably, a test compound that promotes about 50% or more can be selected as a compound or a salt thereof that promotes the expression of the protein gene of the present invention. In the case of the above (X) or ( ⁇ ′), the expression level of the protein gene of the present invention is about 20% or more, preferably 30% or more, compared with the case of the above (ix) or (ix ′). A test compound that preferably inhibits about 50% or more can be selected as a compound that inhibits the expression of the protein gene of the present invention or a salt thereof. (6) A method for screening a compound or a salt thereof that regulates (promotes or inhibits) the expression of the protein gene of the present invention, using the expression of the gene controlled by the protein of the present invention as an index.
- the binding activities of the “protein of the present invention” and the “protein that interacts with the protein of the present invention” are compared.
- the “protein that interacts with the protein of the present invention” may be new or known.
- the binding activity is measured according to a known method, for example, a yeast or mammalian two-hybrid method, a SPA method, an ELISA method, or the like.
- specific examples include (i ′) a) a plasmid that expresses a fusion protein of the protein of the present invention and the DNA-binding domain of the GAL4 gene, and b) a plasmid of the present invention.
- a plasmid that expresses a fusion protein of the protein that interacts with the protein and the transcription activation domain of the VP16 gene, and c) a reporter gene eg, A method for comparing the case where cells into which a plasmid to which luciferase or the like has been introduced is cultured and the case where the above-mentioned cells ( ⁇ ′) are cultured in the presence of a test compound ( ⁇ ′).
- the expression levels of repo overnight genes in (xi ′) and (xii ′) are measured and compared.
- the cells used here may be any cells, and may be cultured in a medium suitable for screening.
- the protein of the present invention used in the SPA method and the ELISA method is, for example, labeled with a radioisotope such as [
- the activity of the protein of the present invention in the above (xii) or (xii ') is about 20% or more, preferably 30% or more, compared to the above (xi) or ()).
- a test compound that promotes about 50% or more can be selected as a compound that promotes the activity of the protein of the present invention or a salt thereof.
- the activity of the protein of the present invention in the case of the above (xii) or (xii ') is about 20% or more, preferably 30% or more, more preferably as compared with the case of the above (xi) or (xi').
- a compound or a salt thereof which promotes or inhibits the activity of the protein of the present invention preferably the protein ⁇ of the present invention
- a compound which promotes or inhibits the expression of the gene of the protein of the present invention preferably the protein ⁇ of the present invention
- a salt thereof, and promote or inhibit expression of the protein of the present invention preferably, protein ⁇ of the present invention
- the compound or a salt thereof is administered during cardiac incomplete chronic phase (heart failure compensation phase) in which the expression of the protein gene of the present invention is enhanced, thereby suppressing excessive compensation mechanism and protecting cardiomyocytes by protecting cardiomyocytes.
- An effect can be expected.
- the screening kit of the present invention may be a protein or partial peptide or a salt thereof used in the present invention, a polypeptide encoding the protein or partial peptide of the present invention, an antibody of the present invention, or a kit for use in the present invention. And cells having the ability to produce the protein or partial peptide to be obtained.
- Compounds or salts thereof obtained using the screening method or screening kit of the present invention include the test compounds described above, for example, peptides, proteins, non-peptidic compounds of biological origin (eg, carbohydrates, lipids, etc.), synthetic compounds, A compound selected from a microorganism culture, a fermentation product, a cell extract, a plant extract, an animal tissue extract, plasma, or the like, or a salt thereof.
- test compounds described above for example, peptides, proteins, non-peptidic compounds of biological origin (eg, carbohydrates, lipids, etc.), synthetic compounds, A compound selected from a microorganism culture, a fermentation product, a cell extract, a plant extract, an animal tissue extract, plasma, or the like, or a salt thereof.
- salt of the compound those similar to the aforementioned salts of the protein of the present invention are used.
- the compound or its salt 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 action is useful as a medicament for preventing and treating heart diseases (eg, cardiomyopathy, myocardial infarction, cardiac failure, angina, etc.).
- heart diseases eg, cardiomyopathy, myocardial infarction, cardiac failure, angina, etc.
- the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention can be safely administered as it is or as an appropriate drug.
- the medicament used for the above administration comprises 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.
- 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 is commonly used in the field of pharmaceuticals. Containing a carrier, diluent or excipient.
- a carrier diluent or excipient.
- lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
- 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.
- 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 (eg, It may be used in combination with propylene glycol, polyethylene glycol), a nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxythylene (50 mol) adduct of hydrogenated castor oil)], and the like.
- alcohol eg, ethanol
- polyalcohol eg, It may be used in combination with propylene glycol, polyethylene glycol
- a nonionic surfactant eg, polysorbate 80, HCO-50 (polyoxythylene (50 mol) adduct of hydrogenated castor oil)
- oily liquid for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used as a
- compositions are conveniently prepared in dosage unit forms to be compatible with the dosage of the active ingredient.
- dosage unit examples include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg / dosage unit for each dosage unit. It is preferred that the drug contains 5 to 100 mg of the above compound, and other dosage forms contain 10 to 25 mg 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, pigs, puppies, pests, birds, cats, dogs, monkeys). , Chimpanzees, etc.) can be administered orally or parenterally.
- warm-blooded animals eg mice, rats, puppies, sheep, pigs, puppies, pests, birds, cats, dogs, monkeys.
- Chimpanzees, etc. can be administered orally or parenterally.
- the dose of the compound or its salt that regulates the activity of the protein of the present invention depends on its production.
- a compound or a salt thereof that modulates the activity of the protein of the present invention for the purpose of treating heart failure preferably, the activity of the protein A of the present invention
- the compound or a salt thereof may be administered in an amount of about 0.1 to 10 mg / day, preferably about 1.0 mg / day. 5050 mg, more preferably about 1.0-20 mg.
- the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like.
- the activity of the protein of the present invention is regulated.
- a compound or a salt thereof preferably a compound of the present invention which inhibits the activity of protein A or a salt thereof
- the compound or a salt thereof is usually administered daily. It is convenient to administer the salt by intravenous injection in an amount of about 0.01 to 3 Omg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg. In the case of other animals, the dose can be administered in terms of weight per 6 O kg.
- 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. It can be used for quantification by sandwich immunoassay.
- the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter, sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
- the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
- the method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. . For example, nephrometry, a competitive method, an immunometric method and a sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
- 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.
- Radioisotopes if example embodiment, [i25 i], (I3i n, [], as.
- the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
- luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
- a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
- the insolubilization of the antigen or antibody physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used.
- the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
- 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 mass of the protein of the present invention in the test wave 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 a time interval.
- the labeling agent and the method of insolubilization can be in accordance with those described above.
- 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.
- 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.
- 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.
- a competition method for example, a competition method, an immunometric method, or a nephrometry.
- the competitive method after the antigen in the test wave 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 in either B or F, and quantify the amount of antigen in the test wave.
- 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
- An immobilization method using a soluble antibody as the first antibody and using an immobilized antibody as the second antibody is used.
- the antigen in the test wave and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated.
- the raw material is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated.
- label either phase The amount is measured and the amount of antigen in the test wave is determined.
- 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 wave is small and only a small amount of sediment is obtained, laser nephrometry utilizing scattering by a laser is preferably used.
- the protein measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to the ordinary conditions and procedures in each method. For details of these general technical means, reference can be made to reviews, documents, etc.
- the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
- 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.
- detecting the protein of the present invention in each fraction at the time of purification analyzing the behavior of the protein of the present invention in test cells, etc. Can be used.
- the DNA of the present invention can be used, for example, in humans or warm-blooded animals (for example, rats, mice, guinea pigs, egrets, birds, higgies, bush, pears, pomas, cats, dogs, Abnormalities (genetic abnormalities) in DNA or mRNA encoding the protein of the present invention or its partial peptide in monkeys, chimpanzees, etc.), for example, damage or sudden mutation of the DNA or mRNA. Les is useful as a diagnostic agent for genes such as decreased expression, increased DNA or mRNA, or overexpression.
- the above-described genetic diagnosis using the DNA of the present invention includes, for example, the well-known Northern Hybridization and the PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences of the United States of America, +86, 2766-2770 (1989)) Can be implemented.
- overexpression is detected by Northern hybridization or DNA mutation is detected by PCR-SSCP method, for example, heart disease (eg, cardiomyopathy, myocardial infarction, heart failure) , Angina pectoris, etc.).
- heart disease eg, cardiomyopathy, myocardial infarction, heart failure
- Angina pectoris etc.
- the antisense polynucleotide of the present invention capable of binding to the DNA of the present invention complementarily and suppressing the expression of the DNA preferably, the protein A of the present invention is Antisense polynucleotides against the polynucleotides that it exerts
- the function of the protein of the present invention or the DNA of the present invention in vivo eg, cardiac function inhibitory activity, cardiomyocyte function inactivating effect, etc.
- It can be used as a prophylactic / therapeutic agent for, for example, heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.).
- the above-mentioned antisense polynucleotide When used as the above-mentioned prophylactic or therapeutic agent, it can be formulated and administered according to a known method.
- the antisense polynucleotide when used, the antisense polynucleotide is used, the antisense polynucleotide is used alone or after being inserted into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. Or orally to humans or other warm-blooded animals (eg, mice, rats, puppies, higgs, bush, puppies, puppies, birds, cats, dogs, monkeys, chimpanzees, etc.) It can be administered parenterally.
- a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like.
- a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like.
- a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like.
- the antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting ingestion, and can be administered by a gene gun or a catheter such as a gel for Hyde mouth gel.
- a physiologically acceptable carrier such as an adjuvant for promoting ingestion
- the dose of the antisense polynucleotide varies depending on the target disease, the subject of administration, the route of administration, and the like.For example, when the antisense polynucleotide of the present invention is orally administered for the purpose of treating heart failure, it is generally used. In an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.
- the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in a tissue or a cell and the state of expression thereof.
- the present invention further provides
- RNAs, ribozymes and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention in the same manner as the antisense polynucleotide, and Can regulate (inhibit) the activity or function of a polynucleotide (eg, DNA) in a patient (eg, cardiac dysfunction-promoting activity, etc.).
- a patient eg, cardiac dysfunction-promoting activity, etc.
- heart disease eg, cardiomyopathy, myocardial infarction, heart failure, stenosis
- It can be used as a preventive and therapeutic agent for heart disease.
- the double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
- the ribozyme 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, pp. 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.
- a part of the RNA encoding the peptide of the present invention includes a portion (RNA fragment) close to the cleavage site on the RNA of the present invention, which can be cleaved by a known lipozyme.
- RNA or ribozyme When the above-described double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.
- the antibody of the present invention which has the effect of neutralizing the activity of the protein of the present invention (preferably, the protein A of the present invention), is useful for treating heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.) It can be used as a prophylactic 'therapeutic.
- heart diseases eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.
- the agent for preventing or treating the above-mentioned diseases containing the antibody of the present invention has low toxicity, and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or other warm-blooded animals (for example, mice, rats, It can be administered orally or parenterally to egrets, sheep, sheep, bush, horses, horses, birds, cats, dogs, monkeys, chimpanzees, etc.
- the dosage varies depending on the administration target, target disease, symptoms, administration route, and the like. For example, treatment of heart failure in adults.
- the invention of the antibody in a single dose, usually about 0.01 to 2 OmgZkg body weight, preferably 0.1 to about 1 OmgZkg body weight, more preferably 0.:! It is convenient to administer about 5 mgZkg body weight by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.
- the antibody of the present invention can be administered by itself or as a suitable pharmaceutical composition.
- the pharmaceutical composition used for the above administration contains the above antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or vehicle.
- Such compositions are provided in dosage forms suitable for oral or parenteral administration.
- 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 field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and tablets for tablets.
- 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.
- aqueous liquid for injection for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, and the like, are used.
- a suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
- compositions are conveniently prepared in dosage unit forms to be compatible with the dosage of the active ingredient.
- dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially injections, for each dosage unit dosage form.
- the antibody contains 5 to 10 O mg, and other dosage forms contain 10 to 25 O mg of the above antibody.
- compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
- the protein of the present invention or the DNA encoding the protein of the present invention includes heart diseases (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.) and the like. It is also useful as a medicament such as a prophylactic or therapeutic agent. Further, the protein of the present invention is used as a vaccine or the like for producing the antibody of the present invention.
- heart diseases eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.
- the protein of the present invention is used as a vaccine or the like for producing the antibody of the present invention.
- the protein of the present invention When the protein of the present invention is used as the above drug, it can be formulated according to a conventional method.
- the DNA of the present invention When the DNA of the present invention is used as the above-mentioned prophylactic or therapeutic agent, the DNA of the present invention is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. After that, it can be carried out according to conventional means.
- the DNA of the present invention can be administered as it is or together with an adjuvant for promoting uptake, using a gene gun or a catheter such as Hyde-mouth gel power catheter.
- the protein of the present invention or the DNA of the present invention is orally acceptable as a sugar-coated tablet, capsule, elixir, microcapsule or the like, or water or other pharmaceutically acceptable, if necessary.
- Sterile solution with liquid, or suspended It can be used parenterally in the form of injections, such as suspensions.
- the protein of the present invention or the DNA of the present invention is required to perform a generally accepted formulation with known carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, etc., which are physiologically recognized. Can be prepared by mixing them in unit dosage forms. The amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
- the dosage 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, in general, for example, in a heart failure patient (with a body weight of 6 O kg), It is about 0.1-100 mg per day, preferably about 1.0-5 Omg, more preferably about 1.0-2 Omg.
- the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
- it is usually, for example, a heart failure patient (body weight 6 O kg )
- the dose can be administered in terms of weight per 60 kg.
- the dosage of the DNA of the present invention varies depending on the administration subject, the target organ, the condition, the administration method, and the like.
- oral administration in general, for example, in a heart failure patient (as 6 O kg), one day About 0.1 to 10 Omg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
- parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
- it is usually used, for example, in heart failure patients (as 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 per day by intravenous injection.
- the dose can be administered in terms of 6 O kg.
- the present invention relates to a DNA encoding the exogenous protein of the present invention (hereinafter referred to as the present invention).
- a non-human mammal having the foreign DNA) or its mutant DNA (sometimes abbreviated as the foreign mutant DNA of the present invention).
- Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
- a calcium phosphate method, an electric pulse method, a Lipofection 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.
- the exogenous DNA of the present invention intended for somatic cells, organs of living organisms, tissue cells, etc. 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 germ cells by a known cell fusion method to produce the DNA-introduced animal of the present invention.
- mice for example, red sea lions, bushes, hidge, goats, blue egrets, dogs, cats, guinea pigs, eight musters, mice, rats and the like are used.
- mice for example, pure strains such as C57B LZ6 and DBA2
- rat eg, Wistar, SD, etc.
- “Mammals” in a recombinant vector that can be expressed in mammals include humans and the like 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.
- 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 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 which is the same or different from the target animal.
- a mammal which is the same or different from the target animal.
- the human DNA of the present invention when introduced, it is derived from various mammals having the DNA of the present invention, which are highly homologous thereto (eg, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.).
- Downstream of various promoters capable of expressing the DNA of the present invention by microinjecting the DNA construct (eg, vector, etc.) to which the human DNA of the present invention is bound into a fertilized egg of a target mammal, for example, a mouse fertilized egg. It is possible to create a DNA-introduced mammal that highly expresses the DNA of the present invention.
- the DNA construct eg, vector, etc.
- Examples of the expression vector of 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 ⁇ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or a baculovirus.
- animal viruses such as Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
- promoters that regulate the expression of DN ⁇ include, for example, virus
- the vector preferably has a sequence that terminates transcription of a target messenger RN ⁇ in a DNA-transfected mammal (generally referred to as terminator), and is, for example, derived from viruses and various mammals.
- terminator a DNA-transfected mammal
- the sequence of each DNA can be used, and preferably, SV40 terminator of Simian virus or the like is used.
- the normal translation region of the protein of the present invention is DNA derived from the liver, kidney, thyroid cells, fibroblasts derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.).
- the foreign abnormal DNA can produce a translation region obtained by mutating the translation region of a normal polypeptide obtained from the above 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 the translation region is ligated downstream of the promoter and, if desired, upstream of the transcription termination site.
- exogenous DNA of the present invention is provided 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 DNA transfer indicates that the progeny of the transgenic animal retains the exogenous DNA of the present invention in all of its germ cells and somatic cells Means to do.
- the progeny of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.
- the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it.
- exogenous DNA of the present invention is provided 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 DNA transfer indicates that all of the offspring of the produced animal carry the exogenous DNA of the present invention in all of its germinal and somatic cells. It means having extra.
- the progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
- the normal DNA of the present invention is highly expressed, and the function of the protein of the present invention is finally enhanced by promoting the function of endogenous normal DNA.
- the disease may develop and can be used as a model animal for the disease. For example, using the normal DNA-introduced animal of the present invention, elucidation of the pathological mechanism of the protein hyperactivity of the present invention and the disease associated with the protein of the present invention, and examination of a method for treating these diseases. Can be performed.
- 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 is also used for a screening test for a therapeutic drug for a disease associated with the protein of the present invention. It is possible.
- a non-human mammal having the foreign abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the foreign DNA is stably maintained by mating. I can do it. Further, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
- a DNA construct with a promoter can be prepared by ordinary DNA engineering techniques. Introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germinal 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 germinal and somatic cells.
- a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed the cells so that all offspring have the DNA.
- the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately reduced by inhibiting the function of endogenous normal DNA.
- Inactive refractory disease may occur and can be used as a model animal for the disease.
- using the abnormal DNA-introduced animal of the present invention to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to treat this disease A study of the method is possible.
- 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 (dominant negative action) in the function-inactive refractory disease of the protein of the present invention. ) Is a model for elucidating.
- the mammal into which the exogenous abnormal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it is also used in a therapeutic drug screening test for the protein of the present invention or a functionally inactive refractory disease thereof. Available.
- the protein-producing cell of the present invention may be specified, associated with apoptosis, differentiation or proliferation, or They can examine the signal transduction mechanism of them, and investigate their abnormalities, etc., and are useful research materials for elucidating the protein of the present invention and its action.
- a test compound is administered to the animal into which the DNA of the present invention has been introduced, and the heart function, electrocardiogram, heart weight, and the like of the animal are measured.
- Heart weight is the parameter of hypertrophy.
- 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.
- the test compound can be evaluated using the suppression of this increase as an index.
- a myocardial infarction is performed, and the heart function, electrocardiogram, heart weight, and the like of the animal are measured.
- the infarct growth inhibitory activity of the test compound can be examined by weighing the infarct layer. Administration of the test compound may be post-infarct surgery.
- the animal can be bred with a genetically hypertensive model rat such as an SHR rat to create a new heart failure model. The compound is administered to the heart failure model prepared as described above, and the animal is examined for cardiac function, electrocardiogram, heart weight, infarct progression inhibitory activity and the like.
- the present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
- the DNA is a reporter gene (eg, from E. coli) 3-galactosidase
- the reporter gene can be expressed under the control of the promoter for the DNA of the present invention
- the present invention provides a method for screening a compound or a salt thereof which promotes or inhibits (preferably inhibits) the promoter activity of DNA of the present invention.
- 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 the DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter referred to as the knockout DNA of the present invention).
- Non-human mammalian embryonic stem cells hereinafter abbreviated as ES cells).
- 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 and inserting or substituting another DNA by a genetic engineering technique.
- the knockout DNA of the present invention may be prepared by, for example, shifting the codon reading frame or disrupting the function of the promoter or exon by these mutations.
- Non-human mammalian embryonic stem cells of the present invention in which DNA is inactivated include, for example, The DNA of the present invention possessed by a non-human mammal to be isolated is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, lacZ (3-galactosidase gene), cat (clo DNA sequence that disrupts the function of exons by inserting a repo allele, represented by the ramphenicol acetyltransferase gene), or terminates gene transcription in the intron between exons.
- a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, lacZ (3-galactosidase gene), cat (clo DNA sequence that disrupts the function of exons by inserting a repo allele, represented by the ramphenicol acetyltransferase gene), or terminates gene transcription in the intron between exons
- ES cells are subjected to Southern hybridization analysis or targeting vector analysis using the DNA sequence on or near the DNA of the present invention as a probe, for example, by the homologous recombination method.
- the above DNA sequence and the DNA sequence of the neighboring region other than the DNA of the present invention used for the production of the targeting vector were analyzed by PCR using primers as primers, and the knockout ES cells of the present invention were selected. Obtainable.
- 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 described above. 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 immunogenic genetic background is used.
- BDFi mice C57BL / 6 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 C57BLZ6 mice as their background.
- the ES cells obtained by using the method described above can be advantageously used when a pathological model mouse is produced, because the genetic background can be replaced with a C57BLZ6 mouse by backcrossing with a C57BLZ6 mouse.
- blastocysts 3.5 days after fertilization are generally used.However, it is more efficient to collect embryos at the 8-cell stage and culture them until blastocysts are used. Many early embryos can be obtained.
- male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
- An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. With this method, the number of ES cells in a colony was about 1 (about 50), compared to about 10 6 cells for karyotype analysis.
- the primary selection of ES cells in the initial stage can be performed by gender discrimination, and the early selection of male cells can greatly reduce the labor required in the initial culture.
- the secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method.
- the embryonic stem cell line obtained in this way usually has very good growth potential, but it must be carefully subcultured because it tends to lose its ability to generate individuals.
- LIF (1-10000) on a suitable feeder cell such as STO fibroblasts
- ES cells can be transformed into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions.
- MJ Evans and MH MJ Evans and MH
- 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.
- non-human mammal the same one as described above is used.
- the non-human mammal deficient in expression of the DNA of the present invention can be obtained, for example, by introducing the evening-targeting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell.
- a cell in which the DNA of the present invention has been knocked out is a DNA sequence on or near the DNA of the present invention, which is used as a probe for Southern hybridization analysis or the DNA sequence on the evening getter vector, and the evening getter vector. Can be determined by PCR analysis using the DNA derived from the mouse and the DNA sequence of the neighboring region other than the DNA of the present invention as a primer.
- a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human cells.
- the chimeric embryo is injected into a mammalian embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal.
- the produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially altered DNA locus of the present invention.
- 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 an individual composed of the added cells having the DNA locus of the present invention, for example, by judging coat color or the like. in this way
- the individual obtained is a heterozygous expression of the protein of the present invention is deficient in the heterozygous expression of the protein of the present invention.
- An expression-deficient individual can be obtained.
- a transgenic non-human mammal having a chromosome into which a getter vector has been introduced can be obtained by injecting a DNA solution into the egg cell nucleus by a microinjection method. Compared to these transgenic non-human mammals, they can be obtained by selecting those having a mutation at the DNA locus of the present invention by homologous recombination of the gene.
- the germline can be obtained and maintained according to a conventional 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 a state where one normal individual and plural homozygous animals are obtained. By crossing 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 very useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
- 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, diseases 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 studying treatment methods.
- the non-human mammal deficient in expression of the DNA of the present invention is used for screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention.
- the present invention is characterized by administering a test compound to a non-human mammal deficient in DNA expression of the present invention, and observing and measuring a change in the animal.
- a method for screening a compound or a salt thereof having a therapeutic / preventive effect on a disease caused by the disease is provided.
- the non-human mammal deficient in DNA expression of the present invention used in the screening method includes the same ones as described above.
- Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound.
- 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 each organ, tissue, disease symptoms, etc. of the animal are used as indicators. Therapeutic and prophylactic effects of test compounds can be tested.
- 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.
- the test compound when screening for a compound having a prophylactic or therapeutic effect on a heart disease (eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.), the test compound is used in a non-human mammal deficient in DNA expression of the present invention. And the heart function, electrocardiogram, heart weight, etc. 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, test compounds can be evaluated using the suppression of this increase as an index.
- a heart disease eg, cardiomyopathy, myocardial infarction, heart failure, angina pectoris, etc.
- Heart weight is the parameter of hypertrophy.
- the heart structure can be examined by calculating the heart weight per body weight, the left ventricular weight per body weight, and the left
- a new heart failure model can be prepared 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, activity for suppressing infarction progression, and the like.
- the compound obtained by the screening method may form a salt.
- the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkalis). Salts with metals and the like are used, and especially preferred are physiologically acceptable acid addition salts.
- 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) And succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
- inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
- 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 above-mentioned drug containing the protein of the present invention.
- the preparations obtained in this way are safe and low toxic, and thus can be used, for example, in humans or non-human mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, horses, 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.
- the compound when the compound is orally administered, generally the heart failure of an adult (assuming a body weight of 6 O kg) is performed.
- the single dose of the compound varies depending on the administration subject, the target disease, etc., but, for example, the compound is usually in the form of an injection and is usually administered to an adult (assuming a body weight of 60 kg) of heart failure.
- the present invention provides a test compound administered to a non-human mammal deficient in expression of a DNA of the present invention, and detects or inhibits the expression of a reporter gene.
- a method for screening a compound or a salt thereof is provided.
- the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention, wherein the DNA of the present invention introduces a repo allele gene.
- a reporter gene which can be expressed under the control of the promoter for the DNA of the present invention is used.
- test compound examples include the same compounds as described above.
- the reporter gene the same one as described above is used, and a galactosidase gene (1 acZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
- the reporter gene is present under the control of the promoter for the DNA of the present invention because the reporter overnight gene is under the control of the promoter for the DNA of the present invention.
- the tissue originally expressing the protein of the present invention may Instead of the protein of the present invention, 3) -galactosidase is expressed. Therefore, for example, staining with a reagent that is a substrate for / 3_galactosidase, such as 5-bromo-4-monocloth-3-indolyl-) 3-galactovyranoside (X-gal)
- a reagent that is a substrate for / 3_galactosidase such as 5-bromo-4-monocloth-3-indolyl-) 3-galactovyranoside (X-gal)
- 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. Near After reacting for about 30 minutes to 1 hour, the 0-galactosidase reaction may be stopped by washing the tissue specimen with an ImM ED TAZ PBS solution, and the coloration may be observed. Further, mRNA encoding 1 ac Z may be detected according to a conventional method.
- PBS phosphate buffered saline
- the compound or a salt thereof obtained by the above screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter activity for DNA of the present invention.
- the compound obtained by the screening method may form a salt.
- the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). Are used, and physiologically acceptable acid addition salts are particularly preferred.
- physiologically acceptable acid addition 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.).
- inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
- organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
- Succinic acid tarta
- the compound of the present invention or a salt thereof that inhibits the activity of a promoter for DNA can inhibit the expression of the protein of the present invention and inhibit the function of the protein.
- heart disease eg, cardiomyopathy, It is useful as a drug for prophylactic and therapeutic agents such as myocardial infarction, heart failure, and angina.
- a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
- the preparations obtained in this way are safe and low toxic, and thus can be used, for example, in humans or non-human mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, horses, horses, cats, cats, Dogs, monkeys, etc.).
- non-human mammals eg, rats, mice, guinea pigs, egrets, sheep, sheep, horses, 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.
- the compound When administered orally, it is generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 mg of the compound per day in an adult (assuming a body weight of 60 kg) heart failure patient. Administer ⁇ 20 mg.
- the single dose of the compound varies depending on the administration subject, target disease, and the like.
- the compound of the present invention that promotes the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to a patient with heart failure (with a body weight of 60 kg), about 0.01 to 3 Omg, preferably about 0.:! To 20 mg, 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.
- the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally, in an adult (assuming a body weight of 60 kg) heart failure patients, the compound is administered in an amount of about 0.1 to: Preferably, about 1.0 to 50 mg, more preferably about 1.0 to 20 mg is administered.
- the single dose of the compound varies depending on the administration subject, target disease, and the like.
- 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.
- the compound When administered to a patient with heart failure (assuming a body weight of 6 O kg), the compound may be administered in an amount of about 0.01 to 3 Omg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg per day. 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.
- 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 of the DNA of the present invention. Investigating or preventing the causes of various diseases caused by inadequate expression of DNA. It can greatly contribute to the development of therapeutic drugs.
- transgenic animal In addition, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) Makes it possible to specifically synthesize the polypeptide and examine its action in living organisms. Further, an appropriate reporter gene is linked to the above promoter, By establishing a cell line that expresses the protein, it can be used as a search system for low-molecular compounds having an action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body.
- bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, and examples thereof are described below.
- an amino acid can have an optical isomer
- the L-form is indicated unless otherwise specified.
- Example 1 shows the nucleotide sequence of primer 98 used in Example 1.
- [SEQ ID NO: 6] 1 shows the nucleotide sequence of Primer-100 used in Example 1.
- Example 1 shows the nucleotide sequence of a reverse primer used in Example 1.
- 1 shows the nucleotide sequence of human 187_2A intron.
- [SEQ ID NO: 20] Shows the nucleotide sequence of the human 187-2B intron.
- the nucleotide sequence of the mouse 187_2B5 'region is shown.
- Example 3 shows the base sequence of the forward primer used in Example 2.
- Example 3 shows the nucleotide sequence of a reverse primer used in Example 2.
- Example 3 shows the base sequence of the forward primer used in Example 2.
- Example 3 shows the nucleotide sequence of a reverse primer used in Example 2.
- Example 3 shows the nucleotide sequence of a primer used in Example 2.
- Example 3 shows the nucleotide sequence of a primer used in Example 2.
- Example 3 shows the base sequence obtained in Example 2.
- Example 3 shows the nucleotide sequence of a primer used in Example 2.
- [SEQ ID NO: 34] 2 shows the amino acid sequence of human 187-2B protein.
- FIG. 2 shows the amino acid sequence of rat 187-2A protein.
- 1 shows the amino acid sequence of rat 187_2B protein.
- Example 7 shows the nucleotide sequence of a primer used in Example 7.
- Example 7 shows the nucleotide sequence of a primer used in Example 7.
- Example 7 shows the nucleotide sequence of a primer used in Example 7.
- Example 7 shows the nucleotide sequence of a primer used in Example 7.
- Example 7 shows the nucleotide sequence of a primer used in Example 7.
- Example 7 shows the nucleotide sequence of a primer used in Example 7.
- the transformant Escherichia coli DH5a / pR187B obtained in Example 6 described below has been used since August 30, 2002, 1-1-1, Higashi, Tsukuba-shi, Ibaraki Prefecture 1 Central It has been deposited with the 6th National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566) Patent Organism Depositary under the accession number FERM BP-8172.
- the transformant Escherichia coli (DH5a / pH187B) obtained in Example 6 described below has been used since August 30, 2002, 1-1 1-1 Higashi, Tsukuba City, Ibaraki Prefecture 1 Chuo No. 6 (Zip code 305-8566) at the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM BP-8173.
- AIST National Institute of Advanced Industrial Science and Technology
- the method of genetic manipulation using Escherichia coli was in accordance with the method described in Molecular cloning.
- the reaction was carried out using the TaKaRa La Taq with GC buf fer (manufactured by Takara Shuzo Co., Ltd.) in the PCR system 9700 (manufactured by PerkinElmer) using the PCR system 9700 (PerkinElmer) and 30 seconds at 95: and 30 at 62: 33 cycles were repeated, with 3 minutes as one cycle at 72 seconds.
- the gene fragment PCR-PCR with Primer 100 and Primer 102 and the reverse primer was cloned into pT7-T vector (Takara Shuzo Co., Ltd.).
- human 187-2 has two splicing variants, Human 187-2A (SEQ ID NO: 17) which is an ortholog of 187-2 (hereinafter sometimes referred to as rat 187-2A) (SEQ ID NO: 13 and SEQ ID NO: 25 of W002 / 36763)
- the human 187-2B gene which is large 5 'upstream (the N-terminal region is large as a protein), could be cloned (SEQ ID NO: 18). Therefore, the human 187-2B cDNA completely contains the human 187-2A cDNA.
- nucleotide sequence represented by SEQ ID NO: 19 is an intron in human 187-2A and the nucleotide sequence represented by SEQ ID NO: 20 is human in 187-2B. This is because it is spliced out as an intron.
- the protein having the amino acid sequence (SEQ ID NO: 33) encoded by the nucleotide sequence represented by SEQ ID NO: 17 is a human 187-2A protein, and the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 18
- the protein having (SEQ ID NO: 34) is designated as human 187-2B protein.
- the nucleotide sequence encoding rat 187-2A protein is shown in SEQ ID NO: 1
- the amino acid sequence is shown in SEQ ID NO: 35.
- the human 187-2A protein and the human 187-2B protein had 53% homology at the amino acid level.
- Human 187-2A protein and AW755252 had 36% homology at the nucleic acid level.
- the human 187-2B protein and AW755252 had 66% homology at the nucleic acid level.
- the reaction is performed by adding 2 ⁇ L of SYBER Green Master Mix 10 // 1 of QuantiTect SYBR Green PCR kit (QIAGEN), 0.4 ⁇ l of each primer, and 71 of sterile distilled water to Type I DNA l. After 95t ⁇ 15 minutes, a cycle of 95 ⁇ 20 seconds, 55 ⁇ 20 seconds, 72 ⁇ 1 minute is repeated 50 times using a thermal cycler gene amp PCR system 9700 (manufactured by PerkinElmer) 50 times, and finally 72 ⁇ The extension reaction was performed for 4 minutes. The sequence of the 5 ′ region of rat 187-2B was obtained by directly sequencing the resulting DNA fragment (SEQ ID NO: 24).
- SEQ ID NO: 24 and rat 187-28 are one gene
- a forward primer SEQ ID NO: 25
- a reverse primer SEQ ID NO: 26
- PCR was performed with PFU polymerase, and the sequence of the obtained fragment was confirmed.
- a cloning primer SEQ ID NO: 27 and SEQ ID NO: 28
- Cloning was performed by PCR.
- the reaction was performed using a marathon lady rat heart cDNA library (Clontech) as type III, and using a QuantiTect SYBR Green PCR kit (QIAGEN) 2X SYBER Green Master Mix 101 and each primer at 0.4 M for type I DNA 1/1. , Sterile distilled water to make a volume of 20 ⁇ 1, and after 95 ⁇ 15 minutes with a thermocycler gene amp PCR system 9700 (PerkinElmer), 95t ⁇ 20 seconds, 5 (TC ⁇ 20 The cycle was repeated 50 times for 2 seconds and 1 minute, and the extension reaction was performed for 72 and 4 minutes at the end.As a result, a band was observed at the expected position. The nucleotide sequence was determined after cloning (SEQ ID NO: 29).
- rat 187 was obtained by using each primer represented by SEQ ID NO: 30 and SEQ ID NO: 31. A fragment containing the base sequence of the latter half of -2B was obtained. After recovering these two fragments, PCR was carried out using the primers of SEQ ID NO: 25 and SEQ ID NO: 31 using the type I, and a fragment of about 1.6 kb was obtained. .
- the reaction was carried out using a cycle sequence kit of PE Applied Biosystems, and a fluorescent DNA sequencer (ABI PRI SM 377, PerkinElmer) was used. And the base sequence was decoded.
- rat 187-2B homologous to human 187-2B was cloned (SEQ ID NO: 32).
- the protein having the amino acid sequence (SEQ ID NO: 36) encoded by the nucleotide sequence represented by SEQ ID NO: 32 is named rat 187-2B protein.
- the rat 187-2B protein and the rat 187-2A protein had 54% homology at the amino acid level.
- the N-terminal region (Nos. 1-70) of the human 187-2B and rat 187-2B proteins has a motif called LIM involved in protein-protein interactions (Mech. Dev. Vol. 91, pp. 5-17, 2000) existed.
- rat 187-2A cDNA SEQ ID NO: 1
- Adeno Custom Service of Takara Shuzo Co., Ltd.
- adenov i rus express ion on vec tor kit (6150) manufactured by Takara Shuzo Co., Ltd.
- the rat was cloned according to the attached method to express rat 187-2A sense and antisense strand adenovirus.
- titer of highly purified virus a known method (Experimental Medicine Supplement New Genetic Engineering Handbook, pp.
- a 14G indwelling needle outer tube (Termo) was introduced into the trachea from the oral cavity and connected to a ventilator.
- the lips and the indwelling needle outer tube were tied using surgical silk (Natsume, braided silk suture 3-0) so that the ventilator would not come off.
- the skin and trunk muscle approximately 5 mm to the left of the rat sternum were incised approximately 4 cm in the median direction.
- an incision was made near the sternum of the second to fifth costal cartilage just below the incision.
- the intercostal muscle was dissected with surgical scissors, and after hemostasis, the thoracic cavity was opened about 3 cm with a thoracotomy device.
- the pericardium was bluntly peeled off with a cotton swab and opened to expose the heart.
- High purity rat 187- 2A sense and antisense expression viral vector solution prepared in Example 3 was diluted with physiological saline to prepare a solution of 2xl0 8 pfu / ml concentration, it gave a 27G injection needle Weighed into lml syringe (Terumo). The virus was administered at 2xl0 fu per kilodalton body weight. As a control, the same amount of null virus without insert was administered.
- an injection needle was attached to the left ventricle, avoiding the coronary arteries and veins, and then the thoracic aorta was stenotic by tightening the Teflon tube. After confirming the stenosis, the virus solution was slowly administered into the left ventricle and allowed to flow back into the left ventricle. Since the left ventricle dilated with administration, the Teflon tube was loosened once every about 10 seconds to allow the virus solution to return to the whole body and prevent the secondary effects of left ventricular dilatation. This administration and the operation of loosening the Teflon tube were repeated three times in total, and the entire amount of the virus solution was refluxed into the left ventricle.
- One of the loops of the thoracic aortic stenosis silk thread was cut off, and the other thread was pulled out to remove the silk thread.
- the chest wall and the skin were sutured with a suture silk thread at intervals of about 5 millimeters.
- the skin incised at the time of the cervical duct and the muscle immediately below it were sutured in the same manner.
- the subject was forced to breath for at least 1 hour using a mechanical respirator. After confirming the spontaneous respiration of the animal while the animal was awake from anesthesia, the conduit was removed. Post-operative animals were housed in breeding cages.
- the heart function was measured as follows. One week after the operation, male Wistar rats (Jcl Wistar rats, 12 weeks old, weighing 350 g to 450 g) were weighed and anesthetized with sodium pentobarbital (25 mg / kg, ip). The abdominal coat of the chest and neck was shaved with a clipper and laid on the operating table in a supine position. An incision is made in the ventral neck with surgical scissors, the muscle immediately below is incised with forceps, and the cervical aorta right next to the trachea is peeled off from the connective tissue.
- Thread 3-0 was applied, the head was tied, and the abdomen was stopped with arterial cleansing.
- the blood vessel was incised with surgical scissors, a mirror catheter (2F, 140 cm, Catalog No. 800-0509, manufactured by Mira Co., Ltd.) was inserted into the blood vessel, and the tip was attached to the left ventricle.
- the mirror catheter was connected to a Polygraph manufactured by NEC, and the pressure fluctuation was monitored with a horoscope to confirm that the mirror catheter was attached to the left ventricle, and the left ventricular pressure was measured.
- MBP mean blood presure
- the measured values of (LVSP: left ventricular sistronic presure), (HR: heart rate), and left ventricular pressure change rate (LV + dp / dt and LV-dp / dt) are measured by MacLab (a waveform analysis software manufactured by NEC). And was calculated. The calibration of the mirror catheter was performed at two points, OmmHg and lOOmmHg, immediately before the start of the experiment using a carrier.
- the chest was opened, the heart was pinched with tweezers, blood vessels and connective tissues were removed, and the heart was excised.
- the heart was washed in saline. Further, a syringe was inserted into the aorta attached to the extracted heart, and physiological saline was perfused throughout the heart to remove blood. Thereafter, the atrium was removed, the ventricular tissue alone was weighed, and the heart weight was measured by correcting for body weight.
- Null (n S) 112 ⁇ 8 442 ⁇ 8 138 ⁇ 9 12.3 ⁇ 1.0 -10.6 ⁇ 0.7 2.67 ⁇ 0.06 Normal rat #
- the virus was administered to the heart of a heart failure rat, and the heart function and the heart weight were measured. Rats 23 weeks after myocardial infarction were administered antisense virus and null virus at pfu / kg. As a result of measuring cardiac function one week later, the antisense group showed significant improvement in heart rate and contractile force (LV + dp / dt) as compared to the null group. (table 1 )
- peptide-recognizing antibody Three types of peptide sequences were extracted from the amino acid sequence of rat 187-2A, and a peptide-recognizing antibody was prepared by Kurabo Industries' custom peptide antibody preparation service.
- the peptide used as the antigen is a peptide having an amino acid sequence represented by SEQ ID NO: 37 in the N-terminal region, SEQ ID NO: 38 in the central region, and SEQ ID NO: 39 in the C-terminal region.
- Western blotting was performed using the rat primary cardiomyocyte extract 3 days after infection with the rat 1872-sense-chain-expressing adenovirus described in Example 3 of WO 02/36763. As a result, it was found that each antibody recognizing the peptides of SEQ ID NO: 37 and SEQ ID NO: 39 can recognize rat 187-2A protein.
- the normal rats and the heart failure model rats obtained in Example 4 were laparotomized under ether anesthesia, killed by exsanguination by transection of the vena cava, and the heart was quickly removed. Phosphate buffer and 4% paraformaldehyde solution were injected in 20 ml portions from the aorta to the left ventricle, and fixed by perfusion. The heart was sliced transversely and fixed at 4 overnight.
- paraffin-embedded blocks were prepared in accordance with the usual method (Histology, Yutaka Sano, Nanzando, 1985).
- the produced block was sliced to a thickness of 4 l ⁇ with a rotating microtome. After slicing, the sections were stretched in a warm bath, mounted on glass slides, and thoroughly dried with a 37 drier. The prepared sections were deparaffinized, stained with hematoxylin and eosin according to a standard method, and mounted (Histology Research, Yutaka Sano, Nanzando, 1985).
- peptide recognition having the amino acid sequence represented by SEQ ID NO: 37 ⁇ Sagi polyclonal antibody and peptide recognition having the amino acid sequence represented by SEQ ID NO: 39 ⁇ Sagi polyclonal antibody was used for immunostaining.
- the immunostaining used the indirect fluorescent antibody method. Immunostaining and instu tu hybrid izat ion were performed according to the method described in Soichi Iijima et al., Pathology and Clinical Extra Edition, 2000. Alexafluor 488 goat anti-Peagle IgG antibody was used as a secondary antibody. Immunostaining was also performed on primary cardiomyocytes prepared from fetal hearts. After completion of the culture, the primary cardiomyocytes cultured in the 4-well culture chamber are fixed with 4% paraformaldehyde solution, and immunostaining is completed using the same antibody as above.
- rat 187-2 ⁇ protein and rat 187-2B protein were expressed in the cytoplasm of cardiomyocytes.
- cardiomyocytes remaining in the infarct transition region of the heart failure model rat their expression is normal in myocardial cells. It was clearly increased compared to the vesicle. Therefore, it was assumed that rat 187-2A protein and rat 187-2B protein were expressed in large amounts in the infarct transition region.
- the positive sites of rat 187-2A protein and rat 187-2B protein in the infarct transition region were alpha-actinin-positive cardiomyocytes, vascular smooth muscle cells such as coronary arteries, and peripheral residual cells.
- the high expression of the gene of rat 187-2 (including both A and B) at the lesion site indicates that the gene of 187-2 is strongly involved in the development of heart failure.
- rat 187-2A protein and rat 187-2B protein were stained using a peptide-recognizing ⁇ sagi polyclonal antibody having the amino acid sequence represented by SEQ ID NO: 37.
- SEQ ID NO: 39 When the cells were stained with cell nuclei of primary cardiomyocytes and using a peptide-recognizing Egret polyclonal antibody having the amino acid sequence represented by SEQ ID NO: 39, an image was observed that was localized in the cytoplasm. Therefore, it is considered that the rat 187-2A protein and the rat 187-2B protein translocate not only to the cytoplasm but also to the nucleus and are functionally expressed.
- the plasmid (containing the nucleotide sequence represented by SEQ ID NO: 18) cloned into the PT7-T vector obtained in Example 1 was named PH187B, and this was named Escherichia coli DH5H.
- Escherichia coli DH5 By transformation, a transformant Escherichia coli DH5 «/ pH187B was obtained.
- Example 7 Production of rat 187-2A transgenic rat and rat 187-2B transgenic rat
- PCR was performed using PFU polymerase, and the obtained fragment was cloned into pCRII T0P0 vector (manufactured by Invitrogen), and the nucleotide sequence was confirmed.
- a Hindi II cleavage site was introduced into the forward primer, and an Xhol cleavage site was introduced into the reverse primer.
- the subcloned rat 187-2A cDNA or rat 187-2B cDNA is excised with HindII and XhoI, the target gene fragment is recovered by 1% agarose gel electrophoresis, and GFX PCR DNA and Gel Band Purification was performed using a purification kit (Amersham Pharmacia Biotech Inc).
- PCR was performed using two types of primers (SEQ ID NO: 42 and SEQ ID NO: 43). That is, PCR was performed using PFU polymerase, and the obtained approximately 220 bp fragment was cloned into pCRII T0P0 vector (manufactured by Invitrogen), and the nucleotide sequence was confirmed.
- mice ⁇ -myosin heavy chain promoter gene 5.4 kpb described in The Journal of Biological Chemistry, 266, 24613 to 24620, 1991 was used.
- two primers SEQ ID NO: 44 and SEQ ID NO: 45 were synthesized from a BAC plasmid (# 22386, Cosmo Bio) containing this region.
- a Hindlll cleavage site was added as a cloning site to the primer represented by SEQ ID NO: 45.
- PCR was carried out using the BAC plasmid (# 22386, Cosmo Bio) using PFU polymerase with PFU polymerase, and the obtained about 1.
- lkbp fragment which is a part of the mouse ⁇ -myosin heavy chain promoter gene, was transformed into invitrogen).
- the 1.lkbp fragment was excised from this plasmid using Ndel and 3 ⁇ 4indIII, recovered by 2% agarose gel electrophoresis, and purified using GFX PCR DNA and Gel Band purification kit (Amersham Pharmacia Biotech Inc). This is called fragment A.
- a 4.3 kbp fragment of the mouse ⁇ -myosin heavy chain promoter gene region other than this 1.lkbp was excised from BAC plasmid (# 22386, Cosmo Bio) using BamHI and Ndel, and recovered by 1% agarose gel electrophoresis. And purified using GFX PCR DNA and Gel Band purification kit (Amersham Pharmacia Biotech Inc). This is called fragment B.
- Fragment A and Fragment B were inserted into the BamHI and Hindlll cloning sites of pBluescriptll SK (+) (Yukara Bio) to construct the pMHC8 plasmid.
- the PMHC8 plasmid containing the mouse ⁇ -myosin heavy chain promoter gene thus prepared was digested with BamHI and Hindlll, and the obtained approximately 5.4 kbp mouse ⁇ -myosin heavy chain promoter gene fragment was subjected to 1% agarose gel electrophoresis. It was recovered and purified using GFX PCR DNA and Gel Band purification kit (Amersham Pharmacia Biotech Inc).
- Digest pCRII T0P0 vector (manufactured by invitrogen) with BamHI and Xhol, recover by 1% agarose gel electrophoresis, and use GFX PCR DNA and Gel Band purification kit. (Amersham Pharmacia Biotech Inc).
- the mouse ⁇ -myosin heavy chain promoter-one gene fragment (having BamHI and Hind111 cleavage sites) obtained in (3) above and the rat 187-2A or rat 187-2A obtained in (1) above were added.
- the 187-2B cDNA gene fragment (having Hindlll and Xhol cleavage sites) was cloned.
- this vector is digested with BamHI and Xhol, and the obtained gene fragment
- PUC118 (Yukakara Bio) was digested with BamHI and EcoRI, recovered by 1% agarose gel electrophoresis, and purified using GFX PCR DNA and Gel Band purification kit (Amersham Pharmacia Biotech Inc). Next, the gene fragment prepared in (4) above (having BamHI and Xhol cleavage sites) and the gene fragment containing the poly A-added signal sequence prepared in (2) above (having Xhol and "EcoRI cleavage sites") were used. Was cloned into the BamHI and EcoRI sites of pUC118 to prepare a rat 187-2A expression plasmid and a rat 187-2B expression plasmid.
- transgenic rats were produced by Oriental Yeast Co., Ltd. according to a known method.
- the expression unit was excised from the rat 187-2A expression plasmid or the rat 187-2B expression plasmid by digestion with BamHI, and microinjected into fertilized rat eggs (collected from Wistar rats purchased with Jcl).
- pseudopregnant rats were fertilized by inserting fertilized eggs to give birth.
- Recombinant animals were selected by PCR.
- blood was collected from the rat tail vein, from which genomic DNA was recovered.
- primers SEQ ID NO: 42 and SEQ ID NO: 4'3 (exogenous poly A-added signal) The primer was used to amplify the null sequence. Individuals showing a band of about 220 bp after electrophoresis were selected as recombinants to obtain rat 187-2A transgenic individuals and rat 187-2B transgenic individuals. The obtained individual was transferred from Oriental Yeast Co., Ltd., and the following analysis was performed.
- RNA derived from the cardiac left ventricle of an F1 heterozygous individual the gene expression level was examined according to the method described in Example 7 of WO 02/36763.
- Compounds or salts thereof that regulate the activity of the protein of the present invention preferably compounds or salts thereof that inhibit the activity of protein A of the present invention
- compounds or salts thereof that regulate the expression of the protein gene of the present invention this
- the compound of the present invention that inhibits the expression of the gene for protein A or a salt thereof), the antibody of the present invention, the antisense polynucleotide of the present invention, and the like can be safely used, for example, as a prophylactic or therapeutic agent for heart disease and the like. it can.
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WO2001092567A2 (en) * | 2000-05-30 | 2001-12-06 | Medigene Ag | Novel target genes for diseases of the heart |
WO2002031111A2 (en) * | 2000-10-12 | 2002-04-18 | Hyseq, Inc. | Novel nucleic acids and polypeptides |
WO2002036763A1 (fr) * | 2000-10-30 | 2002-05-10 | Takeda Chemical Industries, Ltd. | Nouveau gene surexprime dans le coeur et les muscles et son utilisation |
US20020077470A1 (en) * | 1999-04-26 | 2002-06-20 | Walker Michael G. | Cardiac muscle-associated genes |
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US20020077470A1 (en) * | 1999-04-26 | 2002-06-20 | Walker Michael G. | Cardiac muscle-associated genes |
WO2001092567A2 (en) * | 2000-05-30 | 2001-12-06 | Medigene Ag | Novel target genes for diseases of the heart |
WO2002031111A2 (en) * | 2000-10-12 | 2002-04-18 | Hyseq, Inc. | Novel nucleic acids and polypeptides |
WO2002036763A1 (fr) * | 2000-10-30 | 2002-05-10 | Takeda Chemical Industries, Ltd. | Nouveau gene surexprime dans le coeur et les muscles et son utilisation |
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