JP2006298798A - Musklin receptor and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide the identification of the receptors to musklin, the elucidation of the interaction between musklin and the receptor and the screening system for the agonist and/or antagonist of the receptor. <P>SOLUTION: In the method for screening the substance that can change the bondability between an amino acid sequence and musklin, (i) the protein, or its partial peptide or their salts including the amino acid sequence the same as or substantially the same as that represented by the sequence number: 2, and (ii) musklin or its partial peptide or their salts are employed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a receptor for a skeletal muscle-specific secreted protein (musculin), a screening method for a prophylactic / therapeutic agent for a disease involving musclin using the receptor, and the like.

  So-called lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension, and ischemic heart disease are the biggest cause of pressure on medical finance in developed countries including Japan. Modern people are prone to overeating and lack of exercise, and as a result, excess energy accumulates in adipose tissue as neutral fat, causing obesity.

  In recent years, various physiologically active proteins secreted from adipose tissue (collectively referred to as adipocytokines) have been shown to be involved in metabolic regulation and insulin resistance, and application to the treatment of lifestyle-related diseases has been attempted. Fat accumulation during obesity also occurs in the liver, muscles, pancreas, blood vessel walls, etc., causing metabolic abnormalities in these organs, causing insulin resistance, diabetes, hyperlipidemia, hypertension and the like. These organs are thought to be linked to each other through endocrine factors and to carry out complex and advanced metabolic regulation. Therefore, it is presumed that endocrine factors having the same action as adipocytokines are also produced and secreted from muscles, liver, small intestine, blood vessels and the like.

  Shimomura et al. Isolated a gene encoding a secreted protein [named “musclin”] specifically expressed in mouse skeletal muscle using the signal sequence strap (SST) method. 1) Increased markedly by induction of muscle cell differentiation, (2) Decreased / re-increased by fasting / re-feeding, (3) Increased in obese diabetes model, and Musculin is high insulin It has been reported that expression is induced in response to a state and regulates sugar uptake and glycogen synthesis in skeletal muscle cells (Patent Document 1 and Non-Patent Document 1; ). There is also a report that the same protein as muskrin [referred to as osteocrin or bone peptide-1] is expressed bone-specifically and regulates the phenotype of osteoblasts (Patent Document 2 and Non-patent document 2).

In order for musclin to exert its action, it must interact with specific receptors in the target cell. The identification of the musclin receptor greatly clarifies the interaction between musclin and the receptor, the development of receptor agonists and antagonists, and the development of preventive and therapeutic drugs for diabetes, insulin resistance, hypertension, hyperlipidemia, etc. It is thought to contribute. However, no receptor for muscarin (or osteocrine, which is the same protein) has been reported so far.
International Publication No. 2004/111234 Pamphlet (full text) International Publication No. 03/054005 pamphlet (full text) Nishizawa, H. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 279 (19), pp. 19391-19395 (2004) Thomas, G. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 278 (50), pp. 50563-50571 (2003)

  Accordingly, an object of the present invention is to identify a receptor for musclin, elucidate the interaction between musclin and the receptor, and to use the musclin and the receptor to produce an agonist and / or antagonist of the receptor, That is, it is to provide a screening system for drug candidate compounds effective as a preventive / therapeutic agent for diabetes, insulin resistance, hypertension, hyperlipidemia and the like.

  The present inventors include a region in which the musclin protein is homologous to the natriuretic peptide (NP) family (atrial (ANP), brain (BNP), and type C (CNP) are known). As a result of investigating the binding between receptors for these peptides (NPR1, NPR2, and NPR3 are known) and muskrin, muskrin is considered to be involved in ANP clearance. It has been found that it specifically binds to the receptor NPR3 (also referred to as Npr3, Npr-3, NPR-3, Npr-c, NPR-C, referred to herein as “NPR3”). As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] A cell differentiation and / or metabolism regulator comprising a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof,
[2] The cell is selected from the group consisting of skeletal muscle cells, fat cells, pancreatic islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent according to [1] above,
[3] For the prevention and treatment of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia [1] ] The agent according to
[4] A cell differentiation and / or metabolism regulator comprising a nucleic acid encoding a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof,
[5] The cell is selected from the group consisting of skeletal muscle cells, fat cells, pancreatic islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent according to [4] above,
[6] For the prevention and treatment of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia [4] ] The agent according to
[7] Abnormal cell differentiation and / or metabolic abnormality comprising a nucleic acid encoding a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial polynucleotide thereof Diagnostic agent,
[8] The cell is selected from the group consisting of skeletal muscle cells, fat cells, pancreatic islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent according to [7] above,
[9] The above-mentioned [7], which is for diagnosis of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia. Agent,
[10] Abnormal cell differentiation and / or metabolic abnormality, comprising an antibody against a protein comprising the same or substantially the same amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof. Diagnostic agent,
[11] The cells are selected from the group consisting of skeletal muscle cells, adipocytes, islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent according to [10] above,
[12] The above-mentioned [10], which is for diagnosis of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease, and hyperlipidemia. Agent,
[13] A cell differentiation and / or metabolism regulator comprising an antibody against a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof,
[14] The cell is selected from the group consisting of skeletal muscle cells, adipocytes, islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent described in [13] above,
[15] The above-mentioned [13], which is used for prevention / treatment of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia. ] The agent according to
[16] A nucleic acid containing a nucleotide sequence complementary to a nucleic acid encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a part thereof. Cell differentiation and / or metabolic regulators,
[17] The cell is selected from the group consisting of skeletal muscle cells, fat cells, pancreatic islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent described in [16] above,
[18] The above-mentioned [16], which is used for prevention / treatment of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease, and hyperlipidemia. ] The agent according to
[19] A cell differentiation and / or metabolic regulation action characterized by using a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof. Screening method for substances to be shown,
[20] A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof, is provided in the form of a cell that produces it. The method according to [19] above,
[21] A nucleic acid encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial polynucleotide thereof, or the same as the amino acid sequence represented by SEQ ID NO: 2 or The method according to the above [20], further comprising using an antibody against a protein containing substantially the same amino acid sequence or a partial peptide thereof or a salt thereof,
[22] The cell is selected from the group consisting of skeletal muscle cells, adipocytes, islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism, The method according to [19] above,
[23] The above, wherein the substance has prophylactic / therapeutic activity for one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia [19] The method according to
[24] (i) a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof; and (ii) a musclin, a partial peptide thereof, or a salt thereof. A screening method for a substance that changes the binding property between the two,
[25] A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof, is provided in the form of a cell that produces it. The method according to [24] above,
[26] The method according to [25] above, characterized by indicating sugar uptake into cells or glycogen synthesis in cells.
[27] The method according to [24] above, wherein the substance exhibits a cell differentiation and / or metabolic regulation action,
[28] The cell is selected from the group consisting of skeletal muscle cells, adipocytes, islet cells, bone cells, cartilage cells and hepatocytes, and the metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism, The method according to [27] above and [29] one or more diseases wherein the substance is selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia The method of the above-mentioned [27], etc. having the preventive / therapeutic activity of

  Since NPR3 functions as a receptor for musclin, screening for drug candidate compounds that are effective in the prevention and treatment of diseases related to cell differentiation such as skeletal muscle and abnormalities in sugar, lipid, and protein metabolism using NPR3 and musclin can do.

  The muscarinic receptor protein used in the present invention is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. This protein is one of the receptors for the natriuretic peptide (NP) family (ANP, BNP, CNP), and is the same protein as the receptor NPR3 that is involved in the clearance of ANP. Therefore, hereinafter, the maskin receptor protein of the present invention is sometimes referred to as “NPR3” in the present specification.

  NPR3 is, for example, a mammalian cell (eg, human, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.) [eg, hepatocyte, spleen cell, nerve cell, glia. Cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibroblasts, muscle cells, adipocytes, immune cells (eg, macrophages) , T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts , Mammary cells or stromal cells, or progenitor cells, stem cells or cancer cells of these cells] or any tissue in which these cells are present [eg, brain, brain regions (eg, olfactory bulb, amygdala, cerebrum) Basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (eg, Large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, brown adipose tissue, white adipose tissue), skeletal muscle Etc.] and the like. Alternatively, it may be a protein synthesized chemically or by a cell-free translation system, or a recombinant produced from a transformant introduced with a nucleic acid having a base sequence encoding the amino acid sequence. It may be a protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 is about 70% or more, preferably about 80% or more, more preferably about 90%, with the amino acid sequence represented by SEQ ID NO: 2. As mentioned above, amino acid sequences having homology of about 95% or more are particularly preferred. As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue and similar amino acid residues to all overlapping amino acid residues in the case of introducing a gap into one or both). "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
The homology of amino acid sequences in the present specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms are NBLAST and XBLAST Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0), Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is FASTA in the GCG software package. Program It includes built-in 'or the like, may they likewise preferably used.
More preferably, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 is about 60% or more, preferably about 70% or more, more preferably the amino acid sequence represented by SEQ ID NO: 2. Is an amino acid sequence having about 80% or more, particularly preferably about 90% or more identity.

  The protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 includes, for example, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 described above. A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 2 is preferred.

The substantially homogeneous activity includes, for example, a ligand (ie, muscarin) binding activity and a signal signal transduction action. “Substantially homogeneous” indicates that their activities are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that activities such as ligand binding activity and signal signal transduction activity are equivalent (eg, about 0.5 to about 2 times), but quantitative factors such as the degree of these activities and the molecular weight of the protein are different. Also good.
Measurement of activities such as ligand binding activity and signal transduction activity can be carried out according to a method known per se. For example, methods used in screening methods for agonists and antagonists described later, sugar uptake into cells, Glycogen synthesis / degradation in cells, sugar release from cells, fat uptake into cells, fat release from cells, lipolysis in cells, fat synthesis in cells, regulation of protein / non-protein hormone secretion, protein / It can be performed according to a method that uses nonproteinaceous hormone action regulation, appetite regulation, momentum regulation, body weight fluctuation, blood sugar / lipid fluctuation, muscle cell differentiation / proliferation, osteoblast differentiation / proliferation as an index, etc. .

The NPR3 of the present invention includes, for example, (1) one or more of the amino acid sequences represented by SEQ ID NO: 2 (preferably about 1 to 100, preferably about 1 to 50, Preferably about 1 to 10, more preferably a number (1 to 5) of amino acid sequences deleted, (2) one or more amino acid sequences represented by SEQ ID NO: 2 (preferably 1 to 100, preferably about 1 to 50, more preferably about 1 to 10, particularly preferably several (1 to 5) amino acids, (3) SEQ ID NO: 2 An amino acid sequence in which one or two or more (preferably about 1 to 50, preferably about 1 to 10, more preferably a number (1 to 5)) amino acids are inserted into the amino acid sequence represented by (4) One or more of the amino acid sequences represented by SEQ ID NO: 2 (preferably about 1 to 50) Also included is an amino acid sequence in which about 1 to 10 amino acids, more preferably several (1 to 5) amino acids are substituted with other amino acids, or (5) a protein containing an amino acid sequence combining them. It is.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity of the protein is maintained.

  The NPR3 of the present invention is preferably a human NPR3 protein having the amino acid sequence represented by SEQ ID NO: 2 (GenBank accession number: NP_000899), or a homolog thereof in other mammals (eg, accession number to GenBank, respectively) Rat, mouse and bovine homologs registered as NP_037000, NP_032754 and NP_776552 (having about 91%, about 90% and about 93% identity to human NPR3, respectively)).

In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. The NPR3 of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 2, has a C-terminal carboxyl group (—COOH), carboxylate (—COO ⁻ ), amide (—CONH ₂ ) or Any of ester (-COOR) may be sufficient.
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C _6-12 aryl groups such as α-naphthyl; for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl; C _7- such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl; ₁₄ aralkyl group; pivaloyloxymethyl group is used.
When NPR3 of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
Furthermore, in NPR3 of the present invention, the amino group of the N-terminal amino acid residue is protected with a protecting group (for example, a C _1-6 acyl group such as C _1-6 alkanoyl such as formyl group, acetyl group, etc.). N-terminal glutamine residue that can be cleaved in vivo is pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) a suitable protecting group (e.g., formyl group, those protected by C _1-6 an acyl group) such as C _1-6 alkanoyl group such as acetyl group, or a sugar chain-binding Also included are complex proteins such as so-called glycoproteins.

As long as the partial peptide of NPR3 (hereinafter sometimes simply referred to as “partial peptide of the present invention”) is a peptide having the above-mentioned partial amino acid sequence of NPR3 and has substantially the same activity as NPR3, Any one may be used. Here, “substantially the same quality of activity” has the same meaning as described above. Further, “substantially the same quality of activity” can be measured in the same manner as in NPR3.
Specifically, as the partial peptide of the present invention, for example, among the amino acid sequence represented by SEQ ID NO: 2, a region involved in binding to the ligand maskulin and a region involved in signal transduction through the interaction Further, those having a partial amino acid sequence are used.
The partial peptide of the present invention is preferably a peptide having at least 50 or more, preferably 100 or more, more preferably 200 or more amino acids.

  On the other hand, a peptide containing a partial amino acid sequence of NPR3 but having substantially the same quality of activity as NPR3, for example, a region involved in binding to musclin in the amino acid sequence represented by SEQ ID NO: 2, Those having a partial amino acid sequence that does not include a region involved in signal transduction via interaction are not included in the “partial peptide of the present invention”. However, since such a peptide can block the signal transduction action through NPR3 by binding to musclin, it can be useful when it is desired to suppress the signal transduction action.

In the partial peptide of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Here, examples of R in the ester include the same as those described above for NPR3. When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the same ester as the C-terminal ester is used.
Furthermore, in the partial peptide of the present invention, as in NPR3, the amino group of the N-terminal amino acid residue is protected with a protecting group, the N-terminal glutamine residue is pyroglutamine oxidized, Examples include those in which a substituent on the side chain of the amino acid is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.

  Examples of the salt of NPR3 or a partial peptide thereof include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  NPR3 or a salt thereof can be produced from the aforementioned mammalian cells or tissues by a known protein purification method. Specifically, after homogenizing mammalian tissue or cells and removing cell debris by low-speed centrifugation, the supernatant is centrifuged at high speed to precipitate a cell membrane-containing fraction (if necessary, cell membrane fractionation by density gradient centrifugation or the like). NPR3 or a salt thereof can be prepared by subjecting the fraction to chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like.

NPR3 or a partial peptide thereof or a salt thereof (hereinafter sometimes collectively referred to as “NPR3 class”) can also be produced according to a known peptide synthesis method.
The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid capable of constituting NPR3 is condensed with the remaining part and the product has a protecting group, the target protein can be produced by removing the protecting group.
Here, the condensation and the removal of the protecting group are performed according to a method known per se, for example, the methods described in the following (1) to (5).
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV 205 (1977)
(5) Supervised by Haruaki Yajima, Development of follow-up drugs, Volume 14, Peptide synthesis, Hirokawa Shoten

The protein (peptide) thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When the protein (peptide) obtained by the above method is a free form, the free form can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, the protein (peptide) is converted into a salt. When obtained, the salt can be converted into a free form or other salt by a known method or a method analogous thereto.

  For the synthesis of NPR3s, commercially available resins for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein (peptide) according to various known condensation methods. At the end of the reaction, proteins and the like are cut out from the resin, and at the same time, various protecting groups are removed, and an intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the target protein (peptide) or its amide.

  For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOOBt), or the protected amino acid is activated in advance as a symmetric anhydride, HOBt ester or HOOBt ester. Can then be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, N, N-dimethylformamide, N, N-dimethylacetamide, acid amides such as N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range known to be usable for protein bond forming reaction, and is usually selected appropriately from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the removal of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
Examples of the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, etc. are used.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected by, for example, esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.

  Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -Addition of a cation scavenger such as butanedithiol or 1,2-ethanedithiol is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine was removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan was the above 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

  Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.

As another method for obtaining an amide form of a protein (peptide), for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide chain is extended to the desired chain length on the amino group side. And a protein (peptide) from which only the protecting group of the α-amino group at the N-terminus of the peptide chain is removed and a protein (peptide) from which only the protecting group of the carboxyl group at the C-terminal has been removed are produced. ) In a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein (protected peptide) obtained by the condensation, all the protecting groups are removed by the above method to obtain the desired crude protein (crude peptide). This crude protein (crude peptide) can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide form of the desired protein (peptide).
The ester form of the protein (peptide) can be obtained, for example, in the same manner as the amide form of the protein (peptide) after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester. it can.

  The partial peptide of the present invention or a salt thereof can also be produced by cleaving NPR3 or a salt thereof with an appropriate peptidase.

Furthermore, NPR3s can also be produced by culturing a transformant containing a nucleic acid encoding NPR3 or a partial peptide thereof, and separating and purifying NPR3s from the resulting culture. The nucleic acid encoding NPR3 or a partial peptide thereof may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used. The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
As DNA encoding NPR3 or a partial peptide thereof, genomic DNA, genomic DNA library, mammal (eg, human, cow, monkey, horse, pig, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit) , Hamsters, etc.) [eg, spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts Cells, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, Synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells, stem cells or cancer cells of these cells] Or any tissue in which those cells exist [eg, brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland , Stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, Testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, brown adipose tissue, white adipose tissue), skeletal muscle, etc.] derived cDNA, synthetic DNA and the like. Genomic DNA and cDNA encoding NPR3 or its partial peptide can be prepared using Polymerase Chain Reaction (hereinafter referred to as “PCR method”) using the genomic DNA fraction and total RNA or mRNA fraction prepared from the cells and tissues as templates. (Abbreviated) and Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”). Alternatively, genomic DNA and cDNA encoding NPR3 or a partial peptide thereof can be prepared by inserting a genomic DNA prepared from the cells / tissues described above and a total RNA or mRNA fragment into an appropriate vector and It can also be cloned from a cDNA library by colony or plaque hybridization method or PCR method, respectively. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.

Examples of the DNA encoding NPR3 include a DNA containing the nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 under highly stringent conditions. And a DNA encoding a protein containing substantially the same quality as NPR3 (eg, binding activity to muscarin, signal transduction, etc.).
Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 1 under highly stringent conditions include, for example, about 50% or more, preferably about 60% or more, with the base sequence represented by SEQ ID NO: 1. More preferably, DNA containing a base sequence having a homology of about 70% or more, particularly preferably about 80% or more, and most preferably about 90% or more is used.
The homology of the base sequences in the present specification uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gap; filtering = ON; match) It can be calculated by score = 1; mismatch score = -3). As another algorithm for determining the homology of the base sequence, the above-described homology calculation algorithm of amino acid sequences is preferably exemplified as well.

Hybridization is carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to highly stringent conditions.
High stringent conditions include, for example, conditions in which the sodium salt concentration is about 19 to about 40 mM, preferably about 19 to about 20 mM, and the temperature is about 50 to about 70 ° C., preferably about 60 to about 65 ° C. Can be mentioned. In particular, it is preferable that the sodium salt concentration is about 19 mM and the temperature is about 65 ° C.
The DNA encoding NPR3 is preferably a DNA (GenBank accession number: NM_000908) containing a base sequence encoding the human NPR3 protein represented by the base sequence represented by SEQ ID NO: 1, or the other in mammals Homologs (eg, rat, mouse and bovine homologs registered with GenBank as accession numbers NM_012868, NM_008728 and NM_174127, respectively, with about 87%, about 86% and about 89% identity to human NPR3 cDNA, respectively) Etc.).

  The DNA encoding the partial peptide of the present invention is any DNA as long as it contains a base sequence encoding a peptide having the same or substantially the same amino acid sequence as part of the amino acid sequence represented by SEQ ID NO: 2. It may be. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Further, it can be directly amplified by RT-PCR using the mRNA fraction prepared from the cells and tissues described above.

Specifically, examples of DNA encoding the partial peptide of the present invention include (1) DNA having a partial base sequence of the base sequence represented by SEQ ID NO: 1, or (2) SEQ ID NO: 1. Has a base sequence that hybridizes with DNA having a base sequence that is highly stringent, and has substantially the same quality of activity as NPR3 (eg, binding activity to muscarin, signal transduction, etc.) A DNA encoding a peptide is used.
The DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 1 under highly stringent conditions is, for example, about 60% or more, preferably about 70% or more, more preferably about 80% or more with the base sequence. Most preferably, DNA containing a base sequence having about 85% or more identity is used.

  A DNA encoding NPR3 or a partial peptide thereof is amplified by PCR using a synthetic DNA primer having a part of a base sequence encoding the NPR3 or a partial peptide thereof, or a DNA incorporated into an appropriate expression vector, Cloning can be carried out by hybridization with a DNA fragment encoding a part or the entire region of NPR3 protein or a labeled synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning Second Edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

The DNA base sequence can be determined using a known kit such as Mutan ^™ -super Express Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, the Gapped duplex method, Conversion can be performed according to a method known per se such as the Kunkel method or a method analogous thereto.

  The cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

An expression vector containing DNA encoding NPR3 or a partial peptide thereof is produced, for example, by excising a target DNA fragment from DNA encoding NPR3 and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. can do.
Expression vectors include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15); insect cell expression Plasmid (eg, pFast-Bac); animal cell expression plasmid (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophage such as λ phage; insect virus vector such as baculovirus ( Examples: BmNPV, AcNPV); animal virus vectors such as retrovirus, vaccinia virus, adenovirus, etc. are used.
The promoter may be any promoter as long as it is appropriate for the host used for gene expression.
For example, when the host is an animal cell, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex) Virus thymidine kinase) promoter and the like are used. Of these, CMV promoter, SRα promoter and the like are preferable.
When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP _L promoter, lpp promoter, T7 promoter and the like are preferable.
When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable.
When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired. Can do. Examples of the selection marker include a dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter abbreviated as amp ^r ), a neomycin resistance gene ( hereinafter sometimes abbreviated as neo ^r, include G418 resistance) and the like. In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can also be selected using a medium that does not contain thymidine.
In addition, if necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host is added (or replaced with a native signal codon) to the 5 ′ end of DNA encoding NPR3 or a partial peptide thereof. May be. For example, when the host is Escherichia, PhoA signal sequence, OmpA, signal sequence, etc .; when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc .; host is yeast In some cases, MFα • signal sequence, SUC2 • signal sequence, etc .; when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. are used respectively.

NPR3s can be produced by transforming a host with an expression vector containing DNA encoding NPR3 or a partial peptide thereof, and culturing the resulting transformant.
As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells and the like are used.
Examples of the genus Escherichia include, for example, Escherichia coli K12 • DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA) ), 60, 160 (1968)], Escherichia coli JM103 (Nucleic Acids Research, 9, 309 (1981)), Escherichia coli JA221 [Journal of Molecular Biology ( Journal of Molecular Biology), 120, 517 (1978)], Escherichia coli HB101 [Journal of Molecular Biology, 41, 459 (1969)], Escherichia coli C600 [Genetics, 39 Vol., 440 (1954)] is used.
Examples of the genus Bacillus include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], Bacillus subtilis 207-21 [Journal of Biochemistry, 95, 87 (1984)].
Examples of the yeast include Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris (Pichia pastoris) KM71 etc. are used.

Insect cells include, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ^TM cells derived from eggs of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, moth-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used as insect cells. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like.
Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L Cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells and the like are used.

Transformation can be performed according to a known method depending on the type of host. Escherichia bacteria are, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) and Gene (Proceedings of the National Academy of Sciences of the USA). Gene), 17, 107 (1982), and the like.
Bacillus can be transformed according to the method described in, for example, Molecular & General Genetics, 168, 111 (1979).
Yeast is, for example, Methods in Enzymology, 194, 182-287 (1991), Proceedings of the National Academy of Sciences of USA ( Proc. Natl. Acad. Sci. USA), 75, 1929 (1978).
Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, Vol. 6, 47-55 (1988).
Animal cells should be transformed according to the method described in, for example, Cell Engineering Annex 8 New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do.

The culture of the transformant can be performed according to a known method depending on the type of the host.
For example, when culturing a transformant whose host is an Escherichia or Bacillus genus, a liquid medium is preferable as a medium used for the culture. Moreover, it is preferable that a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for the growth of a transformant. Here, as the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc .; As the nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added to the medium. The pH of the medium is preferably about 5 to about 8.
As a medium for culturing a transformant whose host is an Escherichia bacterium, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics ( Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, a drug such as 3β-indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
Culturing of the transformant whose host is Escherichia is usually performed at about 15 to about 43 ° C. for about 3 to about 24 hours. If necessary, aeration or agitation may be performed.
Culturing of a transformant whose host is Bacillus is usually performed at about 30 to about 40 ° C. for about 6 to about 24 hours. If necessary, aeration or agitation may be performed.
As a medium for culturing a transformant whose host is yeast, for example, a Burkholder minimum medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science The USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA, et al., Proceedings of the National Academy -Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably about 5 to about 8. The culture is usually performed at about 20 ° C. to about 35 ° C. for about 24 to about 72 hours. Aeration and agitation may be performed as necessary.
As a medium for culturing a transformant whose host is an insect cell or an insect, for example, 10% bovine serum deactivated in Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] The thing etc. which added additives, such as these, etc. suitably are used. The pH of the medium is preferably about 6.2 to about 6.4. The culture is usually performed at about 27 ° C. for about 3 to about 5 days. You may perform ventilation | gas_flowing and stirring as needed.
As a medium for culturing a transformant whose host is an animal cell, for example, a minimal essential medium (MEM) containing about 5 to about 20% fetal bovine serum [Science, 122, 501 (1952 ], Dulbecco's Modified Eagle Medium (DMEM) [Virology, Vol. 8, 396 (1959)], RPMI 1640 Medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] Used. The pH of the medium is preferably about 6 to about 8. The culture is usually performed at about 30 ° C. to about 40 ° C. for about 15 to about 60 hours. You may perform ventilation | gas_flowing and stirring as needed.
As described above, NPR3s can be produced inside or outside the transformant.

NPR3s can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
For example, when extracting NPR3 from the cytoplasm of cultured cells or cells, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thawing, etc. A method of obtaining a crude extract of soluble protein by centrifuging or filtering after destroying cells or cells is appropriately used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride and a surfactant such as Triton X-100 ^™ . On the other hand, when extracting NPR3s from the membrane fraction, after destroying the cells or cells in the same manner as described above, cell debris is precipitated and removed by low-speed centrifugation, and the supernatant is centrifuged at high speed to precipitate the cell membrane-containing fraction ( A method such as purifying a cell membrane fraction by density gradient centrifugation or the like is used if necessary. When NPR3 is secreted outside the cells (cells), a method of separating the culture supernatant from the culture by centrifugation or filtration is used.
Isolation and purification of NPR3s contained in the thus obtained soluble fraction, membrane fraction or culture supernatant can be performed according to a method known per se. As such methods, methods utilizing the solubility such as salting out and solvent precipitation method; mainly utilizing differences in molecular weight such as dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis method. A method utilizing a difference in charge such as ion exchange chromatography, a method utilizing a specific affinity such as affinity chromatography, a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography, etc. A method using a difference in isoelectric point such as point electrophoresis is used. These methods can be combined as appropriate.

When the protein or peptide thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein or peptide is obtained as a salt, The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.
The NPR3 produced by the transformant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of NPR3s thus obtained can be confirmed by enzyme immunoassay or Western blotting using a specific antibody.

  Furthermore, NPR3s are synthesized in vitro using a cell-free protein translation system consisting of rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc., using RNA corresponding to the DNA encoding NPR3 or a partial peptide thereof as a template. be able to. Alternatively, a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize DNA encoding NPR3 or a partial peptide thereof as a template.

A base sequence encoding a NPR3 protein, that is, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a part thereof, or a base sequence complementary to the base sequence or The nucleic acid containing a part thereof is used in the meaning including not only the nucleic acid encoding the aforementioned NPR3 or a partial peptide thereof but also a base sequence that does not match the frame.
A nucleic acid containing a base sequence complementary to the target region of the target nucleic acid, that is, a nucleic acid that can hybridize with the target nucleic acid can be said to be “antisense” to the target nucleic acid. On the other hand, a nucleic acid containing a base sequence having homology with the target region of the target nucleic acid can be said to be “sense” with respect to the target nucleic acid. Here, “having homology” or “complementary” means about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more between base sequences. Having identity or complementarity.

  A nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence encoding NPR3 protein or a part thereof (hereinafter also referred to as “antisense nucleic acid of the present invention”) is a cloned or determined nucleic acid encoding NPR3 Design and synthesis based on the nucleotide sequence information of Such nucleic acids can inhibit NPR3 gene replication or expression. That is, the antisense nucleic acid of the present invention can hybridize with RNA transcribed from the NPR3 gene, and can inhibit mRNA synthesis (processing) or function (translation into protein).

The length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the antisense nucleic acid hybridizes, and as a result, the translation of the NPR3 protein is inhibited. The short sequence may be about 15 bases, and the long sequence may be the entire mRNA or initial transcription product sequence. In view of ease of synthesis and antigenicity, an oligonucleotide consisting of about 15 to about 30 bases is preferable, but not limited thereto. Specifically, for example, 5 ′ end hairpin loop of nucleic acid encoding NPR3 precursor polypeptide, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, polypeptide translation initiation codon, protein coding region, ORF A translation initiation codon, 3 ′ end untranslated region, 3 ′ end palindromic region, and 3 ′ end hairpin loop can be selected as target regions, but any region within the NPR3 gene can be selected as a target. For example, it is also preferable to use the intron portion of the gene as a target region.
Furthermore, the antisense nucleic acid of the present invention not only hybridizes with NPR3 mRNA or an initial transcription product to inhibit translation into a protein, but also binds to the NPR3 gene, which is a double-stranded DNA, to form a triplex. May be formed so that RNA transcription can be inhibited.

  Antisense nucleic acids are deoxyribonucleotides containing 2-deoxy-D-ribose, ribonucleotides containing D-ribose, other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or non-nucleotides Other polymers with backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a pair of bases as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a ring or a base). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and unmodified polynucleotides (or unmodified oligonucleotides), and more Modified, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotides Modified, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), For example, proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides) , Poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals) , Radioactive metals, boron, oxidizing metals, etc.), alkylating agents, and modified bonds (eg, α-anomeric nucleic acids). . Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.

  Antisense nucleic acids are RNA, DNA, or modified nucleic acids (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to increase the affinity for the target sense strand, and to be antitoxic if toxic Make sense nucleic acids less toxic. Many such modifications are known in the art, such as J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed ., Antisense Research and Applications, CRC Press, 1993, etc.

  Antisense nucleic acids may be altered or contain modified sugars, bases, bonds, donated in specialized forms such as liposomes, microspheres, applied by gene therapy, or added Could be given in form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( For example, hydrophobic substances such as phospholipids and cholesterols). Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of nucleic acids and can be attached via bases, sugars, intramolecular nucleoside linkages. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

  Ribozymes capable of specifically cleaving NPR3 mRNA or the initial transcript within the coding region (including the intron portion in the case of the initial transcript) can also be included in the antisense nucleic acid of the present invention. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligoDNA having the base sequence of the enzyme active site also has a nucleic acid cleaving activity. As long as it has sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate. When NPR3 mRNA itself has a double-stranded structure, the target sequence can be made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, it should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

A double-stranded oligo RNA (siRNA) having a base sequence complementary to NPR3 mRNA or a partial sequence in the coding region of the initial transcript (including an intron in the case of the initial transcript) is also an antisense nucleic acid of the present invention. Can be included. When a short double-stranded RNA is introduced into a cell, mRNA complementary to one strand of the RNA is degraded, so-called RNA interference (RNAi) has been known for a long time in nematodes, insects, plants, etc. However, since it was recently confirmed that this phenomenon also occurs in mammalian cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative technology for antisense and ribozyme. siRNA can be appropriately designed using commercially available software (eg, RNAi Designer; Invitrogen) based on the base sequence information of the target mRNA.
The antisense oligonucleotide and ribozyme of the present invention determine the target region of mRNA or initial transcript based on NPR3 cDNA sequence or genomic DNA sequence information, and are commercially available DNA / RNA automatic synthesizers (Applied Biosystems, Beckman). Etc.) can be prepared by synthesizing a complementary sequence thereto. For siRNA having RNAi activity, sense strand and antisense strand are respectively synthesized by a DNA / RNA automatic synthesizer and denatured in an appropriate annealing buffer at, for example, about 90 to about 95 ° C. for about 1 minute. And annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. In addition, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to alternately overlap, annealing them, and then ligating with ligase.

  The gene expression inhibitory activity of the antisense nucleic acid of the present invention is determined using a transformant containing a nucleic acid encoding NPR3, an in vivo or in vitro NPR3 gene expression system or an NPR3 protein in vivo or in vitro translation system. You can investigate. The nucleic acid can be applied to cells by various methods known per se.

  The present invention also provides an antibody against NPR3 or a partial peptide thereof or a salt thereof. The antibody may be a monoclonal antibody or a polyclonal antibody as long as it has specific affinity for NPR3. An antibody against NPR3 can be produced according to a method for producing an antibody or antiserum known per se, using NPR3 as an antigen.

[Production of monoclonal antibodies]
(A) Preparation of monoclonal antibody-producing cells
NPR3 is administered to a mammal at a site where antibody production is possible by administration, together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually once every 2 to 6 weeks, 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used.
When preparing monoclonal antibody-producing cells, select a mammal with an antigen titer from an antigen-immunized mammal, such as a mouse, and collect the spleen or lymph nodes 2 to 5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody-producing cells to be fused with myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting a labeled NPR3 described below with antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc., and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.

Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which an antigen such as a protein is adsorbed directly or with a carrier, and then radioactive. A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with a substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A; Examples include a method of detecting a monoclonal antibody bound to a solid phase by adding a hybridoma culture supernatant to a solid phase on which an anti-immunoglobulin antibody or protein A is adsorbed, adding a protein labeled with a radioactive substance or an enzyme, etc. .
Selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto, but it can usually be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1-20% fetal bovine serum, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) etc. can be used. The culture temperature is usually 20-40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.

(B) Purification of monoclonal antibody Separation and purification of monoclonal antibodies are carried out in the same manner as in the separation and purification of ordinary polyclonal antibodies (eg, salting-out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis). Method, adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as protein A or protein G, and collecting the antibody to dissociate Specific purification method for obtaining antibody].

[Preparation of polyclonal antibody]
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as a protein) and a carrier protein is prepared, a mammal is immunized in the same manner as the above-described monoclonal antibody production method, and an antibody-containing product against NPR3 is collected from the immunized animal. Thus, the antibody can be produced by separating and purifying the antibody.
Regarding the complex of immune antigen and carrier protein used to immunize mammals, the type of carrier protein and the mixing ratio of carrier and hapten should be such that antibodies can be efficiently produced against haptens that are immunized by cross-linking to carriers. Any ratio may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole, limpet, hemocyanin, etc. are about 0.1 to 20, preferably about 0.1 to 20, in weight ratio to hapten 1. A method of coupling at a ratio of about 1 to 5 is used.
Various coupling agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.
The condensation product is administered to a mammal at a site where antibody production is possible, together with the carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration can be performed about once every 2 to 6 weeks, usually about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably blood.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

  NPR3 functions as a receptor for musclin. Therefore, NPR3, nucleic acid encoding NPR3 or a part thereof, antibody against NPR3 (hereinafter sometimes referred to as “the antibody of the present invention”), antisense nucleic acid against NPR3 nucleic acid (hereinafter referred to as “the present invention”). (Sometimes referred to as "antisense nucleic acid") has the following uses.

(1) Cell differentiation and / or metabolic regulators Musclin is secreted and expressed almost exclusively in skeletal muscle, and is highly expressed in diabetic model mice and refeeding after fasting. (I) cell differentiation and / or (ii) metabolism (eg, liver, adipose tissue, skeletal muscle, pancreatic islet) Clearly, it plays an important role in the regulation of cell differentiation and / or metabolism (eg, sugar, lipid, protein metabolism) in bone, cartilage and the like. Since NPR3 interacts with muskrin to transmit signals related to cell differentiation and / or metabolism, use a) NPR3 or b) DNA encoding NPR3 as a regulator of cell differentiation and / or metabolism Can do.
For example, when there is a patient who cannot expect the physiological effect of the maskulin ligand (NPR3 deficiency) because NPR3 is decreased in vivo, a) NPR3 is administered to the patient and the amount of NPR3 is supplemented. Or b) (b) by administering and expressing DNA encoding NPR3 to the patient, or (b) introducing and expressing DNA encoding NPR3 into the target cell, Can be increased to increase the amount of NPR3 in the patient's body and to fully exert the action of muskrin. That is, NPR3s and DNAs encoding them are useful as preventive and / or therapeutic agents for diseases involving dysfunctional NPR3 that are safe and low in toxicity. Diseases involving NPR3 dysfunction include cell differentiation and / or metabolic abnormalities, preferably cell differentiation and / or metabolism in liver, adipose tissue, skeletal muscle, pancreatic islets, bones, cartilage, etc., preferably sugar, lipid and protein metabolism More specifically, for example, obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, hyperlipidemia, metabolic bone / cartilage disease (eg, osteoporosis, osteomalacia, rickets) , Fibroosteopathies, renal osteodystrophy, osteophetotic disease, ankylosing spinal cord inflammation, marble bone disease, osteoarthritis, rheumatoid arthritis, etc.), but are not limited thereto.

NPR3 and DNA encoding NPR3 (hereinafter sometimes referred to as “DNA of the present invention”) are mixed with a pharmacologically acceptable carrier as necessary to obtain a pharmaceutical composition, and then the above-mentioned pharmaceutical Can be used as
Here, as a pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations , Solubilizers, suspending agents, isotonic agents, buffers, soothing agents and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used.
Suitable examples of excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, light Examples thereof include anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminate metasilicate.
Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Examples thereof include propylmethylcellulose and polyvinylpyrrolidone.
Preferable examples of the disintegrant include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, light anhydrous silicic acid, low substituted hydroxypropyl cellulose and the like.
Preferable examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
Preferable examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. Etc.
Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate; for example, polyvinyl alcohol, polyvinyl Examples include hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like.
Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
Preferable examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate.
Preferable examples of the soothing agent include benzyl alcohol.
Preferable examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Preferable examples of the antioxidant include sulfite and ascorbate.
Suitable examples of colorants include water-soluble food tar dyes (eg, food red Nos. 2 and 3, food yellow Nos. 4 and 5, food blue Nos. 1 and 2), water-insoluble lake dyes ( Examples include aluminum salts of the aforementioned water-soluble edible tar pigments, natural pigments (eg, β-carotene, chlorophyll, Bengala, etc.).
Preferable examples of the sweetening agent include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.

Examples of the dosage form of the pharmaceutical composition include oral preparations such as tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions and suspensions; and injections (eg, subcutaneous injection). Preparations, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), external preparations (eg, nasal preparations, transdermal preparations, ointments, etc.), suppositories (eg, rectal suppositories, vaginal suppositories) Etc.), pellets, drops, sustained-release preparations (eg, sustained-release microcapsules, etc.) and the like.
The pharmaceutical composition can be produced by a method commonly used in the field of pharmaceutical technology, such as the method described in the Japanese Pharmacopoeia. Below, the specific manufacturing method of a formulation is explained in full detail. The content of the active ingredient in the pharmaceutical composition varies depending on the dosage form, the dose of the active ingredient, etc., but is, for example, about 0.1 to 100% by weight.
For example, oral preparations contain active ingredients, excipients (eg, lactose, sucrose, starch, D-mannitol, etc.), disintegrants (eg, carboxymethylcellulose calcium, etc.), binders (eg, pregelatinized starch, gum arabic) , Carboxymethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.) or a lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.), etc., and compression molded, and then if necessary, taste masking, enteric or For the purpose of durability, it is produced by coating with a coating base by a method known per se.

Examples of the coating base include sugar coating base, water-soluble film coating base, enteric film coating base, sustained-release film coating base and the like.
As the sugar coating base, sucrose is used, and one or more selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.
Examples of the water-soluble film coating base include cellulose polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name), Rohm Pharma Co., Ltd.], synthetic polymers such as polyvinylpyrrolidone; polysaccharides such as pullulan.
Examples of enteric film coating bases include cellulose-based polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L [Eudragit L (trade name), Acrylic polymers such as Rohm Pharma Co.], methacrylic acid copolymer LD [Eudragit L-30D55 (trade name), Rohm Pharma Co., Ltd.], and methacrylic acid copolymer S [Eudragit S (trade name), Rohm Pharma Co., Ltd.]; Examples include natural products such as shellac.
Examples of sustained-release film coating bases include cellulose polymers such as ethyl cellulose; aminoalkyl methacrylate copolymer RS [Eudragit RS (trade name), Rohm Pharma Co., Ltd.], ethyl acrylate / methyl methacrylate copolymer suspension Examples thereof include acrylic acid polymers such as suspensions (Eudragit NE (trade name), Rohm Pharma).
Two or more of the coating bases described above may be mixed and used at an appropriate ratio. Moreover, you may use light-shielding agents, such as a titanium oxide, ferric oxide, etc. in the case of coating.

  For injections, the active ingredient is a dispersant (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc.), preservative (eg, methyl paraben, propyl paraben, benzyl alcohol, chlorobutanol, Phenol, etc.), isotonic agents (eg, sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.) and other aqueous solvents (eg, distilled water, physiological saline, Ringer's solution, etc.) or oily solvents (eg, , Olive oil, sesame oil, cottonseed oil, vegetable oils such as corn oil, propylene glycol, etc.) and the like. At this time, additives such as a solubilizer (eg, sodium salicylate, sodium acetate, etc.), a stabilizer (eg, human serum albumin, etc.), a soothing agent (eg, benzyl alcohol, etc.) may be used as desired. The injection solution is usually filled in a suitable ampoule.

Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). .
When the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the DNA of the present invention alone or after being inserted into an appropriate expression vector such as a retrovirus vector, adenovirus vector, adeno-associated virus vector, etc. It can also be administered. In addition, the DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting intake by a gene gun or a catheter such as a hydrogel catheter.

The dose of NPR3 varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, in patients with abnormal sugar / lipid metabolism (with a body weight of 60 kg) About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.
The dose of the DNA of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, in patients with abnormal sugar / lipid metabolism (weight 60 kg) About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.

  On the other hand, an antibody against NPR3 (the antibody of the present invention) inhibits the interaction between muskrin and NPR3 to inactivate (ie neutralize) a signal transduction function involving NPR3, such as cell differentiation and / or metabolic regulatory activity. )can do. On the other hand, nucleic acids (including ribozymes and siRNAs; antisense nucleic acids of the present invention) containing a base sequence complementary to the base sequence encoding NPR3 or a part thereof, NPR3 gene transcription, transcript processing and / or mRNA NPR3 expression can be inhibited by blocking translation from Accordingly, (1) the antibody of the present invention or (2) the antisense nucleic acid of the present invention is used as a cell differentiation and / or metabolic regulator opposite to the above NPR3 or the DNA of the present invention, for example, an excess of NPR3. It can be used as a medicine such as a prophylactic / therapeutic agent for diseases related to expression. Diseases involving overexpression of NPR3 include cell differentiation and / or metabolic abnormalities, preferably cell differentiation and / or metabolism in liver, adipose tissue, skeletal muscle, islet, bone, cartilage, etc., preferably sugar / lipid / protein metabolism More specifically, examples include, but are not limited to, obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, hyperlipidemia, metabolic bone / cartilage disease, and the like.

The antibody of the present invention and the antisense nucleic acid of the present invention can be formulated in the same manner as the NPR3 and the DNA of the present invention, respectively. In addition, the antisense nucleic acid can be administered as it is through a catheter such as a gene gun or a hydrogel catheter.
The dose of the antibody of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, generally, for example, in patients with abnormal sugar / lipid metabolism (weight 60 kg), About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.
Although the dosage of the antisense nucleic acid of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., in the case of oral administration, generally, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) Is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.

(2) Gene diagnostic agent
The nucleic acid encoding NPR3 or a part thereof (hereinafter sometimes referred to as “sense nucleic acid of the present invention”) and the antisense nucleic acid of the present invention can be used as a probe or primer to produce a human or mammal (eg, rat DNA, or mRNA abnormalities (gene abnormalities) encoding NPR3 in mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.) can be detected. It is useful as a genetic diagnostic agent for mutation or decreased expression, increased or excessive expression of the DNA or mRNA.
The above gene diagnosis using the sense or antisense nucleic acid of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874 to 879 (1989), processing. Of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)) be able to.
For example, if a decrease in the expression of NPR3 is detected by Northern hybridization or the like, for example, it is diagnosed that there is a high possibility of suffering from a disease involving NPR3 dysfunction or a high possibility of suffering in the future be able to.
In addition, when overexpression of NPR3 is detected by Northern hybridization or the like, for example, it is diagnosed that there is a high possibility of suffering from a disease involving overexpression of NPR3, or a high possibility of suffering in the future be able to.
Diseases involving dysfunction or overexpression of NPR3 include, for example, cell differentiation and / or metabolic abnormality, preferably cell differentiation and / or metabolism in liver, adipose tissue, skeletal muscle, islet, bone, cartilage, etc., preferably Abnormalities in sugar / lipid / protein metabolism, more specifically, for example, obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, hyperlipidemia, metabolic bone / cartilage disease and the like.

(3) Screening of compounds that change the expression level of NPR3 The sense and antisense nucleic acids of the present invention can be used for screening compounds that change the expression level of NPR3 by using them as probes or primers.
That is, the present invention includes, for example, (i) a non-human mammal (1) blood, (2) a specific organ, (3) a tissue or cell isolated from the organ, or (ii) a transformant. Provided is a method for screening a compound that changes the expression level of NPR3 by measuring the amount of NPR3 mRNA.

Specifically, the amount of NPR3 mRNA is measured as follows.
(I) Normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc. More specifically, obese mouse, diabetic mouse, hypertensive rat, arteriosclerosis Rabbit, cancer-bearing mouse, osteoporosis mouse, rheumatoid arthritis mouse, etc.) (for example, anti-obesity drugs, anti-diabetic drugs, antihypertensive drugs, vasoactive drugs, anti-cancer drugs, anti-osteoporosis drugs, anti-rheumatic drugs) Apply physical stress (eg, water stress, electric shock, light / dark, low temperature, etc.), and after a certain period of time, blood or specific organs (eg, liver, pancreas, kidneys, muscles, bones, joints, etc.) Tissue (eg, brown or white adipose tissue, pancreatic islet, bone tissue, cartilage tissue, etc.) or cells (skeletal muscle cells, adipocytes, pancreatic endocrine cells, hepatocytes, kidney cells) Bone cells, osteoblasts, get osteoclasts, chondrocytes, chondroblasts, such as cartilage absorption cells).
NPR3 mRNA contained in the obtained cells and the like can be quantified by, for example, extracting mRNA from the cells and the like by a normal method and using a technique such as TaqManPCR, and by Northern blotting by means known per se. It is also possible to analyze by performing.
(Ii) A transformant expressing NPR3 or a partial peptide thereof can be prepared according to the above-described method, and NPR3 or the partial peptide mRNA contained in the transformant can be quantified and analyzed in the same manner.

Screening for compounds that alter the expression level of NPR3
(I) A certain time before giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour) Test compound before or 6 hours before or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or simultaneously with drug or physical stress Quantitative analysis of NPR3 mRNA contained in cells after a certain period of time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours) Can be done by
(Ii) When the transformant is cultured according to a conventional method, the test compound is mixed in the medium and cultured for a certain period of time (after 1 to 7 days, preferably 1 to 3 days, more preferably 2 to 3 days). Day later), the amount of mRNA of NPR3 or a partial peptide thereof contained in the transformant can be determined and analyzed.

The compound obtained by using the above screening method or a salt thereof is a compound having an action of changing the expression level of NPR3. Specifically, (i) by increasing the expression level of NPR3, Cell stimulation activity (eg intracellular cAMP production, inositol phosphate production, Ca uptake, cGMP production, glucose uptake into cells, glycogen synthesis / degradation in cells, sugar release from cells, fat into cells) Uptake, fat release from cells, lipolysis in cells, fat synthesis in cells, regulation of protein / non-protein hormone secretion, regulation of protein / non-protein hormone action, regulation of appetite, regulation of momentum, weight fluctuation, blood (B) NPR3 expression level is a compound that enhances medium carbohydrate and lipid fluctuations, regulation of muscle cell differentiation / proliferation, osteoblast differentiation / proliferation, etc. By causing little of a compound that decrease the cell-stimulating activity.
Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These compounds may be novel compounds or known compounds.
The compound that enhances the cell stimulating activity is useful as a safe and low-toxic pharmaceutical for enhancing the physiological activity of NPR3.
The compound that attenuates the cell stimulating activity is useful as a safe and low-toxic pharmaceutical for reducing the physiological activity of NPR3.

When the compound obtained by using the above screening method or a salt thereof is used as a medicine, it can be formulated in the same manner as the NPR3 class.
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). .
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally in patients with abnormal sugar / lipid metabolism (with a body weight of 60 kg) About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.

As described above, NPR3 is expressed specifically in skeletal muscles and functions as a receptor for musclin, which is highly expressed in diabetic model mice and refeeding after fasting, and is expressed in bone and may affect bone and cartilage metabolism. (I) cell differentiation and / or metabolism (for example, cell differentiation and / or metabolism in liver, adipose tissue, skeletal muscle, pancreatic islet, bone, cartilage, etc. (for example, sugar / lipid / protein metabolism) ) Is considered to play an important role. Therefore, a compound that changes the expression level of NPR3 can be used for (i) cell differentiation and / or metabolism (for example, cell differentiation and / or metabolism in liver, adipose tissue, skeletal muscle, islet, bone, cartilage, etc. (for example, , Sugar / lipid / protein metabolism)), preferably cell differentiation and / or metabolic abnormalities (specifically, for example, obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, hyperlipidemia) , Metabolic bone and cartilage diseases, etc.).
When the compound is used as the medicine, it can be formulated in the same manner as the NPR3 class.
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). .
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally in patients with abnormal sugar / lipid metabolism (with a body weight of 60 kg) About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.

(4) Determination of muskrin
Since NPR3 has binding property to muskrin, the concentration of muskrin in vivo can be quantified with high sensitivity.
The quantitative method of the present invention can be used, for example, in combination with a competitive method. That is, the concentration of musclin in the subject can be measured by bringing the subject into contact with the NPR3 class. Specifically, it can be used, for example, according to the method described in the following (1) or (2) or a method analogous thereto.
(1) Hiroshi Irie "Radioimmunoassay" (Kodansha, 1974)
(2) Hiroshi Irie “Continuing radioimmunoassay” (Kodansha, published in 1979)

(5) Screening for compounds (agonists, antagonists, etc.) that change the binding between NPR3 and muskrin
Compounds that change the binding of NPR3 and musclin by using NPR3s or constructing an expression system for recombinant NPR3 and using an affinity assay system using the expression system (for example, peptides, proteins, non-peptides) Sex compounds, synthetic compounds, fermentation products, etc.) or salts thereof can be efficiently screened.
Such compounds include (i) cell stimulating activity via NPR3 (eg, intracellular cAMP production, inositol phosphate production, Ca uptake, cGMP production, glucose uptake into cells, glycogen synthesis / degradation in cells, cell Sugar release from cells, fat uptake into cells, fat release from cells, lipolysis in cells, fat synthesis in cells, regulation of protein / non-protein hormone secretion, regulation of protein / non-protein hormone action, appetite (B) regulation of exercise amount, body weight fluctuation, blood sugar / lipid fluctuation, muscle cell differentiation / proliferation, osteoblast differentiation / proliferation, etc.) Compounds that do not have (antagonists), (c) compounds that increase the binding power between NPR3 and muskrin, or (d) compounds that decrease the binding power between NPR3 and muskrin included.
That is, the present invention comprises (i) a case where NPR3 is contacted with muskrin or a partial peptide thereof or a salt thereof (hereinafter collectively referred to as “musculins”), and (ii) NPR3, muskrin and Provided is a screening method for a compound or a salt thereof that changes the binding property between NPR3 and musclin, which is characterized by comparing with a test compound in contact with the test compound.
The screening method is characterized by measuring and comparing the amount of musclins bound to NPR3, cell stimulating activity, etc. in cases (i) and (ii).

More specifically, the present invention provides:
(1) Measure and compare the amount of labeled musclins bound to NPR3 when the labeled musclins are contacted with NPR3 and when the labeled musclins and test compounds are contacted with NPR3. A screening method for a compound or a salt thereof that changes the binding property between NPR3 and musclin,
(2) When the labeled muskrins are brought into contact with NPR3 producing cells or membrane fractions thereof, and when the labeled muskrins and test compounds are brought into contact with NPR3 producing cells or membrane fractions thereof A method for screening a compound or a salt thereof that changes the binding between NPR3 and muskrin, characterized by measuring and comparing the amount of labeled muskrins bound to the cell or membrane fraction,
(3) When the labeled muskrins are brought into contact with NPR3s expressed on the cell membrane by culturing a transformant containing the DNA of the present invention, the labeled muskrins and the test compound are converted to the DNA of the present invention. NPR3 and musclin, characterized by measuring and comparing the amount of labeled muskrins bound to NPR3s when contacted with NPR3s expressed on the cell membrane by culturing transformants containing A method for screening a compound or a salt thereof that changes the binding property of
(4) A compound that activates NPR3 (for example, muskulins) is contacted with a cell that expresses NPR3 on the cell membrane, and a compound that activates NPR3 and a test compound are expressed on the cell membrane. NPR3 mediated cell stimulating activity (eg, intracellular cAMP production, inositol phosphate production, Ca uptake, cGMP production, glucose uptake into cells, glycogen synthesis / degradation in cells, Sugar release, fat uptake into cells, fat release from cells, lipolysis in cells, fat synthesis in cells, protein / non-protein hormone secretion regulation, protein / non-protein hormone action regulation, appetite regulation Measurement, comparison of momentum regulation, body weight fluctuation, blood sugar and lipid fluctuation, muscle cell differentiation / proliferation, osteoblast differentiation / proliferation, etc. That method of screening a compound or its salt that alters the binding property between NPR3 and musclin, and
(5) A compound that activates NPR3 (eg, muskrins) is brought into contact with NPR3 expressed on the cell membrane by culturing a transformant containing the DNA of the present invention, and NPR3 is activated. NPR3-mediated cell stimulating activity (for example, intracellular cAMP production) when a compound to be converted and a test compound are contacted with NPR3s expressed on the cell membrane by culturing a transformant containing the DNA of the present invention , Inositol phosphate production, Ca uptake, cGMP production, glucose uptake into cells, glycogen synthesis / degradation in cells, sugar release from cells, fat uptake into cells, fat release from cells, lipolysis in cells, cells Fat synthesis, regulation of protein / non-protein hormone secretion, regulation of protein / non-protein hormone action, appetite regulation, momentum regulation, body weight fluctuation, blood sugar and lipid fluctuation Provided is a method for screening a compound or a salt thereof that alters the binding between NPR3 and muskrin, characterized by measuring and comparing muscle cell differentiation / proliferation, osteoblast differentiation / proliferation, etc. .

A specific description of the screening method of the present invention will be given below.
First, the NPR3 used in the screening method of the present invention may be any NPR3 or any of its partial peptides or salts thereof as long as it contains the above-mentioned NPR3. A cell membrane fraction is preferred. However, since human-derived organs are particularly difficult to obtain, human-derived NPR3 and the like that are expressed in large quantities using recombinants are suitable for use in screening.

In order to produce NPR3s, the above-described method is used, and it is preferable to carry out the expression of the DNA of the present invention in mammalian cells or insect cells. CDNA is used as a DNA fragment encoding the target protein portion, but is not necessarily limited thereto. For example, gene fragments or synthetic DNA may be used. In order to introduce a DNA fragment encoding NPR3 or a partial peptide thereof into a host animal (insect) cell and to express them efficiently, the DNA fragment can be expressed using an SV40-derived promoter, a retroviral promoter, a metallothionein promoter, human It is preferably incorporated downstream of a heat shock promoter, a cytomegalovirus promoter, an SRα promoter, a polyhedrin promoter of a nuclear polyhedrosis virus (NPV) belonging to baculoviruses using insects as hosts.
Therefore, the NPR3s used in the screening method of the present invention may be NPR3 purified according to a method known per se, or may be used as a cell producing NPR3s or a cell membrane fraction thereof.

In the above screening method, when cells that produce NPR3s are used, the cells may be fixed with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se.
A cell that produces NPR3 refers to a host cell that expresses NPR3. Examples of the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, and animal cells.
The cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a method known per se after disrupting cells. Cell disruption methods include crushing cells with a Potter-Elvehjem type homogenizer, disrupting with a Waring blender or polytron (manufactured by Kinematica), disrupting with ultrasonic waves, or pressurizing with a French press, etc. Crushing by things. For fractionation of the cell membrane, a fractionation method using centrifugal force such as a fractional centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is centrifuged at low speed (500 rpm to 3000 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours. Is the membrane fraction. The membrane fraction is rich in expressed NPR3s and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of NPR3s in the cell producing NPR3s and the membrane fraction thereof is preferably 10 ³ to 10 ⁸ molecules, more preferably 10 ⁵ to 10 ⁷ molecules per cell. In addition, the higher the expression level, the higher the maskin binding activity (specific activity) per membrane fraction, making it possible not only to construct a highly sensitive screening system, but also to measure a large amount of samples in the same lot. .

In order to carry out the above (1) to (3) for screening a compound that changes the binding property between NPR3 and musclin, for example, an appropriate NPR3 group-containing membrane fraction and labeled musclin are required.
As the NPR3-containing membrane fraction, a natural NPR3-containing membrane fraction or a recombinant NPR3-containing membrane fraction having an activity equivalent to that is desirable. Here, equivalent activity refers to equivalent musculin binding activity, signal information transmission action, and the like.
As labeled muskrins, proteins labeled as “NPR3” in Patent Document 1 or partial peptides thereof or salts thereof labeled according to a conventional method are used. For example, the protein labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like, or a partial peptide thereof or a salt thereof can be used.
Specifically, to screen for compounds that change the binding between NPR3 and muskrin, first the cells that produce NPR3s or their membrane fractions are suspended in a buffer suitable for screening. Prepare a sample. The buffer may be any buffer that does not inhibit the binding of NPR3 and muskrin, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a Tris-HCl buffer. For the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin, deoxycholate and the like can be added to the buffer. Furthermore, protease inhibitors such as PMSF, leupeptin, E-64 (manufactured by Peptide Institute) and pepstatin can be added for the purpose of suppressing the degradation of receptors and ligands by protease. A fixed amount (5000 cpm to 500000 cpm) of labeled musclin is added to 0.01 to ¹⁰ ml of NPR3 class suspension, and 10 ⁻⁴ M to ^{10 −10} M test compound is allowed to coexist at the same time. Prepare a reaction tube with a large excess of unlabeled ligand to determine the amount of non-specific binding (NSB). The reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., for about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and then the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. When the count (B ₀ -NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance is 100%, the specific binding amount (B-NSB) is, for example, The test compound that is 50% or less can be selected as a candidate substance capable of competitive inhibition.

In order to carry out the above methods (4) to (5) for screening a compound that changes the binding property between NPR3 and musclin, for example, NPR3-mediated cell stimulating activity (for example, intracellular cAMP production, inositol phosphorus) Acid production, Ca uptake, cGMP production, glucose uptake into cells, glycogen synthesis / degradation in cells, sugar release from cells, fat uptake into cells, fat release from cells, lipolysis in cells, fat in cells Synthesis, regulation of protein / non-protein hormone secretion, regulation of protein / non-protein hormone action, regulation of appetite, regulation of momentum, body weight fluctuation, blood sugar / lipid fluctuation, muscle cell differentiation / proliferation, in osteoblasts Regulation of differentiation / proliferation and the like) can be measured using a known method or a commercially available measurement kit.
Specifically, first, cells that produce NPR3s are cultured in a multiwell plate or the like. In screening, replace with fresh medium or an appropriate buffer that is not toxic to cells in advance, add test compounds, etc. and incubate for a certain period of time, then extract cells or collect supernatant and generate The product is quantified according to the respective method. When the production of a substance used as an index of cell stimulating activity is difficult to assay with a degrading enzyme contained in cells, an assay may be performed with an inhibitor for the degrading enzyme added. Further, the activity such as cAMP production suppression can be detected as a production suppression effect on the cells whose basic production amount has been increased with forskolin or the like.
In order to perform screening by measuring cell stimulating activity, appropriate cells expressing NPR3s on the membrane are required. The cells expressing NPR3 are preferably a cell line producing natural NPR3, a cell line expressing the aforementioned recombinant NPR3, and the like.
Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like, and these compounds may be novel compounds. It may be a known compound.

A screening kit for a compound that changes the binding property between NPR3 and musclin or a salt thereof contains NPR3s, cells that produce NPR3s, or membrane fractions thereof.
Examples of the screening kit of the present invention include the following.
1. Screening reagents
(1) Measurement buffer and washing buffer
Hanks' Balanced Salt Solution (Gibco) plus 0.05% bovine serum albumin (Sigma).
The solution is sterilized by filtration through a 0.45 μm filter and stored at 4 ° C. or may be prepared at the time of use.
(2) NPR3 standard products
CHO cells expressing NPR3s were subcultured at 5 × 10 ⁵ cells / well in a 12-well plate and cultured for 2 days at 37 ° C., 5% CO ₂ , 95% air.
(3) labeling musclin such commercial ^[3 H], and stored at ^[125 I], ^[14 C], those states of musclin such solution labeled with a ^[35 S] 4 ° C. or -20 ° C., At the time of use, it is diluted to 1 μM with a measuring buffer.
(4) Musculin Standard Solution Dissolve musclin to 1 mM in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) and store at -20 ° C.

2. Measurement method
(1) NPR3 class-expressing CHO cells cultured in a 12-well tissue culture plate are washed twice with 1 ml of measurement buffer, and 490 μl of measurement buffer is added to each well.
(2) After 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M is added, 5 μl of labeled musclin is added and allowed to react at room temperature for 1 hour. To determine the amount of non-specific binding, add 5 μl of 10 ⁻³ M muskulin standard solution instead of the test compound.
(3) Remove the reaction solution and wash 3 times with 1 ml of washing buffer. Labeled musclins bound to cells (or plates) are dissolved in 0.2N NaOH-1% SDS and mixed with 4 ml of liquid scintillator A (Wako Pure Chemical Industries).
(4) The radioactivity is measured using a liquid scintillation counter (manufactured by Beckman), and the Percent Maximum Binding (PMB) is determined by the following equation [Equation 1].

[Equation 1]
PMB = [(B-NSB) / (B ₀ -NSB)] × 100
PMB: Percent Maximum Binding
B: Value when the sample is added
NSB: Non-specific binding
B ₀ : Maximum binding amount

A compound obtained by using the screening method or screening kit or a salt thereof is a compound having an action of changing the binding property between NPR3 and musclin, and specifically, (a) via the musclin-NPR3 interaction. Cell stimulating activity (eg, intracellular cAMP production, inositol phosphate production, Ca uptake, cGMP production, sugar uptake into cells, glycogen synthesis / degradation in cells, sugar release from cells, fat uptake into cells, from cells Fat release, cell lipolysis, cell fat synthesis, protein / non-protein hormone secretion regulation, protein / non-protein hormone action regulation, appetite regulation, exercise quantity regulation, body weight fluctuation, blood sugar Compounds (agonists) having lipid fluctuation, muscle cell differentiation / proliferation, osteoblast differentiation / proliferation, etc.) (b) Compound having no cell stimulating activity (antagonist), a (c) compound that potentiates the binding affinity of the NPR3 and musclin, or (d) a compound that decreases the binding force between NPR3 and musclin.
Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These compounds may be novel compounds or known compounds.
Since the agonist for NPR3 has the same action as the physiological activity of musclin for NPR3, it is useful as a safe and low toxic pharmaceutical according to the musclin activity.
Since antagonists to NPR3 can suppress the physiological activity of musclin for NPR3, they are useful as safe and low-toxic drugs that suppress musclin activity.
A compound that enhances the binding power between NPR3 and musclin is useful as a safe and low toxic pharmaceutical for enhancing the physiological activity of musclin against NPR3.
A compound that decreases the binding power between NPR3 and muskrin is useful as a safe and low-toxic pharmaceutical for reducing the physiological activity of muskrin against NPR3.

(6) Preventive / therapeutic agents for various diseases containing compounds (agonists, antagonists) that change the binding between NPR3 and muskrin
As described above, NPR3 is expressed specifically in skeletal muscles and functions as a receptor for musclin, which is highly expressed in diabetic model mice and refeeding after fasting, and is expressed in bone and may affect bone and cartilage metabolism. (I) cell differentiation and / or metabolism (for example, cell differentiation and / or metabolism in liver, adipose tissue, skeletal muscle, pancreatic islet, bone, cartilage, etc. (for example, sugar / lipid / protein metabolism) ) Is considered to play an important role. Accordingly, compounds (agonists, antagonists) that alter the binding between NPR3 and muskrin are (i) cell differentiation and / or (ii) metabolism (eg in liver, adipose tissue, skeletal muscle, islet, bone, cartilage, etc.) As a regulator of cell differentiation and / or metabolism (for example, sugar / lipid / protein metabolism), preferably cell differentiation and / or metabolic abnormality (specifically, for example, obesity, diabetes, impaired glucose tolerance, arteries) Sclerosis, hypertension, hyperlipidemia, metabolic bone / cartilage disease, etc.).
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). .
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally in patients with abnormal sugar / lipid metabolism (with a body weight of 60 kg) About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.

(7) Quantification of NPR3 Since the antibody of the present invention can specifically recognize NPR3, it can be used for quantification of NPR3 in a test solution, particularly quantification by sandwich immunoassay. . That is, the present invention is characterized by, for example, (i) competitively reacting the antibody of the present invention with a test solution, a labeled protein, etc., and measuring the ratio of the labeled protein, etc. bound to the antibody. (Ii) after reacting the test solution with the antibody of the present invention insolubilized on the carrier and the labeled antibody of the present invention simultaneously or continuously, Provided is a method for quantifying NPR3s in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier.
In the above (ii), it is preferable that the insolubilized antibody and the labeled antibody have an antigen recognition site that does not interfere with the binding to NPR3 (for example, one antibody recognizes the N-terminal part of NPR3. The other antibody reacts with the C-terminal of NPR3 class).

In addition to measuring NPR3 using a monoclonal antibody against NPR3 (hereinafter sometimes referred to as “monoclonal antibody of the present invention”), detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used. The measurement method using an antibody against NPR3 is not particularly limited, and the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (for example, NPR3 amount) in the liquid to be measured is chemically or Any measurement method may be used as long as it is a measurement method that is detected by physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.

In the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), the labeled monoclonal antibody of the present invention is reacted (secondary reaction), and then the labeling agent on the insolubilized carrier By measuring the activity of NPR3, the amount of NPR3 in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. The labeling agent and the insolubilizing method can be based on those described above.
In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
In the method for measuring NPR3s by the sandwich method, the monoclonal antibodies of the present invention used for the primary reaction and the secondary reaction are preferably antibodies having different sites for binding to the NPR3s. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal part of NPR3s, the antibody used in the primary reaction is preferably For example, an antibody that recognizes the N-terminal is used.

The monoclonal antibody of the present invention can be used in measurement systems other than the sandwich method, for example, competitive method, immunometric method, nephrometry, and the like. In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen, (F), and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, a B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or As the first antibody, a soluble one is used, and a solid phase method using a solid phase antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test solution is separated. Are reacted with an excess amount of labeled antibody, and then a solid phased antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.

When these individual immunoassays are applied to the quantification of NPR3 species, no special conditions, procedures, etc. are required. A measurement system for NPR3s may be constructed by adding ordinary technical considerations to those skilled in the art to the normal conditions and operation methods in each method. For details of these general technical means, refer to reviews, books, etc. [For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, issued in 1974), Hiroshi Irie “Continuing radioimmunoassay” (Kodansha, published in 1979), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Medical School, published in 1978), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Second Edition) (Medical School, 1982) Published by Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical School, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques ( Part A)), Vol. 73 (Immunochemical Techniques (Part B)), ibid Vol. 74 (Immunochemical Techniques (Part C)), ibid Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoas say Methods)), Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (published by Academic Press).
As described above, NPR3s can be quantified with high sensitivity by using the antibody of the present invention.
Furthermore, by quantifying NPR3 or a salt thereof in vivo using the antibody of the present invention, various diseases associated with NPR3 dysfunction or enhancement can be diagnosed.
The antibody of the present invention can be used for specifically detecting NPR3 or a salt thereof present in a subject such as a body fluid or a tissue. Further, it can be used for production of an antibody column used for purifying NPR3s, detection of NPR3s in each fraction at the time of purification, analysis of NPR3 behavior in test cells, and the like.

(8) Screening for a compound that changes the amount of NPR3 in the cell membrane Since the antibody of the present invention can specifically recognize NPR3s, it can be used for screening for a compound that changes the amount of NPR3 in the cell membrane.
That is, the present invention is, for example,
(I) After destroying (1) blood, (2) specific organs, (3) tissues or cells isolated from organs of non-human mammals, the cell membrane fraction is isolated and contained in the cell membrane fraction A method for screening a compound that changes the amount of NPR3 in a cell membrane by quantifying NPR3,
(Ii) A compound that alters the amount of NPR3 in the cell membrane by destroying transformants that express NPR3 or a partial peptide thereof, then isolating the cell membrane fraction and quantifying NPR3s contained in the cell membrane fraction Screening method,
(Iii) After sectioning non-human mammal (1) blood, (2) specific organs, (3) tissues or cells isolated from organs, by using immunostaining, A method for screening a compound that changes the amount of NPR3 in the cell membrane by quantifying the degree of staining of NPR3, and confirming NPR3 on the cell membrane,
(Iv) NPR3s on the cell membrane can be obtained by quantifying the degree of staining of NPR3s on the cell surface by using immunostaining after sectioning transformants that express NPR3 or its partial peptides. A method for screening a compound that changes the amount of NPR3s in the cell membrane is provided.

Specifically, NPR3s contained in the cell membrane fraction are quantified as follows.
(I) Normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc. More specifically, obese mouse, diabetic mouse, hypertensive rat, arteriosclerosis Rabbits, cancer-bearing mice, osteoporosis mice, rheumatoid arthritis mice, etc.) (for example, anti-obesity drugs, anti-diabetic drugs, antihypertensive drugs, vasoactive drugs, anti-cancer drugs, anti-osteoporosis drugs, anti-rheumatic drugs) Apply physical stress (eg, water stress, electric shock, light and dark, low temperature, etc.), and after a certain period of time, blood or specific organs (eg, liver, kidney, pancreas, muscle, bone, joint, etc.), Tissue (eg, brown or white adipose tissue, islet, bone tissue, cartilage tissue, etc.) or cells (eg, adipocytes, muscle cells, pancreatic endocrine cells, hepatocytes, Obtain cells, bone cells, osteoblasts, osteoclasts, chondrocytes, chondroblasts, such as cartilage absorption cells). The obtained cells and the like are suspended in, for example, an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, HEPES buffer, etc.), and a surfactant (for example, Triton X-100 ^™ , Tween 20). ^The cells and the like are disrupted using ^TM or the like, and a cell membrane fraction is obtained using techniques such as centrifugation, filtration, and column fractionation.

  The cell membrane fraction refers to a fraction containing a lot of cell membranes obtained by a method known per se after disrupting cells. Cell disruption methods include crushing cells with a Potter-Elvehjem type homogenizer, disrupting with a Waring blender or polytron (manufactured by Kinematica), disrupting with ultrasonic waves, or pressurizing with a French press, etc. Crushing by things. For fractionation of the cell membrane, a fractionation method using centrifugal force such as a fractional centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is centrifuged at low speed (500 rpm to 3000 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours. Is the membrane fraction. The membrane fraction is rich in NPR3s and membrane components such as cell-derived phospholipids and membrane proteins.

NPR3 contained in the cell membrane fraction can be quantified by, for example, sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like.
Such a sandwich immunoassay can be performed in the same manner as described above, and Western blotting can be performed by means known per se.

(Ii) A transformant expressing NPR3 or a partial peptide thereof can be prepared according to the method described above, and NPR3s contained in the cell membrane fraction can be quantified.

Screening for compounds that alter the amount of NPR3 in the cell membrane
(I) A certain time before giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour) Test compound before or 6 hours before or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or simultaneously with drug or physical stress And after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours) after administration, the amount of NPR3 in the cell membrane is quantified. It is possible,
(Ii) When the transformant is cultured according to a conventional method, the test compound is mixed in the medium and cultured for a certain period of time (after 1 to 7 days, preferably 1 to 3 days, more preferably 2 to 3 days). Day later), it can be carried out by quantifying the amount of NPR3s in the cell membrane.

Specifically, NPR3 species contained in the cell membrane fraction are confirmed as follows.
(Iii) Normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc. More specifically, obese mouse, diabetic mouse, hypertensive rat, arteriosclerosis Rabbits, cancer-bearing mice, osteoporosis mice, rheumatoid arthritis mice, etc.) (for example, anti-obesity drugs, anti-diabetic drugs, antihypertensive drugs, vasoactive drugs, anti-cancer drugs, anti-osteoporosis drugs, anti-rheumatic drugs) Apply physical stress (eg, water stress, electric shock, light and dark, low temperature, etc.), and after a certain period of time, blood or specific organs (eg, liver, kidney, pancreas, muscle, bone, joint, etc.), Tissue (eg, brown or white adipose tissue, islet, bone tissue, cartilage tissue, etc.) or cells (eg, adipocytes, muscle cells, pancreatic endocrine cells, hepatocytes, Obtain cells, bone cells, osteoblasts, osteoclasts, chondrocytes, chondroblasts, such as cartilage absorption cells). The obtained cells and the like are made into tissue sections according to a conventional method, and immunostaining is performed using the antibody of the present invention. By quantifying the degree of staining of NPR3 on the cell surface layer, the amount of NPR3s in the cell membrane can be confirmed quantitatively or qualitatively by confirming NPR3 on the cell membrane.
(Iv) It can also be confirmed by taking the same means using a transformant expressing NPR3 or a partial peptide thereof.

The compound obtained by using the above screening method or a salt thereof is a compound having an action of changing the amount of NPR3 in the cell membrane. Specifically, (i) by increasing the amount of NPR3 in the cell membrane, Cell stimulation activity through NPR3 interaction (eg, intracellular cAMP production, inositol phosphate production, Ca uptake, cGMP production, sugar uptake into cells, glycogen synthesis / degradation in cells, sugar release from cells, fat into cells Uptake, fat release from cells, lipolysis in cells, fat synthesis in cells, regulation of protein / non-protein hormone secretion, regulation of protein / non-protein hormone action, regulation of appetite, regulation of momentum, weight fluctuation, blood Compounds that enhance medium carbohydrate and lipid fluctuation, muscle cell differentiation / proliferation, osteoblast differentiation / proliferation, etc.) ) A compound that attenuates the cell stimulating activity by reducing the amount of NPR3 in the cell membrane.
Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. These compounds may be novel compounds or known compounds.
The compound that enhances the cell stimulating activity is useful as a safe and low-toxic pharmaceutical for enhancing the physiological activity of NPR3.
The compound that attenuates the cell stimulating activity is useful as a safe and low-toxic pharmaceutical for reducing the physiological activity of NPR3.

(9) Prophylactic / therapeutic agent for various diseases containing a compound that changes the amount of NPR3 in the cell membrane
As described above, NPR3 is expressed specifically in skeletal muscles and functions as a receptor for musclin, which is highly expressed in diabetic model mice and refeeding after fasting, and is expressed in bone and may affect bone and cartilage metabolism. (I) cell differentiation and / or metabolism (for example, cell differentiation and / or metabolism in liver, adipose tissue, skeletal muscle, pancreatic islet, bone, cartilage, etc. (for example, sugar / lipid / protein metabolism) ) Is considered to play an important role. Thus, a compound that alters the amount of NPR3 in the cell membrane is (i) cell differentiation and / or (ii) metabolism (eg, cell differentiation and / or metabolism in liver, adipose tissue, skeletal muscle, islets, bone, cartilage, etc. ( For example, as a regulator of sugar / lipid / protein metabolism), preferably cell differentiation and / or metabolic abnormality (specifically, for example, obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, hyperlipidemia) Disease, metabolic bone / cartilage disease, etc.).
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). .
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally in patients with abnormal sugar / lipid metabolism (with a body weight of 60 kg) About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.

(10) Production of non-human transgenic animal having DNA encoding NPR3 The present invention is a DNA encoding exogenous NPR3 (hereinafter abbreviated as “exogenous DNA of the present invention”) or a mutant DNA thereof (“ There is provided a non-human mammal having a case that may be abbreviated as “exogenous mutant DNA of the present invention”.
That is, the present invention
[1] A non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
[2] The animal according to [1], wherein the non-human mammal is a rodent
[3] An animal according to [2], wherein the rodent is a mouse or a rat, and [4] a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. Is.
A non-human mammal having an exogenous DNA of the present invention or a mutant DNA thereof (hereinafter abbreviated as a DNA-transferred animal of the present invention) can be used for embryos including unfertilized eggs, fertilized eggs, spermatozoa and primordial cells thereof Preferably, at the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, aggregation method, It can be produced by transferring the target DNA by the microinjection method, particle gun method, DEAE-dextran method, etc. Further, by the DNA transfer method, the target exogenous DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, etc., and can be used for cell culture, tissue culture, etc. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned embryo cells with a cell fusion method known per se.
Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like. Among them, rodents, especially mice (for example, C57BL / 6 strains, DBA2 strains, etc. as pure strains), which have relatively short ontogeny and biological cycle in terms of the creation of disease animal model systems, and are easy to reproduce As the system, B6C3F ₁ system, BDF ₁ system, B6D2F ₁ system, BALB / c system, ICR system, etc.) or rats (for example, Wistar, SD, etc.) are preferable.
Examples of the “mammal” in the recombinant vector that can be expressed in the mammal include humans and the like in addition to the above non-human mammals.

The exogenous DNA of the present invention is not the DNA of the present invention inherently possessed by a non-human mammal but the DNA of the present invention once isolated and extracted from the mammal.
The mutated DNA of the present invention is one in which a mutation (for example, mutation) occurs in the base sequence of the original DNA of the present invention, specifically, addition or deletion of a base, substitution with another base, etc. DNA in which stagnation occurs is used, and abnormal DNA is also included.
The abnormal DNA means DNA that expresses abnormal NPR3. For example, DNA that expresses a protein or the like that suppresses the function of normal NPR3 is used.
The exogenous DNA of the present invention may be derived from mammals of the same species or different species from the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct bound downstream of a promoter that can be expressed in animal cells. For example, when the human DNA of the present invention is transferred, DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto is expressed. The DNA of the present invention is highly expressed by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters into a fertilized egg of a target mammal, such as a mouse fertilized egg. DNA transfer mammals can be created.

Examples of the expression vector carrying the DNA of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, vaccinia viruses and baculoviruses. Animal viruses are used. Of these, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferably used.
Examples of promoters that regulate DNA expression include (1) promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney murine leukemia virus, JC virus, breast cancer virus, poliovirus, etc.) 2) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillarity Acidic protein, glutathione S-transferase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, tartrate-resistant alkaline phosphatase, Atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine triphosphate (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin, α and β myosin heavy chain, Myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A, vasopressin, etc. Or the like. Among these, the cytomegalovirus promoter capable of being highly expressed throughout the body, the human peptide chain elongation factor 1α (EF-1α) promoter, the human and chicken β-actin promoters, and the like are suitable.
The vector preferably has a sequence (generally referred to as a terminator) that terminates transcription of a target messenger RNA in a DNA-transferred mammal. For example, the sequence of each DNA derived from viruses and various mammals The SV40 terminator of simian virus or the like is preferably used.

In addition, the splicing signal of each DNA, enhancer region, part of intron of eukaryotic DNA, etc. 5 'upstream of the promoter region, between the promoter region and the translation region, or the translation region It is also possible to connect it 3 'downstream of the target.
Normal NPR3 translation regions include liver, kidney, adipose tissue, muscle-derived DNA from various mammals (eg, humans, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and various commercially available genomic DNA live sequences. The cDNA can be obtained from the rally as all or part of the genomic DNA, or cDNA prepared by a known method from the RNA derived from the organ or tissue. In addition, exogenous abnormal DNA can be obtained by preparing a translation region obtained by mutating a normal NPR3 translation region 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 metastasized animal by an ordinary DNA engineering technique in which it is linked downstream of the above promoter (and optionally upstream of the transcription termination site).
The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the foreign DNA of the present invention in the embryo cells of the produced animal after the DNA transfer indicates that all the progeny of the produced animal retain the foreign DNA of the present invention in all the germ cells and somatic cells. means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the foreign DNA of the present invention in all of its germ cells and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. .
The transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be excessively present in all germ cells and somatic cells of the target mammal. Excessive exogenous DNA of the present invention is present in the embryo cells of the produced animal after DNA transfer. This means that all the offspring of the produced animal have the exogenous DNA of the present invention in all of the germ cells and somatic cells. Means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has an excess of the foreign DNA of the present invention in all of its germ cells and somatic cells.
By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, it is possible to breed and pass all the offspring to have the DNA in excess.

The non-human mammal having the normal DNA of the present invention has high expression of the normal DNA of the present invention, and eventually develops NPR3 hyperfunction by enhancing the function of the endogenous normal DNA. And can be used as a model animal for the disease state. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathologic mechanism of NPR3 hyperfunction and diseases related to NPR3 and to examine methods for treating these diseases.
Moreover, since the mammal to which the foreign normal DNA of the present invention has been transferred has an increased symptom of free NPR3, it can be used in screening tests for therapeutic agents for diseases related to NPR3.
On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. Furthermore, the target foreign DNA can be incorporated into the aforementioned plasmid and used as a raw material. A DNA construct with a promoter can be prepared by a usual DNA engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germ cell of the produced animal after the DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all the germ cells and somatic cells. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred to have the DNA.

The non-human mammal having the abnormal DNA of the present invention has a high expression of the abnormal DNA of the present invention. Can be used as a model animal for the disease state. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of NPR3 function inactive refractory and to examine a method for treating this disease.
Further, as a specific applicability, the abnormal DNA highly expressing animal of the present invention serves as a model for elucidating the function inhibition (dominant negative action) of normal NPR3 by abnormal NPR3 in NPR3 functional inactive refractory disease.
Moreover, since the mammal to which the foreign abnormal DNA of the present invention is transferred has an increased symptom of free abnormal NPR3, it can also be used in a therapeutic drug screening test for NPR3 functional inactive refractory disease.
In addition, as other applicability of the above two kinds of DNA transfer animals of the present invention, for example,
(1) use as a cell source for tissue culture,
(2) Regarding the relationship with a protein or the like that is specifically expressed or activated by NPR3 by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing produced NPR3 Analysis,
(3) Cell of tissue having DNA is cultured by standard tissue culture technique, and the function of cells from tissues that are generally difficult to culture using these,
(4) Screening for drugs that enhance cell function by using the cells described in (3) above, and
(5) Isolation and purification of mutant NPR3 and antibody production thereof may be considered.
Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of NPR3-related diseases, including NPR3 functional inactive refractories, etc., and in each organ of a disease model related to NPR3 More detailed pathological findings can be obtained, which can contribute to the development of new treatment methods and to the study and treatment of secondary diseases caused by the diseases.
Further, each organ can be taken out from the DNA-transferred animal of the present invention, and after chopping, it is possible to obtain a free DNA-transferred cell, culture it or systematize the cultured cell with a proteolytic enzyme such as trypsin. . In addition, it is possible to investigate the relationship between NPR3 producing cell specification, apoptosis, differentiation or proliferation, or the signal transduction mechanism in them, and investigate their abnormalities. Become.
Furthermore, in order to develop therapeutic agents for diseases related to NPR3, including NPR3 functional inactive refractory using the DNA-transferred animal of the present invention, using the above-described test methods and quantitative methods, It is possible to provide an effective and rapid screening method for a therapeutic drug for the disease. Moreover, it is possible to examine and develop a DNA therapy for a disease associated with NPR3 using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention.

(11) Production of a non-human knockout animal in which a gene encoding NPR3 is inactivated The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and the DNA expression-deficient non-human mammal of the present invention. Provide animals.
That is, the present invention
[1] A non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated,
[2] The embryonic stem cell according to item [1], wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli),
[3] The embryonic stem cell of [1], which is neomycin resistant,
[4] The embryonic stem cell of [1], wherein the non-human mammal is a rodent
[5] The embryonic stem cell according to item [4], wherein the rodent is a mouse,
[6] The non-human mammal deficient in DNA expression, wherein the DNA of the present invention is inactivated,
[7] The DNA can be inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention [6] The non-human mammal according to Item,
[8] The non-human mammal according to item [6], wherein the non-human mammal is a rodent.
[9] A test compound is administered to the non-human mammal according to [8] and [10] the animal according to [7], wherein the rodent is a mouse, and the expression of the reporter gene is detected. The screening method of the compound or its salt which promotes or inhibits the promoter activity with respect to DNA of this invention characterized by these is provided.
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated suppresses the expression ability of DNA by artificially adding mutation to the DNA of the present invention of the non-human mammal, or By substantially losing the activity of NPR3 encoded by the DNA, the DNA has substantially no ability to express NPR3 (hereinafter sometimes referred to as “knockout DNA of the present invention”). Embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same ones as described above are used.
As a method of artificially adding mutation to the DNA of the present invention, for example, a part or all of the DNA sequence can be deleted by using a genetic engineering technique, or another DNA can be inserted or replaced. With these mutations, for example, the knockout DNA of the present invention may be prepared by shifting the reading frame of the codon or destroying the function of the promoter or exon.

Specific examples of non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated (hereinafter abbreviated as “DNA inactivated ES cell of the present invention” or “knockout ES cell of the present invention”) include: Then, the DNA of the present invention possessed by the target non-human mammal is isolated, and a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (clonal A DNA sequence that disrupts the function of an exon by inserting a reporter gene or the like typified by a ramphenicol acetyltransferase gene) or terminates gene transcription at an intron between exons (for example, a poly A addition signal) ), And constructed to disrupt the gene, resulting in the inability to synthesize complete mRNA A DNA strand having a DNA sequence (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cell is subjected to the DNA sequence on or near the DNA of the present invention. Southern hybridization analysis as a probe or analysis by PCR method using the DNA sequence on the targeting vector and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector to select the knockout ES cell of the present invention Can be obtained.
In addition, as the original ES cell for inactivating the DNA of the present invention by homologous recombination method, for example, those already established as described above may be used, or according to the known Evans and Kaufman method. Or newly established ones. For example, in the case of mouse ES cells, currently 129 ES cells are generally used, but the immunological background is unclear, so an alternative purely immunologically genetic background For example, B57 ₁ mice (C57BL / 6 and DBA / 2 and C57BL / 6 mice and C57BL / 6 mice that have had fewer eggs collected by crossing with DBA / 2) well as the F ₁₎ which were established by using the usable good. The BDF ₁ mouse has C57BL / 6 mice in the background, in addition to the advantage that the number of eggs collected is large and the eggs are strong, so when ES cells obtained using this are created pathological model mice The genetic background can be advantageously replaced with C57BL / 6 mice by backcrossing with C57BL / 6 mice.
In addition, when establishing ES cells, blastocysts at the 3.5th day after fertilization are generally used, but in addition to this, an 8-cell stage embryo is collected and cultured to blastocysts for efficient use of many early blastocysts. Embryos can be obtained.
Although both male and female ES cells may be used, male ES cells are usually more convenient for producing germline chimeras. In addition, it is desirable to discriminate between males and females as soon as possible in order to reduce troublesome culture work.

One example of a method for determining the sex of an ES cell is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. If this method is used, the number of ES cells (about 50) is only about 1 colony, while the number of cells required for karyotype analysis is about 10 ⁶ cells. It is possible to perform primary selection of ES cells in the early stage by discriminating between males and females, and the selection of male cells at an early stage makes it possible to greatly reduce the labor of the initial culture.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cell is preferably 100% of the normal number. However, if it is difficult due to physical operations during establishment, the ES cell gene is knocked out and then normal cells (for example, chromosomes in mice). It is desirable to clone again into cells with a number 2n = 40.
The embryonic stem cell line obtained in this way is usually very proliferative, but it tends to lose its ontogenic ability, so it needs to be subcultured carefully. For example, in a carbon dioxide incubator in the presence of LIF (1-10000 U / ml) on a suitable feeder cell such as STO fibroblast (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% Culturing is carried out by a method such as culturing in carbon dioxide gas, 90% air at about 37 ° C., and at the time of passage, for example, trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, preferably about 0.1% trypsin / 1mM EDTA) treatment into single cells and seeding on newly prepared feeder cells. Such passage is usually carried out every 1-3 days. At this time, it is desirable to observe the cells, and if morphologically abnormal cells are found, the cultured cells should be discarded.
ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by monolayer culture to high density or suspension culture until a cell conglomerate is formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin, Proceedings of National Academy of Sciences USA (Proc. Natl. Acad Sci. USA) 78, 7634, 1981; TC Doetschman et al., J. Embryol. Exp. Morphol., 87, 27 1985), the DNA expression-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful for in vitro cell biology of NPR3 or NPR3.

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 comparing the expression level indirectly.
As the non-human mammal, the same ones as described above are used.
The non-human mammal deficient in DNA expression of the present invention can be prepared, for example, by introducing the targeting vector prepared as described above into mouse embryonic stem cells or mouse egg cells, and the DNA of the present invention in the introduced targeting vector is inactivated. The DNA of the present invention can be knocked out by homologous recombination in which the DNA sequence is replaced with the DNA of the present invention on the chromosome of mouse embryonic stem cell or mouse egg cell by gene homologous recombination. Since many of the recombination in mammals are non-homologous, as a means for screening cells that have undergone homologous recombination, for example, a drug resistance gene such as a neomycin resistance gene is inserted into the DNA of the present invention, A targeting vector containing a thymidine kinase (tk) gene in the vicinity of the DNA of the present invention is constructed and introduced into embryonic stem cells or egg cells, and a drug corresponding to the inserted drug resistance gene (for example, G418 or the like for the neomycin resistance gene) And a method for selecting cells that survive in the presence of ganciclovir. That is, when the insertion mutant DNA of the present invention is integrated into the chromosome by homologous recombination, the tk gene is excluded and thus is resistant to ganciclovir, but when integrated by non-homologous recombination, the tk gene is also integrated. Therefore, it becomes ganciclovir sensitivity. If a diphtheria toxin gene or the like is used in place of the tk gene, randomly inserted cells are killed by the production of the toxin, so that selection with a single drug becomes possible.
The final confirmation of the cell in which the DNA of the present invention is knocked out is the Southern hybridization analysis using the DNA sequence of or near the DNA of the present invention as a probe or the DNA sequence on the targeting vector and the mouse used for the targeting vector. The analysis can be performed by analysis by PCR using primers derived from the DNA sequence of the neighboring region other than the DNA of the present invention derived from.
When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cells are placed at a suitable time, for example, the 8-cell non-human mammal. It is injected into animal embryos or blastocysts, and the produced chimeric embryos are transplanted into the uterus of the non-human mammal that is pseudopregnant. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
When some of the germ cells of the chimeric animal have the mutated DNA locus of the present invention, all tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It is obtained by selecting an individual composed of cells having the added DNA locus of the present invention by, for example, determining a coat color. Since the individual thus obtained is usually a hetero-expression-deficient individual, the hetero-expression-deficient individuals can be crossed to obtain a homo-expression-deficient individual of the protein of the present invention from their offspring.

When using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by a microinjection method. It can be obtained by selecting a mammal having a mutation at the DNA locus of the present invention by gene homologous recombination.
Thus, an individual in which the DNA of the present invention is knocked out can be reared in a normal breeding environment after confirming that the animal individual obtained by mating has also been knocked out.
In addition, germline acquisition and retention may be followed in accordance with conventional methods. That is, a homozygous animal having the inactivated DNA in both homologous chromosomes can be obtained by mating male and female animals possessed by the inactivated DNA. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in a state where there are 1 normal individual and multiple homozygous animals. By crossing male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged.
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated is very useful for producing the non-human mammal deficient in DNA expression of the present invention.
Further, since the non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by NPR3, it can be a model for diseases caused by inactivation of the biological activity of NPR3. It is useful for investigating the cause of diseases and examining treatment methods.

(11a) Screening method for compounds having therapeutic / preventive effects on diseases caused by deficiency or damage of the DNA of the present invention The non-human mammal deficient in DNA expression of the present invention may be deficient or damaged of the DNA of the present invention. It can be used for screening for compounds having a therapeutic / prophylactic effect on diseases caused by.
That is, the present invention results from the deficiency or damage of the DNA of the present invention, characterized by administering a test compound to the non-human mammal deficient in DNA expression of the present invention, and observing and measuring changes in the animal. Provided is a screening method for a compound having a therapeutic / prophylactic effect on a disease or a salt thereof.
Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like, and these compounds are novel compounds. It may be a known compound.
Specifically, the DNA expression-deficient non-human mammal of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in the organs, tissues, disease symptoms, etc. of the animal as indicators. The therapeutic / prophylactic effect of the compound can be tested.
As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

In the screening method, when a test compound is administered to a test animal, for example, when the blood glucose level of the test animal decreases by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound Can be selected as a compound having a therapeutic / preventive effect on diseases such as impaired glucose tolerance.
The compound obtained using the screening method is a compound selected from the test compounds described above, and is a disease caused by NPR3 deficiency or damage, such as (i) cell differentiation and / or (ii) metabolism (for example, Abnormalities of cell differentiation and / or metabolism (eg, sugar, lipid, protein metabolism) in liver, adipose tissue, skeletal muscle, islet, bone, cartilage, etc., specifically, for example, obesity, diabetes, glucose tolerance It can be used as a safe and low-toxic therapeutic / preventive agent for abnormalities, arteriosclerosis, hypertension, hyperlipidemia, metabolic bone / cartilage diseases and the like. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals). And the like, and physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
When the compound obtained by the screening method or a salt thereof is used as a medicine, it can be formulated in the same manner as in the NPR3 class.
Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally in patients with abnormal sugar / lipid metabolism (with a body weight of 60 kg) About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.

(11b) Method for screening a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention The present invention comprises administering a test compound to a non-human mammal deficient in DNA expression of the present invention and detecting the expression of a reporter gene The screening method of the compound or its salt which promotes or inhibits the activity of the promoter with respect to DNA of this invention characterized by these is provided.
In the above screening method, the DNA expression deficient non-human mammal of the present invention is inactivated by introducing a reporter gene among the above-mentioned DNA expression deficient non-human mammals of the present invention, A gene capable of expressing the reporter gene under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound are the same as described above.
As the reporter gene, the same ones as described above are used, and β-galactosidase gene (lacZ), soluble alkaline phosphatase gene, luciferase gene, and the like are preferable.
In the non-human mammal of the present invention in which the DNA of the present invention is replaced with the reporter gene, the reporter gene exists under the control of the promoter for the DNA of the present invention, so that the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding NPR3 is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, β-galactosidase is expressed instead of NPR3 in a tissue where NPR3 is originally expressed. Therefore, for example, by staining with a reagent that is a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal), NPR3 The expression state in the animal body can be observed. Specifically, a mouse lacking NPR3 or a tissue section thereof is fixed with glutaraldehyde, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After the reaction for about 30 minutes to 1 hour, the tissue specimen is washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. In addition, mRNA encoding lacZ may be detected according to a conventional method.
The compound obtained by using the screening method or a salt thereof is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). In particular, physiologically acceptable acid addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.

The compound or its salt that promotes the promoter activity for the DNA of the present invention can promote the expression of NPR3 and promote the function of NPR3. It is useful as a pharmaceutical.
The compound or its salt that inhibits the promoter activity for the DNA of the present invention can inhibit the expression of NPR3 and inhibit the function of NPR3. It is useful as a pharmaceutical.
Diseases associated with NPR3 dysfunction or overexpression include, for example, (i) cell differentiation and / or (ii) metabolism (eg, cell differentiation and / or in liver, adipose tissue, skeletal muscle, islet, bone, cartilage, etc. Or abnormal metabolism (for example, sugar / lipid / protein metabolism), specifically, obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, hyperlipidemia, metabolic bone / cartilage disease, etc. Is mentioned.
Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.

When the compound obtained by the screening method or a salt thereof is used as a medicine, it can be formulated in the same manner as in the NPR3 class.
Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally in patients with abnormal sugar / lipid metabolism (with a body weight of 60 kg) About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient with abnormal sugar / lipid metabolism (weight 60 kg) In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. When the administration subject is other than a human, an amount converted per 60 kg body weight can be administered.

Thus, the non-human mammal deficient in DNA expression of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused or the development of preventive / therapeutic drugs.
In addition, by using DNA containing the NPR3 promoter region and linking genes encoding various proteins downstream of it, and injecting them into animal egg cells, so-called transgenic animals (transgenic animals) can be created. It is also possible to synthesize the protein specifically and study its action in the living body. In addition, if a suitable reporter gene is bound to the promoter part and a cell line that expresses it is established, a search system for low molecular weight compounds that specifically promotes or suppresses the production ability of NPR3 itself in the body. Can be used as

In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or common abbreviations in the field, examples of which are described below. In addition, when there are optical isomers with respect to amino acids, L form is shown unless otherwise specified.
DNA: Deoxyribonucleic acid
cDNA: complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: cytosine
RNA: Ribonucleic acid
mRNA: Messenger ribonucleic acid
dATP: Deoxyadenosine triphosphate
dTTP: Deoxythymidine triphosphate
dGTP: Deoxyguanosine triphosphate
dCTP: Deoxycytidine triphosphate
ATP: Adenosine triphosphate
EDTA: Ethylenediaminetetraacetic acid
SDS: Sodium dodecyl sulfate

Gly: Glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: Cysteine
Met: methionine
Glu: Glutamic acid
Asp: Aspartic acid
Lys: Lysine
Arg: Arginine
His: histidine
Phe: Phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
pGlu: pyroglutamic acid
Me: methyl group
Et: ethyl group
Bu: Butyl group
Ph: phenyl group
TC: thiazolidine-4 (R) -carboxamide group

In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Tos: p-toluenesulfonyl
CHO: Formyl
Bzl: benzyl
Cl ₂ Bzl: 2,6-dichlorobenzyl
Bom: benzyloxymethyl
Z: benzyloxycarbonyl
Cl-Z: 2-chlorobenzyloxycarbonyl
Br-Z: 2-bromobenzyloxycarbonyl
Boc: t-Butoxycarbonyl
DNP: Dinitrophenol
Trt: Trityl
Bum: t-Butoxymethyl
Fmoc: N-9-fluorenylmethoxycarbonyl
HOBt: 1-hydroxybenztriazole
HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide
DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
This shows the base sequence of the coding sequence of human NPR3 cDNA.
atgccgtctc tgctggtgct cactttctcc ccgtgcgtac tactcggctg ggcgttgctg 60
gccggcggca ccggtggcgg tggcgttggc ggcggcggcg gtggcgcggg cataggcggc 120
ggacgccagg agagagaggc gctgccgcca cagaagatcg aggtgctggt gttactgccc 180
caggatgact cgtacttgtt ttcactcacc cgggtgcggc cggccatcga gtatgctctg 240
cgcagcgtgg agggcaacgg gactgggagg cggcttctgc cgccgggcac tcgcttccag 300
gtggcttacg aggattcaga ctgtgggaac cgtgcgctct tcagcttggt ggaccgcgtg 360
gcggcggcgc ggggcgccaa gccagacctt atcctggggc cagtgtgcga gtatgcagca 420
gcgccagtgg cccggcttgc atcgcactgg gacctgccca tgctgtcggc tggggcgctg 480
gccgctggct tccagcacaa ggactctgag tactcgcacc tcacgcgcgt ggcgcccgcc 540
tacgccaaga tgggcgagat gatgctcgcc ctgttccgcc accaccactg gagccgcgct 600
gcactggtct acagcgacga caagctggag cggaactgct acttcaccct cgagggggtc 660
cacgaggtct tccaggagga gggtttgcac acgtccatct acagtttcga cgagaccaaa 720
gacttggatc tggaagacat cgtgcgcaat atccaggcca gtgagagagt ggtgatcatg 780
tgtgcgagca gtgacaccat ccggagcatc atgctggtgg cgcacaggca tggcatgacc 840
agtggagact acgccttctt caacattgag ctcttcaaca gctcttccta tggagatggc 900
tcatggaaga gaggagacaa acacgacttt gaagctaagc aagcatactc gtccctccag 960
acagtcactc tactgaggac agtgaaacct gagtttgaga agttttccat ggaggtgaaa 1020
agttcagttg agaaacaagg gctcaatatg gaggattacg ttaacatgtt tgttgaagga 1080
ttccacgatg ccatcctcct ctacgtcttg gctctacatg aagtactcag agctggttac 1140
agcaaaaagg atggagggaa aattatacag cagacttgga acagaacatt tgaaggtatc 1200
gccgggcagg tgtccataga tgccaacgga gaccgatatg gggatttctc tgtgattgcc 1260
atgactgatg tggaggcggg cacccaggag gttattggtg attattttgg aaaagaaggt 1320
cgttttgaaa tgcggccgaa tgtcaaatat ccttggggcc ctttaaaact gagaatagat 1380
gaaaaccgaa ttgtagagca tacaaacagc tctccctgca aatcatgtgg cctagaagaa 1440
tcggcagtga caggaattgt cgtgggggct ttactaggag ctggcttgct aatggccttc 1500
tactttttca ggaagaaata cagaataacc attgagaggc gaacccagca agaagaaagt 1560
aaccttggaa aacatcggga attacgggaa gattccatca gatcccattt ttcagtagct 1620
taa 1623

[SEQ ID NO: 2]
The amino acid sequence of human NPR3 is shown.
Met Pro Ser Leu Leu Val Leu Thr Phe Ser Pro Cys Val Leu Leu Gly
1 5 10 15
Trp Ala Leu Leu Ala Gly Gly Thr Gly Gly Gly Gly Val Gly Gly Gly
20 25 30
Gly Gly Gly Ala Gly Ile Gly Gly Gly Arg Gln Glu Arg Glu Ala Leu
35 40 45
Pro Pro Gln Lys Ile Glu Val Leu Val Leu Leu Pro Gln Asp Asp Ser
50 55 60
Tyr Leu Phe Ser Leu Thr Arg Val Arg Pro Ala Ile Glu Tyr Ala Leu
65 70 75 80
Arg Ser Val Glu Gly Asn Gly Thr Gly Arg Arg Leu Leu Pro Pro Gly
85 90 95
Thr Arg Phe Gln Val Ala Tyr Glu Asp Ser Asp Cys Gly Asn Arg Ala
100 105 110
Leu Phe Ser Leu Val Asp Arg Val Ala Ala Ala Arg Gly Ala Lys Pro
115 120 125
Asp Leu Ile Leu Gly Pro Val Cys Glu Tyr Ala Ala Ala Pro Val Ala
130 135 140
Arg Leu Ala Ser His Trp Asp Leu Pro Met Leu Ser Ala Gly Ala Leu
145 150 155 160
Ala Ala Gly Phe Gln His Lys Asp Ser Glu Tyr Ser His Leu Thr Arg
165 170 175
Val Ala Pro Ala Tyr Ala Lys Met Gly Glu Met Met Leu Ala Leu Phe
180 185 190
Arg His His His Trp Ser Arg Ala Ala Leu Val Tyr Ser Asp Asp Lys
195 200 205
Leu Glu Arg Asn Cys Tyr Phe Thr Leu Glu Gly Val His Glu Val Phe
210 215 220
Gln Glu Glu Gly Leu His Thr Ser Ile Tyr Ser Phe Asp Glu Thr Lys
225 230 235 240
Asp Leu Asp Leu Glu Asp Ile Val Arg Asn Ile Gln Ala Ser Glu Arg
245 250 255
Val Val Ile Met Cys Ala Ser Ser Asp Thr Ile Arg Ser Ile Met Leu
260 265 270
Val Ala His Arg His Gly Met Thr Ser Gly Asp Tyr Ala Phe Phe Asn
275 280 285
Ile Glu Leu Phe Asn Ser Ser Ser Tyr Gly Asp Gly Ser Trp Lys Arg
290 295 300
Gly Asp Lys His Asp Phe Glu Ala Lys Gln Ala Tyr Ser Ser Leu Gln
305 310 315 320
Thr Val Thr Leu Leu Arg Thr Val Lys Pro Glu Phe Glu Lys Phe Ser
325 330 335
Met Glu Val Lys Ser Ser Val Glu Lys Gln Gly Leu Asn Met Glu Asp
340 345 350
Tyr Val Asn Met Phe Val Glu Gly Phe His Asp Ala Ile Leu Leu Tyr
355 360 365
Val Leu Ala Leu His Glu Val Leu Arg Ala Gly Tyr Ser Lys Lys Asp
370 375 380
Gly Gly Lys Ile Ile Gln Gln Thr Trp Asn Arg Thr Phe Glu Gly Ile
385 390 395 400
Ala Gly Gln Val Ser Ile Asp Ala Asn Gly Asp Arg Tyr Gly Asp Phe
405 410 415
Ser Val Ile Ala Met Thr Asp Val Glu Ala Gly Thr Gln Glu Val Ile
420 425 430
Gly Asp Tyr Phe Gly Lys Glu Gly Arg Phe Glu Met Arg Pro Asn Val
435 440 445
Lys Tyr Pro Trp Gly Pro Leu Lys Leu Arg Ile Asp Glu Asn Arg Ile
450 455 460
Val Glu His Thr Asn Ser Ser Pro Cys Lys Ser Cys Gly Leu Glu Glu
465 470 475 480
Ser Ala Val Thr Gly Ile Val Val Gly Ala Leu Leu Gly Ala Gly Leu
485 490 495
Leu Met Ala Phe Tyr Phe Phe Arg Lys Lys Tyr Arg Ile Thr Ile Glu
500 505 510
Arg Arg Thr Gln Gln Glu Glu Ser Asn Leu Gly Lys His Arg Glu Leu
515 520 525
Arg Glu Asp Ser Ile Arg Ser His Phe Ser Val Ala
530 535 540

  The present invention will be described in more detail with reference to the following examples, but these are merely examples and do not limit the scope of the present invention.

Example 1 Cloning of cDNA Encoding Mouse Musclin Protein From the mouse skeletal muscle cDNA library Quick clone cDNA (BD), using the oligo DNA represented by SEQ ID NO: 3 as a sense strand primer, SEQ ID NO: 4 PCR was carried out using the oligo DNA represented by the antisense strand primer to obtain a 5 ′ upstream sequence (SEQ ID NO: 5) starting from each primer.
SEQ ID NO: 3
TTAGCATCAC AGGAGTTTGG AAC 23

SEQ ID NO: 4
TCAGCCTCTG GAACTGGAGA GCC 23

SEQ ID NO: 5
TTAGCATCAC AGGAGTTTGG AACAGCAAGC TTGCAGTCTC CACCCACAGC CAGAGAAGAG 60
AAGTCAGCCA CTGAGCTTTC GGCTAAGCTC CTGCGTCTTG ATGATCTGGT GTCCTTAGAG 120
AATGACGTAT TTGAGACCAA GAAAAAGAGA AGCTTCTCTG GCTTTGGGTC TCCCCTTGAC 180
AGACTCTCAG CTGGGTCTGT AGAGCATAGA GGGAAACAAA GGAAAGCAGT AGATCATTCA 240
AAAAAGCGGT TTGGTATTCC CATGGATCGG ATTGGTAGAA ACCGGCTCTC CAGTTCCAGA 300
GGCTGA 306

Example 2 Preparation of His-tag Tyr Cys Mouse Musculin (HYC-mMusclin) Protein
A gene encoding a mature mMusclin (SEQ ID NO: 7) excluding the signal sequence (SEQ ID NO: 6) of PET-15b (Novagen) was subcloned. Based on the obtained pET-15b-mMusclin plasmid vector, The vector-derived sequence between the His-tag sequence and the sequence encoding mMusclin was modified by PCR-based site-directed mutagenesis to obtain a plasmid vector pET15M-mMusclin having the ORF shown in SEQ ID NO: 8.
The resulting plasmid vector is introduced into competent cells such as BL21 (DE3) (Novagene), and the protein expression is induced by shaking culture and IPTG, thereby accumulating His-Tyr-Cys-mMusclin protein in the cells. The protein was purified by purification using a chelate column using His-tag.
SEQ ID NO: 6
ATGCTGGACT GGAGATTGGC AAGTACACAC TTCATCCTGG CTATGATTGT GATGCTGTGG 60
GGCTCAGGAA AGGCATTCTC TGTGGAC 87

SEQ ID NO: 7
TTAGCATCAC AGGAGTTTGG AACAGCAAGC TTGCAGTCTC CACCCACAGC CAGAGAAGAG 60
AAGTCAGCCA CTGAGCTTTC GGCTAAGCTC CTGCGTCTTG ATGATCTGGT GTCCTTAGAG 120
AATGACGTAT TTGAGACCAA GAAAAAGAGA AGCTTCTCTG GCTTTGGGTC TCCCCTTGAC 180
AGACTCTCAG CTGGGTCTGT AGAGCATAGA GGGAAACAAA GGAAAGCAGT AGATCATTCA 240
AAAAAGCGGT TTGGTATTCC CATGGATCGG ATTGGTAGAA ACCGGCTCTC CAGTTCCAGA 300
GGC 303

SEQ ID NO: 8
ATGGGCAGCA GCCATCATCA TCATCATCAC AGCAGCGGCC TGGTGCCGTA CGGCTGCCAT 60
ATGTTAGCAT CACAGGAGTT TGGAACAGCA AGCTTGCAGT CTCCACCCAC AGCCAGAGAA 120
GAGAAGTCAG CCACTGAGCT TTCGGCTAAG CTCCTGCGTC TTGATGATCT GGTGTCCTTA 180
GAGAATGACG TATTTGAGAC CAAGAAAAAG AGAAGCTTCT CTGGCTTTGG GTCTCCCCTT 240
GACAGACTCT CAGCTGGGTC TGTAGAGCAT AGAGGGAAAC AAAGGAAAGC AGTAGATCAT 300
TCAAAAAAGC GGTTTGGTAT TCCCATGGAT CGGATTGGTA GAAACCGGCT CTCCAGTTCC 360
AGAGGCTGA 369

Example 3 Preparation of full-length mMusclin and C-terminal mMusclin (mCA) proteins
Using the primers shown in SEQ ID NO: 9 and SEQ ID NO: 10 at the LIC multicloning site of pET41-XaLIC vector (Novagene) and introducing the amplified fragment using the gene containing mMUSclin ORF as a template by the LIC-cloning technique Then, an E. coli expression-type plasmid vector encoding a thioredoxin (Trx) fusion mmusclin full-length protein was prepared. Similarly, an Escherichia coli expression type plasmid vector encoding thioredoxin fusion mCA protein was prepared by amplification using SEQ ID NO: 11 and SEQ ID NO: 10.
SEQ ID NO: 9
GGTATTGAGG GTCGCTTAGC ATCACAGGAG TTTGGAACAG 40

SEQ ID NO: 10
AGAGGAGAGT TAGAGCCTCA GCCTCTGGAA CTGGAGAGCC GG 42

SEQ ID NO: 11
GGTATTGAGG GTCGCAGCTT CTCTGGCTTT GGGTCTCCCC 40

The obtained plasmid vector is introduced into competent cells such as BL21 (DE3) (Novagene), and the protein expression is induced by shaking culture and IPTG, so that the Trx-His-mMusclin protein or Trx-His is contained in the cells. The -mCA protein was accumulated, and the protein was purified by purification using a chelate column using His-tag.
Trx-His-mMusclin that binds and binds to the HiTrap SP-HP column equilibrated with a buffer solution consisting of 25 mM HEPES and 100 mM NaCl (pH 7.8). The purified full length mMusclin or mCA separated from the fusion protein was obtained by eluting the protein or Trx-His-mCA protein with 25 mM HEPES, 1000 mM NaCl (pH 7.8).

Example 4 Preparation of biotin-labeled mMusclin The full-length mMusclin obtained in Example 3 was mixed with 2.5 mg / ml x 250 μl of 10 nmol / μl Sulfo-NHS-LC-Biotin 45 μl overnight under ice-cooling. A labeled mMusclin (BK-mMusclin) into which 5 molecules / mMusclin1 molecule was introduced was prepared.
The His-Tyr-Cys-mMusclin 1 mg / mlx500 μl produced in Example 2 was reduced with TCEP (Pierce) in the presence of EDTA, and the final concentration of 0.4 mM Maleimide PEO2 Biotin was mixed overnight at room temperature. A labeled mMusclin (BC-mMusclin) in which about 1 biotin molecule / one molecule was introduced in a group-specific manner was prepared.

Example 5 Preparation of hNpr-3 expressing FreeStyle293 cells
An hNpr3 expression vector (hNpr3 / pCMV6-XL4, NM_000908 Clone # PL1154_A07) was purchased from OriGene. Transiently transfect 0.3 µg (Low) or 1.0 µg (high) of hNpr3 expression vector per 1.0 x 10 ⁶ FreeStyle293 cells by a conventional method, collect cells after 48 hours, and collect PBS- 5x10 ⁶ cells / ml. Suspended in 1.0% BSA. Incubate 1 ml of each cell solution with BK-mMusclin or BC-mMusclin at a final concentration of 0.5 μg / ml for 30 minutes at 4 ° C, wash once, stain with PE-labeled Streptavidin, and use FACS-Aria to identify each Musclin-bound cell. Analyzed. During incubation, 100 μg / ml full-length mMusclin, mCA, or His-Tyr-Cys-mMusclin, or 10 μM ANP (Atrial Natriuretic Peptide, Rat, Mouse), CANP (Atrial Natriuretic Peptide 4-23-NH2 Des- [ Gln18 Ser19 Gly20 Leu21 Gly22], Rat), LANP (Atrial Natriuretic Peptide 104-126 Des- [Cys105 Cys121], Rat) (Phoenix Pharmaceuticals, Inc.) was examined for changes in the binding of labeled mMusclin.
As a result, BK- and BC-labeled mMusclin binds depending on the amount of hNpr3 gene introduced into the cell, and the binding is almost lost by 100 μg / ml full-length mMusclin, mCA, or His-Tyr-Cys-mMusclin. From the results, it was proved that the binding was specific to mMusclin, and that the binding was dependent on NPR3 because it was almost eliminated by 10 μM ANP, CANP and LANP which are known ligands of NPR3 molecule.

  According to the present invention, screening for prophylactic / therapeutic agents and diagnostic agents for diseases associated with cell differentiation such as skeletal muscles and abnormalities in sugar / lipid / protein metabolism, and drug candidates effective for the prophylaxis / treatment of the diseases. Tools can be provided.

Claims (29)

  A cell differentiation and / or metabolism regulator comprising a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof.   The cell is selected from the group consisting of skeletal muscle cells, adipocytes, islet cells, bone cells, cartilage cells and hepatocytes, and the metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent described.   The agent according to claim 1, which is used for prevention / treatment of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia. .   A cell differentiation and / or metabolism regulator comprising a nucleic acid encoding a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial peptide thereof.   5. The cell is selected from the group consisting of skeletal muscle cells, adipocytes, pancreatic islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent described.   The agent according to claim 4, which is used for prevention / treatment of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia. .   A diagnostic agent for abnormal cell differentiation and / or abnormal metabolism comprising a nucleic acid encoding a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial polynucleotide thereof.   The cell is selected from the group consisting of skeletal muscle cells, adipocytes, islet cells, bone cells, cartilage cells and hepatocytes, and the metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent described.   The agent according to claim 7, which is used for diagnosis of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia.   A diagnostic agent for abnormal cell differentiation and / or abnormal metabolism, comprising an antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof.   11. The cell is selected from the group consisting of skeletal muscle cells, adipocytes, pancreatic islet cells, bone cells, cartilage cells and hepatocytes, and the metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent described.   The agent according to claim 10, which is used for diagnosis of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia.   A cell differentiation and / or metabolism regulator comprising an antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof.   14. The cell is selected from the group consisting of skeletal muscle cells, adipocytes, islet cells, bone cells, cartilage cells and hepatocytes, and the metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent described.   The agent according to claim 13, which is used for the prophylaxis or treatment of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia. .   Cell differentiation comprising a nucleic acid containing a nucleotide sequence complementary to a nucleic acid encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a part thereof And / or metabolic regulators.   The cell is selected from the group consisting of skeletal muscle cells, fat cells, islet cells, bone cells, cartilage cells and hepatocytes, and the metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The agent described.   The agent according to claim 16, which is used for prevention / treatment of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia. .   A substance having a cell differentiation and / or metabolic regulation action characterized by using a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof Screening method.   A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof, is provided in the form of a cell that produces it. Item 20. The method according to Item 19.   A nucleic acid encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 or a partial polynucleotide thereof, or the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2 21. The method according to claim 20, further comprising using an antibody against a protein containing the same amino acid sequence or a partial peptide thereof or a salt thereof.   20. The cell is selected from the group consisting of skeletal muscle cells, adipocytes, pancreatic islet cells, bone cells, cartilage cells and hepatocytes, and metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The method described.   20. The prophylactic / therapeutic activity of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia. the method of.   (i) Use of a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof; and (ii) Musclin, a partial peptide thereof, or a salt thereof. A screening method for substances that change the binding properties of the two.   A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a partial peptide thereof, or a salt thereof, is provided in the form of a cell that produces it. Item 25. The method according to Item 24.   26. The method according to claim 25, characterized by indicating sugar uptake into cells or glycogen synthesis in cells.   25. A method according to claim 24, wherein the substance exhibits a cell differentiation and / or metabolic regulatory effect.   28. The cell is selected from the group consisting of skeletal muscle cells, adipocytes, islet cells, bone cells, cartilage cells and hepatocytes, and the metabolism is selected from the group consisting of sugar metabolism, lipid metabolism and protein metabolism. The method described.   28. The prophylactic / therapeutic activity of one or more diseases selected from the group consisting of obesity, diabetes, impaired glucose tolerance, arteriosclerosis, hypertension, metabolic bone / cartilage disease and hyperlipidemia. the method of.