JP2003279567A - Screening method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening preventives and therapeutic agents for a kidney disease or the like. <P>SOLUTION: The screening method uses proteins or their salts having an amino acid arrangement identical or actually identical to an amino acid arrangement represented by the arrangement number of one, and screens the preventive and the therapeutic agent for the disease relating to the protein or its salt. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for screening a preventive / therapeutic drug for renal diseases and the like.

[0002]

2. Description of the Related Art As a protein containing the amino acid sequence represented by SEQ ID NO: 1, Early Growth Response
-1 (sometimes abbreviated as Egr-1 in the present specification) is known. Egr-1 is activated by ischemia,
A transcription factor that induces the expression of key regulators of inflammation, coagulation and vascular permeability (Nature Medicine, vo
l.6, No.12, p.1355). So far, no report has been found on a method for screening a preventive / therapeutic drug for a disease associated with Egr-1, using Egr-1.
Currently, angiotensin-converting enzyme inhibitors and the like are used as therapeutic agents for renal diseases.
It cannot be used in patients with severe renal impairment because it affects renal hemodynamics.

[0003]

[Problems to be Solved by the Invention] A prophylactic / therapeutic agent for renal diseases and the like having excellent effects and having no side effect, and a method capable of screening such a prophylactic / therapeutic agent are demanded. There is.

[0004]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that the expression of renal Egr-1 is remarkably increased in renal disease model animals. It was discovered for the first time, and further, it was found for the first time that a therapeutic effect on renal disease can be obtained by suppressing the expression of renal Egr-1 in a renal disease model animal. The present inventors have completed the present invention as a result of further studies based on these findings.

That is, the present invention is characterized in that 1) a protein containing an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof is used. A method for screening a preventive / therapeutic substance for a disease associated with a salt; 2) A protein containing the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof is used, and a disease associated with the protein or a salt thereof 5. A method for screening a preventive / therapeutic substance; 3) A screening method according to 1) above, wherein the protein has the amino acid sequence represented by SEQ ID NO: 2; 4) A screening method according to 1), wherein the disease is renal disease; ) The screening method according to 4) above, wherein the renal disease is Egr-1-dependent renal disease; 6) Before the renal disease is diabetic nephropathy 4) The screening method described in 7); 7) A case of culturing a cell having the ability to produce a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: when cells having the ability to produce a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 are cultured in the presence of the test compound 1) The screening method according to 1) above, which comprises comparing the production amount of a protein or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 1; 8) the presence of a test compound A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 under or in the absence thereof, or The screening method according to 1) above, which comprises comparing the activities of the salts; 9) A protein containing an amino acid sequence whose activity is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1. Alternatively, the screening method according to the above 8), which has a binding activity to a polynucleotide to which the salt can bind; 10) contains an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1. The screening method according to 8) above, which has an activity of controlling the expression of a gene under the control of transcription by a protein or a salt thereof; 11) an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 When a cell having the ability to produce a protein or a salt thereof is cultured, and the cell is cultured in the presence of a test compound Of the case was,
The production amount of the protein or a salt thereof and the binding activity to a polynucleotide to which the protein or a salt thereof can bind are measured and compared using an antibody against the polynucleotide and the protein or a salt thereof, 1) The screening method described in 1); 12) The compound obtained by the screening method described in 1) above or a salt thereof; 13) The compound obtained by the screening method described in 1) above, or a salt thereof, SEQ ID NO: : 1
A preventive / therapeutic agent for a disease associated with a protein or a salt thereof containing the same or substantially the same amino acid sequence as that shown in 14): 14) The same or substantially the same amino acid sequence as shown in SEQ ID NO: 1. Characterized by using a polynucleotide containing a base sequence encoding a protein or a salt thereof containing the same amino acid sequence or a partial sequence thereof,
A method for screening a preventive / therapeutic substance for a disease associated with a protein or a salt thereof containing the same or substantially the same amino acid sequence as that represented by SEQ ID NO: 1; 15) represented by SEQ ID NO: 1 An amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, characterized in that a polynucleotide containing a nucleotide sequence encoding a protein containing the amino acid sequence or a salt thereof or a partial sequence thereof is used. A method for screening a preventive / therapeutic substance for a disease associated with a protein containing a sequence or a salt thereof; 16) The above-mentioned 14 in which the polynucleotide contains the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 or a partial sequence thereof 17) the screening method described above; 17) the screening method described in 14) above, wherein the disease is renal disease; The 8) renal disease is Egr-1 dependent kidney disease 1
7) the screening method described above; 19) the screening method described in 17) above, wherein the renal disease is diabetic nephropathy; 20) containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. When culturing cells having the ability to produce a protein or a salt thereof, the ability to produce a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 Comparing the amount of RNA encoding a protein or a salt thereof containing an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1, when cells having the same are cultured in the presence of a test compound 21) The screening method described in 14) above; 21) obtained by the screening method described in 14) above. Compound or salt thereof; Compound 22) A 14) was obtained by the screening method described or comprising a salt thereof, SEQ ID NO:
A preventive / therapeutic agent for a disease associated with a protein or a salt thereof containing the same or substantially the same amino acid sequence as that represented by 1; 23) The same or substantially the amino acid sequence represented by SEQ ID NO: 1. 24) An antibody against a protein or a salt thereof, which contains the same amino acid sequence; 24) The same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, which comprises the antibody according to 23) above. 25) A preventive / therapeutic agent for a disease associated with a protein or a salt thereof; 25) complementary to a base sequence encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Identical or substantially to the amino acid sequence represented by SEQ ID NO: 1, comprising a polynucleotide having a specific base sequence or a partial sequence thereof A preventive / therapeutic agent for diseases associated with proteins or salts thereof containing the same amino acid sequence; 26) The same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1, comprising the antibody according to 23) above. 27) a diagnostic agent for a disease associated with a protein containing the same amino acid sequence or a salt thereof; 27) a base encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 A diagnostic agent for a disease associated with a protein or a salt thereof, which comprises a polynucleotide having a sequence or a partial sequence thereof and has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1; 28) SEQ ID NO: 5, which comprises a polynucleotide having the base sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 A preventive / therapeutic agent for a disease associated with a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 1: 29) The prevention according to 28) above, wherein the disease is a renal disease Therapeutic agent; 30) The above, wherein the renal disease is Egr-1-dependent renal disease
9) The preventive / therapeutic drug described above; 31) The preventive / therapeutic drug described in 29) above, wherein the renal disease is diabetic nephropathy; 32) The preventive / therapeutic drug containing diabetic nephropathy, which comprises an Egr-1 inhibitor 33) The preventive / therapeutic drug according to the above 32), wherein the Egr-1 inhibitor is a renal Egr-1 inhibitor; 34) The mammal is characterized in that the Egr-1 inhibitor is administered to the mammal. 35) a method for preventing or treating diabetic nephropathy in 35), the method according to 34) above, wherein the Egr-1 inhibitor is a renal Egr-1 inhibitor; 36) a method for producing a preventive / therapeutic agent for diabetic nephropathy ,
37) Use of Egr-1 inhibitor; 37) Use according to the above 36), wherein the Egr-1 inhibitor is a renal Egr-1 inhibitor;

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter sometimes abbreviated as the protein of the present invention) is , Warm-blooded animals (eg,
Human, mouse, rat, rabbit, sheep, pig, cow,
Horse cells, birds, cats, dogs, monkeys, chimpanzees, etc.) (eg, hepatocytes, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet) Cell, endothelial cell, smooth muscle cell, fibroblast, fiber cell, muscle cell, adipocyte, immune cell (eg, macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, Eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells Etc.) or any tissue in which those cells are present, such as the brain, each part of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, inferior Pituitary Stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung,
Digestive tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen,
Submandibular gland, peripheral blood, prostate, testicles, ovaries, placenta, uterus,
It may be a protein derived from bone, joint, skeletal muscle or the like, or may be a synthetic protein.

The amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 6% of the amino acid sequence represented by SEQ ID NO: 1.
Examples thereof include amino acid sequences having a homology of 0% or more, more preferably about 70% or more, particularly preferably about 80% or more, and most preferably about 90% or more. Sequence number:
Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by 1 include, for example, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 above, A protein having substantially the same activity as the protein containing the amino acid sequence represented by 1: is preferred.

[0008] Examples of substantially the same activity include transcriptional regulatory activity of fibrosis-related genes, cell cycle-related genes, and thrombus-related genes. The term “substantially the same” means that those properties are qualitatively (eg, physiologically or pharmacologically) the same. Therefore, the transcription control activity is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times).
However, quantitative factors such as the degree of activity and the molecular weight of the protein may be different. Fibrosis-related genes include, for example, platelet-derived g
rowth factor-A chain (PDGF-A chain), platelet-der
ived growth factor-B chain (PDGF-B chain), basic
fibroblast growth factor (bFGF), fibroblast growt
h factor 2 (FGF2), tumor growth factor-β1 (TGF-
β1), tumor necrosis factor-α (TNF-α), intercell
ular adhesion molecule 1 (ICAM-1), vascular endothe
lial cell growth factor (VEGF), PDGF β receptor
(PDGF β R), insulin-like growth factor-1 receptor
(IGF-1 R), fibronectin, α2 (1) collagen and the like. Examples of cell cycle-related genes include p53 and the like. Examples of thrombus-related genes include pl
asminogen activator inhibitor-1 (PAI-1), tissue fa
Examples include ctor (TF) and metalloproteinase. The transcription control activity can be measured by a known method, for example, the method of Shi-Fang Yan et al. (Proc. Natl. Acad. Sci. USA 95, 8298-8303).
Page, 1998) or a method similar thereto.

The protein of the present invention includes, for example, 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1, 1 or 2 or more (preferably, in the amino acid sequence represented by SEQ ID NO: 1), preferably an amino acid sequence in which several (1 to 5) amino acids are deleted,
1 to 30 amino acids, preferably 1 to 10 amino acids, and more preferably a number (1 to 5) amino acids added to the amino acid sequence represented by SEQ ID NO: 1 or 2 or more ( Preferably, about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5).
Amino acid sequence in which the amino acid of SEQ ID NO: 1 has been inserted
Substitution of 1 or 2 or more (preferably about 1 to 30, preferably about 1 to 10 and more preferably 1 to 5) amino acids in the amino acid sequence represented by Also included are so-called muteins such as proteins containing the defined amino acid sequences or amino acid sequences combining them. 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. The protein of the present invention is preferably a protein having the amino acid sequence represented by SEQ ID NO: 2, that is, Egr-1.

The protein in the present specification has the N-terminal (amino terminal) at the left end and the C-terminal (carboxyl terminal) at the right end in accordance with the convention of peptide notation. SEQ ID NO: including the protein containing the amino acid sequence represented by 1, the protein of the present invention, C-terminal, carboxyl group (-COOH), a carboxylate (-COO ^-),
Amide (-CONH ₂₎ or an ester (-COOR)
It may be any of Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; a C _3-8 cycloalkyl group such as cyclopentyl or cyclohexyl; , _{C 6-12} aryl group such as α- naphthyl; e.g., benzyl, phenyl _{-C 1-2} alkyl group such as phenethyl; _C, such as α- naphthyl _{-C 1-2} alkyl group such as α- naphthylmethyl _{7- 14} aralkyl group; pivaloyloxymethyl group and the like are used. When the protein of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, 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, the protein of the present invention includes
The amino group of the N-terminal amino acid residue (eg, methionine residue) is a protecting group (eg, formyl group, acetyl group, etc.).
Protected by a C _1-6 acyl group such as _1-6 alkanoyl), the N-terminal glutamine residue produced by cleavage in vivo is pyroglutamine-oxidized, and on the side chain of an amino acid in the molecule. Substituents (eg, -OH, -S
H, amino group, imidazole group, indole group, guanidino group, etc.) a suitable protecting group (e.g., formyl group, such as _{C 1-6} alkanoyl group such as acetyl group _{C 1-6}
Protected by an acyl group) or a complex protein such as a so-called glycoprotein having a sugar chain bonded thereto.

The protein salts of the present invention include physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg,
Alkali metal salts) and the like are used, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,
Methanesulfonic acid, benzenesulfonic acid) and the like are used. The protein of the present invention or a salt thereof can be produced from the cells or tissues of warm-blooded animals described above by a method known per se for protein purification. Specifically, the tissue or cells of a warm-blooded animal is homogenized, and the soluble fraction is separated and purified by chromatography such as reverse phase chromatography or ion exchange chromatography to produce the protein of the present invention or a salt thereof. can do.

The protein of the present invention or a salt thereof can also be produced by 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. The target protein can be produced by condensing the partial peptide or amino acid that can form the protein of the present invention with the residual portion and removing the protecting group when the product has the protecting group. Here, the condensation and the removal of the protective group are carried out by a method known per se,
For example, it is performed according to the methods described in the following. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten The protein 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 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, when the protein is obtained as a salt, The salt can be converted into a free form or another salt by a known method or a method analogous thereto.

The protein of the present invention or a salt thereof is usually synthesized using a commercially available resin for protein synthesis.
Examples of such a resin include chloromethyl resin,
Hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM
Resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ',
4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ', 4'-dimethoxyphenyl-F
moc aminoethyl) phenoxy resin and the like. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to the sequence of the target protein according to various condensation methods known per se. At the end of the reaction, the protein is cleaved from the resin and at the same time various protective groups are removed, and further, an intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein. In the condensation of the protected amino acid described above, various activating reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. Condensation of the protected amino acid can be carried out, for example, by adding the protected amino acid directly to the resin together with an activating reagent and a racemization-inhibiting additive (eg, HOBt, HOOBt), or by previously protecting the protected amino acid with a symmetrical acid anhydride or HOBt ester or HO.
It is carried out by adding it to the resin after activation as an OBt ester.

The solvent used for activation of the protected amino acid or condensation with the resin is appropriately selected from solvents known to be usable for protein condensation reaction. For example, N,
Acid amides such as N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; sulfoxides such as dimethylsulfoxide; pyridine And the like; ethers such as dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. The reaction temperature is appropriately selected from the range known to be used for protein condensation reaction, and is usually appropriately selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient,
Sufficient condensation can be achieved by repeating the condensation reaction without removing the protecting group. When the condensation is insufficient even after repeating the condensation reaction, sufficient condensation can be performed by acetylating the unreacted amino acid with acetic anhydride or acetylimidazole.

A method for protecting a functional group that should not participate in the reaction of the raw material, a protecting group, and a method for removing the protecting group;
The method for activating the functional group involved in the reaction can be appropriately selected from known groups or known means. Examples of the amino group-protecting group of the starting amino acid include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-
Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl,
Fmoc etc. are mentioned. The carboxyl group of the starting amino acid is, for example, alkyl esterified (for example, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, or other linear, branched or Cyclic alkyl esterification), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide formation,
It can be protected by t-butoxycarbonyl hydrazide formation, trityl hydrazide formation and the like. The hydroxyl group of serine can be protected by, for example, esterification or etherification. Examples of groups suitable for esterification include lower (C _1-6 ) alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group. To be Moreover, examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of the protective group for the phenolic hydroxyl group of tyrosine include Bz
1, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z,
t-Butyl or the like is used. Examples of the protecting group for imidazole of histidine include Tos and 4-methoxy-
2,3,6-trimethylbenzenesulfonyl, DNP,
Benzyloxymethyl, Bum, Boc, Trt, Fm
oc or the like is used.

As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon; anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro Acid treatment with methanesulfonic acid, trifluoroacetic acid, or a mixed solution thereof; base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc .; reduction with sodium in liquid ammonia. The elimination reaction by the acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, it is effective to add a cation trapping agent such as anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol. Further, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by the thiophenol treatment. The formyl group used as the indole protecting group of tryptophan is not only deprotected by acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol, etc., but also alkali-treated with dilute sodium hydroxide solution, dilute ammonia or the like. Is also removed by.

Examples of activated carboxyl groups of the starting amino acids include corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,
4-dinitrophenol, cyanomethyl alcohol, para-nitrophenol, HONB, N-hydroxysuccinide, N-hydroxyphthalimide, ester with HOBt)] and the like are used. As the activated amino group of the raw material amino acid, for example, the corresponding phosphoric acid amide is used.

The amide form of the protein is obtained by a known method according to a known method.
It can be produced by amidating a carboxyl group. The ester form of a protein can be produced, for example, by condensing the α-carboxyl group of the carboxy-terminal amino acid of the protein with a desired alcohol.

Further, the protein of the present invention or a salt thereof is obtained by culturing a transformant containing a DNA encoding the protein of the present invention, and separating and purifying the protein of the present invention or a salt thereof from the resulting culture. It can also be manufactured. DN encoding the protein of the present invention
Examples of A include genomic DNA, genomic DNA library, cDNA derived from the cells and tissues described above, cDNA library derived from the cells and tissues described above, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse Transcriptase Polymerase ChainRe can be directly used by preparing total RNA or mRNA fraction from the above cells / tissues.
It can also be amplified by action (hereinafter, abbreviated as RT-PCR method). The DNA encoding the protein of the present invention includes, for example, DNA containing the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. An activity having a nucleotide sequence that hybridizes under high stringent conditions and having substantially the same quality as that of the protein containing the amino acid sequence represented by SEQ ID NO: 1 (eg, fibrosis-related gene, cell cycle-related gene) DNA, etc. which encode a gene and a protein having a transcriptional regulatory activity of a thrombus-related gene and the like. SEQ ID NO: 3
Alternatively, the DNA that can hybridize with the base sequence represented by SEQ ID NO: 4 under high stringent conditions is, for example, about 50% or more, preferably about 50% or more of the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. Is about 60% or more,
More preferably about 70% or more, more preferably about 80%.
%, Particularly preferably about 90% or more, and most preferably about 95% or more homologous DN.
A or the like is used. Hybridization is a method known per se or a method analogous thereto, for example, Molecular Cloning 2nd (J.
Sambrook et al., Cold Spring Harbor Lab. Press, 198
It can be performed according to the method described in 9). When using a commercially available library, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be performed preferably under high stringent conditions. The high stringent condition means, for example, a condition that the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is preferable. The DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 (preferably the amino acid sequence represented by SEQ ID NO: 2) is preferably represented by SEQ ID NO: 3 or SEQ ID NO: 4. For example, DNA containing a base sequence.

D which completely encodes the protein of the invention
NA is amplified by the PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA incorporated into an appropriate expression vector is used for the partial or whole region of the protein of the present invention. It can be cloned by hybridizing with a DNA fragment labeled with a coding DNA fragment or synthetic DNA. Hybridization can be performed, for example, by molecular cloning (Molecula
r Cloning) 2nd (J. Sambrook et al., Cold Spring H
arbor Lab. Press, 1989). When using a commercially available library, hybridization can be performed according to the method described in the instruction manual attached to the library. The nucleotide sequence of DNA can be determined by a known kit, for example, Mutan ^™ -super Express Km (Takara Shuzo Co., Ltd.), Muta
n ^TM -K (Takara Shuzo Co., Ltd.), ODA-LA PCR method, Gap
The conversion can be performed according to a method known per se such as the pedduplex method or the Kunkel method or a method similar thereto. The cloned protein-encoding DNA can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose.
The DNA has an A as a translation initiation codon at the 5'terminal side.
It may have TG, and may have TAA, TGA or TAG as a translation termination codon at the 3'-terminal side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor.

The above-mentioned expression vector may be, for example, a DNA encoding the protein of the present invention
It can be produced by cutting out a fragment and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. As the expression vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC1
2, pUC13); a plasmid derived from Bacillus subtilis (eg, pU
B110, pTP5, pC194); yeast-derived plasmid (eg, pSH19, pSH15); bacteriophage such as λ phage; animal virus such as retrovirus, vaccinia virus, baculovirus; pA1-
11, pXT1, pRc / CMV, pRc / RSV, p
cDNAI / Neo or the like is used. The promoter may be any promoter as long as it is suitable for the host used for gene expression. For example, when the host is an animal cell, SRα promoter, SV4
0 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter and the like are used. Of these, CMV promoter, SRα promoter and the like are preferable. When the host is a bacterium belonging to the genus Escherichia, trp promoter, lac promoter, recA promoter, λP _L promoter, lpp promoter, T7 promoter and the like are preferable. When the host is a bacterium of the genus Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter,
The ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.

As the expression vector, in addition to the above, if desired, an enhancer, splicing signal, poly A
Those containing an additional signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori), and the like can be used. Examples of selection markers include dihydrofolate reductase (hereinafter, dh
fr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter referred to as A
mp ^r ) and a neomycin resistance gene (hereinafter sometimes referred to as Neo ^r , 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 thymidine-free medium. In addition, if necessary, a signal sequence suitable for the host may be added to the N-terminal side of the protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, the PhoA signal sequence, OmpA signal sequence, etc .; When the host is a bacterium belonging to the genus Bacillus, α-
Amylase signal sequence, subtilisin signal sequence, etc .; when host is yeast, MFα signal sequence, SUC2 signal sequence, etc .; when host is animal cell, insulin signal sequence, α-interferon signal sequence , Antibody molecules, signal sequences, etc. are used respectively.

The transformant containing the DNA encoding the protein of the present invention can be prepared according to a known method using the DN
It can be produced by transforming a host with an expression vector containing A. Here, examples of the expression vector include those described above. As a host,
For example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Examples of the genus Escherichia include, for example, Escherichia coli.
hia coli) K12 ・ DH1 [Procedures of
The National Academy of Sciences
Of the USA (Proc. Natl. Acad. Sci. US
A), 60, 160 (1968)], JM103 [Nucleic Acids Resear
ch), Volume 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of
Molecular Biology), 120, 517 (197)
8)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C60.
0 [Genetics, 39, 440
(1954)] or the like is used. Examples of the genus Bacillus include, for example, Bacillus subtili
s) MI114 [Gene, 24, 255 (198
3)], 207-21 [Journal of Biochemistry, Vol. 95, 87]
(1984)] and the like are used. Examples of yeasts include Saccharomyces cerevisii.
revisiae) ^AH22, AH22R ^-, NA87-11
A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1
913, NCYC2036, Pichia pastoris (Pich
ia pastoris) KM71 and the like are used.

Examples of insect cells include virus A
In the case of cNPV, the larvae of the night moth larvae (Spodopte
ra frugiperda cell; Sf cell), Trichoplusia ni midgut-derived MG1 cell, Trichoplusia ni egg-derived Hig
h Five ^TM cells, Mamestra brassicae-derived cells, Estigm
Cells derived from ena acrea are used. The virus is B
In the case of mNPV, silkworm-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 C
RL1711), Sf21 cells (above Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used. Examples of insects include silkworm larvae [Maeda et al., Nature, 315,
592 (1985)]. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter, CHO).
(Abbreviated as (dhfr ⁻ ) cell), mouse L cell, mouse A
tT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

The transformation can be carried out according to a known method depending on the type of host. The genus Escherichia is described in, for example, Proc. Natl. Acad. Sci. USA, Volume 69, Proc.
2110 (1972) and Gene, Volume 17, 107
(1982) and the like for transformation. Bacillus bacteria include, for example, Molecular & General Genetics (Molecular & G
eneral Genetics), 168, 111 (1979) and the like. Yeast is, for example, a method of enzymology (Meth
ods in Enzymology), 194, 182-187 (1)
991), Procedures of the National
Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 75, 19
29 (1978) and the like. Insect cells and insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988) and the like. Animal cells are described in, for example, Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, Volume 52, 456 (1)
It can be transformed according to the method described in 973).

Cultivation of the transformant depends on the type of host.
It can be carried out according to a known method. For example, when a transformant whose host is Escherichia or Bacillus is cultured, a liquid medium is preferable as a medium used for the culture. Further, the medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Here, as the carbon source, for example, glucose, dextrin, soluble starch, sucrose and the like; as the nitrogen source, for example, ammonium salts, nitrates,
Inorganic or organic substances such as corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract; examples of the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factor and the like may be added to the medium. PH of medium
Is preferably about 5-8. As a medium for culturing a transformant whose host is a bacterium of the genus Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Miller) Journalof Expe
riments in Molecular Genetics), 431-433, C
old Spring Harbor Laboratory, New York 1972]
Is preferred. If necessary, a drug such as 3β-indolylacrylic acid may be added to the medium to allow the promoter to work efficiently. Cultivation of a transformant whose host is a bacterium of the genus Escherichia is usually about 15 to 43 ° C.
At about 3 to 24 hours. If necessary, aeration and stirring may be performed. Cultivation of a transformant whose host is a bacterium of the genus Bacillus is usually performed at about 30 to 40 ° C for about 6 to 24 hours. If necessary, aeration and stirring may be performed. As a medium for culturing a transformant whose host is yeast, for example, a Burkholder minimum medium [Bostian, KL et al., Procedures of the
National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA),
77, 4505 (1980)] or SD medium containing 0.5% casamino acid [Bitter, GA et al., Procedures of the National Academy of Sciences of the USA (Proc. Natl. Aca
d. Sci. USA), 81, 5330 (1984)] and the like. The pH of the medium is preferably about 5-8. Culturing is usually performed at about 20 ° C to 35 ° C for about 24 to 72 ° C.
Done on time. 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, Grace's Insect Med
ium (Grace, TCC, Nature, 195,788 (19
62)) to which an additive such as immobilized 10% bovine serum is appropriately added is used. The pH of the medium is preferably about 6.2 to 6.4. Culturing is usually performed at about 27 ° C. for about 3 to 5 days. Aeration and agitation may be performed as necessary. As a medium for culturing a transformant whose host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122] is used.
Vol. 501 (1952)], DMEM medium [Virology, Vol. 8, 396 (1959)], RPMI
1640 medium [The Journal of the American Medical Association (The Journal of the Ame
rican Medical Association) Volume 199, 519 (196
7)], 199 medium [Proceeding of the Society for the Biological Med
icine), Vol. 73, 1 (1950)] and the like are used.
The pH of the medium is preferably about 6-8. Culturing is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours.
Aeration and agitation may be performed as necessary. As described above, the protein of the present invention can be produced inside the transformant, in the cell membrane or outside the cell.

The protein of the present invention can be separated and purified from the culture obtained by culturing the transformant according to a method known per se. For example, when the protein of the present invention is extracted from cultured bacterial cells or cells, the bacterial cells or cells collected from the culture by a known method are suspended in an appropriate buffer solution, and then ultrasonic waves, lysozyme and / or freeze-thawing A method of obtaining a crude protein extract by centrifugation or filtration after disrupting the cells or cells is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . Further, when the protein of the present invention is extracted from the nuclear fraction, the precipitate obtained by the above centrifugation or filtration is treated with, for example, a hypertonic solution, and the supernatant is recovered by centrifugation to recover the crude nuclear protein. A method of obtaining an extract is used. When the protein is secreted into the culture, a method of collecting the culture supernatant from the culture by a known method is used. The protein contained in the thus obtained culture supernatant or the extract can be separated and purified according to a method known per se. As such a method, a method utilizing solubility such as salting-out or solvent precipitation method; mainly utilizing a difference in molecular weight such as dialysis method, ultrafiltration method, gel filtration method and SDS-polyacrylamide gel electrophoresis method Method; method utilizing difference in charge such as ion exchange chromatography; method utilizing specific affinity such as affinity chromatography; method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography; isoelectricity A method utilizing a difference in isoelectric point such as a point electrophoresis method is used. These methods can be combined appropriately.

When the protein thus obtained is obtained as 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 is obtained as a salt. The salt can be converted into a free form or another salt by a method known per se or a method analogous thereto. The protein produced by the transformant can be optionally modified or partially removed by treating it with an appropriate protein-modifying enzyme before or after purification. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The presence of the thus obtained protein of the present invention can be confirmed by an enzyme immunoassay using a specific antibody, Western blotting or the like.

The diseases associated with the protein of the present invention include diseases in which the amount of the protein of the present invention is increased or decreased as compared with the normal condition. Here, as the “disease in which the amount of the protein of the present invention is increased as compared with the normal time”, for example, renal disease (eg, diabetic nephropathy; chronic glomerulonephritis; IgA nephropathy; renal transplantation) Chronic rejection afterwards; renal cancer; peritoneal sclerosis during peritoneal dialysis; cardiovascular disease (eg, arteriosclerosis; myocardial infarction; heart failure; cardiomyopathy; vascular restenosis after PTCA and stent placement; after heart / vessel transplantation) Chronic rejection; thrombosis); cerebrovascular disease (eg, cerebral infarction); lung disease (eg, pulmonary fibrosis, chronic obstructive pulmonary syndrome, lung cancer); liver disease (eg, cirrhosis, hepatitis, liver cancer);
Gastrointestinal disease (eg, colitis, gastric cancer, colon cancer); gonadal disease (eg, prostate cancer); collagen disease (eg, scleroderma, systemic lupus erythematosus); rheumatic disease (eg, rheumatoid arthritis); bone Diseases (eg, osteoporosis) and the like can be mentioned. Examples of the "disease in which the amount of the protein of the present invention is decreased as compared with the normal time" include, for example, gastrointestinal tract disease (eg, gastric ulcer, duodenal ulcer); skin disease (eg, burn, postoperative wound). And so on. The disease associated with the protein of the present invention is preferably “a disease in which the amount of the protein of the present invention is increased when compared with the normal state”. Further, as the disease associated with the protein of the present invention, an Egr-1-dependent disease that develops depending on the amount of Egr-1 in the living body is preferable. The disease associated with the protein of the present invention is preferably renal disease, more preferably Egr-1 dependent renal disease. Particularly, renal Egr-1-dependent renal disease is preferable.

The present invention is characterized in that the protein of the present invention is used to prevent diseases associated with the protein.
The present invention relates to a method for screening a therapeutic substance. The screening method of the present invention includes, for example, 1) culturing cells having the ability to produce the protein of the present invention and culturing cells having the ability to produce the protein of the present invention in the presence of a test compound. so,
Comparing the production amount of the protein of the present invention; 2) culturing cells having the ability to produce the protein of the present invention and culturing cells having the ability to produce the protein of the present invention in the presence of a test compound If you do,
Comparing the activities of the proteins of the invention; 3) with and without the test compound,
Comparing the activity of the proteins of the invention; and the like. The cell having the ability to produce the protein of the present invention is not particularly limited as long as it is the cell having the ability to produce the protein, but is not limited to oxidative stress and growth factors (hereinafter collectively referred to as “Egr-1 expression inducer” In some cases, the production of the protein of the present invention (preferably Egr-1) is induced in response to various stimuli such as treatment. The cell having the ability to produce the protein of the present invention may be the above-mentioned “transformant containing the DNA encoding the protein of the present invention”. Suitable examples of cells capable of producing the protein of the present invention include cells isolated from the kidney of mammals (preferably human, rat, mouse, etc.). These cells may be immortalized. Culturing of cells having the ability to produce the protein of the present invention is carried out in the same manner as the transformant described above. Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like. The protein of the present invention can be quantified by a known method, for example, using an antibody against the protein of the present invention, according to a method such as Western analysis, an ELISA method or the like or a method similar thereto.

The antibody against the protein of the present invention may be either a monoclonal antibody or a polyclonal antibody as long as it is an antibody capable of recognizing the protein of the present invention. The antibody may be the antibody molecule itself, or may be F (ab ') ₂ , Fab', or Fab of the antibody molecule.
It may be a fraction. The antibody may be labeled. As the labeling agent used for labeling the antibody, for example,
Radioisotopes, enzymes, fluorescent substances, luminescent substances, etc. are used. As the radioisotope, for example,
^{[12 5} I], ^[131 I], ^[3 H], ^[14 C] and the like are used. The enzyme is preferably stable and has a large specific activity, for example, β-galactosidase, β-
Glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Furthermore, the biotin-avidin system can be used for binding the antibody or the antigen to the labeling agent.

When quantifying the protein of the present invention, the protein to be quantified may be either contained in the cell or secreted outside the cell, or may be the total of both. Further, when quantifying the protein of the present invention contained in cells, it is preferable that the cells are treated with an appropriate fixative or a membrane permeation enhancer. It is also possible to suspend the cells in an appropriate buffer solution, destroy the cells by ultrasonic waves or freeze-thaw, and then quantify the protein in the disrupted solution. If necessary, the protein in the disrupted solution may be separated and purified, and then the protein may be quantified.

The activity of the protein of the present invention used in the screening method of the present invention includes, for example, transcription control activity. Specific examples thereof include "binding activity for a polynucleotide to which the protein of the present invention can bind", "expression control activity of a gene under the control of transcription control by the protein of the present invention" and the like.
Examples of the polynucleotide include a polynucleotide containing the base sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 described later (preferably the base sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6). A polynucleotide having). The binding activity can be determined by a known method, for example, the method of Shi-Fang Yan et al. (Proc. Natl. Acad. Sci. U
SA 95, 8298-8303, 1998) or a method similar thereto, according to a gel shift assay (electrophoretic mo
bility shift assay).
Alternatively, the above-mentioned polynucleotide to which the protein of the present invention can bind is immobilized on a suitable solid phase (eg, microtiter plate, etc.) (for example, the biotin-labeled polynucleotide is immobilized on (strept) avidin). Phase, etc.), the protein of the present invention (or a fraction containing the same) and a labeled antibody against the protein of the present invention (or an antibody against the protein of the present invention and a labeled secondary antibody against the antibody) are solid-phased. Alternatively, the protein of the present invention bound to the solid phase may be quantified according to a method such as an ELISA method or a method similar thereto to measure the protein of the present invention. Here, when the protein of the present invention is provided from a culture obtained by culturing cells having the ability to produce the protein of the present invention, the production amount of the protein of the present invention in the cells can be simultaneously measured. it can. In addition, the cells can be cultured under stimulation with an Egr-1 expression inducer, if necessary. Examples of the "gene under the control of transcriptional regulation by the protein of the present invention" include fibrosis-related genes, cell cycle-related genes and thrombosis-related genes exemplified as the "substantially the same activity". For the expression control activity of these genes, according to the method described in the Example, prepare appropriate primers from the DNA sequence encoding the gene and perform RT-PCR to measure the amount of the transcription product of the gene. Can be measured by Further, the expression control activity is a known method, for example, the method of Shi-Fang Yan et al. (Proc. Natl.
Acad. Sci. USA 95, 8298-8303, 1998) or a method equivalent thereto, and measured by Northern blotting using a probe prepared by labeling a polynucleotide containing the nucleotide sequence of the DNA encoding the gene. it can. In addition, the expression control activity can be determined by a known method such as Sh
i-Fang Yan et al. (Proc. Natl. Acad. Sci. USA 95
Vol., 8298-8303, 1998) or a method analogous thereto, and SEQ ID NO: on the DNA encoding the “gene under the transcriptional control of the protein of the present invention”:
5 or SEQ ID NO: 6, a vector in which a transcriptional regulatory region containing the nucleotide sequence and an appropriate reporter gene are ligated is prepared, and the vector is introduced into an appropriate cell to express the protein encoded by the reporter gene. It can also be measured by checking.

By the above screening method, a preventive / therapeutic substance for a disease associated with the protein of the present invention, that is, a compound that regulates (promotes or inhibits) the production of the protein of the present invention or the activity of the protein of the present invention ( Compounds that promote or inhibit) can be screened. For example, a test compound that increases the amount of the protein of the present invention by about 20% or more, preferably 30% or more, more preferably about 50% or more is a compound that promotes the production of the protein of the present invention; A test compound that reduces the amount by about 20% or more, preferably by 30% or more, more preferably by about 50% or more can be selected as the compound that inhibits the production of the protein of the present invention. For example, a test compound that increases the activity of the protein of the present invention by about 20% or more, preferably 30% or more, more preferably about 50% or more is a compound that promotes the activity of the protein of the present invention; An activity of about 20% or more, preferably 30% or more,
More preferably, a test compound that decreases by about 50% or more can be selected as a compound that inhibits the activity of the protein of the present invention.

In the above screening method, instead of comparing the production amount of the protein of the present invention in the presence and absence of the test compound, it is suspected that a disease associated with the protein of the present invention or a salt thereof is involved. By quantifying / comparing the amount of the protein of the present invention in cells and other test subjects derived from animals with those from normal control animals, when an increase or decrease in the amount of the protein in the test subject is confirmed, It can be diagnosed that the subject has a disease associated with the protein of the present invention, or that the subject has a high possibility of having the disease. Furthermore, similarly, the activity of the protein of the present invention is measured.
When the increase or decrease in the activity of the protein in the subject is confirmed by comparison, it means that the subject has a disease associated with the protein of the present invention, or the subject may have the disease. It can be diagnosed as highly prone.

The preventive / therapeutic substances for diseases associated with the protein of the present invention obtained by using the screening method of the present invention include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts. It may be any of liquid, animal tissue extract, plasma and the like. These may form a salt, and specific examples of the salt include those similar to the salts of the protein of the present invention described above.

The “prophylactic / therapeutic substance for a disease associated with the protein of the present invention” (compound) obtained by the screening method of the present invention is mixed with a pharmacologically acceptable carrier, if necessary, to give a pharmaceutical composition. Later, the protein of the present invention can be used as a prophylactic / therapeutic drug for diseases associated with the protein. Here, as the pharmacologically acceptable carrier, various organic or inorganic carrier substances conventionally used as formulation materials are used. Excipients, lubricants, binders, disintegrants in solid formulations; solvents in liquid formulations , A solubilizing agent, a suspending agent, a tonicity agent, a buffering agent, a soothing agent, etc. If necessary, formulation additives such as antiseptics, antioxidants, coloring agents and sweeteners can also be used.

Suitable examples of excipients include lactose, sucrose,
D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethyl cellulose, gum arabic, dextrin, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, alumino metasilicate. Magnesium acid etc. are mentioned. Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Suitable examples of binders include pregelatinized starch,
Sucrose, gelatin, gum arabic, methyl cellulose,
Examples thereof include carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone and the like. 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 the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. And so on. Suitable examples of suspending agents include stearyl triethanolamine, sodium lauryl sulfate,
Surfactants such as laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate; hydrophilic such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose Polymers: polysorbates, polyoxyethylene hydrogenated castor oil and the like. Preferable examples of the isotonicity agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like. Preferable examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like. Preferable examples of soothing agents include benzyl alcohol and the like. Preferable examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Preferable examples of the antioxidant include sulfite, ascorbate and the like. 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 and the like, water-insoluble lake dyes ( Examples thereof include aluminum salts of the above water-soluble food tar pigments), natural pigments (eg, β-carotene, chlorophyll, red iron oxide, etc.), etc. Preferable examples of the sweetener include saccharin sodium, dipotassium glycyrrhizinate, and aspartame. , Stevia, etc.

The dosage form of the pharmaceutical composition is, for example, oral preparations such as tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions, suspensions; and injections (eg. , Subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), external preparation (eg,
Nasal administration preparations, transdermal preparations, ointments etc.), suppositories (eg rectal suppositories, vaginal suppositories etc.), pellets, drops, sustained release preparations (eg sustained release microcapsules etc.) Parenteral agents can be mentioned, and these can be safely administered orally or parenterally, respectively. The pharmaceutical composition can be produced by a method conventionally used in the technical field of formulation, such as the method described in the Japanese Pharmacopoeia. Below, the concrete manufacturing method of a formulation is explained in full detail. The content of the compound obtained by the screening method of the present invention in the pharmaceutical composition varies depending on the dosage form, the dose of the compound, etc., and is, for example, about 0.1 to 1.
It is 00% by weight.

For example, an oral preparation includes an active ingredient as an active ingredient, an excipient (eg, lactose, sucrose, starch, D-mannitol, etc.), a disintegrant (eg, carboxymethylcellulose calcium), a binder (eg, pregelatinized starch). , Gum arabic, carboxymethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.) or lubricants (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.), etc. are added for compression molding, and if necessary, taste masking, It is produced by coating with a coating base by a method known per se for the purpose of enteric property or sustainability.

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. Saccharose is used as the sugar coating base, and talc, precipitated calcium carbonate, gelatin,
You may use together 1 type (s) or 2 or more types selected from gum arabic, pullulan, a carnauba wax, etc. As the water-soluble film coating base, for example, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
Cellulose polymers such as hydroxyethyl cellulose and methyl hydroxyethyl cellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name), Rohm Pharma Co.], synthetic polymers such as polyvinylpyrrolidone; polysaccharides such as pullulan And so on. Examples of enteric film coating bases include cellulosic polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L [Eudragit L (trade name), Rohm Pharma Co., Ltd., methacrylic acid copolymer LD [Eudragit L-30D55 (trade name), Rohm Pharma Co.], methacrylic acid copolymer S [Eudragit S]
(Trade name), Rohm Pharma Co., Ltd., and the like; acrylic acid-based polymers; natural products such as shellac. Examples of the sustained-release film coating base include cellulosic polymers such as ethyl cellulose; aminoalkyl methacrylate copolymer RS [Eudragit RS (trade name), Rohm Pharma Co.], ethyl acrylate / methyl methacrylate copolymer suspension. Suspended liquid [Eudragit NE
(Trade name), ROHM Pharma Co., Ltd., and the like. The coating base described above,
You may mix and use 2 or more types in an appropriate ratio. Further, at the time of coating, a light shielding agent such as titanium oxide, iron sesquioxide, etc. may be used.

Injectable preparations are prepared by dispersing active ingredients (eg, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc.), preservatives (eg, methylparaben, propylparaben, benzyl alcohol). ，
Chlorobutanol, phenol, etc., tonicity agent (eg,
Sodium chloride, glycerin, D-mannitol, D-
Soluble and suspended in an aqueous solvent (eg, distilled water, physiological saline, Ringer's solution, etc.) or an oily solvent (eg, olive oil, sesame oil, cottonseed oil, vegetable oil such as corn oil, propylene glycol, etc.) together with sorbitol, glucose, etc. It is produced by turbidity or emulsification. At this time, if desired, additives such as solubilizing agents (eg, sodium salicylate, sodium acetate, etc.), stabilizers (eg, human serum albumin, etc.), soothing agents (eg, benzyl alcohol, etc.) may be used. The injection solution is usually filled in a suitable ampoule.

The preparation thus obtained is safe and has low toxicity. Etc.) or parenterally. The dose of the prophylactic / therapeutic drug for a protein-related disease of the present invention varies depending on the target disease, administration subject, administration route, etc. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, of a compound obtained by the screening method of the present invention which is an active ingredient per day,
More preferably, it is about 1.0 to 20 mg.

An antibody against the protein of the present invention can be produced using the protein as an antigen according to a method known per se for producing an antibody or antiserum. A monoclonal antibody or a polyclonal antibody against the protein of the present invention can be produced, for example, as follows. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal at a site where antibody production can be carried out by administration, itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals used include mammals such as monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats (also chickens other than mammals), but mice and rats are preferably used. To be For example, a warm blooded animal immunized with an antigen, for example, an individual with an antibody titer is selected from mice, and 2 to 5 days after the final immunization, the spleen or lymph node is collected, and antibody-producing cells contained in them are homologous. Alternatively, a monoclonal antibody-producing hybridoma can be prepared by fusing with a myeloma cell of a xenogeneic animal. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation is performed by a known method, for example, the method of Kohler and Milstein [Nature, 25
6, 495 (1975)]. Examples of the fusion accelerator include polyethylene glycol (PE
G) and Sendai virus can be mentioned, but PEG is preferably used.

Examples of myeloma cells include NS-1,
Examples include myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1, but P3U1 is preferably used. The preferable ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG600) is used.
0) is added at a concentration of about 10 to 80%, and 20 to 40
Cell fusion can be efficiently carried out by incubating at 1 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes. Monoclonal antibody-producing hybridomas are prepared, for example, by adding a hybridoma culture supernatant to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed with a carrier, and then adding an anti-immunoglobulin antibody (labeled with a radioactive substance or enzyme) ( When the cells used for cell fusion are mice, an anti-mouse immunoglobulin antibody is used) or protein A and a method for detecting a monoclonal antibody bound to a solid phase; a method in which an anti-immunoglobulin antibody or protein A is adsorbed A hybridoma culture supernatant is added to the phase, a protein labeled with a radioactive substance or an enzyme is added, and the monoclonal antibody bound to the solid phase is detected. The monoclonal antibody can be selected according to a method known per se or a method analogous thereto. Selection of the monoclonal antibody can be carried out usually in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a medium for selecting and breeding the monoclonal antibody, any medium may be used as long as the hybridoma can grow. Examples of such a medium include RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, and GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.). Alternatively, a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culture temperature is
It is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum. The monoclonal antibody thus obtained is a method known per se,
For example, a method for separating and purifying immunoglobulin [eg, salting out method, alcohol precipitation method, isoelectric focusing method, electrophoresis method, adsorption / desorption method using ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, Only the antibody is collected using an antigen-bound solid phase or an active adsorbent such as protein A or protein G,
Specific purification method of dissociating the bond to obtain an antibody].

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

Since the antibody against the protein of the present invention can bind to the protein of the present invention or a salt thereof to inactivate (neutralize) it, a prophylactic / therapeutic agent for diseases associated with the protein of the present invention Can also be used as The prophylactic / therapeutic agent may be an antibody itself against the protein of the present invention, but is preferably a pharmaceutical composition obtained by mixing the antibody with a pharmacologically acceptable carrier. Here, examples of the pharmacologically acceptable carrier include those similar to the above-mentioned "prophylactic / therapeutic substance for diseases associated with the protein of the present invention".
The pharmaceutical composition can be produced in the same manner as in the above-mentioned "prophylactic / therapeutic substance for diseases associated with the protein of the present invention". Since the preparation thus obtained is safe and has low toxicity, it can be used, for example, in mammals (for example, humans, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.). It can be administered orally or parenterally. The dose of the prophylactic / therapeutic drug for the protein-related disease of the present invention varies depending on the target disease, the administration subject, the administration route, etc., but for example, an adult patient (body weight 60)
kg), the amount of the antibody to the protein of the present invention as an active ingredient is about 0.1 to 10 per day.
0 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

Since the expression of the protein of the present invention is increased in, for example, renal diseases (eg, diabetic nephropathy), it is used as a marker for early diagnosis in renal diseases, determination of severity of symptoms, and prediction of disease progression. It is useful. Therefore, the antibody against the protein of the present invention can also be used as a diagnostic agent for diseases associated with the protein of the present invention. That is, the present invention provides (i) a ratio of the labeled protein of the present invention bound to the antibody by competitively reacting the antibody of the present invention with a test solution and the labeled protein of the present invention. The method for diagnosing a disease associated with the protein or a salt thereof, which comprises quantifying the protein of the present invention or a salt thereof in a test solution by measuring After reacting the insolubilized antibody of the present invention and the labeled another antibody of the present invention simultaneously or sequentially, the activity of the labeling agent on the insolubilized carrier is measured to measure the activity of the present invention in the test solution. A method for diagnosing a disease associated with a protein or a salt thereof, which comprises quantifying a protein or a salt thereof.

In the above quantification (ii), when one antibody recognizes the N-terminal portion of the protein of the present invention, the other antibody binds to another portion of the protein of the present invention, for example, the C-terminal portion. An antibody that recognizes is desirable. Also,
In addition to quantifying the protein using a monoclonal antibody against the protein 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.

Quantification of the protein of the present invention or a salt thereof using the antibody of the present invention is not particularly limited, and the amount of the antibody, the antigen or the antibody-antigen complex corresponding to the amount of the antigen in the liquid to be measured. Any measuring method may be used, so long as it is detected by chemical or physical means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used, but from the viewpoint of sensitivity and specificity, the sandwich method described later is particularly preferably used.

As the labeling agent used in the measuring method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H],
[ ¹⁴ C] or the like is used. As the above-mentioned enzyme, those having a stable and large specific activity are preferable, and for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, etc. are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can be used for binding the antibody or the antigen to the labeling agent.

For insolubilizing the antigen or antibody,
Physical adsorption may be used, or a method using a chemical bond usually used for immobilizing and immobilizing proteins may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicone, and glass.

In the sandwich method, an insolubilized monoclonal antibody of the present invention is reacted with a test solution (first reaction), and another labeled monoclonal antibody of the present invention is reacted (secondary reaction), and then insolubilized. By measuring the activity of the labeling agent on the carrier, the amount of the protein of the present invention or its salt in the test solution can be quantified. The primary reaction and the secondary reaction may be carried out in the reverse order, may be carried out simultaneously, or may be carried out at different times. The labeling agent and the method of insolubilization can be the same as those described above. In addition, in the immunoassay method by the sandwich method,
The antibody used for the solid phase antibody or the labeling antibody does not necessarily have to be one type, and a mixture of two or more types of antibodies may be used for the purpose of improving the measurement sensitivity.

In the method of assaying the protein of the present invention or its salt by the above-mentioned sandwich method, the monoclonal antibodies of the present invention used in the primary reaction and the secondary reaction are antibodies having different binding sites for the protein of the present invention. It is preferably used. That is, when the antibody used in the secondary reaction recognizes the C-terminal portion of the protein of the present invention, the antibody used in the primary reaction and the secondary reaction is preferably an antibody used in the primary reaction. An antibody that recognizes an N-terminal other than the C-terminal is used.

The monoclonal antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competitive method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen is reacted.
(F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), the labeled amount of either B or F is measured, and the amount of antigen in the test liquid is quantified. In this reaction method, a soluble antibody is used as the antibody, a liquid phase method using polyethylene glycol, a second antibody against the antibody is used for B / F separation, and a solid phase antibody is used as the first antibody, or The first antibody is soluble and the second antibody is a solid-phased antibody. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are competitively reacted with a fixed amount of the labeled antibody and then the solid phase and the liquid phase are separated, or the antigen in the test liquid is separated. After reacting with an excess amount of the labeled antibody and then adding the immobilized antigen to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated.
Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry utilizing laser scattering is preferably used. In applying each of these immunological assay methods to the quantification method of the present invention, no special conditions, operations, etc. are required to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like. For example, Hiroshi Irie's "Radioimmunoassay"
(Kodansha, published in 1974), Hiroshi Irie edited by "Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa, et al. "Enzyme Immunoassay" (medical school, published in 1978), Eiji Ishikawa, et al. "Enzyme-linked immunosorbent assay" (2nd edition) (Medical Shoin, published in 1982), Eiji Ishikawa et al. "Enzyme-linked immunosorbent assay" (3rd edition) (medical Shoin, published in 1987), "Me
thods in ENZYMOLOGY '' Vol. 70 (Immunochemical Techniq
ues (Part A)), ibid Vol. 73 (Immunochemical Techniq
ues (Part B)), ibid Vol. 74 (Immunochemical Techniq
ues (Part C)), ibid Vol. 84 (Immunochemical Techniq
ues (Part D: Selected Immunoassays)), ibid Vol. 9
2 (Immunochemical Techniques (Part E: Monoclonal An
tibodies and General Immunoassay Methods)), ibid.
Vol. 121 (Immunochemical Techniques (Part I: Hybrid
oma Technology and Monoclonal Antibodies)) (above,
Academic Press, Inc.) etc. can be referred to. As described above, the protein of the present invention or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention.

In the above-mentioned quantification method using the antibody of the present invention, a biopsy sample (eg, kidney cells) of a subject animal is used as a subject, and the concentration of the protein of the present invention or a salt thereof in the subject is quantified. Thus, when overexpression or reduction of the protein is detected, for example, it is diagnosed that a disease related to the protein of the present invention or a salt thereof, such as renal disease, is involved or is likely to be affected in the future. be able to.

The present invention is characterized by further using a polynucleotide containing a nucleotide sequence encoding the protein of the present invention or a partial sequence thereof (hereinafter sometimes abbreviated as the polynucleotide of the present invention). The present invention relates to a method for screening a preventive / therapeutic substance for a disease associated with the protein. The polynucleotide of the present invention comprises a nucleotide sequence (DNA or R) encoding the protein of the present invention.
It may be any as long as it contains NA (preferably DNA) or a partial sequence thereof. Examples of the polynucleotide include DNA encoding the protein of the present invention and RNA such as mRNA, which may be double-stranded or single-stranded. When double-stranded, double-stranded DNA, double-stranded RNA or DNA: RNA
It may be a hybrid. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). Examples of the DNA encoding the protein of the present invention include those mentioned above. The polynucleotide of the present invention may be in a free form, a salt, an amide form or an ester form. Examples of the salt include those similar to the salts of the protein of the present invention, and examples of the amide and ester include those in which the terminal phosphate group is amidated or esterified.

The screening method using the polynucleotide of the present invention can be carried out by, for example, culturing cells having the ability to produce the protein of the present invention and testing cells having the ability to produce the protein of the present invention in the presence of a test compound. The amount of RNA encoding the protein of the present invention is compared with that in the case of culturing in the following manner; Here, as the cell having the ability to produce the protein of the present invention, the method of culturing the cell, and the test compound, the same ones as the screening method using the protein of the present invention described above can be mentioned. RNA encoding the protein of the present invention can be prepared by known methods, for example, molecular
Cloning (Molecular Cloning) 2nd (J. Sambrook
et al., Cold Spring Harbor Lab. Press, 1989) or a method similar thereto. For example, the RNA can be obtained by a known method, for example, Northern hybridization using a nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 as a probe, or a part thereof; SEQ ID NO: as a primer. 3 or a PCR method using a nucleic acid containing a part of the nucleotide sequence represented by SEQ ID NO: 4 or a method analogous thereto; and the like.
For example, the amount of RNA (preferably mRNA) encoding the protein of the present invention is about 20% or more, preferably 30%.
%, More preferably about 50% or more, as a compound that promotes the expression of RNA encoding the protein of the present invention; the amount of RNA (preferably mRNA) encoding the protein of the present invention is about 20. A test compound that reduces by more than%, preferably more than 30%, more preferably more than about 50% can be selected as a compound that inhibits the expression of RNA encoding the protein of the present invention.

Prophylactic / therapeutic substances for diseases associated with the protein of the present invention obtained by the screening method using the polynucleotide of the present invention include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, It may be any of a plant extract, an animal tissue extract, plasma and the like. These may form a salt, and specific examples of the salt include those similar to the salts of the protein of the present invention described above. The preventive / therapeutic substance for a disease associated with the protein of the present invention obtained by the screening method is mixed with a pharmacologically acceptable carrier as necessary to form a pharmaceutical composition, and then the disease associated with the protein of the present invention. It can be used as a preventive / therapeutic drug for. Here, examples of the pharmacologically acceptable carrier include those similar to the case of the preventive / therapeutic substance for the diseases associated with the protein of the present invention obtained by the screening method using the protein of the present invention. The pharmaceutical composition is
It can be produced in the same manner as in the case of the preventive / therapeutic substance for diseases associated with the protein of the present invention obtained by the screening method using the protein of the present invention. Since the preparation thus obtained is safe and has low toxicity,
For example, mammals (eg, human, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey,
Chimpanzee) and can be administered orally or parenterally. The dose of the prophylactic / therapeutic drug for the diseases related to the protein of the present invention is a target disease, an administration target,
Depending on the administration route, etc., for example, in an adult patient (body weight 60 kg) suffering from renal disease, the compound obtained by the screening method of the present invention, which is an active ingredient, is about 0.1 to 100 mg per day. ,
It is preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

The preventive / therapeutic substance for diseases associated with the protein of the present invention can also be screened by detecting the promoter activity of the gene encoding the protein. DN encoding the protein of the present invention
In a cell in which A is replaced with a reporter gene or in a non-human mammal, the reporter gene exists under the control of the promoter of the gene encoding the protein of the present invention. By confirming the expression of the encoded substance, the activity of the promoter can be detected.
Further, the activity of the promoter can be detected in cells and non-human mammals having a vector formed by linking the transcriptional regulatory region of the gene encoding the protein of the present invention and a reporter gene. Here, as the reporter gene, for example, β-galactosidase gene (lac
Z), soluble alkaline phosphatase gene, luciferase gene and the like. For example, when a part of the DNA region encoding the protein of the present invention is replaced with the β-galactosidase gene (lacZ) derived from Escherichia coli, originally, in the tissue in which the protein of the present invention is expressed,
Β-galactosidase is expressed instead of the protein of the present invention. Thus, for example, 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-g
The expression state of the protein of the present invention can be easily confirmed by staining with a reagent such as al) that serves as a substrate for β-galactosidase. Specifically, cells or tissue sections are fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. for about 30 minutes or longer. 1
After reacting for a period of time, the tissue sample was treated with 1 mM EDTA / PB.
By washing with S solution, the β-galactosidase reaction is stopped, and by observing the color development, the expression state of the protein of the present invention in cells or tissues can be confirmed. In addition, mRNA encoding lacZ may be detected according to a conventional method. The compound that promotes or inhibits the promoter activity of the gene encoding the protein of the present invention regulates the expression of the protein and the activity of the protein, and thus is useful as a prophylactic / therapeutic agent for diseases associated with the protein of the present invention. is there.

Since the antisense nucleotide of the DNA encoding the protein of the present invention hybridizes with the polynucleotide encoding the protein of the present invention and inhibits the translation into the protein, the compound obtained by the screening method of the present invention Similarly to the above, the protein of the present invention is useful as a prophylactic / therapeutic drug for diseases associated with the protein.
Here, the antisense nucleotide is complementary to the base sequence of the DNA encoding the protein of the present invention,
Alternatively, an antisense DNA is preferable, as long as it has a substantially complementary base sequence or a partial sequence thereof and has an action of suppressing the expression of the DNA. The nucleotide sequence substantially complementary to the nucleotide sequence of the DNA encoding the protein of the present invention includes, for example, a nucleotide sequence complementary to the nucleotide sequence of the DNA (that is, a complementary strand of the DNA encoding the protein of the present invention. And a partial base sequence of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. In particular, of the entire base sequence of the complementary strand of the DNA encoding the protein of the present invention, the base sequence of the portion encoding the N-terminal portion of the protein (eg, the base sequence near the start codon)
Antisense nucleotides having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with the complementary strand thereof are preferable. Specifically, an antisense nucleotide having a base sequence complementary to or substantially complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or a part thereof; Preferably,
Examples include an antisense nucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or a part thereof. The number of bases of the antisense nucleotide is not particularly limited as long as it can specifically hybridize with the polynucleotide encoding the protein of the present invention and inhibit translation into the protein, but is usually about 10 to 40, preferably Is about 15 to 30. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleotide is, for example, phosphorothioate,
It may be substituted with a chemically modified phosphate residue such as methylphosphonate and phosphorodithionate. The sugar (deoxyribose) of each nucleotide may be replaced with a chemically modified sugar structure such as 2′-O-methylation,
The base portion (pyrimidine, purine) may also be chemically modified, and may be any as long as it hybridizes to the DNA having the base sequence represented by SEQ ID NO: 2. These antisense nucleotides can be produced using a known DNA synthesizer or the like. The antisense nucleotide of the DNA encoding the protein of the present invention has low toxicity and suppresses the function of the protein of the present invention or the DNA encoding the protein (eg, thyroxine de5′-iodination enzyme activity) in vivo. Therefore, the protein of the present invention can be used, for example, as a prophylactic / therapeutic agent for diseases associated with the protein. The antisense nucleotide is formulated into a mammal (eg, human, mouse, rat) in the same manner as in the case of a prophylactic / therapeutic substance for a disease associated with the protein of the present invention obtained by a screening method using the protein of the present invention , Rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.) or parenterally. Also,
The antisense nucleotide can also be administered after inserting it into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector. The antisense nucleotide may be administered by a gene gun or a catheter such as a hydrogel catheter, or may be locally administered as an inhalant in the trachea after aerosolization.
The dose of the antisense nucleotide varies depending on the target disease, administration subject, administration route, etc., but for example, in an adult patient (body weight 60 kg) suffering from renal disease, about 0.1 to 100 mg per day , Preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. Furthermore, the antisense nucleotide of the DNA encoding the protein of the present invention can also be used as a diagnostic oligonucleotide probe for investigating the presence of the DNA or its expression in tissues and cells. Similarly to the above antisense polynucleotide, double-stranded RNA containing a part of RNA encoding the protein of the present invention, ribozyme containing a part of RNA encoding the protein of the present invention, and the like are also proteins of the present invention. Alternatively, since the function of DNA encoding the protein can be suppressed, it can be used, for example, as a prophylactic / therapeutic agent for renal diseases. Here, the double-stranded RNA can be prepared by a known method, for example, Nature, 411, 494.
According to the method described on page 2001, it can be designed and manufactured based on the sequence of the polynucleotide of the present invention. Ribozymes can be prepared by known methods, for example TRENDS in Molecular.
According to the method described in Medicine, Volume 7, 221 pages, 2001,
It can be designed and manufactured based on the sequence of the polynucleotide of the present invention. For example, a desired ribozyme can be produced by linking a known ribozyme to a part of RNA encoding the protein of the present invention. As a part of RNA encoding the protein of the present invention,
RNA of the present invention that can be cleaved by known ribozymes
The part (RNA fragment) close to the above cleavage site is mentioned. When the above double-stranded RNA or ribozyme is used as a prophylactic / therapeutic agent for diseases associated with the protein of the present invention, it can be formulated and administered in the same manner as the above-mentioned antisense polynucleotide.

The polynucleotide of the present invention can be used, for example, in mammals (eg,
Human, rat, mouse, guinea pig, rabbit, sheep,
It is possible to detect an abnormality (gene abnormality) of the DNA or mRNA encoding the protein of the present invention in pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.). It is useful as a gene diagnostic agent for mutations or decreased expression, increased DNA or mRNA, or excessive expression. The above-mentioned gene diagnosis using the polynucleotide of the present invention can be carried out, for example, by known Northern hybridization or PCR-SSCP method (Genomics (Genomics)).
s), Vol. 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences of USA.
s of the National Academy of Sciences of the Unite
d States of America), Vol. 86, 2766-277.
0 (1989)) and the like. For example, when overexpression or decrease is detected by Northern hybridization, or PCR-SSCP
When a DNA mutation is detected by the method, it can be diagnosed that the subject is suffering from a disease related to the protein of the present invention such as renal disease or is likely to be affected in the future.

The present invention further comprises a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6, which is a preventive / therapeutic agent for diseases associated with the protein of the present invention. Regarding Here, the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is D to which the protein of the present invention binds.
It is a decoy nucleotide for NA. The polynucleotide can be produced according to a method known per se. The polynucleotide has low toxicity and suppresses the function of the protein of the present invention or the DNA encoding the protein in vivo (eg, transcriptional regulatory activity of fibrosis-related gene, cell cycle-related gene, and thrombus-related gene). Therefore, the protein of the present invention can be used as a prophylactic / therapeutic drug for diseases associated with the protein.
The polynucleotide having the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 is the same as in the case of a prophylactic / therapeutic substance for a disease associated with the protein of the present invention obtained by the screening method using the protein of the present invention. To formulate a mammal (e.g., human, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog,
It can be administered orally or parenterally to monkeys, chimpanzees, etc.). The polynucleotide can also be administered after inserting it into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. The polynucleotide may be administered by a gene gun or a catheter such as a hydrogel catheter, or may be locally administered as an inhalant into the trachea after aerosolization. The dose of the prophylactic / therapeutic drug for a protein-related disease of the present invention varies depending on the target disease, administration subject, administration route, etc., but for example, in an adult patient (body weight 60 kg) suffering from renal disease, About 0.1 to 100 mg, preferably about 1.0 to 5, per day of the polynucleotide having the base sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 which is an active ingredient.
It is 0 mg, more preferably about 1.0 to 20 mg.

The present invention further relates to a prophylactic / therapeutic drug for diabetic nephropathy, which comprises an Egr-1 inhibitor. Eg
The r-1 inhibitor is not particularly limited as long as it is a substance capable of suppressing the production or expression of Egr-1 in the body; or the activity of Egr-1, in vivo, peptides, proteins,
It may be any of non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. These may form a salt, and specific examples of the salt include those similar to the salts of the protein of the present invention described above. The Egr-1 inhibitor is preferably a substance capable of suppressing the production or expression of Egr-1 in the kidney; or the activity of Egr-1, that is, a renal Egr-1 inhibitor. The Egr-1 inhibitor may be a substance capable of suppressing the production or expression of factors such as fibrosis-related gene, cell cycle-related gene, thrombosis-related gene and the like in vivo; or the activity of these factors. Here, examples of the fibrosis-related gene, the cell cycle-related gene and the thrombus-related gene include those exemplified as the above-mentioned “substantially the same activity”. A prophylactic / therapeutic drug for diabetic nephropathy can be formulated by using an Egr-1 inhibitor as in the case of the prophylactic / therapeutic substance for the diseases associated with the protein of the present invention. The preventive / therapeutic agent for diabetic nephropathy of the present invention is safe and has low toxicity, and therefore, for example, mammals (for example, human, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey) , Chimpanzees, etc.) or parenterally. The dose of the prophylactic / therapeutic drug for diabetic nephropathy of the present invention varies depending on the target disease, administration subject, administration route, etc., but for example, in an adult patient (body weight 60 kg), Egr which is the active ingredient per day is the active ingredient. The amount of the -1 inhibitor is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

In this specification, when an abbreviation is used for a base or amino acid, IUPAC-IUB Commission
Based on the abbreviations based on on Biochemical Nomenclature or abbreviations commonly used in this field, examples are given below. When amino acids may have optical isomers, the L form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Cysteine Met: Cysteine Met : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine T r: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Sec: selenocysteine (selenocystein
e)

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the partial sequence of the amino acid sequence of Egr-1, which is conserved among human, mouse, and rat. [SEQ ID NO: 2] This shows the amino acid sequence of human Egr-1. [SEQ ID NO: 3] This shows the base sequence of DNA encoding human Egr-1 having the amino acid sequence represented by SEQ ID NO: 2. [SEQ ID NO: 4] This shows the base sequence of DNA encoding human Egr-1 having the amino acid sequence represented by SEQ ID NO: 2. [SEQ ID NO: 5] This shows the base sequence of decoy nucleotide of Egr-1. [SEQ ID NO: 6] This shows the base sequence of decoy nucleotide of Egr-1. [SEQ ID NO: 7] This shows the base sequence of Egr-1 antisense oligonucleotide. [SEQ ID NO: 8] This shows the base sequence of a primer for amplifying Egr-1 cDNA. [SEQ ID NO: 9] This shows the base sequence of a primer for amplifying Egr-1 cDNA. [SEQ ID NO: 10] Double-stranded DN capable of binding to Egr-1
The base sequence of the sense strand of A is shown. [SEQ ID NO: 11] Double-stranded D that cannot bind to Egr-1
The base sequence of the sense strand of NA is shown.

[0067]

EXAMPLES The present invention will be more specifically described below with reference to Examples and Experimental Examples, but the present invention is not limited thereto.

Experimental Example 1 Early growth respo in Wistar fatty rat kidney
Increased nse-1 (Egr-1) mRNA expression Non-insulin-dependent diabetes mellitus (NIDD) at 13, 22 and 40 weeks of age
Male Wistar f with M) and spontaneous diabetic nephropathy (DN)
Using atty rats (Takeda Ravix) and its normal control male Wistar lean rats of the same age (Takeda Ravix), 24-hour urine collection and tail vein blood sampling were performed, and urinary albumin excretion and plasma glucose concentration were measured. And plasma insulin concentration was measured. Urinary albumin excretion is A / G
B-test Wako (Wako Pure Chemical Industries, Ltd.) was used to measure plasma glucose levels using SYNCHRON CX5 Delta (Beckman Coulter).
Was measured using. The insulin concentration was measured by the RIA method (Shionogi & Co.). Kidneys were collected from each of 5 rats and stored at -80 ° C, and then the kidneys were crushed to extract total RNA. Primers and fluorescent probes were prepared based on previously reported mRNA sequences, and various mRNA expression levels were measured by the real-time quantitative RT-PCR method using ABI PRISM 7700 (Applied Biosystems). The results are shown in [Table 1] and [Table 2]. Wistar f
Atty rats exhibit NIDDM at 13 weeks of age and DN at 22 weeks of age.
, And urinary albumin excretion increased. Increased expression of Egr-1 mRNA was observed after 22 weeks of age, and Tran at 40 weeks of age.
sforming growth factor-β1 (TGF-β1) and fibronectin
An increase in mRNA expression was observed. [Table 1] Urinary albumin excretion, plasma glucose concentration and plasma insulin concentration Wistar lean rats Wistar fatty rats 13 weeks 22 weeks 40 weeks 13 weeks 22 weeks 40 weeks urinary albumin 5.1 4.0 10.4 6.7 55.7 182.0 excretion (mg / day) in the plasma glucose 138.9 137.1 134.3 319.2 402.9 319.4 concentration (mg / dl) Plasma insulin 123.0 125.6 158.7 1091.9 1019.2 1674.4 Concentration (μUnits / ml) (n = 5, Mean) [Table 2] mRNA expression levels of Egr-1, TGF-β1 and fibronectin in Wistar fatty rat kidney (Relative value when the expression level in the kidney of Wistar lean rats at each week of age is set to 1) 13-week-old 22-week-old 40-week - old Egr-1 1.1 2.2 2.4 TGF-β1 1.1 1.0 1.7 Fibronectin 1.2 1.0 2.1 (n = 5, Mean)

Experimental Example 2 Early growth respo in Zucker fatty rat kidney
Changes in nse-1 (Egr-1) mRNA expression Male Zu with hyperinsulinemia and spontaneous renal failure
Candesartan cilexetil (angiotensin II type 1 receptor antagonist) suspended in 0.5% methylcellulose 100 cP was orally administered once daily for 9 weeks to cker fatty rats (ZF rats, 18 weeks old, Charles River Japan). The same-week-old male Zucker lean rats (ZL rat, Charles River Japan), which were the control group and the normal control group, were orally administered with 0.5% methylcellulose 100 cP (Vehicle) once a day for consecutive days. Eight weeks after administration, urine was collected for 24 hours, and urinary albumin excretion was measured using A / GB Test Wako (Wako Pure Chemical Industries, Ltd.). Nine weeks after the administration, the kidney was collected, stored at -80 ° C, and then crushed to extract total RNA. ABI PRISM770 was prepared by preparing primers and fluorescent probes based on the previously reported mRNA sequences.
Real-time quantitative RT-using 0 (Applied Biosystems)
The expression levels of various mRNAs were measured by the PCR method. The results are shown in [Table 3].
And [Table 4]. In the kidneys of Zucker fatty rats with increased urinary albumin excretion, Egr-1 mRNA expression increased and Transforming growth factor-β1 (TGF-
β1), platelet-derived growth factor-B (PDGF-
B), fibronectin and α1 (I) collagen each mRNA expression was also increased. Furthermore, when the increase in urinary albumin excretion was suppressed by administration of candesartan cilexetil, any increase in the mRNA expression level was suppressed. [Table 3] Urinary albumin excretion, blood glucose concentration, and blood insulin concentration ZL rat ZF rat ZF rat Vehicle Vehicle candesartan cilexetil Urinary albumin 48.5 401.5 61.7 Excretion (mg / day) (n = 8-9, Mean) [Table 4] Egr-1, TGF-β1, in the kidney of Zucker fatty rat, MRNA expression level of PDGF-B, fibronectin and α1 (I) collagen (relative value when the expression level in kidney of ZL rat is 1) ZF Rat ZF Rat Vehicle Candesartan Egr-1 2.9 1.3 TGF-β1 2.1 1.2 PDGF-B 1.5 1.0 Fibronectin 2.1 1.1 α1 (I) collagen 2.3 1.6 (n = 8-9, Mean)

Experimental Example 3 Early growth in rat renal glomerular mesangial cells
Increased expression of response-1 (Egr-1) mRNA Expression of renal glomerular mesangial cells (pas) isolated and cultured from 4-week-old male Sprague-Dawley rats (SD rats, CLEA Japan)
sege 7) in DMEM medium containing 20% fetal calf serum (FCS).
After culturing for 3 days, use 3% DMEM containing 0.2% bovine serum albumin.
Cultured for a day. FCS (20%) or angio
Tensin II (AII) (10 ⁻⁶ M) was added and reacted at 37 ° C. for 30 minutes. Candesartan, an AII type 1 receptor antagonist, is FCS
Alternatively, 5 minutes before AII stimulation, the cells were added to the medium so that the final concentration was 10 ⁻⁵ M. Total RNA was extracted 30 minutes after FCS or AII stimulation. A primer and a fluorescent probe were prepared based on a previously reported mRNA sequence, and the Egr-1 mRNA expression level was measured by a real-time quantitative RT-PCR method using ABI PRISM 7700 (Applied Biosystems). The results are shown in [Table 5] and [Table 6]. Egr-1
The mRNA expression level was markedly increased 30 minutes after FCS stimulation and partially suppressed by candesartan. A slight increase in Egr-1 mRNA expression was observed 30 minutes after AII stimulation, and most was suppressed by candesartan. [Table 5] Increase in Egr-1 mRNA expression and suppression of candesartan by FCS stimulation (relative value when expression level before stimulation is 1) [Table 6] AII stimulation increases Egr-1 mRNA expression and candesartan suppressive effect (relative value when expression level before stimulation is 1)

Experimental Example 4 ea in kidney of spontaneously hypercholesterolemic rat
Increased expression of rly growth response-1 (Egr-1) mRNA Male spontaneously hypercholesterolemic rats (SHC rats, 11 weeks old, Takeda Ravix) spontaneously developing renal impairment and normal control group of the same age group. Using male Sprague-Dawley rats (SD rats, CLEA Japan, Inc.), 24-hour urine collection and tail vein blood collection were performed, and urinary albumin excretion and plasma total cholesterol concentrations were measured. Urinary albumin excretion
Plasma total cholesterol concentration measured by A / GB Test Wako (Wako Pure Chemical Industries) was measured by SYNCHRON CX5 Delta (Beckm
an Coulter). At 12 weeks of age, kidneys were collected from rats, stored at -80 ° C, and then crushed tot
al RNA was extracted. ABI PRISM7700 (Applied B
iosystems) for real-time quantitative RT-PCR
The gr-1 mRNA expression level was measured. 12-week-old SHC rat and SD
Urinary albumin excretion in rats is 179.2 and 9.2, respectively.
(Mg / day, Mean (n = 5)) and plasma cholesterol levels were 92.4 and 43.5 (mg / dl, Mean (n = 5)), respectively, and all parameters were high in SHC rats. . Egr-1 mRNA in SHC rat kidney at this time
The expression level was compared with that in the SD rat kidney,
It increased to 3.8 times (Mean (n = 5)).

Experimental Example 5 Early growth res in human embryonic kidney-derived HEK-293 cells
Induction of renal fibrosis-related gene expression by overexpression of ponse (Egr) -1 Human embryonic kidney-derived HEK-293 cells (CRL-1573, ATCC, passege
40) was cultured in DMEM medium containing 10% fetal bovine serum for 1 day, and then human Egr-1 expression plasmid was introduced into the cells at a concentration of 2 μg / dish using Polyfect (QIAGEN) 20 μl / 3.5 cm dish. The Egr-1 expression plasmid is the pBK-CMV vector.
A plasmid in which cDNA of the coding region of human Egr-1 was incorporated into (STRATAGENE) was used. Only the pBK-CMV vector used for Egr-1 plasmid construction was introduced into control cells. Twenty-four hours after the introduction, cells were collected and proteins and total RNA were extracted. Expression of Egr-1 protein
After performing SDS-PAGE, Egr-1 specific antibody (Anti-Egr-1 (C-1
9), Santa Cruz) was used for Western blotting, and bands were detected by chemiluminescence method (PIERCE). The brightness integrated value of the detected band was calculated by CS Analyzer 1.00a (ATTO). In addition, the mRNA expression level of the renal fibrosis-related gene was measured by a quantitative RT-PCR method using ABI PRISM 7700 (Applied Biosystems) by preparing a primer and a fluorescent probe based on the previously reported mRNA sequence. The results are shown in [Table 7]. Egr
-1 expression plasmid introduction increased Egr-1 protein expression, and also introduced tissue factor, fibronectin, intercel
The expression of renal fibrosis-related gene of lular adhesion molecule (ICAM) -1 mRNA was increased. [Table 7] Increased Egr-1 protein expression and renal fibrosis-related gene expression due to introduction of Egr-1 plasmid (relative value when control expression level is 1)

Example 1 Early growth in rat glomerular mesangial cells
response-1 (Egr-1) antisense oligodeoxynucleotide
(AS-ODN) action The renal glomerular mesangial cells (passege 4-5) isolated and cultured from 4-week-old male SD rats were treated with D containing 20% fetal bovine serum.
After culturing in MEM medium for 3-4 days until it becomes subconfluent, replace with DMEM containing 0.2% bovine serum albumin.
Performed um starvation. 48 hours after the start of Serum starvation, Egr-1 AS-ODN (SEQ ID NO: 7) or Control-ODN
(A scrambl that matches the size of AS-ODN and each number of bases
ed ODN, both of which were requested to be synthesized by BIOGNOSTIK) were added to the medium at a concentration of 20 μM, left to stand in a CO ₂ incubator for 24 hours, and then basic fibroblast growth factor (bFGF) (3 ng / m
It was stimulated with l) and reacted at 37 ° C for 1-2 hours. Protein was extracted from cells 1 hour after bFGF stimulation, and total RNA was extracted from cells 2 hours after bFGF stimulation. Expression of Egr-1 protein was carried out by SDS-PAGE, followed by Egr-1 specific antibody (Anti-Egr-1
Western blotting using (C-19), Santa Cruz)
Bands were detected by chemiluminescence method (PIERCE). The brightness integrated value of the detected band was calculated by CS Analyzer 1.00a (ATTO). The mRNA expression level of the tissue factor was measured by a quantitative RT-PCR method using ABI PRISM 7700 (Applied Biosystems) by preparing a primer and a fluorescent probe based on a previously reported mRNA sequence. The results are shown in [Table 8]. bFG
Ehr-1 protein expression increased 5.1-fold 1 hour after F stimulation,
This increase was suppressed by Egr-1 AS-ODN treatment. Contr
No inhibitory effect was observed with ol-ODN treatment. Tissue factor mRNA increased 2.5-fold 2 hours after bFGF stimulation, but this increase was almost completely suppressed by Egr-1 AS-ODN treatment. No inhibitory effect was observed in the Control-ODN treated group. No cytotoxicity due to the introduction of Egr-1 AS-ODN was observed. [Table 8] Effects of Egr-1 AS-ODN introduction on Egr-1 protein expression and tis sue factor mRNA expression after bFGF stimulation (relative value when control expression level is 1) Stimulation Control bFGF (3 ng / ml) treatment vehicle vehicle AS-ODN Control-ODN Egr-1 protein 1.0 5.1 2.0 7.0 Tissue factor mRNA 1.0 2.5 1.1 4.6 (n = 2-3, Mean)

Example 2 Egr-1 utilizing DNA binding activity
Protein quantification A synthetic DNA having an Egr-1 binding sequence and an anti-Egr-1 antibody were combined to set up a system for quantifying the amount of Egr-1 protein on a microplate. (1) Construction of Egr-1 expression plasmid and expression in animal cells To obtain human Egr-1 cDNA, cDNA derived from human adipose tissue was used.
(Clontech) as a template and synthetic chain (reason chain number 8 and 9) as a primer
ction. The end portion of the amplified cDNA was cleaved with restriction enzymes EcoRI and PstI, and cloned into plasmid pcDNA3.1 (+) (Invitrogen) cleaved with EcoRI and PstI.
The nucleotide sequence was confirmed. Culture Petri dish (FALCON 3003)
, DMEM containing 10% FCS and 100 μg / ml kanamycin was used as a medium, 2 × 10 ⁶ human-derived cultured cells HEK293 were seeded, and the next day, 20 μg of the above Egr-1 expression plasmid and Lipofe were added.
The gene was introduced into the cells using ctamine2000 (Invitrogen). After 48 hours, remove the medium and wash with PBS.
ml solution (25 mM Tris-HCl (pH 7.4), 0.4 M NaCl, 100
1/1 in an aqueous solution containing μM EDTA, 1 mM DTT, 1% Triton X-100
00 amount of Protease inhibitor cocktail (SIGMA, P8340)
Was added thereto to lyse the cells, and the cells were collected in a sample tube. After allowing to stand on ice for 5 minutes, the mixture was stirred with a vortex mixer for 10 seconds and centrifuged at 12000 xg at 4 ° C for 5 minutes to obtain a supernatant. This was used as a standard sample. Similarly, gene transfer of vector pcDNA3.1 (+) was carried out, and the obtained cell lysate was used as a control.

(2) Detection of Egr-1 Synthesis containing Egr-1 binding sequence in which 5 ′ end was labeled with biotin
DNA (SEQ ID NO: 10) was allowed to form a double strand with its complementary strand synthetic DNA (the complementary strand is not biotinylated). In addition, synthetic D introduced with base substitution that makes Egr-1 unable to bind.
A double strand was also formed for NA (SEQ ID NO: 11) and used as a control. Block A diluted 4 times with PBS
It was diluted to a final concentration of 50 nM with ce (containing 1 mM EDTA) (manufactured by Snow Brand Milk Products Co., Ltd., purchased from Dainippon Pharmaceutical Co., Ltd.), and 100 μl of each was added to a 96-well plate (IWAKI) on which streptavidin was immobilized.
After standing still at room temperature for 30 minutes, the solution was removed by suction, 300 μl of Block Ace (containing 1 mM EDTA) diluted 4-fold with PBS was added, and the mixture was left standing at room temperature for 3 hours. Remove the solution and remove 300 μl
Wash three times with PBS, and use the measurement buffer (50 mM Tris-HCl (pH 7.
4), 100 mM NaCl, 10% Block Ace, 200 μM ZnSO _4, 250 μ
M DTT, 10 μg / ml poly dI-dC (Amersham), 0.05% Tr
100 μl of standard sample or control sample (prepared in (1) above) diluted 50 times with itonX-100] was added to each well. After standing at room temperature for 40 minutes, 300 μl of washing solution (2 mM i
midazole-buffered saline, 0.02% Tween 20, 200 μM Z
Wash 3 times with nSO ₄ ) and anti-Egr-1 antibody (rabbit, polyclonal antibody, SANTA CRUZ, sc-189) diluted 500 times with poly dI-dC-free measurement buffer, 100 μl per well After addition, the mixture was allowed to stand at 4 ° C. for 5 hours. 3 with 300 μl wash
Washed and anti-rabbit IgG antibody (HRP labeled, Cell Signaling, diluted 2000 times with poly dI-dC-free measurement buffer)
100 μl) was added to each well, and the mixture was allowed to stand at 4 ° C. overnight. Wash 6 times with 300 μl of washing solution, and add TMB solution (DAKO) to 10
After adding 0 μl each to develop color, 100 μl of 2N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured using an absorptiometer (TECAN, Spectra Rainbow). Egr
-1 on a plate on which synthetic DNA with a sequence capable of binding is fixed
The absorbance when using a lysate of cells into which gr-1 cDNA was introduced was 0.751. When the same sample was measured on a plate with unbound DNA immobilized, the absorbance was 0.016, DNA
The value was 0.005 for the plate without fixing. Egr-1 c
The value is 0.0 when measured with a plate on which the DNA of the binding sequence is immobilized using a lysate of cells into which DNA has not been introduced.
It was 05.

[0076]

EFFECT OF THE INVENTION According to the screening method of the present invention, it is possible to screen a preventive / therapeutic drug having excellent effects and having no side effect, such as renal disease.

[0077]

[Sequence list] SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Screening Method <130> B03007 <150> JP 2002-003769 <151> 2002-01-10 <160> 11 <170> PatentIn version 3.1 <210> 1 <211> 123 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> Partial amino acid sequence of Egr-1 protein which is conserved        between human, mouse and rat. <400> 1 Tyr Gln Ser Gln Leu Ile Lys Pro Ser Arg Met Arg Lys Tyr Pro Asn 1 5 10 15 Arg Pro Ser Lys Thr Pro Pro His Glu Arg Pro Tyr Ala Cys Pro Val             20 25 30 Glu Ser Cys Asp Arg Arg Phe Ser Arg Ser Asp Glu Leu Thr Arg His         35 40 45 Ile Arg Ile His Thr Gly Gln Lys Pro Phe Gln Cys Arg Ile Cys Met     50 55 60 Arg Asn Phe Ser Arg Ser Asp His Leu Thr Thr His Ile Arg Thr His 65 70 75 80 Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala                 85 90 95 Arg Ser Asp Glu Arg Lys Arg His Thr Lys Ile His Leu Arg Gln Lys             100 105 110 Asp Lys Lys Ala Asp Lys Ser Val Val Ala Ser         115 120 <210> 2 <211> 543 <212> PRT <213> Homo sapiens <400> 2 Met Ala Ala Ala Lys Ala Glu Met Gln Leu Met Ser Pro Leu Gln Ile 1 5 10 15 Ser Asp Pro Phe Gly Ser Phe Pro His Ser Pro Thr Met Asp Asn Tyr             20 25 30 Pro Lys Leu Glu Glu Met Met Leu Leu Ser Asn Gly Ala Pro Gln Phe         35 40 45 Leu Gly Ala Ala Gly Ala Pro Glu Gly Ser Gly Ser Asn Ser Ser Ser     50 55 60 Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Asn Ser Ser 65 70 75 80 Ser Ser Ser Ser Thr Phe Asn Pro Gln Ala Asp Thr Gly Glu Gln Pro                 85 90 95 Tyr Glu His Leu Thr Ala Glu Ser Phe Pro Asp Ile Ser Leu Asn Asn             100 105 110 Glu Lys Val Leu Val Glu Thr Ser Tyr Pro Ser Gln Thr Thr Arg Leu         115 120 125 Pro Pro Ile Thr Tyr Thr Gly Arg Phe Ser Leu Glu Pro Ala Pro Asn     130 135 140 Ser Gly Asn Thr Leu Trp Pro Glu Pro Leu Phe Ser Leu Val Ser Gly 145 150 155 160 Leu Val Ser Met Thr Asn Pro Pro Ala Ser Ser Ser Ser Ala Pro Ser                 165 170 175 Pro Ala Ala Ser Ser Ala Ser Ala Ser Gln Ser Pro Pro Leu Ser Cys             180 185 190 Ala Val Pro Ser Asn Asp Ser Ser Pro Ile Tyr Ser Ala Ala Pro Thr         195 200 205 Phe Pro Thr Pro Asn Thr Asp Ile Phe Pro Glu Pro Gln Ser Gln Ala     210 215 220 Phe Pro Gly Ser Ala Gly Thr Ala Leu Gln Tyr Pro Pro Pro Ala Tyr 225 230 235 240 Pro Ala Ala Lys Gly Gly Phe Gln Val Pro Met Ile Pro Asp Tyr Leu                 245 250 255 Phe Pro Gln Gln Gln Gly Asp Leu Gly Leu Gly Thr Pro Asp Gln Lys             260 265 270 Pro Phe Gln Gly Leu Glu Ser Arg Thr Gln Gln Pro Ser Leu Thr Pro         275 280 285 Leu Ser Thr Ile Lys Ala Phe Ala Thr Gln Ser Gly Ser Gln Asp Leu     290 295 300 Lys Ala Leu Asn Thr Ser Tyr Gln Ser Gln Leu Ile Lys Pro Ser Arg 305 310 315 320 Met Arg Lys Tyr Pro Asn Arg Pro Ser Lys Thr Pro Pro His Glu Arg                 325 330 335 Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe Ser Arg Ser             340 345 350 Asp Glu Leu Thr Arg His Ile Arg Ile His Thr Gly Gln Lys Pro Phe         355 360 365 Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu Thr     370 375 380 Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile 385 390 395 400 Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg His Thr Lys                 405 410 415 Ile His Leu Arg Gln Lys Asp Lys Lys Ala Asp Lys Ser Val Val Ala             420 425 430 Ser Ser Ala Thr Ser Ser Leu Ser Ser Tyr Pro Ser Pro Val Ala Thr         435 440 445 Ser Tyr Pro Ser Pro Val Thr Thr Ser Tyr Pro Ser Pro Ala Thr Thr     450 455 460 Ser Tyr Pro Ser Pro Val Pro Thr Ser Phe Ser Ser Pro Gly Ser Ser 465 470 475 480 Thr Tyr Pro Ser Pro Val His Ser Gly Phe Pro Ser Pro Ser Val Ala                 485 490 495 Thr Thr Tyr Ser Ser Val Pro Pro Ala Phe Pro Ala Gln Val Ser Ser             500 505 510 Phe Pro Ser Ser Ala Val Thr Asn Ser Phe Ser Ala Ser Thr Gly Leu         515 520 525 Ser Asp Met Thr Ala Thr Phe Ser Pro Arg Thr Ile Glu Ile Cys     530 535 540 <210> 3 <211> 1629 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (1629) <223> <400> 3 atg gcc gcg gcc aag gcc gag atg cag ctg atg tcc ccg ctg cag atc 48 Met Ala Ala Ala Lys Ala Glu Met Gln Leu Met Ser Pro Leu Gln Ile 1 5 10 15 tct gac ccg ttc gga tcc ttt cct cac tcg ccc acc atg gac aac tac 96 Ser Asp Pro Phe Gly Ser Phe Pro His Ser Pro Thr Met Asp Asn Tyr             20 25 30 cct aag ctg gag gag atg atg ctg ctg agc aac ggg gct ccc cag ttc 144 Pro Lys Leu Glu Glu Met Met Leu Leu Ser Asn Gly Ala Pro Gln Phe         35 40 45 ctc ggc gcc gcc ggg gcc cca gag ggc agc ggc agc aac agc agc agc 192 Leu Gly Ala Ala Gly Ala Pro Glu Gly Ser Gly Ser Asn Ser Ser Ser     50 55 60 agc agc agc ggg ggc ggt gga ggc ggc ggg ggc ggc agc aac agc agc 240 Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Asn Ser Ser 65 70 75 80 agc agc agc agc acc ttc aac cct cag gcg gac acg ggc gag cag ccc 288 Ser Ser Ser Ser Thr Phe Asn Pro Gln Ala Asp Thr Gly Glu Gln Pro                 85 90 95 tac gag cac ctg acc gca gag tct ttt cct gac atc tct ctg aac aac 336 Tyr Glu His Leu Thr Ala Glu Ser Phe Pro Asp Ile Ser Leu Asn Asn             100 105 110 gag aag gtg ctg gtg gag acc agt tac ccc agc caa acc act cga ctg 384 Glu Lys Val Leu Val Glu Thr Ser Tyr Pro Ser Gln Thr Thr Arg Leu         115 120 125 ccc ccc atc acc tat act ggc cgc ttt tcc ctg gag cct gca ccc aac 432 Pro Pro Ile Thr Tyr Thr Gly Arg Phe Ser Leu Glu Pro Ala Pro Asn     130 135 140 agt ggc aac acc ttg tgg ccc gag ccc ctc ttc agc ttg gtc agt ggc 480 Ser Gly Asn Thr Leu Trp Pro Glu Pro Leu Phe Ser Leu Val Ser Gly 145 150 155 160 cta gtg agc atg acc aac cca ccg gcc tcc tcg tcc tca gca cca tct 528 Leu Val Ser Met Thr Asn Pro Pro Ala Ser Ser Ser Ser Ala Pro Ser                 165 170 175 cca gcg gcc tcc tcc gcc tcc gcc tcc cag agc cca ccc ctg agc tgc 576 Pro Ala Ala Ser Ser Ala Ser Ala Ser Gln Ser Pro Pro Leu Ser Cys             180 185 190 gca gtg cca tcc aac gac agc agt ccc att tac tca gcg gca ccc acc 624 Ala Val Pro Ser Asn Asp Ser Ser Pro Ile Tyr Ser Ala Ala Pro Thr         195 200 205 ttc ccc acg ccg aac act gac att ttc cct gag cca caa agc cag gcc 672 Phe Pro Thr Pro Asn Thr Asp Ile Phe Pro Glu Pro Gln Ser Gln Ala     210 215 220 ttc ccg ggc tcg gca ggg aca gcg ctc cag tac ccg cct cct gcc tac 720 Phe Pro Gly Ser Ala Gly Thr Ala Leu Gln Tyr Pro Pro Pro Ala Tyr 225 230 235 240 cct gcc gcc aag ggt ggc ttc cag gtt ccc atg atc ccc gac tac ctg 768 Pro Ala Ala Lys Gly Gly Phe Gln Val Pro Met Ile Pro Asp Tyr Leu                 245 250 255 ttt cca cag cag cag ggg gat ctg ggc ctg ggc acc cca gac cag aag 816 Phe Pro Gln Gln Gln Gly Asp Leu Gly Leu Gly Thr Pro Asp Gln Lys             260 265 270 ccc ttc cag ggc ctg gag agc cgc acc cag cag cct tcg cta acc cct 864 Pro Phe Gln Gly Leu Glu Ser Arg Thr Gln Gln Pro Ser Leu Thr Pro         275 280 285 ctg tct act att aag gcc ttt gcc act cag tcg ggc tcc cag gac ctg 912 Leu Ser Thr Ile Lys Ala Phe Ala Thr Gln Ser Gly Ser Gln Asp Leu     290 295 300 aag gcc ctc aat acc agc tac cag tcc cag ctc atc aaa ccc agc cgc 960 Lys Ala Leu Asn Thr Ser Tyr Gln Ser Gln Leu Ile Lys Pro Ser Arg 305 310 315 320 atg cgc aag tat ccc aac cgg ccc agc aag acg ccc ccc cac gaa cgc 1008 Met Arg Lys Tyr Pro Asn Arg Pro Ser Lys Thr Pro Pro His Glu Arg                 325 330 335 cct tac gct tgc cca gtg gag tcc tgt gat cgc cgc ttc tcc cgc tcc 1056 Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe Ser Arg Ser             340 345 350 gac gag ctc acc cgc cac atc cgc atc cac aca ggc cag aag ccc ttc 1104 Asp Glu Leu Thr Arg His Ile Arg Ile His Thr Gly Gln Lys Pro Phe         355 360 365 cag tgc cgc atc tgc atg cgc aac ttc agc cgc agc gac cac ctc acc 1152 Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu Thr     370 375 380 acc cac atc cgc acc cac aca ggc gaa aag ccc ttc gcc tgc gac atc 1200 Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile 385 390 395 400 tgt gga aga aag ttt gcc agg agc gat gaa cgc aag agg cat acc aag 1248 Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg His Thr Lys                 405 410 415 atc cac ttg cgg cag aag gac aag aaa gca gac aaa agt gtt gtg gcc 1296 Ile His Leu Arg Gln Lys Asp Lys Lys Ala Asp Lys Ser Val Val Ala             420 425 430 tct tcg gcc acc tcc tct ctc tct tcc tac ccg tcc ccg gtt gct acc 1344 Ser Ser Ala Thr Ser Ser Leu Ser Ser Tyr Pro Ser Pro Val Ala Thr         435 440 445 tct tac ccg tcc ccg gtt act acc tct tat cca tcc ccg gcc acc acc 1392 Ser Tyr Pro Ser Pro Val Thr Thr Ser Tyr Pro Ser Pro Ala Thr Thr     450 455 460 tca tac cca tcc cct gtg ccc acc tcc ttc tcc tct ccc ggc tcc tcg 1440 Ser Tyr Pro Ser Pro Val Pro Thr Ser Phe Ser Ser Pro Gly Ser Ser 465 470 475 480 acc tac cca tcc cct gtg cac agt ggc ttc ccc tcc ccg tcg gtg gcc 1488 Thr Tyr Pro Ser Pro Val His Ser Gly Phe Pro Ser Pro Ser Val Ala                 485 490 495 acc acg tac tcc tct gtt ccc cct gct ttc ccg gcc cag gtc agc agc 1536 Thr Thr Tyr Ser Ser Val Pro Pro Ala Phe Pro Ala Gln Val Ser Ser             500 505 510 ttc cct tcc tca gct gtc acc aac tcc ttc agc gcc tcc aca ggg ctt 1584 Phe Pro Ser Ser Ala Val Thr Asn Ser Phe Ser Ala Ser Thr Gly Leu         515 520 525 tcg gac atg aca gca acc ttt tct ccc agg aca att gaa att tgc 1629 Ser Asp Met Thr Ala Thr Phe Ser Pro Arg Thr Ile Glu Ile Cys     530 535 540 <210> 4 <211> 1629 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (1629) <223> <400> 4 atg gcc gcg gcc aag gcc gag atg cag ctg atg tcc ccg ctg cag atc 48 Met Ala Ala Ala Lys Ala Glu Met Gln Leu Met Ser Pro Leu Gln Ile 1 5 10 15 tct gac ccg ttc gga tcc ttt cct cac tcg ccc acc atg gac aac tac 96 Ser Asp Pro Phe Gly Ser Phe Pro His Ser Pro Thr Met Asp Asn Tyr             20 25 30 cct aag ctg gag gag atg atg ctg ctg agc aac ggg gct ccc cag ttc 144 Pro Lys Leu Glu Glu Met Met Leu Leu Ser Asn Gly Ala Pro Gln Phe         35 40 45 ctc ggc gcc gcc ggg gcc cca gag ggc agc ggc agc aac agc agc agc 192 Leu Gly Ala Ala Gly Ala Pro Glu Gly Ser Gly Ser Asn Ser Ser Ser     50 55 60 agc agc agc ggg ggc ggt gga ggc ggc ggg ggc ggc agc aac agc agc 240 Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Asn Ser Ser 65 70 75 80 agc agc agc agc acc ttc aac cct cag gcg gac acg ggc gag cag ccc 288 Ser Ser Ser Ser Thr Phe Asn Pro Gln Ala Asp Thr Gly Glu Gln Pro                 85 90 95 tac gag cac ctg acc gca gag tct ttt cct gac atc tct ctg aac aac 336 Tyr Glu His Leu Thr Ala Glu Ser Phe Pro Asp Ile Ser Leu Asn Asn             100 105 110 gag aag gtg ctg gtg gag acc agt tac ccc agc caa acc act cga ctg 384 Glu Lys Val Leu Val Glu Thr Ser Tyr Pro Ser Gln Thr Thr Arg Leu         115 120 125 ccc ccc atc acc tat act ggc cgc ttt tcc ctg gag cct gca ccc aac 432 Pro Pro Ile Thr Tyr Thr Gly Arg Phe Ser Leu Glu Pro Ala Pro Asn     130 135 140 agt ggc aac acc ttg tgg ccc gag ccc ctc ttc agc ttg gtc agt ggc 480 Ser Gly Asn Thr Leu Trp Pro Glu Pro Leu Phe Ser Leu Val Ser Gly 145 150 155 160 cta gtg agc atg acc aac cca ccg gcc tcc tcg tcc tca gca cca tct 528 Leu Val Ser Met Thr Asn Pro Pro Ala Ser Ser Ser Ser Ala Pro Ser                 165 170 175 cca gcg gcc tcc tcc gcc tcc gcc tcc cag agc cca ccc ctg agc tgc 576 Pro Ala Ala Ser Ser Ala Ser Ala Ser Gln Ser Pro Pro Leu Ser Cys             180 185 190 gca gtg cca tcc aac gac agc agt ccc att tac tca gcg gca ccc acc 624 Ala Val Pro Ser Asn Asp Ser Ser Pro Ile Tyr Ser Ala Ala Pro Thr         195 200 205 ttc ccc acg ccg aac act gac att ttc cct gag cca caa agc cag gcc 672 Phe Pro Thr Pro Asn Thr Asp Ile Phe Pro Glu Pro Gln Ser Gln Ala     210 215 220 ttc ccg ggc tcg gca ggg aca gcg ctc cag tac ccg cct cct gcc tac 720 Phe Pro Gly Ser Ala Gly Thr Ala Leu Gln Tyr Pro Pro Pro Ala Tyr 225 230 235 240 cct gcc gcc aag ggt ggc ttc cag gtt ccc atg atc ccc gac tac ctg 768 Pro Ala Ala Lys Gly Gly Phe Gln Val Pro Met Ile Pro Asp Tyr Leu                 245 250 255 ttt cca cag cag cag ggg gat ctg ggc ctg ggc acc cca gac cag aag 816 Phe Pro Gln Gln Gln Gly Asp Leu Gly Leu Gly Thr Pro Asp Gln Lys             260 265 270 ccc ttc cag ggc ctg gag agc cgc acc cag cag cct tcg cta acc cct 864 Pro Phe Gln Gly Leu Glu Ser Arg Thr Gln Gln Pro Ser Leu Thr Pro         275 280 285 ctg tct act att aag gcc ttt gcc act cag tcg ggc tcc cag gac ctg 912 Leu Ser Thr Ile Lys Ala Phe Ala Thr Gln Ser Gly Ser Gln Asp Leu     290 295 300 aag gcc ctc aat acc agc tac cag tcc cag ctc atc aaa ccc agc cgc 960 Lys Ala Leu Asn Thr Ser Tyr Gln Ser Gln Leu Ile Lys Pro Ser Arg 305 310 315 320 atg cgc aag tac ccc aac cgg ccc agc aag acg ccc ccc cac gaa cgc 1008 Met Arg Lys Tyr Pro Asn Arg Pro Ser Lys Thr Pro Pro His Glu Arg                 325 330 335 cct tac gct tgc cca gtg gag tcc tgt gat cgc cgc ttc tcc cgc tcc 1056 Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe Ser Arg Ser             340 345 350 gac gag ctc acc cgc cac atc cgc atc cac aca ggc cag aag ccc ttc 1104 Asp Glu Leu Thr Arg His Ile Arg Ile His Thr Gly Gln Lys Pro Phe         355 360 365 cag tgc cgc atc tgc atg cgc aac ttc agc cgc agc gac cac ctc acc 1152 Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu Thr     370 375 380 acc cac atc cgc acc cac aca ggc gaa aag ccc ttc gcc tgc gac atc 1200 Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile 385 390 395 400 tgt gga aga aag ttt gcc agg agc gat gaa cgc aag agg cat acc aag 1248 Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg His Thr Lys                 405 410 415 atc cac ttg cgg cag aag gac aag aaa gca gac aaa agt gtt gtg gcc 1296 Ile His Leu Arg Gln Lys Asp Lys Lys Ala Asp Lys Ser Val Val Ala             420 425 430 tct tcg gcc acc tcc tct ctc tct tcc tac ccg tcc ccg gtt gct acc 1344 Ser Ser Ala Thr Ser Ser Leu Ser Ser Tyr Pro Ser Pro Val Ala Thr         435 440 445 tct tac ccg tcc ccg gtt act acc tct tat cca tcc ccg gcc acc acc 1392 Ser Tyr Pro Ser Pro Val Thr Thr Ser Tyr Pro Ser Pro Ala Thr Thr     450 455 460 tca tac cca tcc cct gtg ccc acc tcc ttc tcc tct ccc ggc tcc tcg 1440 Ser Tyr Pro Ser Pro Val Pro Thr Ser Phe Ser Ser Pro Gly Ser Ser 465 470 475 480 acc tac cca tcc cct gtg cac agt ggc ttc ccc tcc ccg tcg gtg gcc 1488 Thr Tyr Pro Ser Pro Val His Ser Gly Phe Pro Ser Pro Ser Val Ala                 485 490 495 acc acg tac tcc tct gtt ccc cct gct ttc ccg gcc cag gtc agc agc 1536 Thr Thr Tyr Ser Ser Val Pro Pro Ala Phe Pro Ala Gln Val Ser Ser             500 505 510 ttc cct tcc tca gct gtc acc aac tcc ttc agc gcc tcc aca ggg ctt 1584 Phe Pro Ser Ser Ala Val Thr Asn Ser Phe Ser Ala Ser Thr Gly Leu         515 520 525 tcg gac atg aca gca acc ttt tct ccc agg aca att gaa att tgc 1629 Ser Asp Met Thr Ala Thr Phe Ser Pro Arg Thr Ile Glu Ile Cys     530 535 540 <210> 5 <211> 9 <212> DNA <213> Artificial <220> <221> misc_feature <223> Oligonucleotide designed to act as decoy for Egr-1. <400> 5 gcgtgggcg 9 <210> 6 <211> 9 <212> DNA <213> Artificial <220> <221> misc_feature <223> Oligonucleotide designed to act as decoy for Egr-1. <400> 6 gcgggggcg 9 <210> 7 <211> 20 <212> DNA <213> Artificial <220> <221> misc_feature <223> Oligonucleotide designed to act as antisense DNA for Egr-1 mRNA. <400> 7 gcggggtgca ggggcacact 20 <210> 8 <211> 30 <212> DNA <213> Artificial <220> <221> misc_feature <223> Oligonucleotide designed to act as primer for amplifying Egr-1        cDNA. <400> 8 ccgaattcag tgttccccgc gccccgcatg 30 <210> 9 <211> 29 <212> DNA <213> Artificial <220> <221> misc_feature <223> Oligonucleotide designed to act as primer for amplifying Egr-1        cDNA. <400> 9 ggctcgagaa cctccatctg acctaagag 29 <210> 10 <211> 26 <212> DNA <213> Artificial <220> <221> misc_feature <223> Oligonucleotide designed to act as sense strand of        double-stranded DNA capable of binding with Egr-1. <400> 10 tgactcgccc tcgcccccgc gccggg 26 <210> 11 <211> 26 <212> DNA <213> Artificial <220> <221> misc_feature <223> Oligonucleotide designed to act as sense strand of        double-stranded DNA incapable of binding with Egr-1. <400> 11 tgactcgccc tcgacccagc gccggg 26

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 49/00 A61K 49/00 A 4H045 A61P 3/10 A61P 3/10 13/12 13/12 43/00 111 43/00 111 C07K 16/18 C07K 16/18 C12Q 1/02 C12Q 1/02 1/68 1/68 A G01N 33/15 G01N 33/15 Z 33/53 33/53 D M 33/566 33 / 566 // C12N 15/09 C12N 15/00 AF term (reference) 2G045 AA35 AA40 BA11 BB50 DA13 DA36 FB02 FB03 4B024 AA01 AA11 BA80 CA04 CA12 DA02 EA04 HA11 4B063 QA01 QA05 QA18 QQ08 QQ42 QQ53 QR77 QR02 QRQQR QRQQ QRQR AA02 AA07 AA13 AA14 AA17 BA02 BA08 BA23 BA35 BA44 CA53 CA56 CA59 NA13 NA14 ZA812 ZC022 ZC352 4C085 AA13 AA14 BB11 CC01 CC02 CC04 CC05 CC12 CC17 CC21 CC23 EE01 HH13 JJ01 KA03 KA26 KB82 LL20 4A045 CA40 DA75 EA20 EA50

Claims (37)

[Claims] 1. Use of a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, for diseases associated with the protein or its salt Screening method for preventive and therapeutic substances. 2. Use of a protein containing the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof to prevent a disease associated with the protein or a salt thereof.
Method for screening therapeutic substance. 3. The screening method according to claim 1, wherein the protein has the amino acid sequence represented by SEQ ID NO: 2. 4. The screening method according to claim 1, wherein the disease is renal disease. 5. The screening method according to claim 4, wherein the renal disease is Egr-1-dependent renal disease. 6. The screening method according to claim 4, wherein the renal disease is diabetic nephropathy. 7. When cells having the ability to produce a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 are cultured, Represented by SEQ ID NO: 1 when a cell having the ability to produce a protein or a salt thereof containing an amino acid sequence identical or substantially identical to the represented amino acid sequence is cultured in the presence of a test compound The screening method according to claim 1, wherein the production amount of a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence is compared. 8. A method for comparing the activity of a protein or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 in the presence and absence of a test compound. Claim 1 characterized by
The screening method described. 9. The activity according to claim 8, which is a binding activity to a polynucleotide to which a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 can bind. Screening method. 10. The activity is the expression control activity of a gene under the control of transcriptional control by a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof. The screening method according to claim 8. 11. A case of culturing a cell having the ability to produce a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, and the test compound The amount of production of the protein or its salt and the binding activity to the polynucleotide to which the protein or its salt can bind when cultured in the presence of -The screening method according to claim 1, wherein the screening is performed for comparison. 12. A compound or a salt thereof obtained by the screening method according to claim 1. 13. A protein containing the compound obtained by the screening method according to claim 1 or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Or a prophylactic / therapeutic drug for a disease associated with the salt. 14. Use of a polynucleotide containing a base sequence encoding a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof. A method for screening a preventive / therapeutic substance for a disease associated with a protein or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 15. A polynucleotide represented by SEQ ID NO: 1, which is characterized in that a polynucleotide containing a nucleotide sequence encoding a protein containing the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof or a partial sequence thereof is used. For screening a preventive / therapeutic substance for a disease associated with a protein or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence described above. 16. The screening method according to claim 14, wherein the polynucleotide contains the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 or a partial sequence thereof. 17. The screening method according to claim 14, wherein the disease is renal disease. 18. The screening method according to claim 17, wherein the renal disease is Egr-1-dependent renal disease. 19. The renal disease is diabetic nephropathy.
The screening method described. 20. When a cell having the ability to produce a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 is cultured, Represented by SEQ ID NO: 1 when a cell having the ability to produce a protein or a salt thereof containing an amino acid sequence identical or substantially identical to the represented amino acid sequence is cultured in the presence of a test compound R encoding a protein or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence
The screening method according to claim 14, which comprises comparing the amount of NA. 21. A compound or a salt thereof obtained by the screening method according to claim 14. 22. A protein containing the compound obtained by the screening method according to claim 14 or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Or a prophylactic / therapeutic drug for a disease associated with the salt. 23. An antibody against a protein or its salt, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 24. The antibody according to claim 23,
A prophylactic / therapeutic agent for a disease associated with a protein or its salt containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 25. A polynucleotide having a base sequence complementary to a base sequence encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof A preventive / therapeutic agent for a disease associated with a protein or a salt thereof, which comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 26. An antibody, which comprises the antibody according to claim 23,
A diagnostic agent for a disease associated with a protein or a salt thereof which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. 27. A SEQ ID NO: comprising a polynucleotide having a nucleotide sequence encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial sequence thereof. : A diagnostic agent for a disease associated with a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by 1: or a salt thereof. 28. The same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1, which comprises a polynucleotide having the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6. A preventive / therapeutic agent for a disease associated with a protein containing the amino acid sequence of or a salt thereof. 29. The preventive / therapeutic drug according to claim 28, wherein the disease is renal disease. 30. The preventive / therapeutic drug according to claim 29, wherein the renal disease is Egr-1-dependent renal disease. 31. The method according to claim 29, wherein the renal disease is diabetic nephropathy.
The preventive and therapeutic drugs listed. 32. A preventive / therapeutic drug for diabetic nephropathy, which comprises an Egr-1 inhibitor. 33. The preventive / therapeutic drug according to claim 32, wherein the Egr-1 inhibitor is a renal Egr-1 inhibitor. 34. A method for preventing or treating diabetic nephropathy in a mammal, which comprises administering an Egr-1 inhibitor to the mammal. 35. The method according to claim 34, wherein the Egr-1 inhibitor is a renal Egr-1 inhibitor. 36. Use of an Egr-1 inhibitor for producing a prophylactic / therapeutic drug for diabetic nephropathy. 37. The use according to claim 36, wherein the Egr-1 inhibitor is a renal Egr-1 inhibitor.