JP2004269512A - Preventing and treating agent of renal disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preventing and treating agent of renal diseases such as diabetic nephropathy. <P>SOLUTION: This preventing and treating agent of the renal diseases such as diabetic nephropathy is provided with a compound adjusting (preferably inhibiting) the activity of a protein having the same or substantially the same amino acid sequence with the amino acid sequence expressed by sequence number: 1 (in the specification) or the expression of the gene of the protein, an antisense polynucleotide containing a base sequence complementary to or substantially complementary to a base sequence of a DNA encoding the protein or its partial peptide or containing the part of the same, an antibody against the protein or its partial peptide, etc. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a prophylactic / therapeutic agent for a renal disease such as diabetic nephropathy, a diagnostic agent, and a screening for a prophylactic / therapeutic agent for a renal disease such as diabetic nephropathy.

The kidney maintains the body's homeostasis by regulating the concentration of body fluid components through excretion and reabsorption, and also functions as an endocrine organ that produces and secretes bioactive substances such as active vitamin D, erythropoietin, and renin It is a very important organization. When the function of the kidney, which plays such an important role, is impaired, various disease states are caused, and a wide variety of kidney diseases occur. Among these renal diseases, there is also a nephropathy closely related to diabetes, obesity and abnormal lipid metabolism.In particular, in diabetic nephropathy caused by diabetes, strict control of diabetes is required after diagnosis of nephropathy. In many cases, it is difficult to stop the progress, and there are many cases of renal failure, and the number of patients is not small. As a model animal for renal disease, Wistar Fatty rats (WF rats) exhibit symptoms characteristic of diabetes in addition to symptoms of renal dysfunction, and therefore, as a model for diabetic nephropathy (Frontiers in diabetes research, lessons from animal). Diabetes II, 535-541, 1988), Zucker Fatty rats (ZF rats) exhibit renal dysfunction and diabetic symptoms in the same manner as WF rats. Therefore, as a diabetic nephropathy model (Kidney international, 52 Vol., S218-S220, 1997), spontaneously hypercholesterolemic rats (SHC rats) exhibit symptoms similar to human focal glomerulosclerosis, and are used as focal glomerulosclerosis models (Japan Journal of Nephrology, 37, 91-99, 1995).
On the other hand, human Type IIb Na / Pi cotransporter (sodium phosphate cotransporter type IIb) is a molecule belonging to the transporter family that has sodium-dependent phosphate transport activity. High activity at pH is consistent with the results of a phosphate uptake assay using rabbit or mouse small intestine, suggesting that it may be a molecule responsible for phosphorus uptake in the small intestine (Biochemical and Biophysical Research Communications 258, 578-582, 1999). Further, there is disclosed a method for treating a disease state related to the function of human Type IIb Na / Pi cotransporter, for example, a method for causing an abnormal serum phosphate concentration such as hypophosphatemia and hyperphosphatemia, and the like. It is disclosed in JP-A-2000-245488 (Patent Document 1). US Pat. No. 6,319,688 (Patent Document 2) (JP-A-2000-78991) discloses that antagonists and agonists of human Type IIb Na / Pi cotransporter are used for chronic renal failure, end stage renal disease, uremic bone disease. It is described as useful for the prophylactic treatment of cancer.
JP 2000-245488 A USP 6,319,688

  Regarding renal lesions caused by the above primary diseases, studies incorporating cell biology and molecular biology techniques have been vigorously conducted with the aim of elucidating the pathogenesis and pathophysiology, and new findings are being obtained. However, there are still a number of unsolved issues at the moment. There is a need for safe and excellent agents for preventing and treating renal diseases.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, analyzed model animals such as WF rat, ZF rat, SHC rat, and found a gene whose expression is significantly increased. As a result of further studies based on the results, the present invention has been completed.

That is, the present invention
(1) a renal disease comprising a compound having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a compound inhibiting the activity of a salt thereof, or a salt thereof Prophylactic and therapeutic agents,
(1a) the prophylactic / therapeutic agent according to the above (1), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis;
(2) a protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a compound thereof or a salt thereof which inhibits the expression of a gene thereof; Prophylactic / therapeutic agent for kidney disease,
(2a) the prophylactic / therapeutic agent according to the above (2), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis;
(3) The prophylactic / therapeutic agent according to the above (1) or (2), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis;
(4) a base complementary or substantially complementary to the base sequence of a polynucleotide encoding a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 An antisense polynucleotide containing the sequence or a portion thereof,
(5) a medicine comprising the antisense polynucleotide according to the above (4),
(6) The medicament according to the above (5), which is a prophylactic / therapeutic agent for kidney disease.
(6a) the prophylactic / therapeutic agent according to the above (6), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis;
(7) an antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof;
(8) a medicine comprising the antibody according to (7),
(9) The medicament according to the above (8), which is a prophylactic / therapeutic agent for kidney disease.
(9a) The prophylactic / therapeutic agent according to the above (9), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis,
(10) a diagnostic agent comprising the antibody according to the above (7),
(11) The diagnostic agent according to the above (10), which is a diagnostic agent for kidney disease.
(12) a diagnostic agent for renal disease, comprising a polynucleotide encoding a protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1,
(13) Screening of a prophylactic / therapeutic agent for renal disease, which comprises using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. Method,
(13a) (i) A labeled inorganic phosphate is added to a cell capable of expressing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. The amount of the inorganic phosphate incorporated into the cell is measured when the salt is added and (ii) when the labeled inorganic phosphate is added to the cell in the presence of the test compound. ) Described screening method,
(13b) The screening method according to the above (13a), wherein the labeled inorganic phosphate is an inorganic phosphate labeled with a radioisotope ([ ³² P], [ ³³ P], etc.),
(13c) (i) An inorganic phosphate is added to a synthetic lipid bilayer containing 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 peptide thereof or a salt thereof. And (ii) in the presence of the test compound, the inorganic lipid transport activity was measured when the synthetic lipid bilayer was contacted with a buffer containing inorganic phosphate. The screening method according to the above (13),
(13d) The screening method according to (13), wherein the kidney disease is diabetic nephropathy or focal glomerulosclerosis,
(13e) Activity of 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 peptide thereof or a salt thereof obtained by using the screening method described in the above (13). A prophylactic / therapeutic agent for renal disease comprising a compound that inhibits or a salt thereof,
(14) A prophylactic / therapeutic agent for renal disease, comprising 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 peptide thereof or a salt thereof. Screening kit,
(15) A prophylactic / therapeutic agent for renal disease, characterized by using a polynucleotide 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 peptide thereof. Screening method,
(15a) The screening method according to (15), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis,
(16) Prevention and treatment of renal disease, characterized by containing a polynucleotide 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 peptide thereof. Agent screening kit,
(17) The screening method according to the above (13) or (15), wherein the kidney disease is diabetic nephropathy or focal glomerulosclerosis,
(17a) a compound having a prophylactic or therapeutic effect on renal disease, or a salt thereof, obtained by using the screening method according to (13) or (15) or the screening kit according to (14) or (16);
(17b) A prophylactic / therapeutic agent for renal disease, comprising the compound according to (17a) or a salt thereof,
(18) a compound or a salt thereof that inhibits the activity of a protein or a partial peptide thereof 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, for a mammal; Or a method for preventing or treating renal disease, which comprises administering an effective amount of a compound or a salt thereof that inhibits the expression of the gene of the protein or its partial peptide or a salt thereof.
(18a) The method according to the above (18), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis,
(19) inhibits the activity of a protein or a partial peptide thereof 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, or inhibits the activity of the protein or partial peptide thereof or a salt thereof A method for preventing and treating renal disease, characterized by inhibiting the expression of
(19a) The method of the above (19), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis,
(20) Inhibits the activity of 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 peptide thereof or a salt thereof for producing a prophylactic / therapeutic agent for kidney disease Use of a compound or a salt thereof, or a compound or a salt thereof that inhibits gene expression of the protein or a partial peptide thereof or a salt thereof;
(20a) The use according to (20) above, wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis.

  The protein (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 peptide thereof or a salt thereof) and the polynucleotide of the present invention include, for example, renal diseases (eg, diabetes Nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescent formation Markers for inflammatory nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.) A modulator (preferably an inhibitor), which is useful and obtained by screening using the protein, polynucleotide or an antibody against the protein; Antibodies against the substance, the antisense polynucleotide of the present invention, and the like, have low toxicity, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferation) Nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure , Renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.). Further, the polynucleotide of the present invention, the antisense polynucleotide of the present invention and the like include, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative Nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, Renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.) are useful for diagnosis, prevention or treatment.

  A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter, also referred to as the protein of the present invention or the protein used in the present invention) may be human or warm-blooded. Animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) cells (eg, hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, obesity) Cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or Plasma cells, or precursors of these cells, stem cells or cancer cells) or any tissue in which those cells are present, for example, the brain, each part of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, Hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), It may be a protein derived from blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., or may be a synthetic protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes about 60% or more, preferably about 70% or more, preferably about 80% or more as the amino acid sequence represented by SEQ ID NO: 1. Preferably, an amino acid sequence having about 90% or more, preferably about 95% or more homology is exemplified.
Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 described above. And proteins having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1. Examples of the protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include a protein containing the amino acid sequence represented by SEQ ID NO: 3, and a protein represented by SEQ ID NO: 5 Examples include proteins containing an amino acid sequence.
Examples of substantially the same activity include an inorganic phosphate uptake activity and an inorganic phosphate transport activity. Substantially homogenous indicates that the properties are homogenous (eg, physiologically or pharmacologically). Therefore, inorganic phosphate uptake activity, inorganic phosphate transport activity, etc. are 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 these activities and the molecular weight of the protein may be different.
Inorganic phosphate uptake activity can be measured according to a method known per se, for example, the method described in Methods in Enzymology, Vol. 191, pp. 494-505, 1990 or a method analogous thereto.
Specifically, a labeled inorganic phosphate (eg, an inorganic phosphate labeled with a radioisotope, etc.) is added to cells expressing the protein of the present invention, and the mixture is incubated for a certain period of time. It is performed by measuring the amount of phosphoric acid. The reaction is performed in an appropriate buffer, and the inorganic phosphate not taken up by the cells is washed away before measurement. When the labeling agent is a radioisotope, the uptake activity is measured by a known method, for example, using a radioactivity measuring device.
The inorganic phosphate transport activity can be measured according to a method known per se, for example, the method described in Biochimica et Biophysica Acta, Vol. 1236, pp. 339-344, 1995 or a method analogous thereto.
Specifically, a buffer containing an inorganic phosphate is added to the synthetic lipid bilayer containing the protein of the present invention, and the change in membrane current that occurs when inorganic phosphate is transported by the protein of the present invention. Measure. The membrane current is measured using a known method, for example, a membrane potential fixing method. The synthetic lipid bilayer containing the protein of the present invention is prepared, for example, by using a purified protein obtained from a membrane fraction of cells expressing the protein of the present invention according to a known membrane protein purification method, using a phospholipid or the like as a material. Then, it can be manufactured according to a known method, for example, a method described in "Patch Clamp Experimental Technique" (issued by Yoshioka Shoten, 1996).

Examples of the protein used in the present invention include, for example, (i) one or two or more (for example, about 1 to 100) in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. (Preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5) amino acids, (ii) SEQ ID NO: 1, SEQ ID NO: 3 or 1 or 2 or more (eg, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 to 5) in the amino acid sequence represented by SEQ ID NO: 5 And (iii) one or more (eg, about 1 to 100, preferably 1 to 100) amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. 30 or so, preferred (Iv) about 1 to 10, more preferably number (1 to 5) amino acids, and (iv) the amino acid represented by SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. One or more amino acids in the sequence (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably number (1 to 5)) are other amino acids So-called muteins such as proteins containing the substituted amino acid sequence or (v) an amino acid sequence combining them are also included.
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.

In the protein in this specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) in accordance with the convention of peptide labeling. The proteins used in the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, have a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), or an amide (—CONH ₂ ) at the C-terminus. Alternatively, any of ester (—COOR) may be used.
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl and n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl , C _6-12 aryl groups such as α-naphthyl, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or C _7- alkyl groups such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl. _{14 An} aralkyl group, a pivaloyloxymethyl group and the like are used.
When the protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Furthermore, the protein used in the present invention, the amino acid residue (eg, methionine residue) of the N-terminal amino group protecting group (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _{1- 6-} acyl group), N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, substituent on the side chain of amino acid in the molecule (eg, -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc. are protected with a suitable protecting group (eg, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group, acetyl group). Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
Specific examples of the protein used in the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 1, a protein containing the amino acid sequence represented by SEQ ID NO: 3, and a protein containing the amino acid sequence represented by SEQ ID NO: 5. And a protein containing the amino acid sequence to be used.

The partial peptide of the protein used in the present invention is a partial peptide of the protein used in the present invention described above, and is preferably any peptide having the same properties as the protein used in the present invention described above. It may be something.
Specifically, for the purpose of preparing the antibody of the present invention described below, the amino acid sequence of the 1st to 101st, 187th to 360th, and 575th to 690th amino acids in the amino acid sequence represented by SEQ ID NO: 1 A peptide having the amino acid sequence of positions 1-101, 187-360, and 575-695 in the amino acid sequence represented by SEQ ID NO: 3, and a peptide having the amino acid sequence of SEQ ID NO: 5 Peptides having the amino acid sequence at positions 1-101, 187-360, and 575-697 are exemplified. For example, it has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the protein used in the present invention. Peptides and the like are used.
Further, the partial peptide used in the present invention has one or more (for example, about 1 to 20, preferably about 1 to 10, more preferably number (1 to 5)) amino acids in its amino acid sequence. Is deleted, or one or more (eg, about 1 to 20, preferably about 1 to 10, more preferably number (1 to 5)) amino acids are added to the amino acid sequence, or One or two or more (eg, about 1 to 20, preferably about 1 to 10, more preferably number (1 to 5)) amino acids are inserted into the amino acid sequence, or One or more (for example, about 1 to 20, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids) may be substituted with another amino acid.

The partial peptide used in the present invention may have a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ) or an ester (—COOR) at the C-terminus.
Further, similar to the protein used in the present invention, the partial peptide used in the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, and the N-terminal amino acid residue (eg, , A methionine residue) in which the amino group is protected by a protecting group, a glutamine residue produced by cleavage of the N-terminal side in vivo and pyroglutamine oxidation, and a substituent on the side chain of an amino acid in the molecule. Also included are those protected with an appropriate protecting group, and complex peptides such as so-called glycopeptides to which sugar chains are bonded.
The partial peptide used in the present invention can also be used as an antigen for preparing an antibody.

As the salt of the protein or partial peptide used in the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metals) are used. Acceptable acid addition salts are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
The protein or its partial peptide or its salt used in the present invention can be produced from the aforementioned human or warm-blooded animal cells or tissues by a known protein purification method, or contains a DNA encoding the protein. It can also be produced by culturing a transformant. Further, it can be produced according to the peptide synthesis method described later.
When producing from human or mammalian tissues or cells, after homogenizing human or mammalian tissues or cells, extraction is performed with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.

For the synthesis of the protein or partial peptide or a salt thereof, or an amide thereof used in the present invention, a commercially available resin for protein synthesis can be generally used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. I do.
For the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid was directly added to the resin together with a racemization inhibitor additive (for example, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetric acid anhydride or HOBt ester or HOOBt ester. It can be added later to the resin.

  The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a 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, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.

Examples of the protecting group for the amino group of the raw material include, for example, Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazide, trityl hydrazide and the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for this esterification include lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, and groups derived from carbonic acid such as benzyloxycarbonyl groups and ethoxycarbonyl groups. Used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
As the protecting group for the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.
As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide 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, or hydrogen fluoride anhydride, methanesulfonic acid, trifluoromethane, etc. Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4 -Addition of a cation scavenger such as butanedithiol, 1,2-ethanedithiol and the like is effective. In addition, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of the functional group that should not be involved in the reaction of the raw materials, the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a protein or a partial peptide, for example, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After the elongation, a protein or partial peptide from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the C-terminal carboxyl group has been removed, and these are produced. The protein or peptide is condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. This crude protein or peptide is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein or peptide.
In order to obtain an ester of a protein or peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein or An ester of the peptide can be obtained.

The partial peptide or a salt thereof used in the present invention can be produced according to a peptide synthesis method known per se, or by cleaving the protein used in the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the target peptide. it can. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (i) to (v).
(I) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments on Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)
(V) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten After the reaction, conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. Can be used to purify and isolate the partial peptide used in the present invention. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide obtained by a salt is a known method or analogous method It can be converted into a free form or another salt by a method.

The polynucleotide encoding the protein used in the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence encoding the protein used in the present invention. Preferably it is DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be performed directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a preparation of a total RNA or mRNA fraction from the cells and tissues described above.
As the DNA encoding the protein used in the present invention, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a nucleotide sequence hybridized with the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions Any DNA may be used as long as it contains a soybean base sequence and encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1.

Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 60% or more, preferably about 70% or more with the base sequence represented by SEQ ID NO: 2. DNA containing a base sequence having a homology of about 80% or more, preferably about 90% or more, and preferably about 95% or more is used. Specific examples include a DNA containing the base sequence represented by SEQ ID NO: 4, a DNA containing the base sequence represented by SEQ ID NO: 6, and the like.
Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). . When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringent conditions.
The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 is a DNA containing the base sequence represented by SEQ ID NO: 2, and represented by SEQ ID NO: 3. Examples of the DNA encoding the protein containing the amino acid sequence include a DNA containing the base sequence represented by SEQ ID NO: 4, and a DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 5 include the sequence DNA containing the base sequence represented by No. 6 is used.

The polynucleotide (eg, DNA) encoding the partial peptide used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide used in the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the aforementioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
As the DNA encoding the partial peptide used in the present invention, for example, a DNA having a part of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or the nucleotide sequence represented by SEQ ID NO: 2 and a high string DNAs containing a base sequence that hybridizes under gentle conditions and a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention are used.
The DNA hybridizable to the base sequence represented by SEQ ID NO: 2 has the same significance as described above.
The same hybridization method and high stringency conditions as described above are used.

Means for cloning a DNA that completely encodes the protein or partial peptide used in the present invention (hereinafter, in the description of the cloning and expression of the DNA encoding the same, these may be simply abbreviated as the protein of the present invention) Amplification by a PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or encoding a DNA incorporated into an appropriate vector into a part or the entire region of the protein of the present invention. And a DNA fragment labeled with a DNA fragment or a synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
The DNA base sequence can be converted by ODA-LA PCR using PCR, a known kit, for example, Mutan ^™ -super Express Km (Takara Bio Inc.), Mutan ^™ -K (Takara Bio Inc.), or the like. Method, a gapped duplex method, a Kunkel method, or the like, or a method analogous thereto.
The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
The expression vector for the protein of the present invention is, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by the following.

Examples of the vector include plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.
The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter, etc. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used, if desired. it can. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r), neomycin resistant gene (hereinafter sometimes abbreviated as Neo ^r, G418 resistance). In particular, when the dhfr gene is used as a selection marker using dhfr gene-deficient Chinese hamster cells, the target gene can also be selected using a thymidine-free medium.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.
A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12.DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] Are used.
As the Bacillus bacterium, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)] and the like are used.
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036P, and so on. Used.

Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; Sf cell), MG1 cells derived from the midgut of Trichoplusia ni, and High Five ^™ derived from eggs of Trichoplusia ni. Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used.
As insects, for example, silkworm larvae are used [Nature, vol. 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells and the like are used.
Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982) and the like can be used to transform Escherichia.

Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
To transform yeast, for example, the method described in Methods in Enzymology, vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) can be used. it can.
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, Vol. 52, 456 (1973).
Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.
When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and among them, a carbon source necessary for growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid (Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently.
When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
When culturing a transformant in which the host is yeast, examples of the medium include Burkholder's minimum medium [Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and 0.5%. SD medium containing casamino acid [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration or stirring is added.
When culturing a transformant in which the host is an insect cell or an insect, the medium may be Grace's Insect Medium (Nature, 195, 788 (1962)) to which additives such as immobilized 10% bovine serum are appropriately added. Is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration and / or agitation are added.
When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], etc. Is used. Preferably, the pH is between about 6 and 8. The cultivation is usually carried out at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added.
As described above, the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.

The protein of the present invention can be separated and purified from the culture by, for example, the following method.
When extracting the protein of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.
Purification of the protein contained in the culture supernatant or extract thus obtained can be carried out by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and use of difference in hydrophobicity such as reverse phase high performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.

When the protein thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when the protein obtained is a salt, a method known per se or analogous thereto Can be converted to a free form or another salt.
The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
The presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody, Western blotting, or the like.

The antibody against the protein or partial peptide or a salt thereof used in the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention.
An antibody against a protein or a partial peptide or a salt thereof (hereinafter, these may be simply referred to as the protein of the present invention) used in the present invention uses the protein of the present invention as an antigen and is known per se. Can be produced according to the method for producing an antibody or antiserum described in (1).
[Preparation of monoclonal antibody]
(A) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal by itself or together with a carrier and a diluent at a site where antibody production is possible by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained therein. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently.
Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed together with a carrier, and then radioactive substances or A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with an enzyme or the like (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A; A method in which a hybridoma culture supernatant is added to a solid phase to which globulin antibodies or protein A is adsorbed, a protein labeled with a radioactive substance, an enzyme, or the like is added, and a monoclonal antibody bound to the solid phase is detected.
Selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of monoclonal antibody Monoclonal antibody can be separated and purified by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchange method Absorption / desorption method using body (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific antibody that collects only antibody by active adsorbent such as protein A or protein G, and dissociates the bond to obtain antibody Purification method].
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting a substance and separating and purifying the antibody.
Regarding the complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of the carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by crosslinking the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ~ 5 is used.
Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. 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, or the like, preferably from the blood of a warm-blooded animal immunized by the above method.
The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.

The nucleotide sequence of a polynucleotide encoding a protein or a partial peptide used in the present invention (eg, DNA (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of antisense polynucleotide)) As an antisense polynucleotide having a complementary or substantially complementary base sequence or a part thereof, a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof is used. Any antisense polynucleotide may be used as long as it has a portion and has an action of suppressing the expression of the DNA, but antisense DNA is preferable.
The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. In particular, in the case of an antisense polynucleotide directed to translation inhibition in the entire base sequence of the complementary strand of the DNA of the present invention, the base sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, An antisense polynucleotide 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 a complementary strand of a base sequence near a codon, etc.) B) In the case of an antisense polynucleotide which directs RNA degradation by RNase H, it is about 70% or more, preferably about 80% or more, more preferably about 90% with the complementary strand of the entire base sequence of the DNA of the present invention including introns. As described above, most preferably, antisense polynucleotides each having about 95% or more homology are suitable.
Specifically, a base sequence complementary to or substantially complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a part thereof is An antisense polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of a DNA containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a part thereof Nucleotides (more preferably, an antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a part thereof), etc. No.
The antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is replaced with, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate or phosphorodithionate. It may be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base portion (pyrimidine, purine) may also be chemically modified. Or any one that hybridizes to DNA having the base sequence represented by SEQ ID NO: 2. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, the antisense polynucleotide (nucleic acid) corresponding to the protein gene of the present invention, which can inhibit the replication or expression of the gene, is cloned or the base of DNA encoding the determined protein is determined. It can be designed and synthesized based on sequence information. Such antisense polynucleotides can hybridize to the RNA of the protein gene of the present invention and inhibit the synthesis or function of the RNA, or through interaction with the protein-related RNA of the present invention. The expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of the protein-associated RNA of the present invention, and that can specifically hybridize with the protein-associated RNA of the present invention, can be used in vivo and in vitro to produce the protein gene of the present invention. It is useful for regulating and controlling the expression of, and also useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. The "correspondence" between a nucleotide, base sequence or nucleic acid and a protein usually refers to the amino acid of the protein (as indicated) derived from the nucleotide (nucleic acid) sequence or its complement. 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' Although a terminal palindrome region or a 3′-end hairpin loop can be selected as a preferable target region, any region within a protein gene can be selected as a target.
Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize with the target region, the target nucleic acid is Can be said to be "antisense." Antisense polynucleotides are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, N-glycosides of purine or pyrimidine bases and other types of polynucleotides. , Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, such as those found in DNA or RNA. Base pairing and nucleotides having a configuration that allows base attachment). They may be double stranded DNA, single stranded DNA, double stranded RNA, single stranded RNA, DNA: RNA hybrids, and may further comprise unmodified polynucleotides (or unmodified oligonucleotides), known modifications. Additions, e.g., those with labels known in the art, capped, methylated, substituted for one or more natural nucleotides with analogs, modified with intramolecular nucleotides Having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), having a charged or sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.) Such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Nal peptides, poly-L-lysine, etc.) or sugars (eg, monosaccharides, etc.) having side chain groups, interactive compounds (eg, acridine, psoralen, etc.), chelating compounds (eg, , Metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) Is also good. Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, or the like, or with functional groups such as ethers, amines, and the like. It may have been converted.
The antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include a sulfur derivative of a nucleic acid, a thiophosphate derivative, a nucleic acid which is resistant to degradation of polynucleoside amide and oligonucleoside amide, and the like. The antisense polynucleotide of the present invention can be designed, for example, as follows. That is, the antisense polynucleotide in a cell is made more stable, the cell permeability of the antisense polynucleotide is increased, the affinity for a target sense strand is increased, and if the toxicity is reduced. In some cases, the toxicity of the antisense polynucleotide is reduced. Many such modifications have been reported, for example, in Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Applications, CRC Press, 1993.
The antisense polynucleotides of the present invention may contain altered or modified sugars, bases, bonds, and may be provided in special forms such as liposomes, microspheres, or applied by gene therapy. , Can be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( Hydrophobic substances such as, for example, phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via bases, sugars, intramolecular nucleoside bonds. Other groups include cap groups specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of the antisense polynucleotide can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. .

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

  Since the expression of the protein of the present invention is increased in kidney tissues of Wistar Fatty rats (WF rats), Zucker Fatty rats (ZF rats) and spontaneously hypercholesterolemic rats (SHC rats), it can be used as a disease marker. I can do it. That is, it is useful as a marker for early diagnosis of renal dysfunction, determination of the severity of symptoms, and prediction of disease progression. The compound or its salt that regulates (preferably inhibits) the activity of the protein of the present invention, the antibody of the present invention, the antisense polynucleotide of the present invention, and the like can be used as a prophylactic / therapeutic agent for renal diseases such as diabetic nephropathy. Can be used.

(1) Screening of drug candidate compounds for diseases The protein of the present invention has increased expression in kidney tissues of Wistar Fatty rats (WF rats), Zucker Fatty rats (ZF rats) and spontaneously hypercholesterolemic rats (SHC rats). Therefore, a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein of the present invention may be, for example, a renal disease (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, microvariable type) Nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubulointerstitial nephritis, acute tubule Necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.). Preferably, it is a prophylactic / therapeutic agent for diabetic nephropathy, focal glomerulosclerosis and the like.
Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (inhibits or promotes) the activity of the protein of the present invention.
That is, the present invention relates to a compound that regulates (promotes or inhibits) the activity of the protein of the present invention (eg, an inorganic phosphate uptake activity, an inorganic phosphate transporting activity, etc.) characterized by using the protein of the present invention, or a compound thereof. A method for screening a salt is provided.
Specifically, a comparison was made between (i) the inorganic phosphate uptake activity of a cell containing the protein of the present invention and (ii) the inorganic phosphate uptake activity of a mixture of a cell containing the protein of the present invention and a test compound. A method for screening a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention,
(I ') the inorganic phosphate transport activity of the synthetic lipid bilayer containing the protein of the present invention, and (ii') the inorganic phosphate transport activity of the mixture of the synthetic lipid bilayer containing the protein of the present invention and the test compound. A method of screening for a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention, which is characterized by comparison, and the like, may be mentioned.
In the above-mentioned screening method, for example, in the case of (i) and (ii), the inorganic phosphate uptake activity can be determined by a known method, for example, the method described in Methods in Enzymology, Vol. 191, pp. 494-505, 1990, or Measure and compare according to an equivalent method.
Specifically, (i) a cell capable of expressing the protein of the present invention to which a labeled inorganic phosphate is added, and (ii) a cell capable of expressing the protein of the present invention in the presence of a test compound. , The amount of inorganic phosphate taken into cells when a labeled inorganic phosphate is added is measured, and a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention is screened. I do. This reaction is performed in an appropriate buffer, and inorganic phosphate that has not been taken up by cells is washed away before measurement.
Examples of the labeled inorganic phosphate include an inorganic phosphate labeled with a radioisotope (eg, [ ³² P], [ ³³ P], etc.).
When an inorganic phosphate labeled with a radioisotope is used, the activity of the cells to take up the inorganic phosphate is measured by a known method, for example, using a radioactivity measuring device or the like.
The labeled inorganic phosphate may be reacted with cells expressing the protein of the present invention after being mixed with the test compound, or the labeled inorganic phosphate may be contacted with cells expressing the protein of the present invention. After that, a test compound may be added.
The cells capable of expressing the protein of the present invention can be obtained by transfecting the gene encoding the protein of the present invention into appropriate cells, for example, animal cells such as COS-7 cells, CHO cells, HEK293 cells, and Xenopus oocytes. By introducing and culturing, the protein of the present invention is expressed on the cell membrane. The method for culturing cells capable of expressing the protein of the present invention is the same as the method for culturing the transformant of the present invention described above.
In the above-mentioned screening method, for example, in the case of (i ′) and (ii ′), the inorganic phosphate transport activity is determined by a known method, for example, Biochimica et Biophysica Acta, Vol. 1236, pp. 339-344, 1995 and the like. The measurement is performed according to the method described in (1) or a method analogous thereto, and the results are compared.
Specifically, (i ′) the case where the buffer containing inorganic phosphate is brought into contact with the synthetic lipid bilayer containing the protein of the present invention, and (ii) the protein of the present invention in the presence of the test compound. A compound that modulates (inhibits or promotes) the activity of the protein of the present invention by measuring the inorganic phosphate transport activity when a buffer solution containing an inorganic phosphate is brought into contact with a synthetic lipid bilayer containing Screen the salt.
In the measurement of the inorganic phosphate transporting activity, a change in membrane current that occurs when inorganic phosphate is transported by the protein of the present invention is measured by a known method, for example, a membrane potential fixing method.
The synthetic lipid bilayer containing the protein of the present invention is prepared, for example, by using a purified protein obtained from a membrane fraction of cells expressing the protein of the present invention according to a known membrane protein purification method, using a phospholipid or the like as a material. Then, it can be manufactured according to a known method, for example, a method described in "Patch Clamp Experimental Technique" (issued by Yoshioka Shoten, 1996).
The inorganic phosphate may be reacted with the lipid bilayer containing the protein of the present invention after being mixed with the test compound, and the inorganic phosphate ion may be added to the lipid bilayer containing the protein of the present invention. After contact, the test compound may be added.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
For example, a test compound that increases the inorganic phosphate uptake activity in the case of the above (ii) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the above case (i), As a compound that promotes the activity of the protein of the present invention, the activity of taking up inorganic phosphate in the case of the above (ii) is about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (i). A test compound which reduces the activity by at least% can be selected as a compound that inhibits the activity of the protein of the present invention.
For example, a test compound that increases the inorganic phosphate transport activity in the case of the above (ii ′) by about 20% or more, preferably 30% or more, more preferably about 50% or more compared to the case of the above (i ′) As the compound that promotes the activity of the protein of the present invention, the inorganic phosphate transport activity in the case of the above (ii ′) is about 20% or more, preferably 30% or more, as compared with the case of the above (i ′). A test compound that preferably reduces by about 50% or more can be selected as a compound that inhibits the activity of the protein of the present invention.

The compound having the activity of promoting the activity of the protein of the present invention is useful as a safe and low-toxic drug for enhancing the physiological activity of the protein of the present invention.
The compound having the activity of inhibiting the activity of the protein of the present invention is a safe and low-toxic drug for suppressing the physiological activity of the protein of the present invention, for example, renal diseases (eg, diabetic nephropathy, focal glomeruli) Sclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney , Tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumor, chronic rejection after renal transplantation, etc.). Preferably, it is a prophylactic / therapeutic agent for diabetic nephropathy, focal glomerulosclerosis and the like.
Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts And compounds selected from plasma and the like. As the salt of the compound, those similar to the aforementioned salts of the peptide of the present invention are used.

Furthermore, the expression of the gene encoding the protein of the present invention also increases in the renal tissues of Wistar Fatty rats (WF rats), Zucker Fatty rats (ZF rats) and spontaneously hypercholesterolemic rats (SHC rats). Compounds or salts thereof that regulate (preferably inhibit) the expression of the gene encoding the protein of the present invention include, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change type) Nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubulointerstitial nephritis, acute tubule Necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.).
Therefore, the polynucleotide (eg, DNA) of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (preferably, inhibits) the expression of a gene encoding the protein of the present invention.
The screening method includes (iii) culturing cells capable of producing the protein of the present invention and (iv) culturing cells capable of producing the protein used in the present invention in the presence of the test compound. A screening method characterized by performing a comparison with the case.
In the above method, the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases (iii) and (iv) is measured and compared.
Examples of the test compound and cells having the ability to produce the protein of the present invention include the same cells as described above.
The protein amount is measured by a known method, for example, using an antibody that recognizes the protein of the present invention, and measuring the protein present in a cell extract or the like according to a method such as Western analysis, ELISA, or a method analogous thereto. can do.
The amount of mRNA is measured according to a known method, for example, Northern hybridization or PCR using a nucleic acid containing SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 or a part thereof as a probe or a method analogous thereto. be able to.
For example, a test compound which increases the gene expression level in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii), according to the present invention As a compound that promotes the expression of the gene encoding the protein of the present invention, a test compound that inhibits about 20% or more, preferably 30% or more, more preferably about 50% or more suppresses the expression of the gene encoding the protein of the present invention. Can be selected as the compound.

The screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide used in the present invention.
Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention are test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts. Or a salt thereof, or a compound selected from animal tissue extract, plasma and the like. As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
A compound or a salt thereof that regulates (preferably inhibits) the activity of the protein of the present invention and a compound or a salt thereof that regulates (preferably inhibits) the expression of a gene encoding the protein of the present invention may be, for example, a renal disease ( For example, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, Prevention of crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after kidney transplantation, etc. -Useful as a therapeutic agent.
When the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.

For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As compositions for parenteral administration, for example, injections, suppositories and the like are used. Dosage forms such as agents. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used. Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, or the like is used, and benzyl benzoate, benzyl alcohol, or the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories used for rectal administration are prepared by mixing the above compound or a salt thereof with a conventional suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg, especially 5 to 100 mg, for each dosage unit. Other dosage forms preferably contain from 10 to 250 mg of the above compound.
Each of the above-mentioned compositions may contain other active ingredients as long as the composition does not cause an undesirable interaction with the compound.
The preparations obtained in this way are safe and low toxic, for example, human or warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Can be administered orally or parenterally.
The dose of the compound or a salt thereof varies depending on its action, target disease, subject of administration, administration route and the like. When a compound or a salt thereof is orally administered, the compound or a salt thereof is generally used in an adult (with a body weight of 60 kg) in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 50 mg per day. Administer 1.0-20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like. When the (preferably inhibitory) compound or a salt thereof is administered to an adult (as a body weight of 60 kg) in the form of an injection, usually about 0.01 to 30 mg, preferably about 0.1 to 20 mg, of the compound or a salt thereof per day. Conveniently, preferably about 0.1 to 10 mg is administered by intravenous injection. In the case of other animals, the dose can be administered in terms of the weight per 60 kg.

(2) Quantification of the protein of the present invention, its partial peptide or its salt An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. Since it can be used, it can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay.
That is, the present invention
(I) reacting the antibody of the present invention with a test solution and the labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody. And (ii) simultaneously or sequentially reacting the test solution with the antibody of the present invention insolubilized on a carrier and another antibody of the present invention labeled on a carrier. The present invention provides a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction.
In the quantification method (ii), it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.

The protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, or F (ab ') ₂ , Fab' or Fab fraction of the antibody molecule may be used.
The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited. Any measurement method may be used as long as the amount is detected by chemical or physical means and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competitive method, an immunometric method, and a sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
Examples of the labeling agent used in the measurement method using a labeling substance include radioisotopes (eg, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C], [ ³² P], [ ³³ P ], [ ³⁵ S], etc.), fluorescent substances [eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)], fluorescamine, fluorescein isothiocyanate, etc. ], Enzymes (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), lanthanide elements and the like are used. . Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving the measurement sensitivity and the like. You may.
In the method for measuring the protein of the present invention by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different site to which the protein of the present invention binds is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably An antibody that recognizes, for example, the N-terminal other than the C-terminal is used.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like.
In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. As the first antibody, a soluble antibody is used, and an immobilization method using an immobilized antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction against a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.
For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 70 (Immunochemical Techniques (Part C)) 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies). )) (The above is from Academic Press) ) And the like can be referred to.
As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
Furthermore, when an increase or decrease in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, renal diseases (eg, diabetic nephropathy, Focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus Nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc. Can be diagnosed as high.
Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. Further, for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.

(3) Gene Diagnostics The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog) , Monkeys, chimpanzees, etc.) can detect abnormalities (genetic abnormalities) in DNA or mRNA encoding the protein of the present invention or partial peptides thereof, for example, damage, mutation or reduced expression of the DNA or mRNA, It is useful as a diagnostic agent for a gene such as an increase or an overexpression of the DNA or mRNA.
The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization, PCR-SSCP method (Genomics, Vol. 5, pp. 874-879, 1989, Proceedings of the National Academy of Sciences of the United States). States of America, 86, 2766-2770, 1989).
For example, when overexpression or decrease is detected by Northern hybridization, or when DNA mutation is detected by PCR-SSCP method, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis) , Chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescent-forming nephritis, lupus nephritis, amyloid kidney, urine Tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumor, chronic rejection after renal transplantation, etc.) can be diagnosed as being highly likely.

(4) Pharmaceuticals Containing Antisense Polynucleotide The antisense polynucleotide of the present invention, which can complementarily bind to the DNA of the present invention and suppress the expression of the DNA, has low toxicity and exhibits the present invention in vivo. Can suppress the function of the protein of the present invention or the DNA of the present invention, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative Nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, Renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.).
When the antisense polynucleotide is used as the prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se.
Also, for example, after inserting the antisense polynucleotide alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, a human or mammal (eg, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, and the like). The antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered using a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered locally into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of intracellular uptake, the above antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection), and is used for intravenous, subcutaneous, lesion, Etc. may be administered.
The dose of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense polynucleotide of the present invention is administered for the purpose of treating diabetic nephropathy, a general method is used. For an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.
Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of expression thereof.
Like the above-mentioned antisense polynucleotide, a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a ribozyme containing a part of the RNA encoding the protein of the present invention, and the like also include the gene of the present invention. Can be suppressed, and the function of the protein used in the present invention or the DNA used in the present invention in a living body can be suppressed. Thus, for example, renal diseases (eg, diabetic nephropathy, focal thread) Glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescent-forming nephritis, lupus nephritis, amyloid Renal, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumor, chronic rejection after renal transplantation, etc.) It can be used.
The double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
A ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known ribozyme to a part of RNA encoding the protein of the present invention. As a part of the RNA encoding the protein of the present invention, a portion (RNA fragment) close to a cleavage site on the RNA of the present invention, which can be cleaved by a known ribozyme, can be mentioned.
When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.

(5) Medicine containing the antibody of the present invention The antibody of the present invention having an action of neutralizing the activity of the protein of the present invention includes, for example,
Kidney disease (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferation Nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after kidney transplantation) It can be used as a prophylactic / therapeutic agent.
The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.
That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). ), Syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections and the like. Is included. Such injections are prepared according to a known method, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used. Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, or the like is used, and benzyl benzoate, benzyl alcohol, or the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg, especially 5 to 100 mg, for each dosage unit. Other dosage forms preferably contain 10 to 250 mg of the above antibody.
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the antibody.
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity, and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form in humans or mammals (eg, rats, rabbits, sheep, pigs, It can be administered orally or parenterally (eg, intravenously) to cattle, cats, dogs, monkeys, and the like. The dose varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for the treatment of diabetic nephropathy in adults, the antibody of the present invention is usually administered as a single dose. About 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day, injection It is conveniently administered as In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.
In addition, the antibody of the present invention can be used, for example, for renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA kidney Disease, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, kidney Tumors, chronic rejection after kidney transplantation, etc.).

(6) DNA-transferred animal The present invention relates to a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (hereinafter abbreviated as the exogenous mutant DNA of the present invention). Is provided.
That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) The animal according to (2), wherein the rodent is a mouse or rat, and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. Things.

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by transferring a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. In addition, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like by the DNA transfer method, and can be used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Above all, rodents with relatively short ontogeny and biological cycle in terms of the creation of disease animal model systems and easy reproduction are particularly useful in mice (especially pure strains such as C57BL / 6 strain, DBA2 strain, etc.). as a system, B6C3F ₁ strain, BDF ₁ strain, B6D2F ₁ strain, BALB / c strain, ICR strain, etc.) or rats (e.g., Wistar, etc. SD) and the like are preferable.
"Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by non-human mammals, but to the DNA of the present invention once isolated and extracted from the mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA and the like are used, and also include abnormal DNA.
The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is transferred, DNA derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto is expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, a vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-transferring mammals can be created.

Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.
Examples of the promoter for controlling the DNA expression include, for example, (i) a promoter of a DNA derived from a virus (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acid Protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen types I and II, cyclic AMP-dependent protein kinase βI subunit, dystrophy Fin, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin , Α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α Aku Down, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoters and the like are preferable.
The above vector preferably has a sequence that terminates transcription of a messenger RNA of interest in a DNA-transferred mammal (generally called a terminator). A simian virus SV40 terminator or the like is preferably used.

In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region for the purpose of further expressing the desired foreign DNA. It is also possible to connect 3 ′ downstream of the above depending on the purpose.
The normal translation region of the protein of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From the various genomic DNA libraries described above, or as a starting material, a complementary DNA prepared by a known method from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above-described cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA.
The translation region can be prepared as a DNA construct that can be expressed in a transposed animal by a conventional DNA engineering technique in which it is ligated downstream of the above-mentioned promoter and, if desired, upstream of the transcription termination site.
Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal cells and somatic cells. means. The progeny of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as a DNA-carrying animal by confirming that the exogenous DNA is stably maintained by mating. .
Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.
By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred to have the DNA in excess.

The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and eventually promotes the hyperactivity of the protein of the present invention by promoting the function of endogenous normal DNA. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.
Further, since the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the free protein of the present invention, a prophylactic / therapeutic agent for a disease related to the protein of the present invention, for example, a renal disease ( For example, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, Prevention of crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after kidney transplantation, etc.・ Can be used for screening tests for therapeutic agents.
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The progeny of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all progeny have the DNA.

The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of the endogenous normal DNA, and finally the functionally inactive form of the protein of the present invention. It can be refractory and can be used as a model animal for the disease. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
Also, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention can inhibit the abnormal protein of the present invention (dominant negative action) by the abnormal protein of the present invention in the inactive refractory disease of the protein of the present invention. A model to elucidate.
In addition, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the free protein of the present invention, a prophylactic / therapeutic agent for the protein of the present invention or a functionally inactive refractory disease, such as kidney Diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membrane proliferative Nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, kidney tumor, chronic rejection after kidney transplantation, etc.) It can also be used for screening tests for prophylactic and therapeutic agents.
In addition, other possible uses of the above two kinds of DNA transgenic animals of the present invention include, for example,
(I) use as a cell source for tissue culture,
(Ii) A peptide specifically expressed or activated by the protein of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or analyzing a peptide tissue expressed by the DNA Analysis of the relationship with
(Iii) culturing cells of DNA-bearing tissue by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture;
(Iv) Screening of a drug that enhances the function of the cell by using the cell described in (iii) above, and (v) Isolation and purification of the mutant protein of the present invention and production of an antibody thereof are conceivable.

Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention. More detailed pathological findings in each organ of a disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.
In addition, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain the released DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to identify the protein-producing cell of the present invention, examine its relevance to apoptosis, differentiation or proliferation, or investigate the signal transduction mechanism thereof, and investigate their abnormalities. It is an effective research material for
Further, in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, using the DNA-transferred animal of the present invention, It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using a method or the like. In addition, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for diseases associated with the protein of the present invention.

(7) Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention.
That is, the present invention
(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
(2) the embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli);
(3) the embryonic stem cell according to (1), which is neomycin-resistant;
(4) the embryonic stem cell according to (1), wherein the non-human mammal is a rodent;
(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,
(6) a non-human mammal deficient in expression of the DNA of the present invention, wherein the DNA is inactivated;
(7) The DNA is inactivated by introducing a reporter gene (eg, a β-galactosidase gene derived from Escherichia coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention (6). The non-human mammal according to the item,
(8) the non-human mammal according to (6), wherein the non-human mammal is a rodent;
(9) Administering a test compound to the non-human mammal according to (8), wherein the rodent is a mouse, and (10) the animal according to (7), and detecting the expression of a reporter gene. And a method of screening for a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention.

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA which is artificially mutated to the DNA of the present invention possessed by the non-human mammal, thereby suppressing the expression ability of the DNA, or By substantially eliminating the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter sometimes referred to as the knockout DNA of the present invention). ) Non-human mammal embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same one as described above is used.
The method of artificially mutating the DNA of the present invention can be carried out, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene represented by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); Inability to synthesize complete messenger RNA Thus, a DNA strand having a DNA sequence constructed so as to eventually destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the resulting ES cells PCR using a DNA sequence on or near the DNA of the present invention as a probe and a DNA sequence on a targeting vector and a DNA sequence in a neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as a primer And selecting the knockout ES cells of the present invention.
As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known Evans and Kaufma method. It may be a newly established one. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. for example the purpose of acquiring the obvious ES cells, the BDF ₁ mice (C57BL / 6 and DBA / 2 for the egg collection number of lack of C57BL / 6 mice and C57BL / 6 were improved by crossing with DBA / 2 Those established using F ₁ ) can also be used favorably. BDF ₁ mice are often egg number, and, in addition to the advantage that the egg is tough, since with C57BL / 6 mice in the background, when the ES cells obtained therefrom, which were generated pathological model mice , C57BL / 6 mice can be advantageously used in that their genetic background can be replaced with C57BL / 6 mice by backcrossing.
In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. However, a large number of blastocysts can be efficiently obtained by collecting embryos at the 8-cell stage and culturing them up to blastocysts. Early embryos can be obtained.
Although either male or female ES cells may be used, male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.

As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a PCR method can be mentioned. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture The primary selection of ES cells in the early stage can be performed by discriminating between male and female, and the early stage of culture can be greatly reduced by enabling the selection of male cells at an early stage.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the cells are knocked out of normal cells (for example, chromosomes in mice). It is desirable to clone again into cells (number 2n = 40).
The embryonic stem cell line obtained in this manner usually has very good growth properties, but it must be carefully subcultured because it tends to lose its ontogenic ability. For example, on a suitable feeder cell such as STO fibroblast, in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% oxygen The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, preferably about 0.1% trypsin) / 1mM EDTA) treatment, and seeding on freshly prepared feeder cells. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.
ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985]. The DNA-deficient cells of the present invention obtained by differentiation are useful in in vitro cell biology studies of the proteins of the present invention.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
As the non-human mammal, those similar to the aforementioned can be used.
The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.
Cells in which the DNA of the present invention has been knocked out can be used as a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the DNA of the present invention derived from the mouse used for the targeting vector. The determination can be made by analysis by a PCR method using a DNA sequence of a nearby region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. The chimeric embryo is injected into an animal embryo or a blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individual thus obtained is usually an individual with a heterozygous expression of the protein of the present invention, which is crossed with an individual with a heterozygous expression of the protein of the present invention, and homozygous expression of the protein of the present invention from their offspring. Defective individuals can be obtained.

When an egg cell is used, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
In this manner, the individual in which the DNA of the present invention has been knocked out can be bred in an ordinary breeding environment after confirming that the DNA has been knocked out in the animal individual obtained by mating.
Furthermore, the germline can be obtained and maintained in accordance with a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by breeding the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.
In addition, since the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a model of a disease caused by inactivation of the biological activity of the protein of the present invention. Therefore, it is useful for investigating the causes of these diseases and examining treatment methods.

(7a) Method of screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention The non-human mammal deficient in DNA expression of the present invention is deficient or damaged of the DNA of the present invention Can be used for screening for a compound having a therapeutic / preventive effect on a disease caused by the above.
That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Diseases, for example, kidney diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy , Membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumor, renal transplantation, A method for screening a compound or a salt thereof having a therapeutic / preventive effect against chronic rejection and the like.
Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same ones as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. These compounds are novel compounds. Or a known compound.
Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

For example, when screening for a compound having a therapeutic / preventive effect on diabetic nephropathy, etc., a test compound is administered to a non-human mammal deficient in expression of DNA of the present invention, and a test compound non-administration group is used as an indicator of renal function Changes in the amount of urinary protein excretion, glomerular filtration rate, or glomerular sclerosis rate, which is an indicator of renal dysfunction, are observed over time.
In the screening method, when the test compound is administered to a test animal, if the disease symptom of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound is It can be selected as a compound having a therapeutic / preventive effect on the above diseases.
The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect on a disease caused by deficiency or damage of the protein of the present invention. It can be used as a drug for safe and low toxic prophylactic / therapeutic agents. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered, generally an adult (assuming a body weight of 60 kg) diabetic nephropathy In about, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. In the case of parenteral administration, the single dose of the compound varies depending on the administration subject, the target disease and the like. For example, the compound is usually in the form of an injection and is usually administered to an adult (with a body weight of 60 kg) of diabetic nephropathy. When administered to a patient, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. In the case of other animals, the dose can be administered in terms of the weight per 60 kg.

(7b) Method for screening for a compound that promotes or inhibits the activity of the promoter for the DNA of the present invention The present invention comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and detecting the expression of a reporter gene. And a method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for DNA of the present invention.
In the above-described screening method, the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound include the same compounds as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, a tissue that expresses the protein of the present invention should be used instead of the protein of the present invention. Β-galactosidase is expressed. Therefore, for example, the present invention can be easily performed by staining with a reagent serving as a substrate for β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal). Of the protein in an animal body can be observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.
The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metals). And the like, and especially preferred are physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, For example, salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.

A compound or a salt thereof that promotes or inhibits (preferably inhibits) the promoter activity of the DNA of the present invention can regulate the expression of the protein of the present invention and regulate the function of the protein. , Diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, half a month Prevention of somatic nephritis, lupus nephritis, amyloid kidney, tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumor, chronic rejection after kidney transplantation, etc. Useful as a therapeutic.
Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the subject to be administered, the administration route, and the like. For example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally the adult (body weight) is used. For a diabetic nephropathy patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that inhibits the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to a diabetic nephropathy patient (with a body weight of 60 kg), it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. is there. In the case of other animals, the dose can be administered in terms of the weight per 60 kg.
As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to the investigation of the cause of various diseases caused or the development of preventive / therapeutic agents.
Further, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene-transferred animal). Once created, it will also be possible to specifically synthesize the protein and study its effects in living organisms. Furthermore, if a suitable reporter gene is bound to the above promoter portion, and a cell line that expresses the reporter gene is established, a low-molecular compound having an action of specifically promoting or suppressing the production ability of the protein itself of the present invention in the body. Can be used as a search system.

In the present specification, when bases, amino acids, and the like are indicated by abbreviations, they are based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate 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: Threin Crythine : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe Phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Sec: selenocysteine (selenocysteine)

Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine-4 (R) -carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl
Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitro Phenyl Trt: Trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBT: 3,4-dihydro-3-hydroxy-4-oxo-
1-2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
1 shows the amino acid sequence (GenBank Accession No. AAC98695) of human Type IIb Na / Pi Cotransporter.
[SEQ ID NO: 2]
1 shows the nucleotide sequence of human Type IIb Na / Pi Cotransporter (GenBank Accession No. AF111856).
[SEQ ID NO: 3]
2 shows the amino acid sequence of rat Type IIb Na / Pi Cotransporter (GenBank Accession No. AAF76291).
[SEQ ID NO: 4]
1 shows the nucleotide sequence of rat Type IIb Na / Pi Cotransporter (GenBank Accession No. AF157026).
[SEQ ID NO: 5]
2 shows the amino acid sequence of mouse Type IIb Na / Pi Cotransporter (GenBank Accession No. AAC80007).
[SEQ ID NO: 6]
The nucleotide sequence of mouse Type IIb Na / Pi Cotransporter (GenBank Accession No. AF081499) is shown.
[SEQ ID NO: 7]
3 shows the base sequence of a primer used in Example 2.
[SEQ ID NO: 8]
3 shows the base sequence of a primer used in Example 2.
[SEQ ID NO: 9]
4 shows the nucleotide sequence of a probe used in Example 2.
[SEQ ID NO: 10]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 11]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 12]
7 shows the nucleotide sequence of a probe used in Example 3.

本実験では、各群の個体別腎臓Total RNAを等量ずつ混合した総量40μgのTotal RNAをひとつの試料として（例えば、生後40週のWFラットの場合、この群の個体数は5であるから1個体当たり8μgを混合して総量40μgとした）、Oligonucleotide microarray (Rat Genome U34A, U34B, U34C; Affymetrix社) を用いて遺伝子発現解析を行った。実験方法は、Affymetrix社の実験手引き書 (Expression analysis technical manual) に従った。実験で得られたMicroarray掲載各配列の発現測定値をExpression analysis software（GeneSpring：Silicon Genetics社）で解析し、発現変動を示す配列を抽出した。
その結果、ラットType IIb Na/Pi Cotransporter（Genbank Accession No. AF157026）が3種のモデルいずれでも発現亢進していた。特にSHCラットではラットType IIb Na/Pi Cotransporterの変動が顕著であった。 Hereinafter, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto.
Example 1
The following experiments were conducted to discover drug targets from channel transporters whose expression fluctuates in renal disease model rat kidney tissue.
As disease model animals, Wistar Fatty rats (WF rats), Zucker Fatty rats (ZF rats), and spontaneously hypercholesterolemic rats (SHC rats) were used. Wistar Lean Rat (WL rat), Zucker Lean Rat (ZL rat) for ZF rat, and SD Rat (SD rat) for SHC rat. Disease model animals and control animals were bred under the same conditions, with WF and WL rats at 13 weeks, 20 weeks, 40 weeks, and 68 weeks, and ZF and ZL rats at 8 weeks and 27 weeks, SHC rats and At 6 weeks, 12 weeks, 20 weeks and 30 weeks after birth, kidneys were collected from at least 5 individuals in each group of SD rats, and total RNA was prepared for each individual according to the method described in the instruction manual of ISOGEN (Nippon Gene). Among the prepared samples, in WF rats and WL rats, ZF rat For ZL rats and ZF rats, a 27-week-old sample with renal impairment in ZF rats was developed. It was used for expression analysis experiments. Table 1 shows the experimental configuration.

In this experiment, a total amount of 40 μg of total RNA obtained by mixing equal amounts of individual kidney total RNA of each group was used as one sample (for example, in the case of WF rats aged 40 weeks, the number of individuals in this group is 5 Gene expression analysis was performed using an oligonucleotide microarray (Rat Genome U34A, U34B, U34C; Affymetrix) using a mixture of 8 μg per individual to give a total amount of 40 μg. The experimental method was in accordance with the experimental manual (Expression analysis technical manual) of Affymetrix. The expression measurement value of each sequence on the Microarray obtained in the experiment was analyzed by Expression analysis software (GeneSpring: Silicon Genetics), and a sequence showing an expression variation was extracted.
As a result, the expression of rat Type IIb Na / Pi Cotransporter (Genbank Accession No. AF157026) was enhanced in all three models. In particular, the fluctuation of rat Type IIb Na / Pi Cotransporter was remarkable in SHC rats.

Example 2
For the purpose of closely examining the results of the analysis using the Oligonucleotide microarray shown in Example 1, the expression level of the rat Type IIb Na / Pi Cotransporter (Genbank Accession No. AF157026) in each individual kidney was measured by the TaqMan PCR System. Using the individual total kidney RNA used in Example 1 as the template RNA, the expression fluctuation was analyzed by comparing the expression levels of the experimental group and the control group. The experimental groups were WF rats, ZF rats and SHC rats, and the corresponding control groups were WL rats, ZL rats and SD rats.
First, a reverse transcription reaction was performed using the individual RNA prepared in Example 1 as a template with a TaqMan Gold RT-PCR Kit (Applied Biosystems) to prepare cDNA. The composition of the reaction solution was as follows: 1 μg of individual total kidney RNA prepared in Example 1, 5.5 mM MgCl ₂ , 0.5 mM dNTPs, 2.5 μM Ramdom Hexamers, 0.4 U / μl RNase Inhibitor, 1.25 U / μl MultiScribe Reverse Transcriptase and kit. The total volume was 50 μl with the attached buffer for reverse transcription reaction. The reaction was carried out using a thermal cycler (PE Biosystems) by keeping the temperature at 25 ° C for 10 minutes, at 48 ° C for 30 minutes, and at 95 ° C for 5 minutes. Next, the expression amount of the rat Type IIb Na / Pi Cotransporter gene was measured by TaqMan PCR using the cDNA prepared by the above method as a template. The composition of the reaction solution was 5 μl of cDNA prepared by the above method (corresponding to 2.4 ng of Total RNA), 0.2 μM of a synthetic DNA primer (SEQ ID NO: 7), 0.2 μM of a synthetic DNA primer (SEQ ID NO: 4), and 0.2 μM of a synthetic DNA probe (sequence). No. 5) 0.2 μM and 7.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems) were added to make a total volume of 15 μl. The reaction was carried out using a 7900HT Sequence Detection System (Applied Biosystems) at 50 ° C. for 2 minutes and at 95 ° C. for 10 minutes, followed by a cycle of 95 ° C. for 15 seconds and 60 ° C. for 1 minute 40 times. In order to correct for the amount of template cDNA added between samples, the expression level of glyceraldehyde triphosphate dehydrogenase (GAPDH) was measured for each sample, and the rat Type IIb Na / Pi Cotransporter gene expression level was measured. It was calculated as the relative expression level to the GAPDH expression level. The relative expression amount was expressed as a value obtained by multiplying the value obtained by dividing the copy number of the rat Type IIb Na / Pi Cotransporter gene per unit liquid volume by the copy number of the rat GAPDH gene per unit liquid amount, by 100. TaqMan Rodent GAPDH Control Reagents VIC Probe (Applied Biosystems) was used to measure the expression value of rat GAPDH. For both the rat Type IIb Na / Pi Cotransporter gene and the rat GAPDH gene, measurement was performed twice for each sample, and the average value was used as the expression value.
The results are shown in FIGS.
As a result of analyzing the relative expression levels obtained in the above experiments, the expression level of the rat Type IIb Na / Pi Cotransporter gene with the progression of the disease state in any of the experimental group WF, ZF, SHC renal disease model rats Was confirmed to be higher than that in the control group, and the results of Example 1 were supported.
TCV116 (generic name: Candesartan cilexetil) reported to have a renal protective effect in ZF rats (Kidney international 49, S115-S118, 1996, Clinical Experimental Nephrology 4, 207-214, 2000, Japan Journal of Pharmacology 88, 300-306, 2002) was administered daily at 10 mg / kg body weight daily for 18 to 9 weeks after birth. The non-administration group received only the same amount of excipient (0.5% methylcellulose 100 cP solution) as the administration group. Kidneys were collected at 27 weeks of age from the three groups of vehicle-administered ZF rats, TCV116-administered ZF rats, and vehicle-administered ZL rats. Total kidneys were collected by individual according to the method described in the instruction manual for ISOGEN (Nippon Gene). RNA was prepared and the relative expression was analyzed in the same manner as described above.
The results are shown in FIG.
From the results in FIG. 4, when TCV116 was administered to the ZF rat before the onset of renal injury, the expression level of the rat Type IIb Na / Pi Cotransporter gene became almost the same as that of the control ZL rat. It can be seen that the enhanced expression as observed in ZF rats does not occur. On the other hand, in ZF rats to which TCV116 was administered, the urinary protein level showed a normal value similar to that of ZL rats, and renal function decline was suppressed. As described above, since the increased expression of the rat Type IIb Na / Pi Cotransporter gene is observed in conjunction with the decreased renal function, the increased expression of the rat Type IIb Na / Pi Cotransporter gene is associated with decreased renal function. Conceivable.

各細胞を75cm²培養フラスコにサブコンフルエントになるよう培養して、トリプシン-EDTA処理により細胞を回収した。回収した細胞から、ISOGEN（ニッポンジーン社製）またはRNeasy Mini Kit（キアゲン社製）を用いてtotal RNAを調製した。調製したtotal RNA に対してTaqMan Revease Transcription Reagents（アプライドバイオシステムズ社製）を用いて逆転写反応を行いcDNAを調製した。次いで、以上の方法で調製したcDNAを鋳型としてヒトType IIb Na/Pi Cotransporter遺伝子の発現量をTaqMan PCRで測定した。反応液の組成は、各種正常ヒト細胞由来cDNA溶液 2μl（Total RNA 4ng相当）、合成DNAプライマー（配列番号：１０）0.4μM、合成DNAプライマー（配列番号：１１）0.4μM、合成DNAプローブ（配列番号：１２）0.2μMおよびTaqMan Universal PCR Master Mix（Applied Biosystems 社）を5μl加え、総液量を10μlとした。反応は、7900HT Sequence Detection System（Applied Biosystems 社）を用い、50℃・2分間、95℃・10分間の保温の後、95℃・15秒、60℃・1分のサイクルを40回繰り返した。試料間での鋳型cDNA添加量の多少を補正するため、各試料についてグリセルアルデヒド3リン酸デヒドロゲナーゼ（GAPDH）の発現値を測定し、ヒトType IIb Na/Pi Cotransporter遺伝子発現量をヒトGAPDH発現値に対する相対的発現量として算出した。相対的発現量は、単位液量あたりのヒトType IIb Na/Pi Cotransporter遺伝子のコピー数を同単位液量あたりのヒトGAPDH遺伝子のコピー数で除した値を100倍した値として表した。ヒトGAPDHの発現値測定には、TaqMan GAPDH Control Reagents（Applied Biosystems 社）を用いた。また、ヒトType IIb Na/Pi Cotransporter遺伝子およびヒトGAPDH遺伝子ともに、測定は各試料2回行い、その平均値を発現値とした。
結果を図５に示す。
24種類の正常ヒト細胞の中では、腎臓近位尿細管細胞で、ヒトType IIb Na/Pi Cotransporter遺伝子の比較的高い発現が認められた（GAPDHの約2％）。これより、ヒトType IIb Na/Pi Cotransporter遺伝子が腎臓で機能していると考えられる。 Example 3
In order to examine the expression of human Type IIb Na / Pi Cotransporter at the cellular level, expression analysis in various normal human cultured cells was performed. Normal human cells were purchased from Cambrex BioScience Walkersvill and cultured according to the method described in the instruction manual attached to the product. Table 2 shows the cells used in the experiment and the medium used to culture each cell.

Each cell was cultured in a 75 cm ² culture flask so as to become subconfluent, and the cells were recovered by trypsin-EDTA treatment. From the collected cells, total RNA was prepared using ISOGEN (Nippon Gene) or RNeasy Mini Kit (Qiagen). Reverse transcription was performed on the prepared total RNA using TaqMan Revease Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA. Next, the expression level of the human Type IIb Na / Pi Cotransporter gene was measured by TaqMan PCR using the cDNA prepared by the above method as a template. The composition of the reaction solution was 2 μl of various normal human cell-derived cDNA solutions (corresponding to 4 ng of Total RNA), 0.4 μM of synthetic DNA primer (SEQ ID NO: 10), 0.4 μM of synthetic DNA primer (SEQ ID NO: 11), and synthetic DNA probe (SEQ ID NO: 11). No. 12) 0.2 μM and 5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems) were added to make a total volume of 10 μl. The reaction was carried out using a 7900HT Sequence Detection System (Applied Biosystems) at 50 ° C. for 2 minutes and at 95 ° C. for 10 minutes, followed by a cycle of 95 ° C. for 15 seconds and 60 ° C. for 1 minute 40 times. To correct for the amount of template cDNA added between samples, the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured for each sample, and the expression level of the human Type IIb Na / Pi Cotransporter gene was compared to the expression level of human GAPDH. Was calculated as the relative expression level with respect to. The relative expression level was expressed as a value obtained by multiplying the value obtained by dividing the copy number of the human Type IIb Na / Pi Cotransporter gene per unit liquid volume by the copy number of the human GAPDH gene per unit liquid volume, by 100 times. TaqMan GAPDH Control Reagents (Applied Biosystems) was used for measuring the expression value of human GAPDH. In addition, both the human Type IIb Na / Pi Cotransporter gene and the human GAPDH gene were measured twice for each sample, and the average value was used as the expression value.
FIG. 5 shows the results.
Among 24 normal human cells, relatively high expression of human Type IIb Na / Pi Cotransporter gene was observed in renal proximal tubule cells (about 2% of GAPDH). This suggests that the human Type IIb Na / Pi Cotransporter gene functions in the kidney.

It is a figure showing the time-dependent change of rat Type IIb Na / Pi Cotransporter expression level. In the figure, the vertical axis shows the relative expression level of the rat Type IIb Na / Pi Cotransporter gene expression value to the rat GAPDH expression value, and the horizontal axis shows the age at which the kidney was collected. ■ shows the results of the experimental group (WF rats) and □ shows the results of the control group (WL rats). It is a figure showing the time-dependent change of rat Type IIb Na / Pi Cotransporter expression level. In the figure, the vertical axis shows the relative expression level of the rat Type IIb Na / Pi Cotransporter gene expression value to the rat GAPDH expression value, and the horizontal axis shows the age at which the kidney was collected. ■ indicates the results of the experimental group (ZF rats), and □ indicates the results of the control group (ZL rats). It is a figure showing the time-dependent change of rat Type IIb Na / Pi Cotransporter expression level. In the figure, the vertical axis shows the relative expression level of the rat Type IIb Na / Pi Cotransporter gene expression value to the rat GAPDH expression value, and the horizontal axis shows the age at which the kidney was collected. ■ indicates the results of the experimental group (SHC rats), and □ indicates the results of the control group (SD rats). FIG. 4 is a graph showing the effect of TCV116 on rat Type IIb Na / Pi Cotransporter expression level. In the figure, the vertical axis indicates the relative expression level of the rat Type IIb Na / Pi Cotransporter gene expression value to the rat GAPDH expression value. The horizontal axis indicates the type of rat used in the experiment and the presence or absence of TCV116 administration. ZF (Vehicle) shows the results of a ZF rat administered with a vehicle, ZF (TCV116) shows the results of a ZF rat administered with TCV116, and ZL (Vehicle) shows the results of a ZL rat administered with a vehicle. It is a figure showing the expression level of human Type IIb Na / Pi Cotransporter in various normal human cells. In the figure, the vertical axis indicates the relative expression level of the human Type IIb Na / Pi Cotransporter gene expression value to the GAPDH expression value. Lane 1 on the horizontal axis is umbilical vein endothelial cells, lane 2 is aortic vascular endothelial cells, lane 3 is coronary artery endothelial cells, lane 4 is skin microvascular endothelial cells, lane 5 is lung microvascular endothelial cells, and lane 6 is pulmonary artery blood vessels. Endothelial cells, lane 7 aortic smooth muscle cells, lane 8 coronary artery smooth muscle cells, lane 9 uterine smooth muscle cells, lane 10 bronchial smooth muscle cells, lane 11 skeletal muscle cells, lane 12 mammary epithelial cells, Lane 13 is bronchial epithelial cells, lane 14 is bronchiolar epithelial cells, lane 15 is lung fibroblasts, lane 16 is kidney proximal tubule cells, lane 17 is mesangial cells, lane 18 is renal cortical cells, lane 19 is kidney Epithelial cells, lane 20 shows mesenchymal stem cells, lane 21 shows chondrocytes, lane 22 shows osteoblasts, lane 23 shows prostate stromal cells, and lane 24 shows epidermal keratinocytes.

Claims (20)

Prevention and treatment of renal diseases comprising a protein or a partial peptide thereof or a compound thereof which inhibits the activity of 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, or a salt thereof Agent. A renal disease comprising a compound or a salt thereof, which inhibits the expression of a protein or a partial peptide thereof, 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; Prophylactic and therapeutic agents. The agent according to claim 1 or 2, wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis. A nucleotide sequence complementary or substantially complementary to the nucleotide sequence of a polynucleotide encoding a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or An antisense polynucleotide containing a portion. A pharmaceutical comprising the antisense polynucleotide according to claim 4. The medicament according to claim 5, which is a prophylactic / therapeutic agent for kidney disease. An antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. A medicament comprising the antibody according to claim 7. The medicament according to claim 8, which is a prophylactic / therapeutic agent for kidney disease. A diagnostic agent comprising the antibody according to claim 7. The diagnostic agent according to claim 10, which is a diagnostic agent for kidney disease. A diagnostic agent for renal disease comprising a polynucleotide 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 peptide thereof. A method for screening for a prophylactic / therapeutic agent for renal disease, comprising using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. A kit for screening a prophylactic / therapeutic agent for renal disease, comprising 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 peptide thereof, or a salt thereof. . A method for screening for a prophylactic / therapeutic agent for renal disease, comprising using a polynucleotide encoding a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. . Screening of a prophylactic / therapeutic agent for renal disease, comprising a polynucleotide 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 peptide thereof. Kit. 16. The screening method according to claim 13, wherein the kidney disease is diabetic nephropathy or focal glomerulosclerosis. A compound or a salt thereof, which inhibits the activity of a protein or a partial peptide thereof 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, Alternatively, a method for preventing or treating renal disease, which comprises administering an effective amount of a compound or a salt thereof that inhibits the expression of a partial peptide or a salt thereof gene. Inhibits the activity of a protein or a partial peptide thereof 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, or a gene of the protein or its partial peptide or a salt thereof A method for preventing or treating renal disease, characterized by inhibiting expression. A compound that inhibits the activity of 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 peptide thereof or a salt thereof for producing a prophylactic / therapeutic agent for kidney disease, or Use of a salt thereof, or a compound or a salt thereof that inhibits the expression of the gene of the protein or its partial peptide or a salt thereof.

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