WO2004071534A1

WO2004071534A1 - Preventives/remedies for kidney diseases

Info

Publication number: WO2004071534A1
Application number: PCT/JP2004/001645
Authority: WO
Inventors: Hiroyuki Miya; Takanori Matsuo; Hiroko Tsuge
Original assignee: Takeda Pharmaceutical Company Limited
Priority date: 2003-02-17
Filing date: 2004-02-16
Publication date: 2004-08-26

Abstract

As preventives/remedies for kidney diseases such as diabetic nephropathy, use can be made of a compound controlling (preferably inhibiting) the activity of a protein having an amino acid sequence, which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:1, or the expression of a gene of this protein; an antisense polynucleotide having a base sequence, which is complementary or substantially complementary with the base sequence of DNA encoding the above protein or its peptide fragment, or a part thereof; an antibody against the above protein or its peptide fragment, and so on.

Description

Description Prevention and treatment of renal disease Technical field

The present invention relates to a preventive / therapeutic agent for renal diseases such as diabetic nephropathy, a diagnostic agent, and a screening of a preventive / therapeutic agent for renal diseases such as diabetic nephropathy. Background art

· The kidneys excrete · Maintain the body's homeostasis by regulating the concentration of body fluid components through reabsorption, and activate vitamins! ), Is an extremely important tissue that also functions as an endocrine organ that produces and secretes physiologically active substances such as erythropoietin and renin. Impaired renal function, which plays such an important role, can lead to a variety of conditions and a wide variety of renal diseases. These kidney diseases include nephropathy closely related to diabetes, obesity, and abnormal lipid metabolism.In particular, diabetic nephropathy, which is a primary disease of diabetes, requires strict control of diabetes after diagnosis of nephropathy. However, it is difficult to prevent progress, and in many cases renal failure occurs, and the number of patients is not small. As a model animal for renal disease, Wistar Fatty rats (WF rats) show symptoms characteristic of diabetes in addition to renal dysfunction, so they are used as a diabetic nephropathy model (Front iers in diabetes research). , lessons from animal diabetes II, 535-541, 1988), and Zucker Fatty rats (ZF rats) show diabetic nephropathy because they exhibit renal dysfunction and diabetes as well as WF rats. As a model (Kidney international, 52, S218-S220, 1997), spontaneously hypercholesterolemia rats (SHC rats) show symptoms similar to those of human focal glomerulosclerosis. It has been reported as a model of glomerulosclerosis (Japanese Journal of Nephrology, Vol. 37, pp. 91-99, 1995).

On the other hand, human type lib Na / Pi cotransporter (sodium phosphate cotransporter type lib) is a molecule belonging to transpomf amyl, which has sodium-dependent phosphate transport activity, and is expressed in the small intestine. And high activity at low pH This point is consistent with the results of the phosphate uptake assay using the rabbit and mouse small intestine, suggesting that it may be a molecule responsible for phosphorus uptake in the small intestine (Biochemical and Biophysical Research Communi cat ions, 258, 578-582, 1999). In addition, treatment of diseases related to the function of human Type Lib Na / Picot ransporter, such as treatment of abnormal conditions of serum phosphate concentration such as hypophosphatemia and hyperphosphatemia, etc. Are disclosed in Japanese Patent Application Laid-Open No. 2000-245488. U.S. Pat.No. 6,319,688 states that angiogonists and agonists of human Type I ib Na / Picotransporter are useful in treating chronic renal failure, end-stage renal disease, uremic bone disease, and cancer. It is described as useful for preventive care.

Regarding renal lesions caused by the above-mentioned underlying diseases, studies that incorporate techniques of cell biology and molecular biology are being vigorously conducted with the aim of elucidating the pathogenesis and pathophysiology, and new knowledge is being obtained. However, there are still many unsolved issues at this time. There is a need for safe and excellent prevention of renal diseases << therapeutic agents. Disclosure of the invention

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 this finding, the present invention has been completed.

That is, the present invention

(1) A renal disease comprising a compound which inhibits the activity of 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, or a salt thereof Prevention and treatment of

(1a) the prophylactic or therapeutic agent according to (1), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis;

(2) a compound containing 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 gene expression Prevention and treatment of kidney disease, (2a) the prophylactic or therapeutic agent according to (2) above, wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis;

(3) The prophylactic and / or therapeutic agent according to (1) or (2) above, 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 having 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 (4),

(6) the medicament according to the above (5), which is an agent for preventing or treating renal disease;

(6a) the prophylactic or therapeutic agent according to (6) above, 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 pharmaceutical comprising the antibody according to (7) above,

(9) The medicament according to the above (8), which is an agent for preventing or treating kidney disease.

(9a) The preventive / therapeutic agent according to (9) above, wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis.

(10) a diagnostic agent comprising the antibody according to (7),

(11) The diagnostic agent according to the above (10), which is a diagnostic agent for renal disease,

(12) an agent for diagnosing 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 polynucleotide encoding a partial peptide thereof,

(13) Screening of a prophylactic and therapeutic agent for renal disease, characterized by using 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. Method,

(13a) (i) identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 (A) adding a labeled inorganic phosphate to a cell capable of expressing a protein containing the same amino acid sequence or its partial peptide or a salt thereof, and (ii) applying the labeled amino phosphate to the cell in the presence of a test compound. The screening method according to the above (13), wherein the amount of the inorganic phosphate taken into the cells when the labeled inorganic phosphate is added is measured,

(13 b) labeled inorganic phosphate, radioisotope ^([32 P], ^[33 P], etc.) screening how the (13 a), wherein the labeled inorganic phosphate in,

(13c) (i) 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. Inorganic phosphate transport activity was measured when the buffer containing salt was brought into contact with the buffer containing inorganic phosphate, and (ii) when the buffer containing inorganic phosphate was brought into contact with the synthetic lipid bilayer in the presence of the test compound. Above to measure

(13) the screening method described,

(13d) the screening method according to (13), wherein the kidney disease is diabetic nephropathy or focal glomerulosclerosis;

(13e) 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 obtained by using the screening method described in the above (13). A prophylactic / therapeutic agent for kidney disease comprising a compound that inhibits the activity of a salt or a salt thereof,

(14) A prophylactic and / or therapeutic agent for renal disease characterized by 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. Screening kit,

(15) Prevention and treatment of 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. How to screen the agent,

(15a) The renal disease is diabetic nephropathy or focal glomerulosclerosis (1) 5) the screening method described,

(16) Prevention 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, Kit for screening therapeutic agents,

(17) The screening method according to the above (13) or (15), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis,

(17a) A compound having a prophylactic or therapeutic effect on renal disease or a salt thereof, which is 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 or salt thereof according to (17a) above,

(18) a compound that inhibits the activity of a protein, or a partial peptide 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 in mammals Or an effective amount of a compound or 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, or a method for preventing or treating renal disease,

(18a) The method of (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 an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, or inhibits the activity of the protein or a partial thereof; Prevention and treatment of renal disease, characterized by inhibiting the expression of the peptide or its salt gene. (19a) The renal disease is diabetic nephropathy or focal glomerulosclerosis (19) ) Described prevention, treatment,

(20) Prevention of renal disease · A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof for producing a therapeutic agent A compound that inhibits the activity or its Use of a salt, or a compound or a salt thereof that inhibits expression of the gene for the protein or its partial peptide or a salt thereof;

(20a) The use according to (20), wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the time course of the expression level of rat Typelib Na / Pi Cotransporter. In the figure, the vertical axis represents the relative expression level of rat Typelib Na / Pi Cotransporter gene expression value to rat GAPDH expression value, and the horizontal axis represents the age at which kidneys were collected.鱷 indicates the results of the experimental group (WF rats), and the mouth indicates the results of the control group (WL rats).

FIG. 2 is a graph showing the time course of the expression level of rat Typelib Na / Pi Cotransporter. In the figure, the vertical axis indicates the relative expression level of the rat Type Lib Na / Pi Co transporter gene expression value to the rat GAPDH expression value, and the horizontal axis indicates the age at which the kidney was collected. ■ shows the results of the experimental group (ZF rats) and the mouth shows the results of the control group (ZL rats).

FIG. 3 is a diagram showing a time-dependent change in the expression level of rat Typelib Na / Pi Cotransporter. In the figure, the vertical axis indicates the relative expression level of the rat Type Lib Na / Pi Cotransporter gene expression value to the rat GAPDH expression value, and the horizontal axis indicates the age at which the kidney was collected. The results are for the experimental group (SHC rats) and the control group (SD rats) for the mouth.

FIG. 4 is a graph showing the effect of TCV116 on rat Typelib Na / Pi Cotransporter expression levels. In the figure, the vertical axis indicates the relative expression level of the rat Typelib Na / Pi Cotransporter gene expression value to the rat GAPDH expression value. The horizontal axis shows the type of rat used in the experiment and the presence or absence of TCV116 administration. ZF (Vehicle) shows the results of ZF rats to which vehicle was administered, ZF (TCV116) shows the results of ZF rats to which TCV116 was administered, and ZL (Vehicle) shows the results of ZL rats to which vehicle was administered.

FIG. 5 is a diagram showing the expression levels of human Typelib Na / Pi Cotransporter in various normal human cells. In the figure, the vertical axis indicates the relative expression level of the human Typelib 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 endothelial cells, lane 3 is coronary 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 flat muscle cells, lane 9 uterine smooth muscle cells, lane 10 bronchial smooth muscle cells, lane 11 skeletal muscle cells, lane 12 mammary epithelium Cells, lane 13 is bronchial epithelial cells, lane 14 is bronchiolar epithelial cells, lane 15 is lung fibroblasts, lane 16 is renal proximal tubule cells, lane 17 is mezzanine cells, lane 18 is renal cortical cells, lane 19 shows renal 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. BEST MODE FOR CARRYING OUT THE INVENTION

A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter, also referred to as the protein of the present invention or the protein used in the present invention) may be a human or a human. Cells of warm-blooded animals (eg, guinea pigs, rats, mice, chicks, etc., egrets, pigs, sheep, pigs, monkeys, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells, kidney) / 3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells

(Eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts , Osteoclasts, mammary gland cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells, or any tissue in which those cells are present, for example, the brain, various parts of the brain (eg, olfactory bulb) , Amygdala, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary, stomach, victory, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin , Muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. The protein May it, may be a synthetic protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 60% or more, preferably about 70% or more, preferably about 80% or more of the amino acid sequence represented by SEQ ID NO: 1. % Or more, preferably about 90% or more, preferably about Amino acid sequences having a homology of 95% or more.

Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described above. However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable. Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 3, and a protein represented by SEQ ID NO: 5 And a protein containing an amino acid sequence.

Examples of substantially the same activity include an inorganic phosphate uptake activity and an inorganic phosphate transport activity. Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) equivalent. 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). ), But the 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, 191: 494-505, 1990 or a method analogous thereto.

Specifically, a labeled inorganic phosphate (eg, an inorganic phosphate labeled with a radioisotope) is added to cells expressing the protein of the present invention, and after incubation for a certain time, intracellular This is performed by measuring the amount of inorganic phosphoric acid. The reaction is performed in an appropriate buffer, and the inorganic phosphate that has not been taken up by 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 Biochimi ca et Biophysica ca Acta, Vol. 1236, pp. 339-344, 1995, or a method analogous thereto. Can be.

Specifically, inorganic phosphate is added to the synthetic lipid bilayer containing the protein of the present invention. A buffer containing a salt is added, and the change in membrane current that occurs when inorganic phosphate is transported by the protein of the present invention is measured. 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 can be obtained by, for example, 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. The material can be manufactured according to a known method, for example, a method described in "Patch Clamp Experimental Technique" (Yoshioka Shoten, published in 1996).

Examples of the protein used in the present invention include: (i) one or two or more (for example, 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1, 'SEQ ID NO: 3 or SEQ ID NO: 5; An amino acid sequence in which about 0, preferably about 1 to 30, preferably about 1 to 10, and more preferably about (1 to 5) amino acids have been deleted; (ii) SEQ ID NO: 1, 1 or 2 or more amino acid sequences represented by SEQ ID NO: 3 or SEQ ID NO: 5 (for example, about 1 to 100, preferably about 1 to 30 and preferably about 1 to 10) (Iii) one or two amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and more preferably (1 to 5) amino acids. Above (for example, about 1 to 100 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, Preferably, a 7-amino acid sequence into which a number (1 to 5) of amino acids have been inserted, (iv) one or more of the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 Or two or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids. So-called mucins such as proteins containing an amino acid sequence substituted with an amino acid of (a) or (v) an amino acid sequence obtained by 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 the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (potassium terminus) according to the convention of peptide notation. The protein used in the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal lipoxyl group (-C00H), It may be any of (—COO—), amide (—C0NH ₂ ) and ester (—C00R).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl Le, isopropyl, CM alkyl group, such as n- butyl, cyclopentyl Le, C ₃ _ ₈ cycloalkyl group such as cyclohexyl, for example, phenyl, alpha-Na C ₆ _ ₁₂ 7 aryl group such as Fuchiru, for example, benzyl, C _7, such as single Nafuchiru C _M Al kill groups such as phenyl one CM alkyl group or single naphthylmethyl such phenethyl - ₁₄ Ararukiru group, A pivaloyloxymethyl group or the like is used.

When the protein used in the present invention has a lipoxyl group (or carboxylate) other than the C-terminal, the protein in which the lipoxyl 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 or the like is used.

Furthermore, the protein used in the present invention, the amino acid residue (e.g., Mechionin residues) of N-terminal Amino group protecting groups (e.g., c Bok such c Bok ₆ Al force Noiru such formyl group, Asechiru group _6- Glycyl group), N-terminal glutamine residue generated by cleavage in vivo, and pyroglutamine oxidation, substituent on the side chain of amino acid in the molecule (e.g.,- 0H, -SH, amino group, imidazole group, indole group, guanidino group, etc. are protected with appropriate protecting groups (for example, C-silyl groups such as alkanoyl groups such as formyl group and acetyl group). And complex proteins such as so-called glycoproteins to which sugar chains are 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. Proteins containing the represented amino acid sequence are exemplified.

The partial peptide of the protein used in the present invention is the partial peptide of the protein used in the present invention described above, and is preferably any one having the same properties as the protein used in the present invention described above. Even things Good.

Specifically, for the purpose of preparing the antibody of the present invention described later, the amino acids represented by SEQ ID NO: 1 to 1 to 101, 1 87 to 360, and 57 A peptide having an amino acid sequence of the 5th to 69th amino acid sequence, the 1st to 101st, the 187th to 360th, and the 575th to 6995th amino acid sequence represented by SEQ ID NO: 3 A peptide having the amino acid sequence at position No. 1-101, 187-360, and 575-699 in the amino acid sequence represented by SEQ ID NO: 5; And the like. For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably, of the constituent amino acid sequences of the protein used in the present invention. Is a peptide having 200 or more amino acid sequences.

The partial peptide used in the present invention may have one or two or more amino acids in its amino acid sequence (for example, about 1 to 20, preferably about 1 to 10, more preferably about 1 to 5). Amino acids are deleted, or one or more amino acids are added to the amino acid sequence (for example, about 1 to 20, preferably about 1 to 10, more preferably about 1 to 5 )) Or one or more amino acids (eg, about 1 to 20, preferably about 1 to 10, more preferably number (1 to 5)) to the amino acid sequence. Or one or more amino acids in the amino acid sequence (for example, about 1 to 20 amino acids, preferably about 1 to 10 amino acids, more preferably about 1 to 5 amino acids). An amino acid may be substituted with another amino acid.

The partial peptide used in the present invention, the C-terminus force Rupokishiru group (- C00H), Karupokishireto (-COO "), amido (- C0NH ₂₎ or ester (- C00R) may be either.

Further, similar to the protein used in the present invention, the partial peptide used in the present invention has a lipoxyl group (or carboxylate) other than the C-terminal, and the N-terminal amino acid residue. When the amino group of a group (eg, methionine residue) is protected by a protecting group, the dal formed by cleavage of the N-terminal side in vivo The amino acid residue has been oxidized with pip dar, the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or the so-called sugar peptide to which a sugar chain is bound. Complex peptides and the like are also included.

The partial peptide used in the present invention can also be used as an antigen for producing 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, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.

The protein or its partial peptide or a salt thereof used in the present invention can be produced from the above-mentioned human or warm-blooded animal cells or tissues by a known method for purifying a protein, or a protein encoding D It can also be produced by culturing a transformant containing NA. It can also be produced according to the peptide synthesis method described below.

When producing from human or mammalian tissues or cells, the human or mammalian tissues or cells are homogenized, then extracted with an acid or the like, and the resulting extract is subjected to reversed phase chromatography, ion exchange chromatography, etc. Purification and isolation can be performed by combining the above chromatography.

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 usually used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4 —Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2,, 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2,, 4,1-dimethoxyphenyl) F mo Ami Noethyl) phenoxy resin and the like. Using such a resin, amino acids having a suitably protected amino group and side chain functional group are condensed on the resin in accordance with the sequence of the target protein in accordance with 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 protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the disulfide diimides, DCC, N, N, -diisopropylcarpo- imide, N-ethyl-Ν '-(3-dimethylaminoprolyl) carbopimide, and the like are used. For these activations, the protected amino acid may be added directly to the resin with a racemization inhibitor additive (e.g., HOB t, HOOB t), or may be pre-protected as a symmetrical anhydride or HOB t ester or H〇OB t ester The amino acid can be added to the resin after activation.

The solvent used for activating the protected amino acid or condensing with the resin may 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-dimethylacetoamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, trifluoroethanol, etc. Alcohols, sulphoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof. Used. The reaction temperature is appropriately selected from a range known to be usable for the 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, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, use acetic anhydride or By acetylating unreacted amino acids using cetyl imidazole, it is possible to prevent the subsequent reactions from being affected.

Examples of the protecting group for the amino group of the raw material include τ, Boc, t-pentyloxycarponyl, isopolnyloxycarbonyl, 4-methoxybenzyloxyl-ponyl, C 1 _Z, Br—Z, and adaman Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

Lepoxyl groups are, for example, alkyl esterified (e.g., methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cyclopentyl, cyclooctyl, 2-adamantyl, etc.). Branched or cyclic alkyl esterification), aralkyl esterification (eg, benzyl ester, 412 trobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phene It can be protected by nasylation, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide, or the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for the esterification, for example, a group derived from carbonic acid such as a lower (C ₆ ) alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a benzyloxycarbonyl group, and an ethoxycarbonyl group can be used. Can be 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, Bz1, C12-Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used. As the protecting group for imidazole of histidine, for example, Tos, 4-methoxy-1,2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the activated carbonyl group of the raw material include, for example, a corresponding acid anhydride, azide, and an active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2 , 4-dinitrophenol, shear Nomethyl alcohol, p-nitrophenol, H〇NB, N-hydroxysuccinimide, N-hydroxyfurimide, ester with HOBt)]. As the activated amino group of the raw material, for example, a corresponding phosphoramide is used.

Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid. Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia, etc. Used. The elimination reaction by the above acid treatment is generally carried out at a temperature of about 120 ° C. to about 40 ° C. In the acid treatment, for example, anisol, phenol, thioanisole, methacrylol, paracresol, etc. It is effective to add a cation scavenger such as toluene, dimethyl sulfide, 1,4-butanedithiol, or 1,2-ethanedithiol. 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 substituted with 1,2-ethanedithiol, 1,4- In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc.

The protection of the functional group which should not be involved in the reaction of the raw material, the protection group, the elimination of the protective group, and the activation of the functional group involved in the reaction can be appropriately selected from known groups or known means. ·

Another method for obtaining an amide form of a protein or partial peptide is, for example, first protecting a carboxy-terminal amino acid with the desired amino acid by amidating and protecting the amino acid side of the peptide (protein) chain. After lengthening, the protein or partial peptide from which only the ο; -amino protecting group at the N-terminus of the peptide chain has been removed, and the protein or partial protein from which only the protecting group at the C-terminal carbonyl group has been removed. A peptide is produced, and these proteins or peptides are condensed in a mixed solvent as described above. The details of the condensation reaction are the same as above. is there. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above method, and a desired crude protein or peptide can be obtained. This crude protein or peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide of the desired protein or peptide.

To obtain an ester of a protein or peptide, for example, after condensing the α-lipoxyl group of the terminal amino acid with a desired alcohol to form an amino acid ester, in the same manner as the amide of a protein or peptide, The desired ester of the protein or peptide can be obtained.

The partial peptide used in the present invention or a salt thereof 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 desired peptide. can do. Examples of the known condensation method and elimination of the protecting group include the methods described in the following [omega] to (V).

(0 M. Bodanszky and M.A. Ondet t i, Pept i de Synthes is, Interscience Publ ishers, New York (1966)

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

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

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

(V) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten In addition, after the reaction, conventional purification methods, for example, solvent extraction, distillation, column chromatography, 'liquid chromatography' recrystallization The partial peptides used in the present invention can be purified and isolated by combining these methods. When the partial peptide obtained by the above method is a free form, it may be obtained by a known method or a method analogous thereto. The compound can be converted to an appropriate salt, or conversely, when obtained as a salt, can be converted to a free form or another salt by a known method or a method analogous thereto.

The polynucleotide encoding the protein used in the present invention may be any polynucleotide containing 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, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA.

The vector used for the library may be any of pacteriophage, plasmid, cosmid, and phagemid. Alternatively, it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells and tissues described above.

The DNA encoding the protein used in the present invention includes, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a DNA hybridizing 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 nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 60% or more, preferably about 70% or more of the nucleotide 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 is carried out by a method known per se or a method analogous thereto, for example, 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, the reaction can be performed under high stringent conditions.

The high stringent conditions refer to, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 t: preferably about 60 to 65. In particular, it is most preferable that the sodium concentration is about 19 mM and the temperature is about 65 ° C. More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, the DNA containing the base sequence represented by SEQ ID NO: 2; As the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 4, a DNA containing the nucleotide sequence represented by SEQ ID NO: 4 and a protein containing the amino acid sequence represented by SEQ ID NO: 5 are encoded. As the DNA to be used, a DNA containing the base sequence represented by SEQ ID NO: 6 and the like are used.

The polynucleotide (e.g., DNA) encoding the partial peptide used in the present invention may be any polynucleotide containing a base sequence encoding the partial peptide used in the present invention described above. Is also good. Further, it may be any of genomic DNA, genomic DNA library, the above-described cell-tissue-derived cDNA, the above-described cell-tissue-derived cDNA library, and synthetic DNA. Examples of the DNA encoding the partial peptide used in the present invention include, for example, a DNA having a part of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or SEQ ID NO: 2. DNAs that contain a base sequence that hybridizes with a base sequence under high stringency conditions and that contain a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention are used.

DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 2 has the same significance as described above.

The same hybridization method and eight stringent conditions as described above are used. The DNA and the partial peptide used in the present invention (hereinafter, in the description of the cloning and expression of the DNAs encoding them, these may be simply abbreviated to the protein of the present invention) may be used to completely encode the DNA. As a means for cloning, the DNA is amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA incorporated into an appropriate vector is used as the cloning DNA of the present invention. Selection can be carried out by hybridization with a DNA fragment coding for a part or the entire region of the quality or labeled with a synthetic DNA. The hybridization method 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 using the ODA-LA PCR method using PCR, a known kit, for example, Mutan ™ -super Express Km (Takara Shuzo Co., Ltd.), Mutan ™ -K (Takara Shuzo Co., Ltd.), etc. It can be carried out according to a method known per se such as the gapped duplex method, the Kunke method, or a method analogous thereto.

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

The expression vector for the protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) downstream of the DNA fragment from a promoter in an appropriate expression vector. It can be manufactured by connecting to

Examples of the vector include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast ( Example, pSHl 9, pSH 15), Bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, pXT1, pRc / CMV, pRc / RSV, pcDNA I / Neo, etc. Is 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, SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.

Of these, it is preferable to use the CMV (cytomegalovirus) promoter, the SRa promoter, and the like. If the host is a genus Escherichia, the host is a Bacillus genus such as _trp promoter, _lac promoter, _recA promoter, λΡ ^ promoter, ι _ρρ promoter, and _c7 promoter. In this case, the SΡΟ1 promoter overnight, the SPO2 promoter, the penP promoter, and the like. When the host is yeast, the PH05 promoter, the PGK promoter, the GAP promoter, the 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.

The expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like, if desired. Can be used. The selection marker one, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistant], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r) , Ne Omaishin resistant gene (hereinafter sometimes abbreviated as Ne 0 ¹ ", G418 resistant) and the like. in particular, when used as a selection marker dh fr gene using dh fr gene-deficient Chinese hamster cells Alternatively, the target gene can be selected using a thymidine-free medium.

In addition, if necessary, a signal sequence suitable for the host may be added to the protein of the present invention. Add to N terminal side. If the host is a genus Escherichia, the PhoA 'signal sequence, OmpA signal sequence, etc., if the host is a Bacillus genus, the amylase * signal sequence, subtilisin signal sequence, etc. In some cases, MFa signal sequence, SUC2 signal sequence, etc. In the case where the host is an animal cell, insulin signal sequence, -interferin signal sequence, antibody molecule, signal sequence, etc. can be used, respectively. .

Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant can be produced.

As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells and the like are used.

Specific examples of the genus Escherichia include, for example, Escherichia coli.

(Escherichia coli) K 12 · DH 1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM 103 [Nucleic Acids Research, 9, 309 (1981)], J A221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like are used.

As the Bacillus sp., 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, AH22 R-, NA87-11A, DKD-5D, 2OB-12, Schizosaccharomyces pombe N CYC 1913, NCYC 2036, Pichia pastori (Pichia pastoris) KM 71 or the like is used.

Insect cells include, for example, when the virus is Ac NPV, a cell line derived from a larva of night moth (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, High derived from egg of Trichoplusia ni Five ™ cells,

Cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. If the virus is BmNP V, a silkworm-derived cell line (Bombyx mori N cells) Vesicles; BmN cells). Examples of the Sf cells include Sf9 cells (ATCC CRL1711) and Sf21 cells (Vaughn, JL et al., In Vipo).

(In Vivo), 13, 213-217, (1977)).

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 CH〇 (hereinafter abbreviated as CHO cells), dh fr gene-deficient Chinese hamster cells CHO (hereinafter, CHO (dh fr—) cells Abbreviations), mouse L cells, mouse AtT-20, mouse myeloid cells, mouse ATDC 5 cells, rat GH3, human FL cells, etc. are used.

Transformation of a genus Escherichia can be performed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982). it can.

Transformation of Bacillus spp. Can be carried out, for example, according to the method described in Molecular 'and General Genetics (Molecular & General Genetics), vol. 168, 111 (1979).

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

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

To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, 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 bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for cultivation. It contains carbon sources, nitrogen sources, inorganic substances, etc., necessary for the growth of sorghum. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn chips, lica, peptone, casein, meat extract, and soybean meal. Inorganic or organic substances such as potato extract and inorganic substances include, for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8. As a medium for culturing a bacterium belonging to the genus Escherichia, for example, an M9 medium containing glucose and casamino acids (Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. If necessary, a drug such as, for example, 3; 8-indolylacrylic acid can be added 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 may be applied.

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 can be applied.

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

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

When culturing an insect cell or a transformant whose host is an insect, the culture medium may be supplemented with Grace's Insect Medium (Nature, 195, 788 (1962)) with appropriate addition of immobilized 10% serum or other additives. Are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, the medium may be, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science, 122 vol., 501 (1952)], DM EM medium [Virology, 8 volumes, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium

(Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)), etc. The pH is preferably about 6 to 8. Culture is usually performed at about 30 to 40 ° C. Run for 15-60 hours, adding aeration and / or agitation as needed.

As described above, the protein of the present invention can be produced in the cells of the transformant, in the cell membrane, or outside the cells.

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 the cultured cells or cells, after the culture, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme, and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude protein extract by centrifugation or filtration is appropriately used. A protein modifier such as urea or hydrochloric guanidine in the buffers may contain a surfactant such as preparative Litton X- 1 0 0 ^TM. When the protein is secreted into the culture solution, after the culture is completed, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.

The protein contained in the culture supernatant or extract obtained in this manner can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly for molecular weight analysis. Method using difference, method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, hydrophobicity such as reversed-phase high-performance liquid chromatography, etc. A method utilizing a difference, a method utilizing an isoelectric point difference such as an isoelectric focusing method, and the like are 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 it is obtained as a salt, a method known per se or analogous thereto Depending on the free form or other Can be converted to a 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, dalicosidase and the like are used.

The presence of the thus-produced protein of the present invention can be measured by 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 a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. Is also good.

An antibody against the protein or 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 is obtained by using the protein of the present invention as an antigen. The antibody or antiserum can be produced according to a known method for producing an antibody or antiserum.

[Preparation of monoclonal antibody]

(a) Preparation of monoclonal antibody-producing cells

The protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or a diluent at a site capable of producing an antibody upon administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of warm-blooded animals to be used include monkeys, rabbits, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. Allogeneic or antibody-producing cells contained in By fusing with a myeloma cell of a heterologous animal, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.

Examples of myeloma cells include 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 PEG 1000 to PEG 6000) is used at a concentration of about 10 to 80%. Cell fusion can be carried out efficiently by adding the mixture and incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes.

Various methods can be used to screen 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 adsorbed directly or together with a carrier. An anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or protein A is added, and the monoclonal antibody bound to the solid phase is added. Hybridoma culture supernatant is added to the solid phase to which the anti-immunoglobulin antibody or protein A is adsorbed, and proteins labeled with radioactive substances or enzymes are added, and the monoclonal antibody bound to the solid phase is detected. And the like.

The selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. For example, RP containing 1-20%, preferably 10-20% fetal calf serum MI 1640 medium, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd. )) Etc. can be used. The culture temperature is usually from 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 antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (e.g., salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (e.g., DEAE) Adsorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method of collecting antibody only with antigen-bound solid phase or active adsorbent such as protein A or protein G and dissociating the bond to obtain antibody) Can do it.

(Preparation of polyclonal antibody)

The polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, an 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 method for producing a monoclonal antibody described above. The antibody can be produced by collecting an antibody-containing substance against the protein of the present invention and separating and purifying the antibody.

Regarding the complex of immunizing antigen and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio between carrier and hapten depend on the efficiency of the antibody against hapten immunized by cross-linking with a carrier. If possible, any kind may be crosslinked at any ratio.For example, serum serum albumin, thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 1 for hapten. A method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used.

In addition, various condensing agents must be used for force coupling between the hapten and carrier. However, there can be used, for example, an active ester reagent containing daltaraldehyde, carbodiimide, a maleimide active ester, a thiol group, or a dithiopyridyl group.

The condensation product is administered to a warm-blooded animal at a site where antibody production is possible or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every about 2 to 6 weeks, for a total of about 3 to 10 times. The polyclonal antibody can be preferably collected from blood, such as blood or ascites, of a warm-blooded animal immunized by the above method.

The polyclonal antibody titer in the antiserum can be measured 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-described separation and purification of the monoclonal antibody.

Polynucleotides encoding the protein or partial peptide used in the present invention (e.g., DNA (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of antisense polynucleotides) The antisense polynucleotide having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of)) or a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA of the present invention. Any antisense polynucleotide may be used as long as it has a basic nucleotide sequence or a part thereof and has an action capable 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, the entire nucleotide sequence or a part of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). A base sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology with the base sequence is exemplified. In particular, of the total nucleotide sequence of the complementary strand of the DNA of the present invention, (a) in the case of an antisense polynucleotide directed to translation inhibition, the nucleotide sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, , A base sequence near the start codon, etc.) and about 70% or more, preferably about 80% or more, more preferably about 70% or more. When the antisense polynucleotide having a homology of 90% or more, most preferably about 95% or more is (mouth) an antisense polynucleotide which directs RNA degradation by RNase H, the present invention containing an intron About 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology with the complementary strand of the entire base sequence of the DNA. Nucleotides are respectively preferred.

Specifically, a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a part thereof And preferably a nucleotide sequence complementary to the nucleotide sequence of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a part thereof. Antisense polynucleotide having a nucleotide sequence (more preferably, a nucleotide sequence complementary to the nucleotide sequence of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or a part thereof) Sense polynucleotide) and the like.

An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.

To prevent degradation by hydrolases such as nucleases, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted by a group. In addition, the sugar (deoxy lipose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-dimethylation, and the base (pyrimidine, purine) may also be chemically modified. And 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 D-encoding the determined protein. NA base sequence Can be designed and synthesized based on information. Such antisense polynucleotides can hybridize to RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the protein-related RNA of the present invention through interaction with the RNA. Thus, the expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides complementary to the selected sequence of the protein-linked RNA of the present invention, and polynucleotides capable of specifically hybridizing with the protein-related RNA of the present invention, can be used in vivo and in vitro. It is useful for regulating and controlling the expression of protein genes, and is also useful for treating or diagnosing diseases. The term "corresponding" means having homology or being complementary to a nucleotide, base sequence or a specific sequence of a nucleic acid including a gene. The "correspondence" between a nucleotide, nucleotide sequence or nucleic acid and a protein generally refers to the amino acid of the protein (as specified in the directive) 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, The terminal palindrome region or the 3, terminal hairpin loop can be selected as a preferable target region, but any region in the protein gene can be selected as the 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 be hybridized with the target region, the target nucleic acid is included in the polynucleotide of the target region. On the other hand, it can be said that it is “antisense”. Antisense polynucleotides include polydexoxyribonucleotides containing 2-deoxy D-report, polyliponucleotides containing D-lipose, and other types of N-glycosides of purine or pyrimidine bases. Or other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that the polymer is DNA or RNA) (Including nucleotides having a configuration that allows base pairing and base attachment as found in the above). It May be a double-stranded DNA, a single-stranded DNA, a double-stranded RNA, a single-stranded RNA, a DNA: RNA hybrid, and may further comprise an unmodified polynucleotide (or an unmodified oligonucleotide). ), Those with known modifications, such as those with a label, capped, methylated, and one or more natural nucleotides replaced with analogs known in the art Those having an intramolecular nucleotide modification, for example, those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, calcium phosphate, etc.), a charged bond or a sulfur-containing bond ( (Eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (eg, nucleases, nucleases' inhibitors, toxins, antibodies, signa) Peptides, poly-L-lysine, etc.) or sugars (eg, monosaccharides, etc.) with side-chain groups, inter-current compounds (eg, acridine, psoralen, etc.), chelating compounds (eg, For example, those containing metals, radioactive metals, boron, oxidizing metals, etc., those containing alkylating agents, those with modified bonds (eg, alpha-anomeric nucleic acids, etc.) There may be. Where "nucleoside",

“Nucleotides” and “nucleic acids” may include those containing not only purine and pyrimidine bases, but also other modified heterocyclic bases. Such modifications may include those containing 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, etc., or functional groups such as ethers, amines, etc. It may be converted to.

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 sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides that are resistant to degradation of oligonucleoside amides. The antisense polynucleotide of the present invention can be designed, for example, as follows. That is, an antisense polynucleotide that makes an antisense polynucleotide more stable in a cell. Increase the cell permeability of the polynucleotide, increase the affinity for the sense strand of interest, and, if so, reduce the toxicity of the antisense polynucleotide. Many such modifications have been reported, for example, in Pharm Tech Japan, vol. 8, p. 247 or p. 395, 1992, Antisense Research and Appli- cations, CRC Press, 1993.

The antisense polynucleotide of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in a special form such as ribosome or microsphere, applied by gene therapy, It can be provided in an added form. Such additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes and increase nucleic acid uptake.

(Eg, phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside linkage. Other groups include capping groups specifically located at the 3 'or 5' end of the nucleic acid that prevent degradation by nucleases such as exonucleases and RNases. . 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 measured using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the protein in vivo or in vitro translation system of the present invention. You can find out. Hereinafter, a 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 DNA) May be abbreviated as the DNA of the present invention))), the protein or partial peptide of the present invention or (Hereinafter sometimes abbreviated as the antibody of the present invention) and a DNA antisense polynucleotide of the present invention (hereinafter sometimes abbreviated as the antisense polynucleotide of the present invention). The application will be described.

The expression of the protein of the present invention is increased in the kidney tissues of Wistar Fatty rats (WF rats), Zucker Fatty rats (ZF rats) and spontaneously hypercholesterolemic rats (SHC rats). It can be used as one of the best. In other words, it is useful as a marker for early diagnosis of renal dysfunction, judgment of symptom severity, and prediction of disease progression. A compound or a salt thereof 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, for example, for the prevention and treatment of renal diseases such as diabetic nephropathy It can be used as an agent.

(1) Screening of drug candidate compounds for diseases

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), Compounds or salts thereof that regulate (preferably inhibit) the activity of the protein of the present invention include, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, Mesangium Proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic adult nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, It can be used as a preventive or therapeutic agent for acute renal failure, renal sclerosis, renal edema, renal tumor, chronic rejection after renal transplantation, etc. Preferred are prophylactic and therapeutic agents 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 provides a compound which 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. How to screen salt Provide the law.

Specifically, a comparison between (i) the inorganic phosphate uptake activity of cells containing the protein of the present invention and (ii) the inorganic phosphate uptake activity of a mixture of cells 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,

(ί ′) Inorganic phosphate transport activity in the synthetic lipid bilayer containing the protein of the present invention, and (ii ′) Inorganic phosphate transport activity of the mixture of the synthetic lipid bilayer containing the protein of the present invention and the test compound And a method of screening for a compound or its salt that regulates (promotes or inhibits) the activity of the protein of the present invention, which is characterized by comparing the above.

In the above screening method, for example, in the case of (0 and (ii), inorganic phosphate uptake activity is 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, ω is obtained by adding a labeled inorganic phosphate to cells capable of expressing the protein of the present invention; and (ii) cells 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. . Perform this reaction in an appropriate buffer, and wash off inorganic phosphate that has not been taken up by cells 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 measuring the amount of radioactivity, for example, using a radioactivity measuring device.

The labeled inorganic phosphate may be reacted with cells expressing the protein of the present invention after being mixed with the test compound, and the labeled inorganic phosphate may be added to cells expressing the protein of the present invention. After contacting, the test compound may be added. The cells capable of expressing the protein of the present invention described above include cells encoding a gene encoding the protein of the present invention in appropriate cells, for example, COS-7 cells, CH0 cells, HEK293 cells, African Xenopus oocytes, and the like. The protein of the present invention is expressed on a cell membrane by being introduced into an animal cell or the like and cultured. The method for culturing cells capable of expressing the protein of the present invention is the same as the above-described method for culturing the transformant of the present invention.

In the above screening method, for example, in the case of (ΐ ′) and (if), the inorganic phosphate transport activity can be determined by a known method, for example, Biochimica et al.

Measure and compare according to the method described in Biophysica Acta, Vol. 1236, pp. 339-344, 1995, or a method analogous thereto.

Specifically, (i ′) the case where a buffer solution containing an inorganic phosphate is brought into contact with the synthetic lipid bilayer containing the protein of the present invention; and (ii) the case where the present invention is used in the presence of a test compound. A compound that regulates (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 protein-containing synthetic lipid bilayer. Or screen the salt.

In the measurement of the inorganic phosphate transport activity, a change in the membrane current that occurs when inorganic phosphate is transported by the protein of the present invention is measured using a known method, for example, a membrane potential fixing method.

The synthetic lipid bilayer containing the protein of the present invention can be prepared by, for example, using a purified protein obtained from a membrane fraction of a cell expressing the protein of the present invention according to a known membrane protein purification method, using a phospholipid or the like. The material can be produced according to a known method, for example, a method described in "Patch Clamp Experimental Technique" (Yoshioka Shoten, published in 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 bringing into 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 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 uptake activity in the case of the above (ii) is about 20% or more, preferably 30% or more, more preferably A test compound that reduces about 50% or more 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 as compared with the case of the above (II), 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, more preferably as compared with the case of the above (Γ). Can be selected as compounds that inhibit 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 an 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 disease

(E.g., diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimally altered Nef osteomyelitis, 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 kidney transplantation It is useful as a preventive and therapeutic agent. Preferably, it is a prophylactic / therapeutic agent for glycemic nephropathy, focal glomerulosclerosis and the like.

Compounds or salts thereof obtained using the screening method or the screening kit of the present invention include, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissues It is a compound selected from extracts, plasma, etc. As the salt of the compound, those similar to the aforementioned salts of the peptide of the present invention are used. Furthermore, the gene encoding the protein of the present invention is also expressed in kidney tissues of Wistar Fatty rats (WF rats), Zucker Fatty rats (ZF rats) and spontaneously hypercholesterolemia (SHC rats). Compounds that modulate (preferably inhibit) the expression of the gene encoding the protein of the present invention because they are elevated include, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, Chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, intraluminal proliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, urine It can be used as a prophylactic / therapeutic agent for tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.).

Accordingly, the polynucleotide (eg, DNA) of the present invention can be used 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. Useful as

The screening methods include (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 a test compound. A screening method characterized by performing a comparison with the case where the method is performed.

In the above method, the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of the 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 amount of the protein is measured by a known method, for example, using an antibody recognizing the protein of the present invention, and analyzing the protein present in a cell extract or the like according to a method such as Western analysis, ELISA, or a method analogous thereto. Can be measured.

The amount of mRNA can be measured by 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 PCR. Measure according to the method Can be

For example, a test compound that 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 compared to the case of the above (iii), 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, is obtained by coding the protein of the present invention. Can be selected as a compound that suppresses the expression of a gene that causes the expression.

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 using the screening method or screening kit of the present invention include the test compounds described above, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, and plants. It is a compound selected from an extract, an animal tissue extract, and plasma, or a salt thereof. 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 ( E.g., 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, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.) It is useful as a prophylactic and therapeutic agent.

When a 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 into a preparation according to a conventional method. '

For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). ), Syrups, emulsions, suspensions, etc. I can do it. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As compositions for parenteral administration, for example, injections, suppositories, etc. are used. Injections are intravenous, subcutaneous, intradermal, intramuscular, intravenous, intraarticular. Includes dosage forms such as injections. 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. Examples of the aqueous liquid for injection include physiological saline, isotonic solution containing pudose and other adjuvants, and a suitable solubilizing agent such as alcohol (eg, ethanol), Use in combination with polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylen (50iol) adduc tof hydrogenated cas tor oil)), etc. You may. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. Suppositories used for rectal administration are prepared by mixing the above compound or a salt thereof with a usual suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in the form of a dosage unit so as to conform to the dose of the active ingredient. Examples of the dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially 5 to 100 mg for each dosage unit. Preferably, other dosage forms contain 10 to 250 mg of the above compound.

Each of the above-mentioned compositions may contain other active ingredients as long as the compound and the above-mentioned compound do not cause an undesirable interaction.

The preparations obtained in this way are safe and low toxic, for example, in humans or warm-blooded animals (eg, mice, rats, puppies, higgies, bush, puppies, puppies, To birds, cats, dogs, monkeys, chimpanzees, etc.) orally or parenterally.

The dose of the compound or a salt thereof varies depending on its action, target disease, subject of administration, route of administration, and the like. For example, the activity of the protein of the present invention is regulated for the purpose of treating diabetic nephropathy ( When a compound or a salt thereof is orally administered, the compound or a salt thereof is generally administered in an amount of about 0.1 to 100 mg, preferably about 1. 0 to 50 mg, more preferably about 1.0 to 20 mg is administered. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like.For example, the activity of the protein of the present invention is regulated for the purpose of treating diabetic nephropathy. When a compound (or a salt thereof) which is preferably (inhibited) is administered to an adult (with a body weight of 60 kg) usually in the form of an injection, the compound or a salt thereof is used in an amount of about 0.01 to 30 mg, preferably about 0.1 to 30 mg per day. Conveniently, 1-20 mg, more preferably about 0.1-10 mg, will be administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 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, so that the quantification of the protein of the present invention in a test solution can be performed. In particular, it can be used for quantification by the San Germanti 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. A method for quantifying the protein of the present invention in a test solution, and

(ii) Measuring the activity of the labeling agent on the insolubilized carrier after simultaneously or consecutively reacting the test wave with the antibody of the present invention insolubilized on the carrier and another labeled antibody of the present invention on the carrier. A method for quantifying the protein of the present invention in a test wave is provided.

In the quantification method (ii), one of the antibodies is the N-terminal of the protein of the present invention. Preferably, the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention.

In addition, 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, and may be an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any method can be used as long as the amount is determined by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, a competitive method, an immunometric method, and a sandwich method are preferably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.

Examples of the labeling agent used in the assay method using the labeling substance, for example, a radioactive isotope (e.g., ^[125 iota], ^[131 iota], [¾], TO, ™, ^[33 P], a ^[35 s] ), Fluorescent substances (eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience)), fluorescamine, fluorescein isothiocynate, etc.), enzymes (examples) , Β-lactosidase,) 3-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc., luminescent substances (eg, luminol, luminol derivatives, reluciferin, lucigenin, etc.), element of laninide 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, test solution was added to the insolubilized monoclonal antibody of the present invention. (Primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction), and then the activity of the labeling agent on the insolubilized carrier is measured. Can be quantified. 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 the same as those described above. In addition, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and two or more kinds of antibodies are used for the purpose of improving the measurement sensitivity and the like. Mixtures may be used.

In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is an antibody having a different site to which the protein of the present invention binds. It is preferably used. That is, when the antibody used in the primary reaction and the secondary reaction is, for example, 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 Is an antibody that recognizes other than the C-terminal, for example, the N-terminal.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry method.

In the competitive method, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (BZF separation) The labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble phase is used as the antibody, B / F separation is carried out using polyethylene glycol, a liquid phase method using a second antibody against the above antibody, or a solid phase antibody is used as the first antibody, or An immobilization method using a soluble antibody as the first antibody and an immobilized antibody as the second antibody is used.

In the immunometric method, the ability to separate the solid phase from the liquid phase after a competitive reaction between the antigen in the test wave and the immobilized antigen for a certain amount of the labeled antibody, or the antigen in the test solution Is allowed to react with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Then either In nephrometry, the amount of insoluble sediment 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.

'No special conditions, procedures, etc. are required to apply these individual immunological assays to the quantification method of the present invention. The system for measuring the protein of the present invention may be constructed by adding ordinary technical considerations to those skilled in the art to the ordinary conditions and procedures in each method. For details of these general technical means, reference can be made to reviews and documents.

For example, edited by Hiro Irie "Radio Nono Atsue" (Kodansha, published in 1949), edited by Hiro Irie "Radio Imno Atsue" (Kodansha, published in 1954), edited by Eiji Ishikawa et al. "Immunoassay" (Medical Shoin, published in 1975), edited by Eiji Ishikawa et al., "Enzyme Immunoassay" (2nd edition) (Medical Publishing, published in 1977), Eiji Ishikawa, edited by "Enzyme Immunoassay" Law (3rd edition) (Issue Shoin, published in 1962), "Methods in ENZYMOLOGYJ Vol. 70 (Immunochemical Techniques (Part A))", Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)) ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)), ibid. Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and

Monoclonal Ant ibodi es)) (above, published by Academic Press).

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 disease (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 Renal disease, tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc., or may be affected in the future It 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. In addition, 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 for (3) Gene diagnostics

The DNA of the present invention can be used, for example, in humans or warm-blooded animals (eg, rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, monkeys) by using them as probes. , Chimpanzees, etc.) can detect abnormalities (gene abnormalities) in DNA or mRNA encoding the protein of the present invention or a partial peptide thereof.

It is useful as a gene diagnostic agent for A damage, mutation or decreased expression, and increase or excessive expression of the DNA or mRNA.

The above-described gene 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 of America, Vol. 86, pp. 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 disease (eg, diabetic nephropathy, focal thread) Glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, Diagnosed as having a high likelihood of amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumor, chronic rejection after kidney transplantation, etc. Can be. (4) a drug containing an 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 functions of the protein of the present invention or the DNA of the present invention in vivo. 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, tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema齓 Can be used as a prophylactic or therapeutic agent for kidney tumors, chronic rejection after kidney transplantation, etc.).

When the antisense polynucleotide is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se.

Further, for example, the above-mentioned antisense polynucleotide is used alone or in a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, and thereafter, human or mammal ( For example, it can be administered orally or parenterally to rats, puppies, sheep, sheep, bush, puppies, cats, dogs, monkeys, etc.). 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 by a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered topically into the trachea as an inhalant.

Furthermore, in order to improve pharmacokinetics, prolong the half-life, and improve the efficiency of intracellular uptake, the above antisense polynucleotide is formulated alone or together with a carrier such as ribosome (injection), and is intravenously, subcutaneously, It may be administered to a lesion or the like. The dosage 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, In general, for adults (body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day. You.

Furthermore, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.

Similarly to the above-mentioned antisense polynucleotide, a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a lipozyme containing a part of the RNA encoding the protein of the present invention, etc. Since the expression of 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, for example, renal diseases (eg, Nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome group, mesangial proliferative nephritis, endoproliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescent formation Prevention and treatment of 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.) It can be used as such.

The double-stranded RNA can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001). The lipozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, p. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of RNA encoding the protein of the present invention. A part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) adjacent to the cleavage site on the RNA of the present invention which can be cleaved by a known lipozyme.

When the above-described double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.

(5) a drug containing the antibody of the present invention

The antibody of the present invention, which has the activity of neutralizing the activity of the protein of the present invention, includes, for example, For example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, microdegenerative 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 after renal transplantation Rejection) can be used as a prophylactic or therapeutic agent.

The antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.

That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules) And syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As compositions for parenteral administration, for example, injections, suppositories, etc. are used. Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, etc. Include shapes. Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. Examples of the aqueous liquid for injection include physiological saline, isotonic solution containing pudose and other adjuvants, and a suitable solubilizing agent such as alcohol (eg, ethanol) and polyalcohol ( For example, propylene glycol, polyethylene glycol), a nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) addoct of hydrogenated catalyst)], etc. may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually Filled into ampoules. A suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a usual suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in the form of a dosage unit so as to conform to the dose of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories, and usually 5 to 500 mg per dosage unit form, especially 5 to 500 mg for injections.

It is preferred that 100 mg, and other dosage forms, 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 above-mentioned 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.

It can be administered orally or parenterally (eg, intravenously) to animals (eg, rats, egrets, sheep, sheep, bush, squirrels, cats, dogs, monkeys, etc.). The dose varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for the treatment of diabetic nephropathy in adults, the antibody of the present invention is used as a single dose. Usually, 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 1 to 5 times a day It is convenient to administer as an injection about three times. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.

Further, 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 nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal It is also useful as a diagnostic agent for edema, kidney tumors, chronic rejection after kidney transplantation, etc.). (6) DNA transfer animal

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

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 a rat, and

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

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA transgenic animal of the present invention) include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. In contrast, preferably, during the stage of embryonic development in the development of a non-human mammal (more preferably, at the stage of a single cell or a fertilized egg and generally before the 8-cell stage), a calcium phosphate method, an electric pulse method, a ribofection method, It can be produced by transferring the desired DNA by an agglutination method, microinjection method, particle gun method, DEAE-dextran method, or the like. Further, by the DNA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, 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 cells with the above-mentioned germ cells by a cell fusion method known per se.

As non-human mammals, for example, porcupines, pigs, higgins, goats, magpies, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Above all, rodents with relatively short ontogeny and biological cycles in terms of the creation of disease animal model systems, and easy to breed rodents, especially mice (for example, pure strains such as C57BL / 6 strain, DBA2 strain, etc.) As the crossing strain, B6C3F, strain, BDF [strain, B6D2F, strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD, etc.) is preferable. "Mammals" in a recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by non-human mammals but to the DNA of the present invention once isolated and extracted from the mammal. Examples of the mutant DNA of the present invention include those in which a mutation (for example, mutation) has occurred in the base sequence of the original DNA of the present invention, specifically, addition or deletion of a base, substitution with another base, or the like. The DNA in which the occurrence has occurred is used, and the abnormal DNA is also included. The abnormal DNA refers to 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 downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is transferred, various mammals having the DNA of the present invention having a high homology with the human (for example, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.) Microinjection of a DNA construct (e.g., vector, etc.) to which a human DNA of the present invention is bound downstream of one of various promoters capable of expressing DNA of origin from a fertilized egg of a target mammal, for example, a mouse fertilized egg By doing so, a DNA-transferred mammal that highly expresses the DNA of the present invention can be produced.

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 Moroni leukemia virus, and vaccinia viruses. Alternatively, animal viruses such as baculovirus are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.

Examples of promoters that regulate the above DNA expression include: (i) virus (eg, simian virus, cytomegalovirus, Moroni leukemia virus) Promoters of DNA derived from humans, JC virus, breast cancer virus, poliovirus, etc.), and (ii) Promoters derived from various mammals (human, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.) 1.For example, albumin, insulin II, perobrakin II, elas ubiquitin, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, gliubithion S-transferase, platelet-derived growth factor / 3, Keratin Kl, K10 and Κ14, collagen type I and type II, cyclic AMP-dependent protein kinase i3 I subunit, dystrophin, tartrate-resistant lipophilic phosphatase, atrial sodium diuretic factor, endothelial receptor ostium synkinase ( (Generally abbreviated as Tie 2) Pharmacium adenosine 3-phosphate oxidase (Na, K-ATPase), neurofilament light chain, metallotionin I and IIA, meloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), 1 -1—ras, renin, dopamine jS—hydroxyl, thyroid peroxidase (TPO), peptide chain elongation factor la (EF-1), β-actin, «and | S myosin heavy chain, myosin light chain 1 and 2, myelin base protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin II, smooth muscle actin-. Preproenkephalin A And vasopressin and the like. Among them, a cytomegalovirus promoter that can be highly expressed in the whole body, a promoter of a human peptide chain elongation factor 1a (EF-1), a human and a chicken / 3-actin promoter are preferable. is there.

The vector preferably has a sequence that terminates transcription of a messenger RNA of interest in a DNA-transferred mammal (generally called terminator). For example, each vector may be derived from a virus or a mammal. A DNA sequence can be used, and preferably, SV40 terminator of simian virus or the like is used.

In addition, splicing signals of each DNA, enhancer region, and part of eukaryotic DNA introns for the purpose of further expressing the desired foreign DNA. It is also possible to connect a promoter 5 ′ upstream of the promoter region, between the promoter region and the translation region, or 3 ′ downstream of the translation region.

The normal translation region of the protein of the present invention is human or various mammals (eg, egrets, dogs, cats, guinea pigs, eight musters, rats, mice, etc.). Liver, kidney, thyroid cells, fibroblasts All or part of genomic DNA from cell-derived DNA and various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from dried, kidney, thyroid, or fibroblast-derived RNA. It can be obtained as a raw material. In addition, a foreign abnormal DNA can produce a translation region obtained by mutating the translation region of a normal protein obtained from the above-described cells or tissues by a point mutagenesis method.

The translation region can be prepared as a DNA construct that can be expressed in a 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 germ 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 germ cells and somatic cells Means that. The progeny of such animals inheriting the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.

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

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 the germinal cells of the animal after transfer of the DNA indicates that the offspring of the animal in which the animal is produced excessively contains the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to have. The offspring of an animal of this species that has inherited the exogenous DNA of the present invention, In excess.

By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes, and mating the male and female animals, it is possible to breed so that all offspring 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 function of endogenous normal DNA to thereby finally produce the protein of the present invention. May develop hyperfunction, and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the 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.

In addition, since the mammal to 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 associated with the protein of the present invention, such as kidney Diseases (e.g., diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimally altered nephritis 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, after kidney transplantation It can also be used for screening tests for prophylactic and therapeutic agents such as chronic rejection.

On the other hand, the non-human mammal having the foreign abnormal DNA of the present invention should be subcultured in a normal breeding environment as an animal having the DNA after confirming that the foreign DNA is stably retained by the crossing. Can be done. Further, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a source substance. The DNA construct with the promoter can be prepared by ordinary DNA engineering techniques. 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 a produced animal after DNA transfer means that the offspring of the produced animal have the abnormal DNA of the present invention in all of its germinal and somatic cells. The offspring of this type of animal that inherited the exogenous DNA of the present invention All germ cells and somatic cells have the abnormal DNA of the present invention. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes, and crossing the male and female animals, it is possible to breed subculture so that all offspring have the DNA.

In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately reduced by inhibiting the function of endogenous normal DNA. Inactive refractory disease may occur, and it can be used as a disease model animal. For example, it is possible to elucidate the pathological mechanism of the functionally inactive refractory state of the protein of the present invention and to examine a method for treating this disease using the abnormal DNA transgenic animal of the present invention.

In addition, as a specific possibility, the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of a normal protein by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention (dominant negative activity). Action).

In addition, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, the preventive / therapeutic agent for the protein or functionally inactive type refractory of the present invention is used. , 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 adult nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, renal tumor, after kidney transplantation It can also be used for screening tests for preventive and therapeutic agents such as chronic rejection.

In addition, other possible uses of the above two types of DNA transgenic animals of the present invention include, for example,

(i) use as a cell source for tissue culture,

(ii) Direct analysis of DNA or RNA in the tissue of the DNA-transferred animal of the present invention, or analysis of peptide tissue expressed by the DNA to specifically express or express the protein of the present invention. Analysis of the relationship with the activating peptide, (iii) culturing cells of a tissue having DNA by standard tissue culture techniques, and using these to study the function of cells from tissues that are generally difficult to culture,

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

(V) Isolation and purification of the mutant protein of the present invention and production of its antibody may be considered.

Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine the clinical symptoms of diseases related to the protein of the present invention, including the inactive type refractory type of the protein of the present invention, and the like. It is possible to obtain more detailed pathological findings in each organ of the disease model related to the protein of the present invention, to develop new treatment methods, and to contribute to the research and treatment of secondary diseases caused by the disease. it can.

In addition, it is possible to take out each organ from the DNA-transferred animal of the present invention, shred it, and then use a protease such as trypsin to obtain free DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to identify the protein-producing cells of the present invention, examine their relationship with apoptosis, differentiation or proliferation, or investigate their signal transduction mechanisms, and investigate their abnormalities. It is an effective research material for elucidating its action.

Further, in order to use the DNA-transferred animal of the present invention to develop a therapeutic agent for a disease associated with the protein of the present invention, including the inactive type refractory type of the protein of the present invention, Using a test method and a quantitative method, it is possible to provide an effective and rapid screening method for a therapeutic agent for the disease. Furthermore, using the DNA transfer product of the present invention or the exogenous DNA expression vector of the present invention, it is possible to study and develop a method for treating a DNA associated disease of the present invention for DNA.

(7) Knockout animal

The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been 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, an ιδ-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 DN の of the present invention, wherein the DNΑ is inactivated;

(7) The DNA is inactivated by introducing a repo overnight gene (eg, an 8-galactosidase gene derived from Escherichia coli), and the repo overnight gene is under the control of a promoter for the DNA of the present invention. A non-human mammal according to (6), which is capable of expressing;

(8) the non-human mammal according to (6), wherein the non-human mammal is a rodent;

(9) the non-human mammal according to (8), wherein the rodent is a mouse; and

(10) A compound that promotes or inhibits the activity of the promoter of DNA of the present invention, which comprises administering a test compound to the animal described in (7) and detecting the expression of a repo overnight gene; A method for screening the salt is provided. A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated refers to a DNA of the present invention possessed by the non-human mammal, which is artificially mutated to suppress the expression ability of the DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA substantially does not have the ability to express the protein of the present invention (hereinafter, referred to as the knockout DNA of the present invention). Refers to 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 performed, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. By these mutations, for example, the knockout DNA of the present invention may be prepared by shifting the reading frame of codons or disrupting the function of the promoter or exon.

Non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter referred to as the present invention) Specific examples of the DNA-inactivated ES cells or the knockout ES cells of the present invention) include, for example, isolating the DNA of the present invention from a non-human mammal of interest, and adding a neomycin resistance gene to its exon portion. A drug resistance gene typified by the idalomycin resistance gene, or a repo allele gene typified by lacZ (3-galactosidase gene) and cat (chloramphenicol acetyltransferase gene) Or the insertion of a DNA sequence that terminates gene transcription (for example, a po1yA addition signal) into the intron between the exons, resulting in a complete messenger RNA. A DNA strand having a DNA sequence constructed so as to result in the disruption of the gene by preventing the synthesis of ) Is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells are subjected to Southern hybridization using the DNA sequence on or near the DNA of the present invention as a probe. The knockout ES cells of the present invention are selected by analyzing the DNA sequence on the targeting vector and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector by using the primers as primers. Can be obtained by 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 the method described in the known Evans and Kaufma method may be used. A newly established one may be used. For example, in the case of mouse ES cells, generally, 129 ES cells are currently used, but since the immunological background is not clear, it is a pure line that substitutes the immunological genetic background. For example, for the purpose of obtaining ES cells for which C57BLZ6 and C57BLZ6 mice have a low number of eggs, the BDFt mice (C57BLZ6 and DBAZ2 BDFi mice can be used satisfactorily because they have the advantage of high number of eggs collected and robust eggs, and have C57BLZ6 mice as a background. ES cells can be advantageously used when a pathological model mouse is created, because the genetic background can be replaced by C57BLZ6 mice by backcrossing with C57BLZ6 mice. In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used, but efficiency is also improved by collecting embryos at the 8-cell stage and culturing them up to blastocysts. Many early embryos can be obtained well.

Either male or female ES cells may be used, but male ES cells are generally more convenient for producing a breeding line chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.

An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. Using this method, conventionally, for example G-banding method, it requires about 1 0 ⁶ cells for karyotype analysis, the need in ES cell number of about 1 colony (about 5 0) Therefore, the primary selection of ES cells in the early stage of culture 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 preferably 100% of the normal number, but if it is difficult due to physical operations at the time of establishment, knock out the ES cell gene and On the other hand, it is desirable to clone again into cells in which the number of chromosomes is 2 n = 40 in mice).

Embryonic stem cell lines obtained in this way usually have very good proliferative potential, but must be carefully subcultured because they tend to lose their ontogenetic potential. For example, on a suitable feeder cell, such as STO fibroblasts, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen) in the presence of LIF (1-10000 U / ml) , 5% CO 2, 90% air) at about 37. At the time of subculture, for example, trypsin ZEDTA solution (usually 0.001 to 0.5% trypsin ZO. L) To 5 mM EDTA (preferably, about 0.1% trypsin / ImM EDTA), and seeding the cells on freshly prepared feeder cells. Such subculture is usually performed every 11 to 13 days. At this time, it is desirable to observe the cells and discard the cultured cells if morphologically abnormal cells are found. ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature, Vol. 292, pp. 154, 1981; GR Martin Proceedings of the National Academy of Sciences' Ob Science Sciences Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embrio Morphology, Vol. 87, p. 27. 1985]. The DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in the cell biology of the protein of the present invention in the mouth of in vivo.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA amount of the animal using a known method and indirectly comparing the expression levels.

As the non-human mammal, the same one as described above is used.

The non-human mammal deficient in expression of the DNA of the present invention may be obtained, for example, by introducing the evening-getting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell, and introducing the evening-getting vector into the DNA of the present invention. Knockout of the DNA of the present invention by homologous recombination in which the DNA sequence in which the DNA has been inactivated replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination. Can be.

The cells in which the DNA of the present invention has been knocked out are subjected to Southern hybridization analysis using the DNA sequence on or near the DNA of the present invention as a probe or the DNA sequence on the overnight getter vector, and the target DNA. The DNA can be determined by a PCR method using the DNA sequence of a neighboring region other than the DNA of the present invention derived from the mouse used as the primer vector as a primer. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is used at an appropriate time, for example, at the 8-cell stage. The chimeric embryo is injected into a non-human mammal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal. The produced animal also has a normal DNA locus of the present invention. This is a chimera animal composed of both an artificial cell and an artificially mutated cell having the 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 obtained from the population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the DNA locus of the present invention to which a mutation has been added, for example, by judging coat color. The individual obtained in this manner is usually an individual having a heterozygous expression of the protein of the present invention, which is crossed with an individual having a heterozygous expression of the protein of the present invention. A homozygous deficient individual can be obtained.

When using an egg cell, for example, a transgenic non-human mammal having a chromosome into which the evening-getting vector has been introduced can be obtained by injecting a DNA solution into the nucleus of the egg cell by a microinjection method. Compared to transgenic non-human mammals, the homologous recombination of the D

Obtained by selecting those with mutations at the NA locus.

When the DNA of the present invention is knocked out in this manner, the animal obtained by the crossing should be confirmed to be knocked out of the DNA and subjected to rearing in a normal rearing environment. Can be.

Furthermore, the germline can be obtained and maintained according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and a plurality of homozygous animals are obtained. By mating male and female heterozygous goats, a homozygote having the inactivated DNA is obtained.

-Breeding and breeding wild and heterozygous animals.

The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are the DN of the present invention.

It is very useful in creating non-human mammals deficient in A expression.

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, the non-human mammal may be inactivated by the inactivation of the biological activity of the protein of the present invention. Can be a model of the disease It is useful for investigation and treatment.

(7a) A method for screening 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 can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention.

That is, the present invention is characterized in that a test compound is administered to a non-human mammal deficient in expression of the DNA of the present invention, and changes in the animal are observed and measured. Diseases caused by, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimally altered nephrotic syndrome, mesangium, proliferative nephritis, endoproliferative nephritis, IgA nephropathy , Membranous nephropathy, membranous proliferative nephritis, crescentic adult nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, kidney To provide a method for screening a compound or a salt thereof having a therapeutic / preventive effect on tumors, chronic rejection after kidney transplantation, etc.).

Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.

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

Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound and compared with a non-treated control animal to examine changes in organs, tissues, disease symptoms and the like of the animal. The therapeutic / preventive effect of the test compound can be tested as an index.

As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection, etc. are used, and the method 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 the DNA of the present invention, and the test compound non-administration group and the renal function Observe, over time, differences in urine protein excretion and glomerular filtration rate, which are indicators, or glomerular sclerosis rate, which is an indicator of renal dysfunction.

In the screening method, when the test compound is administered to a test animal, 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. 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 against a disease caused by deficiency or damage of the protein of the present invention. It can be used as a medicament such as a safe and low toxic prophylactic / therapeutic agent for the disease. Further, a compound derived from the compound obtained by the above screening can also be used.

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals). And the like, and especially preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, etc.). Acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).

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

The preparations obtained in this way are safe and have low toxicity, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, butter, horseshoe, horses, cats, cats). And monkeys). The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, generally the diabetic kidney of an adult (with a body weight of 60 kg) is used. For patients with sickness, 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, target disease and the like. For example, the compound is usually in the form of an injection and is usually used for adult (as 60 kg) sugar urine. When administered to patients with pathologic nephropathy, about 0.01 to 30 mg, preferably about 0.1 to 2 Omg, and more preferably about 0.1 to 1 Omg of the compound is administered intravenously per day. It is conveniently administered by In the case of other animals, it is also possible to administer the amount converted per 60 kg.

(7b) A method for screening a conjugate which promotes or inhibits the activity of a promoter for DNA of the present invention

The present invention relates to the activity of a promoter for the DNA of the present invention, which 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 repo allele. To provide a method for screening a compound or a salt thereof that promotes or inhibits cell growth.

In the above screening method, the non-human mammal deficient in expression of the DNA of the present invention includes, among the aforementioned non-human mammals deficient in the expression of the DNA of the present invention, the DNA of the present invention obtained by introducing a repo overnight gene. Those inactivated and capable of expressing the repo overnight gene under the control of the promoter for the DNA of the present invention are 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 (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is preferable.

In a non-human mammal deficient in DNA expression of the present invention in which the DNA of the present invention has been replaced with a reporter overnight gene, the reporter gene is under the control of the promoter of the DNA of the present invention. By tracing the expression of the coding substance, 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 by the iS_galactosidase gene (1 ac Z) derived from Escherichia coli, a tissue that originally expresses the protein of the present invention may 3-galactosidase is expressed instead of this protein. Therefore, for example, by staining with a reagent that serves as a substrate for i3-galactosidase, such as 5-bromo-4-chloro-3-indolyl-j8-galactopyranoside (X-gal), it is convenient In addition, the expression state of the protein of the present invention in an animal body can be observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with dartalaldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at room temperature. After reacting at about 7 ° C for about 30 minutes to 1 hour, the / 3_-galactosidase reaction can be stopped by washing the tissue specimen with 1 mM ED TAZ PBS solution, and the coloration can be observed. . In addition, 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 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 physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, Salts such as maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. 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. Kidney disease (e.g., 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 kidney transplantation, etc.) Useful as a prophylactic 'therapeutic.

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

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

The preparations obtained in this way are safe and low toxic, for example, in humans or mammals (e.g., rats, mice, guinea pigs, egrets, sheep, sheep, horses, horses, cats, cats, cats). And monkeys and monkeys). The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the route of administration, and the like.For example, when the compound of the present invention that inhibits the promoter activity against DNA is orally administered, it is generally used. In an adult (assuming a body weight of 60 kg) diabetic nephropathy, about 0.1 to 100 mg, preferably about 1.0 to 50 nig, 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 against DNA is usually in the form of an injection. When administered to an adult (as 60 kg) diabetic nephropathy patient, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Onig of the compound per day is administered intravenously. It is convenient to administer by injection. In the case of other animals, the dose can be administered in terms of 60 kg.

As described above, the non-human mammal deficient in DNA expression of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter of DNA of the present invention, The present invention can greatly contribute to the investigation of the cause of various diseases caused by DNA expression deficiency or the development of preventive and therapeutic agents. In addition, using a DNA containing a promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) For example, it would be possible to specifically synthesize the protein and examine its action in living organisms. Furthermore, by binding an appropriate reporter gene to the above promoter portion and establishing a cell line capable of expressing the same, a low molecule having an action of specifically promoting or suppressing the production ability of the protein of the present invention itself in the body. It can be used as a search system for compounds. In the present specification, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the relevant field, and examples thereof are described below. When amino acids can have optical isomers, the L-form is indicated unless otherwise specified.

DNA: Deoxylipo nucleic acid

c DNA: Complementary deoxylipo nucleic acid

A: Adenine

T: Thymine

G: Guanin

C: Shitoshin

RNA: ribonucleic acid

mRNA: messenger ribonucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

d CTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

EDTA: Ethylenediaminetetraacetic acid

SDS: Sodium dodecyl sulfate

G 1 y: glycine

A 1 a: Alanine

V a 1: Palin

Le u: Leucine I 1 e: Isoleucine

S e r: Serine

T r: Threonine

Cy s: Cystine

Me t: Methionin

G 1 u: Glutamic acid

As: Aspartic acid

L y s: lysine

A r g: Arginine

H i s: Histidine

Ph e: Phenylalanine

Ty r: Tyrosine

T r p: Tributofuan

Pro: Proline

A s n: Asparagine

G 1 n: Guru Yu Min

P G 1 u: Pyroglutamic acid

S e c: selenocysteine Also, a substituent frequently used in this specification f

Write.

Me methyl group

Etethyl group

Bu butyl group

Ph phenyl group

TC thiazolidine-1 (R) One-pot lipoxamide group To s p-toluenesulfonyl

CHO Holmill

B z 1

Cl ₂ -Bzl 2, 6-dichlorobenzyl Bom: benzyloxymethyl

Z: benzyloxycarponyl

C 1 z: 2-cyclobenzoyloxycarbonyl

B r -Z: 2-bromobenzyloxycarbonyl

B o c: t-butoxycarponyl

DNP: dinitrophenyl

Trt: Trityl

Bum: t-butoxymethyl

Fmo c: N—9_fluorenylmethoxycarbonyl

HOB t: 1-hydroxybenztriazole

HOOB t: 3,4-dihydro-3-hydroxy-1-oxo

1-2, 3 Benzotriazine

HONB: Tohydroxy-5-norporene-2,3-dicarboximide DC C The sequence numbers in the sequence listing in the present specification show the following sequences.

[SEQ ID NO: 1]

The amino acid rooster sequence (GenBank Accession No. AAC98695) of human Type lib Na / Pi Cotransporter is shown.

[SEQ ID NO: 2]

Nucleotide sequence of human Type lib Na / Pi Cotransporter (GenBank Accession No.

AF111856).

[SEQ ID NO: 3]

2 shows the amino acid sequence (GenBank Accession No. AAF76291) of rat Type lib Na / Pi Cotransporter.

[SEQ ID NO: 4]

The nucleotide sequence of rat Type lib Na / Pi Cotransporter (GenBank Accession No. AF157026) is shown.

[SEQ ID NO: 5]- The amino acid sequence (GenBank Accession No. AAC80007) of mouse Type lib Na / Pi Cotransporter is shown.

[SEQ ID NO: 6]

The nucleotide sequence of mouse Type lib Na / Pi Cotransporter (GenBank Accession No. AF081499) is shown.

[SEQ ID NO: 7]

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

[SEQ ID NO: 8]

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

[SEQ ID NO: 9]

3 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]

3 shows the nucleotide sequence of a probe used in Example 3. 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 performed to discover drug targets from the channels and transport pathways that fluctuate in the kidney tissue of rat kidney model.

The disease model animals are Wistar Fatty rat (WF Rad), Zucker Fatty rat (ZF rat), and spontaneously hypercholesterolemic rat (SHC rat). 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 are bred under the same conditions. Rats and WL rats are 13 weeks, 20 weeks, 40 weeks and 68 weeks old. At week 5, kidneys were collected from a minimum of 5 individuals in each group at 8 and 27 weeks of age for ZF and ZL rats and at 6, 12, 20, and 30 weeks of age for SHC and SD rats. Total RNA was prepared for each individual according to the method described in the instruction manual of IS0GEN (Nitsubon Gene). Among the prepared samples, in WF rats and WL rats, samples from the WF rats at the age of 40 weeks, at which renal damage was progressing in the WF rats, and 68 weeks after birth, at which kidney function was further advanced and renal function was reduced, were compared with the ZF rat. For ZL rats and ZL rats, a 27-week-old sample with renal impairment in ZF rats was developed, and for SHC and SD rats, a 30-week-old sample with decreased renal function in SHC rats was used for the following comprehensive genes: It was used for expression analysis experiments. Table 1 shows the experimental configuration.

〔table 1〕

In this experiment, a total of 40 g of total RNA obtained by mixing equal amounts of kidney total RNA for each individual in each group was used as one sample (for example, in the case of a WF rat 40 weeks old, the number of individuals in this group is 5 Therefore, gene expression analysis was performed using an Oligonucleotide microarray (Rat Genome U34A, U34B, U34C; Af fymetrix) in which 8 ng per individual was mixed to give a total amount of 40 nig. The experimental method followed Affymetrix's experiment guide (Expression analysis technical manual). Obtained in the experiment

Expression analysis software

(GeneSpring: Silicon Genetics) to extract sequences showing expression fluctuations.

As a result, the expression of rat Type lib Na / Pi Cotransporter (Genbank Accession No. AF157026) was enhanced in all three models. Especially in the SHC rats, the rat Type lib Na / Pi Cotransporter fluctuated significantly. Example 2

In order to refine the analysis results using the Oligonucleotide microarray described in Example 1, rat type lib Na / Pi Cotransporter was used by TaqMan PCR System.

(Genbank Accession No. AF157026) in each individual kidney was measured. Using the total kidney RNA of each individual used in Example 1 as type II RM, 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 RNA for each individual prepared in Example 1 as a type III using a TaqMan Gold RT-PCR Kit (Applied Biosystems) to prepare cDNA. The composition of the reaction solution, Individual kidney Total RNA lng prepared in Example _{1, 5.5mM MgCl 2, 0.5mM dNTPs} , 2.5 zM Ramdoni Hexamers, 0.4U / 1 RNase Inhibitor,

1.25 U / / 1 MultiScribe Reverse Transcriptase and reverse transcription application buffer supplied with the kit were used to make the total volume 50 xl. The reaction was performed using a thermal cycler (PE

Using Biosystems), the temperature was kept at 25 ° C for 10 minutes, at 48 ° C for 30 minutes, and at 95 ° C for 5 minutes. Next, the cDNA prepared by the above method was used as type III, and the expression level of the rat Type lib Na / Pi Cotransporter gene was measured by TaqMan PCR. The composition of the reaction solution was cDNA 5 Ι (corresponding to 2.4 ng of Total RNA) prepared by the above method, synthetic DNA primer — (SEQ ID NO: 7) 0.2 M, synthetic DNA primer (SEQ ID NO: 4), synthetic DNA probe (SEQ ID NO: 4). : 5) 0.2 M and TaqMan Universal PCR Master Mix

(Applied Biosystems) and 7.5 1 were added to make the total volume 151. 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 ° 15 seconds and 60 ° C for 1 minute 40 times. I returned. In order to compensate for the amount of type II cDNA added between samples, the expression value of dariceraldehyde triphosphate dehydrogenase (GAPDH) was measured for each sample, and rat Type lib Na / Pi Cotransporter was used. The gene expression level was calculated as the relative expression level to the rat GAPDH expression level. The relative expression level is calculated as the number of copies of the Type lib Na / Pi Cotransporter gene per unit volume. The value was divided by one copy of the GAPDH gene and multiplied by 100. TaqMan Rodent GAPDH Control Reagents VIC Probe

(Applied Biosystems) was used. Also, rat Type lib Na / Pi

For both the 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, it was found that the expression level of the rat Type lib Na / Pi Cotransporter Was confirmed to be higher than that in the control group, confirming the results of Example 1.

TCV116 (generic name: Candesartan cilexetil) (Kidney international 49, SI 15-SI 18, 1996, CI inical Experimental Nephrology 4, 207-214, So-called 0 year, Japan Journal of Pharmacology, 88, 300-306, 2002) was administered daily at 10 mg / kg body weight daily for 18 weeks to 9 weeks after birth. The non-administration group received only the same amount of vehicle (0.5% methylcellulose lOOcP solution) as the administration group. Kidneys were collected at 27 weeks of age from the three groups of ZF rats, vehicle-administered ZF rats, vehicle-administered ZF rats, and vehicle-administered ZL rats. Total RNA was prepared for each individual, and the relative expression was analyzed in the same manner as above.

The results are shown in FIG.

From the results in Fig. 4, when TCV116 was administered to ZF rats before the onset of renal injury, the expression level of the rat Type lib Na / Pi Cotransporter gene was similar to that of the control ZL rats, indicating that TCV116 was not present. It can be seen that the expression enhancement as observed in the administered ZF rats does not occur. On the other hand, in ZF rats to which TCV116 was administered, the urinary protein level was as normal as that of ZL rats, and renal function decline was suppressed. As described above, increased expression of the rat Type lib Na / Pi Cotransporter gene is observed in association with decreased renal function, and therefore, increased expression of the rat Type lib Na / Pi Cotransporter gene is associated with decreased renal function. it is conceivable that. Example 3

For the purpose of examining the expression of human Type Lib Na / Pi Cotransporter at the cell level, expression analysis was performed on various normal human cultured cells. Normal human cells were purchased from Cambrex Bioscience Walkersville 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.

(Table 2)

No. Cell name Medium

1 Umbilical vein vascular endothelial cell CC-25Π bullet kit EGM CC-3124

2 Aortic vascular endothelial cells CC-2535 Bullet kit EGM-2 CC-3162

3 Coronary vascular endothelial cells CC-2585 Bullet kit EGM-2MV CC-3202

4 Skin microvascular endothelial cells CC-2543 Pret kit EGM-2MV CC-3202

5 Lung microvascular endothelial cells CC-2527 Bullet kit EGM-2MV CC-3202

6 Pulmonary artery endothelial cells CC-2530 Bullet kit EGM CC-3124

7 Aortic smooth muscle cells CC-2571 Pret kit SmGM-2 CC-3182

8 Coronary artery smooth muscle cells CC-2583 Bullet kit SmGM-2 CC-3182

9 Uterine smooth muscle cells CC-2562 Bullet kit SmGM-2 CC-3182

10 Bronchial smooth muscle cells CC-2576 Bullet Kit SmGM-2 CC-3182

11 Skeletal muscle satellite cells CC-2561 Bullet kit SkGM CC-3160

12 Breast epithelial cells CC-2551 Bullet kit MEGM CC-3150

13 Bronchial epithelial cells CC-2541 Bullet kit SAGM CC-3118

14 Lung bronchiolar epithelial cells CC-2547 Pret kit SAGM CC-3118

15 Lung fibroblasts CC-2512 Bullet kit FGM-2 CC-3132

16 Kidney proximal tubular epithelial cells CC-2553 Bullet kit REGM CC-3190

17 Mesangium cells CC-2559 Bullet kit MsGM CC-3146

18 Kidney cortical epithelial cells CC-2554 Bullet kit REGM CC-3190

19 Renal epithelial cell CC-2556 Bullet kit REGM CC-3190

20 Mesenchymal stem cells PT-2501 Bullet kit MSCGM PT-3001

21 Knee chondrocytes of the knee joint CC-2550 Bullet kit CGM CC-3216

22 Osteoblast CC-2538 Preset kit 0GM CC-3207

23 Prostate stromal cells CC-2508 Bullet kit SCGM CC-3205

24 Epidermal keratinocytes CC-2501 Bullet kit KGM-2 CC-3107 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. _{6 From the} recovered cells, IS0GEN (manufactured by Futtsubon Gene) or RNeasy Mini. Kit (Qiagen) Was used to prepare total RNA. The prepared total RNA was subjected to a reverse transcription reaction using TaqMan Revease Transcription Reagents (manufactured by Applied Biosystems) to prepare cDNA. Next, the expression level of the human Type lib Na / Pi Cotransporter gene was measured by TaqMan PCR using the cDNA prepared by the above method as type III. The composition of the reaction solution was as follows: various normal human cell-derived cDNA solutions 21 (equivalent to 4 ng of total RNA), synthetic DNA primers (SEQ ID NO: 10) 0.4 M, synthetic DNA primers (SEQ ID NO: 11) 0.4 · 0.2 l of the synthetic DNA probe (SEQ ID NO: 12) and 51 of TaqMan Universal PCR Master Mix (Applied Biosystems) were added to make the total volume 10 1. 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. Repeated times. To compensate for the amount of type II cDNA added between samples, the expression value of dariceraldehyde triphosphate dehydrogenase (GAPDH) was measured for each sample, and the expression level of the human Type lib Na / Pi Cotransporter gene was measured. It was calculated as the relative expression level to the human GAPDH expression level. The relative expression level was expressed as 100 times the value obtained by dividing the number of copies of the human Type lib Na / Pi Cotransporter gene per unit liquid volume by the number of copies of the human GAPDH gene per unit liquid volume. TaqMan GAPDH Control Reagents (Applied Biosystems) was used to measure the expression value of human GAPDH. In addition, for both the human Type lib Na / Pi Cotransporter gene and the human GAPDH gene, measurement was performed twice for each sample, and the average value was used as the expression value.

Fig. 5 shows the results.

Among the 24 types of normal human cells, kidney proximal tubule cells and human type lib

Relatively high expression of the Na / Pi Cotransporter gene was observed (about 2% of GAPDH). This suggests that the human Type lib Na / Pi Cotransporter gene functions in the kidney. Industrial applicability

The proteins and polynucleotides of the present invention include, for example, renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome group, mesangial proliferative nephritis, endoproliferative nephritis) , IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, Rubus nephritis, amyloid kidney, tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis And a renal edema, a renal tumor, a chronic rejection after kidney transplantation, etc.), and a modulator obtained by screening using the protein, the polynucleotide, or an antibody against the protein. Preferably an inhibitor), an antibody against the protein, an antisense polynucleotide of the present invention, and the like, are less toxic, for example, a renal disease (eg, diabetic nephropathy, Glomerulosclerosis, chronic glomerulonephritis, minimal change nephrotic syndrome, mesangial proliferative nephritis, intraluminal proliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescent-forming nephritis , Lupus nephritis, amyloid kidney, tubulointerstitial nephritis, acute tubular necrosis, acute renal failure, renal sclerosis, renal edema, kidney tumor, chronic rejection after kidney transplantation, etc.) Can be used. Furthermore, the polynucleotide of the present invention, the antisense polynucleotide of the present invention, and the like can be used, for example, in renal diseases (eg, diabetic nephropathy, focal glomerulosclerosis, chronic glomerulonephritis, microdegenerative nephrotic syndrome, mesangium) Proliferative nephritis, intraluminal proliferative nephritis, IgA nephropathy, membranous nephropathy, membranous proliferative nephritis, crescentic nephritis, lupus nephritis, amyloid kidney, tubular interstitial nephritis, acute tubular necrosis, acute kidney It is useful for diagnosis, prevention or treatment of insufficiency, renal sclerosis, renal edema, renal tumors, chronic rejection after renal transplantation, etc.).

Claims

The scope of the claims

1. Kidney comprising a compound or a salt thereof that inhibits the activity of a protein having the same or substantially the same amino acid sequence as SEQ ID NO: 1 or a partial peptide thereof or a salt thereof Disease prevention and treatment.

2. Contains a compound or a salt thereof that inhibits the expression of a protein or a partial peptide thereof or a salt thereof that has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Prevention and treatment of renal disease.

3. The preventive / therapeutic agent according to claim 1, wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis.

4. Complementary or substantially complementary to the base sequence of a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof Antisense polynucleotide containing a unique base sequence or a part thereof.

5. A medicament comprising the antisense polynucleotide according to claim 4.

6. The medicament according to claim 5, which is an agent for preventing or treating renal disease.

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 drug comprising the antibody according to claim 7.

9. The medicament according to claim 8, which is an agent for preventing and treating renal disease.

10. A diagnostic agent comprising the antibody according to claim 7.

11. The diagnostic agent according to claim 10, which is a diagnostic agent for kidney disease.

12. 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.

1 3. 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 Prevention of renal disease characterized by using a method for screening a therapeutic agent.

14. A prophylactic or therapeutic agent for renal disease characterized by 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. Screening kit.

15 5. Prevention of renal disease characterized by using a polynucleotide encoding a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof · Screening method for therapeutic agents.

16. Prevention of renal disease characterized by containing a polynucleotide encoding a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof · Kit for screening therapeutic agents.

17. The screening method according to claim 13 or claim 15, wherein the renal disease is diabetic nephropathy or focal glomerulosclerosis.

18. A compound that inhibits the activity of a protein, a partial peptide thereof, or a salt thereof having the same or substantially the same amino acid sequence as that shown in SEQ ID NO: 1, in mammals Or a method for preventing or treating renal disease, which comprises administering an effective amount of a compound or a salt thereof, or a compound or a salt thereof, which inhibits the expression of a gene of the protein or its partial peptide or a salt thereof.

1 9. Inhibits the activity of a protein or a partial peptide or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, or the protein or a partial peptide thereof Or a method for preventing or treating kidney disease, which comprises inhibiting the expression of a salt gene.

20. A protein or a partial peptide or a partial peptide thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 for producing an agent for preventing or treating renal disease Use of a compound that inhibits the activity of a salt or a salt thereof, or a compound or a salt thereof that inhibits the expression of a gene of the protein or its partial peptide or a salt thereof.