JP2004121246A - Preventives/remedies for neurodegenerative diseases - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe and excellent prophylactic or therapeutic agent for neurodegenerative diseases, diabetes or the like. <P>SOLUTION: The protein which is a compound or its salt inhibiting activity of Neuronal cell death inducible putative kinase, a compound inhibiting expression of the gene of the protein, the antisense polynucleotide, the antibody, etc., can be used as a prophylactic or therapeutic agent for neurodegenerative diseases, diabetes, etc., and a compound or its salt promoting activity of the protein, a compound promoting expression of the gene of the protein can be used as a prophylactic and therapeutic agent for cancer, rheumatic disease, etc. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a preventive / therapeutic agent and a diagnostic agent for a neurodegenerative disease. More specifically, the present invention relates to a prophylactic / therapeutic agent, a diagnostic agent, and a screening for the prophylactic / therapeutic agent for diabetes, cancer, and rheumatic diseases.

Alzheimer's disease is a typical neurodegenerative disease with progressive dementia and cognitive dysfunction, but no effective treatment has been found so far. Needless to say, Alzheimer's disease is one of the most important diseases in the aging society, and the development of its therapeutic agent is of great significance in terms of medical economics.

On the other hand, it is known that abnormal proteins accumulate in the endoplasmic reticulum due to factors that affect protein biosynthesis, such as heat shock and glucose starvation, and stress on the endoplasmic reticulum (ER stress). When endoplasmic reticulum stress is applied to a living body, a group of endoplasmic reticulum stress response genes including chaperone molecules are expressed, and abnormal proteins are repaired or degraded to maintain homeostasis.

In recent years, the importance of the relationship between various neurodegenerative diseases and endoplasmic reticulum stress has been emphasized. Parkin has been identified as a pathogenic gene for autosomal recessive juvenile parkinsonism (AR-JP), a hereditary Parkinson's disease (Nature, 392, 605-608, 1998) and is involved in the proteolytic system It has been reported that it is a ubiquitin ligase (Nat. Genet, 25, 302-305, 2000). Furthermore, Pael (Parkin associated endothelin receptor-like) receptor was identified as a Parkin substrate. This receptor is a protein whose higher-order structure is difficult to form, and the entire lower-order structure formation of this protein is usually rapidly degraded by the action of Parkin. It has been reported that the cells accumulate in the endoplasmic reticulum and the cells undergo cell death due to endoplasmic reticulum stress (Non-patent Document 1, Cell, 105, 891-902, 2001). Furthermore, cells with a mutation in presenilin 1 that is a causative gene of familial Alzheimer's disease become vulnerable to ER stress, and β-amyloid production is increased by deletion of Ire1, which is involved in the ER stress response (Non-Patent Document 2 Biochem. Biophysic. Acta, 1536, pp. 85-96, 2001; Non-Patent Document 3 J. Biol. Chem., 276, 2108-2114, 2001). .

On the other hand, in order to comprehensively analyze gene expression, a microarray method in which cDNA or oligonucleotides are immobilized has been developed, and the technology for finding disease-specific changes in gene expression has become widespread, and its usefulness has been confirmed. . For example, Affymetrix's GeneChip system is being widely used for diagnosing diseases such as cancer and finding drug discovery target genes.

It has been reported that neuronal cell death inducible putative kinase (NIPK) is up-regulated in rat-derived cells by NGF starvation or calcium ionophore stimulation and has a kinase domain in its translation region (Non-Patent Document 4, BBRC, vol. 258). , 260-264, 1999). Recently, NIPK binds to the transcription factor ATF4 and regulates transcriptional activity (Oncogene 22, 2823-2835, 2003; Experimental Cell Research 286, 308-320, 2003). Binding to Akt1, which is a signal transduction molecule, and negatively controlling the signal (Non-patent Document 5, Science 300, 1574-1577, 2003). Activation of the Akt1 signaling pathway has been reported to play an important role in the pathogenesis of cancer and rheumatic diseases. PTEN, a tumor suppressor gene, inactivates Akt1, but in many cancers, DNA mutations delete the function of PTEN and activate Akt1 constantly, which leads to canceration and malignancy of cells. It plays an important role (Non-Patent Document 6 Nature Reviews Cancer 2, 489-501, 2002). In addition, since Akt1 activity is enhanced in synovial cells of rheumatoid patients and apoptosis can be induced by suppressing the function of Akt1, activation of Akt1 leads to the pathology of rheumatic diseases accompanied by synovial cell proliferation. It has been suggested that it plays an important role in the formation change (Non-Patent Document 7 Arthritis Rheum. 44, 1555-1567, 2001).
Cell, 105, 891-902, 2001 Biochem. Biophysic. Acta, 1536, 85-96, 2001 J. Biol. Chem., 276, 2108-2114, 2001 Biochemical and Biophysical Research Communications, 258, 260-264, 1999 Science 300, 1574-1577, 2003 Nature Reviews Cancer 2, 489-501, 2002 Arthritis Rheum. 44, 1555-1567, 2001

There is a need for safe and superior agents for preventing and treating neurodegenerative diseases or diabetes.

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, found a gene whose expression is significantly increased at the time of induction of neuronal cell death accompanied by ER stress. The inventors have also found that they promote dependent cell death, and as a result of further studies based on these findings, they have completed the present invention.

That is, the present invention
(1) A neurodegeneration 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 Prophylactic / therapeutic agents for diseases or diabetes,
(2) a protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a compound thereof or a salt thereof which inhibits the expression of a gene thereof; Prophylactic / therapeutic agents for neurodegenerative diseases or diabetes,
(3) a base complementary or substantially complementary to a base sequence of a polynucleotide encoding a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 An antisense polynucleotide containing the sequence or a portion thereof,
(4) a medicine comprising the antisense polynucleotide according to (3),
(5) the medicament according to (4), which is a prophylactic / therapeutic agent for a neurodegenerative disease or diabetes;
(6) an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof;
(7) a medicine comprising the antibody according to the above (6),
(8) the medicament according to (7), which is a prophylactic / therapeutic agent for a neurodegenerative disease or diabetes;
(9) a diagnostic agent comprising the antibody of (6) above,
(10) The diagnostic agent according to the above (9), which is a diagnostic agent for a neurodegenerative disease or diabetes,
(11) a diagnostic agent for a neurodegenerative disease or diabetes, 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,
(12) The protein, its partial peptide, or a salt thereof, which comprises using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, its partial peptide, or a salt thereof. A method for screening a compound or a salt thereof that inhibits the activity of a salt,
(13) a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof, A kit for screening a compound that inhibits the activity of the salt or a salt thereof,
(14) a compound or a salt thereof obtainable by using the screening method according to (12) or the screening kit according to (13);
(15) The expression of the above protein gene, 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. A method for screening a compound that inhibits or a salt thereof,
(16) Expression of the protein gene described above, which comprises a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. A kit for screening a compound that inhibits or a salt thereof,
(17) a compound or a salt thereof obtainable by using the screening method according to (15) or the screening kit according to (16);
(18) a medicament comprising the compound according to (14) or (17) or a salt thereof;
(19) The medicament according to the above (18), which is a prophylactic / therapeutic agent for a neurodegenerative disease or diabetes.
(20) a method for preventing or treating neurodegenerative disease or diabetes, which comprises administering to a mammal an effective amount of the compound according to (14) or (17) or a salt thereof;
(21) inhibiting the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, or inhibiting the expression of the protein gene; Prevention and treatment of neurodegenerative diseases or diabetes,
(22) Use of the compound of (14) or (17) or a salt thereof for producing a prophylactic / therapeutic agent for neurodegenerative disease or diabetes,
(23) a cancer comprising a compound or a salt thereof which promotes the activity of a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or Prophylactic / therapeutic agent for rheumatic diseases,
(24) A compound or a salt thereof which promotes the expression of a protein or a partial peptide thereof or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof. Prophylactic / therapeutic agents for cancer or rheumatic diseases,
(25) a medicine comprising a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof or a salt thereof,
(26) The medicament according to the above (25), which is an agent for preventing or treating cancer or rheumatic disease.
(27) a medicine comprising a polynucleotide encoding a protein or a partial peptide thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1,
(28) The medicament according to the above (27), which is an agent for preventing or treating cancer or rheumatic disease.
(29) a diagnostic agent for cancer or rheumatic 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,
(30) The above protein, its partial peptide, or a salt thereof, which comprises using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, its partial peptide, or a salt thereof. A method for screening a compound or a salt thereof that promotes the activity of a salt,
(31) a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof, A kit for screening a compound or a salt thereof that promotes the activity of the salt,
(32) a compound or a salt thereof obtainable by using the screening method according to (30) or the screening kit according to (31);
(33) The expression of the above protein gene, which comprises 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, A method for screening a compound to promote or a salt thereof,
(34) Expression of the above protein gene, which comprises a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. A kit for screening for a compound that promotes
(35) a compound or a salt thereof obtainable by using the screening method according to (33) or the screening kit according to (34);
(36) a medicament comprising the compound according to (32) or (35) or a salt thereof;
(37) The medicament according to the above (36), which is a prophylactic / therapeutic agent for cancer or rheumatic disease.
(38) a method for preventing or treating cancer or rheumatic diseases, which comprises administering to a mammal an effective amount of the compound according to (32) or (35) or a salt thereof;
(39) promoting 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 promoting the expression of the protein gene; Prevention or treatment of cancer or rheumatic diseases,
(40) Use of the compound described in (32) or (35) or a salt thereof for producing a prophylactic / therapeutic agent for cancer or rheumatic disease is provided.

Compounds or salts thereof that inhibit the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, compounds that inhibit the gene expression of the protein, or salts thereof, Antibodies, antisense polynucleotides of the gene, etc., for example, include neurodegenerative diseases [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic side) Chordosclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.], diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, Diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.) As any prophylactic / therapeutic agent, a compound or a salt thereof which promotes the activity of the protein, a compound or a salt thereof which promotes the expression of a gene of the protein, the protein, a polynucleotide encoding the protein, etc. (E.g., colon, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, testis, thyroid, pancreas, brain, ovary, It can be used as a prophylactic / therapeutic agent for rheumatic diseases (eg, rheumatoid arthritis, osteoarthritis, gout, etc.). In addition, various polynucleotides of the present invention are useful, for example, for diagnosing the above-mentioned diseases.

The protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter, sometimes referred to as the protein of the present invention or the protein used in the present invention) used in the present invention is Cells of humans and warm-blooded animals (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow) Cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, Natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts , Mammary gland cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells) or any tissue in which those cells are present, for example, the brain, various parts of the brain (eg, olfactory bulb, amygdala, Basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (Eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testes, ovaries, placenta, uterus, bones, joints, proteins derived from skeletal muscle, etc. It may be a synthetic protein.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 60% or more, more preferably about 70% or more as the amino acid sequence represented by SEQ ID NO: 1. And more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology.

(4) The homology of amino acid sequences can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool).

Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing the same amino acid sequence as the above-mentioned amino acid sequence represented by SEQ ID NO: 1. And proteins having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1. Specifically, the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20 And the like.

活性 Substantially the same activity includes, for example, a neurofibrillary tangle change promoting activity, a nerve cell death promoting activity, and the like. Substantially homogenous indicates that the properties are homogenous (eg, physiologically or pharmacologically). Therefore, it is preferable that the above activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Quantitative factors such as the molecular weight of the protein may be different.

To measure the activity of promoting neurofibrillary tangles, for example, after introducing an expression vector for the protein of the present invention into cells, the nerve fibers are stained by an immunostaining method (eg, using an anti-tau antibody or the like), and the stained image is observed under a microscope. By measuring the degree of change in nerve fibers.

To measure the activity of promoting neuronal cell death, for example, an expression vector for the protein of the present invention is introduced into cells, and a cell death inducer (eg, tunicamycin, thapsigargin, 2-deoxyglucose, β-amyloid, okadaic acid, homocysteine, etc.) Can be measured by measuring the amount of degeneration of axons using calcein staining, the respiratory activity of mitochondria, the amount of LDH (lactate dehydrogenase) in the culture supernatant, or the amount of DNA cleavage accompanying cell death.

Examples of the protein used in the present invention include (1) (i) one or more (for example, about 1 to 100, preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1. (Preferably about 1 to 10, more preferably number (1 to 5) amino acids), and (ii) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 ( For example, an amino acid sequence to which about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids have been added, (iii) SEQ ID NO: 1. One or more amino acids (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5)) are inserted into the represented amino acid sequence. Amino acid sequence (Iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably about 1 So-called muteins, such as proteins containing an amino acid sequence in which 〜5) amino acids are substituted with other amino acids, or (v) an amino acid sequence obtained by combining them.

場合 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 (carboxyl terminus) according to the convention of peptide labeling. The protein used in the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1 has a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), and an amide (—CONH ₂ ) at the C-terminus. Alternatively, any of ester (—COOR) may be used.

Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl and n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl , C _6-12 aryl groups such as α-naphthyl, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or C _7- alkyl groups such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl. _{14 An} aralkyl group, a pivaloyloxymethyl group and the like are used.

場合 When the protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Furthermore, the protein used in the present invention, the amino acid residue (eg, methionine residue) of the N-terminal amino group protecting group (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _{1- 6-} acyl group), N-terminal glutamine residue formed by cleavage in vivo, pyroglutamine-oxidized, substituent on the side chain of amino acid in the molecule (for example, -OH,- SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected with a suitable protecting group (eg, C _1-6 acyl group such as C _1-6 alkanoyl group such as formyl group and acetyl group). Or complex proteins such as so-called glycoproteins to which sugar chains are bound.

具体 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.

The partial peptide of the protein used in the present invention is a partial peptide of the protein used in the present invention described above, and is preferably any peptide having the same properties as the protein used in the present invention described above. It may be something.

Specifically, for the purpose of preparing the antibody of the present invention described below, a peptide having the amino acid sequence of the 1st to 30th and 250th to 280th amino acids in the amino acid sequence represented by SEQ ID NO: 1 and the like can be mentioned. . For example, it has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more amino acid sequences among the constituent amino acid sequences of the protein used in the present invention. Peptides and the like are used.

In the partial peptide used in the present invention, one or more (preferably about 1 to 10, more preferably a number (1 to 5)) amino acids in the amino acid sequence are deleted, or One or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are added to the amino acid sequence, or the amino acid One or more (preferably about 1 to 20, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are inserted into the sequence, or One or more (preferably about 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids may be substituted with another amino acid.

The partial peptide used in the present invention may have a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ) or an ester (—COOR) at the C-terminus.

Further, the partial peptides used in the present invention include those having a carboxyl group (or carboxylate) in addition to the C-terminus and the N-terminal amino acid residues (eg, , A methionine residue) in which the amino group is protected with a protecting group, a glutamine residue generated by cleavage of the N-terminal side in vivo and pyroglutamine oxidation, a substituent on the side chain of the amino acid in the molecule is Also included are those protected with an appropriate protecting group, and complex peptides such as so-called glycopeptides to which sugar chains are bound.

部分 The partial peptide used in the present invention can also be used as an antigen for preparing an antibody.

As the salt of the protein or partial peptide used in the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used. Are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.

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

When producing from human or mammalian tissues or cells, the homogenized human or mammalian tissues or cells are extracted with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.

市 販 A commercially available resin for protein synthesis can be generally used for the synthesis of the protein or partial peptide, its salt, or its amide used in the present invention. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or an amide thereof. I do.

For the condensation of the above protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, the protected amino acid was directly added to the resin together with a racemization inhibitor additive (for example, HOBt, HOOBt), or the protected amino acid was previously activated as a symmetric acid anhydride or HOBt ester or HOOBt ester. Later it can be added to the resin.

(4) The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole, so that the subsequent reaction is not affected.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.

The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for the esterification include lower (C _1-6 ) alkanoyl groups such as an acetyl group, aroyl groups such as a benzoyl group, and groups derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group. Used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.

As the protecting group for the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.

@ As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

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

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

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

As another method for obtaining an amide form of a protein or a partial peptide, for example, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After the elongation, a protein or partial peptide from which only the protecting group for the N-terminal α-amino group of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the C-terminal carboxyl group has been removed, and these are produced. The protein or peptide is condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. This crude protein or peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein or peptide.

In order to obtain an ester of a protein or peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired protein or An ester of the peptide can be obtained.

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

ポ リ 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, cDNA derived from the above-described cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA.

ベ ク タ ー The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be performed directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR) 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 nucleotide sequence hybridized with the nucleotide sequence represented by SEQ ID NO: 2 under high stringency conditions. Any DNA may be used as long as it contains a soybean base sequence and encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1 described above.

Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 under high stringency conditions include, for example, about 50% or more, preferably about 60% or more with the base sequence represented by SEQ ID NO: 2. More preferably, a DNA containing a base sequence having a homology of about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used. Specifically, the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 And the like.

The homology of the nucleotide sequences was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gap allowed; filtering = ON; match score = 1; It can be calculated by mismatch score = -3).

Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). . When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.

High stringency conditions refer to, for example, conditions of a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.

More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, a DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used.

ポ リ As the polynucleotide (eg, DNA) encoding the partial peptide used in the present invention, any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide used in the present invention may be used. Further, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.

As the DNA encoding the partial peptide used in the present invention, for example, a DNA having a part of the DNA containing the base sequence represented by SEQ ID NO: 2, or the base sequence represented by SEQ ID NO: 2 and a high string DNAs containing a base sequence that hybridizes under gentle conditions and a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention are used.

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

は The same hybridization method and high stringency conditions as described above are used.

Means for cloning DNA that completely encodes proteins and partial peptides used in the present invention (hereinafter, in the description of cloning and expression of DNAs encoding them, these may be simply abbreviated as the protein of the present invention) Amplification by a PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or encoding a DNA incorporated in an appropriate vector into a part or the entire region of the protein of the present invention. And a DNA fragment labeled with a DNA fragment or a synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted using PCR, a known kit, for example, Mutan ^™ -super Express Km (Takara Shuzo Co., Ltd.), Mutan ^™ -K (Takara Shuzo Co., Ltd.), etc., using the ODA-LA PCR method. The method can be carried out according to a method known per se, such as the gapped duplex method and the Kunkel method, or a method analogous thereto.

ＤＮＡ 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 stop codon at the 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

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

Examples of the vector include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus, and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, and the like are used.

プ ロ モ ー タ ー The promoter used in the present invention may be any promoter that is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.

Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter, etc. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used, if desired. it can. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r), neomycin resistant gene (Hereinafter, may be abbreviated as Neo ^r , G418 resistance). In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells deficient in dhfr gene, the target gene can be selected using a thymidine-free medium.

(4) If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, or the like is used. When the host is a bacterium belonging to the genus Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used.

形 質 A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed.

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

Specific examples of Escherichia bacteria include, for example, Escherichia coli K12.DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] Are used.

As 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 Saccharomyces cerevisiae ^{(Saccharomyces cerevisiae) AH22, AH22R -} , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe (Schizosaccharomyces pombe) NCYC1913, NCYC2036, such as Pichia pastoris (Pichia pastoris) KM71 is Used.

Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of night-moth (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, and High Five ^™ derived from egg of Trichoplusia ni. Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example, Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like are used.

As the insect, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter, CHO cells), dhfr gene deficient Chinese hamster cell CHO (hereinafter, CHO (dhfr ^-) cell), mouse L Cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells and the like are used.

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

Transformation of Bacillus sp. Can be performed, for example, according to the method described in Molecular & General Genetics, vol. 168, 111 (1979).

To transform yeast, for example, the method described in Methods in Enzymology, vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) can be used. it can.

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

{To transform animal cells, see, eg, Cell Engineering Separate Volume 8} New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, vol. 52, 456 (1973).

Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.

When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the culturing, and a carbon source necessary for growth of the transformant is used therein. Nitrogen sources, inorganic substances, etc. are included. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc., as a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract and the like Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. . If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently.

場合 When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.

場合 When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added.

When culturing a transformant in which the host is yeast, for example, Burkholder minimum medium [Bostian, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] And SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.

When culturing an insect cell or a transformant whose host is an insect, a medium such as 10% bovine serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) is used as appropriate. The added one is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration or stirring is added.

When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], etc. Is used. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration and stirring are added.

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.

分離 The protein of the present invention can be separated and purified from the culture by, for example, the following method.

When extracting the protein of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to sonication, lysozyme and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after the culture is completed, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.

培養 The protein contained in the culture supernatant or extract obtained in this manner can be purified by appropriately combining known separation / purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Using differences in charge, methods using charge differences such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, and using differences in hydrophobicity such as reversed-phase high-performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.

When the protein thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or analogous thereto Can be converted to a free form or another salt.

The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed before or after purification by the action of an appropriate protein modifying enzyme. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody, Western blotting, or the like.

The antibody against the protein or partial peptide or a salt thereof used in the present invention may be a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention.

Antibodies against the protein or partial peptide used in the present invention or a salt thereof (hereinafter sometimes simply referred to as the protein of the present invention in the description of the antibody) use the protein of the present invention as an antigen and are known per se. Can be produced according to the antibody or antiserum production method described in
[Preparation of monoclonal antibody]
(A) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal by itself or together with a carrier and a diluent at a site where antibody production is possible by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

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

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. By incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes, cell fusion can be carried out efficiently.

Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is directly or adsorbed together with a carrier, and then radioactive substances or A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with an enzyme or the like (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A; A method in which a hybridoma culture supernatant is added to a solid phase to which globulin antibodies or protein A is adsorbed, a protein labeled with a radioactive substance, an enzyme, or the like is added, and a monoclonal antibody bound to the solid phase is detected.

Selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture A medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
(B) Purification of monoclonal antibody Monoclonal antibody can be separated and purified by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchange method Absorption / desorption method using body (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific antibody that collects only antibody by active adsorbent such as protein A or protein G, and dissociates the bond to obtain antibody Purification method].
(Preparation of polyclonal antibody)
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting a substance and separating and purifying the antibody.

Regarding the complex of an immunizing antigen and a carrier protein used to immunize a warm-blooded animal, the type of the carrier protein and the mixing ratio between the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by crosslinking the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ~ 5 is used.

Various condensing agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.

(4) The condensation product is administered to a warm-blooded animal at a site capable of producing an antibody itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.

The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.

ポ リ Polyclonal antibody titer in antiserum can be measured in the same manner as in the above-described measurement of antibody titer in antiserum. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.

The nucleotide sequence of a polynucleotide encoding a protein or a partial peptide used in the present invention (eg, DNA (hereinafter, these DNAs may be abbreviated to DNA of the present invention in the description of antisense polynucleotide)) The antisense polynucleotide having a complementary or substantially complementary base sequence or a part thereof includes a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof. Any antisense polynucleotide may be used as long as it has a portion 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, about 70% of the total nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more. In particular, in the case of an antisense polynucleotide directed to translation inhibition, of the entire base sequence of the complementary strand of the DNA of the present invention, the base sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, An antisense polynucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand of a base sequence near a codon, etc. B) In the case of an antisense polynucleotide that directs RNA degradation by RNase H, it is about 70% or more, preferably about 80% or more, more preferably about 90% with the complementary strand of the entire base sequence of the DNA of the present invention including introns. As described above, most preferably, antisense polynucleotides having about 95% or more homology are suitable.

Specifically, an antisense polynucleotide having a base sequence complementary to or substantially complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2, or a part thereof, preferably, for example, An antisense polynucleotide having a base sequence complementary to the base sequence of the DNA containing the base sequence represented by SEQ ID NO: 2 or a part thereof (more preferably, a DNA sequence containing the base sequence represented by SEQ ID NO: 2 A base sequence complementary to the base sequence, or an antisense polynucleotide having a part thereof).

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

In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense DNA is replaced with, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate or phosphorodithionate. It may be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base portion (pyrimidine, purine) may also be chemically modified. Or any one that hybridizes to the 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, an antisense polynucleotide capable of inhibiting the replication or expression of the protein gene of the present invention is cloned or designed based on the nucleotide sequence information of DNA encoding the determined protein, and synthesized. Can. Such nucleotides (nucleic acids) can hybridize with the RNA of the protein gene of the present invention and inhibit the synthesis or function of the RNA, or through interaction with the protein-related RNA of the present invention. The expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides that are complementary to a selected sequence of the protein-associated RNA of the present invention, and that can specifically hybridize with the protein-associated RNA of the present invention, can be used in vivo and in vitro to produce the protein gene of the present invention. It is useful for regulating and controlling the expression of, and also useful for treating or diagnosing diseases and the like. The term “corresponding” means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. "Corresponding" between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) as directed by the nucleotide (nucleic acid) sequence or its complement. . 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation termination codon, 3' end untranslated region, 3 ' The terminal palindrome region and the 3'-end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as a target.

関係 The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region can be said to be that the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target is “antisense”. Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are 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 a base such as found in DNA or RNA And nucleotides having a configuration that allows base attachment). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides), or even known. Such as labeled, capped, methylated, one or more naturally-occurring nucleotides replaced by analogs, intramolecular nucleotides Modified, eg, those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charged or sulfur-containing bonds (eg, phosphorothioate, phosphorodithioate, etc.) ), Such as proteins (nucleases, nuclease inhibitors, toxic , Antibodies, signal peptides, poly-L-lysine, etc.), sugars (eg, monosaccharides), etc., having side chain groups, interacting compounds (eg, acridine, psoralen, etc.), chelates Those containing compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) It may be. Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or by functional groups such as ethers, amines, etc. It may have been converted.

(4) 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, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids.

Thus, many modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed. , Antisense Research and Applications, CRC Press, 1993.

The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It could be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Examples of other groups include cap groups specifically arranged at the 3 'end or 5' end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

(4) The antisense nucleic acid inhibitory activity can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various methods known per se.

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

The expression of the protein of the present invention is increased by nervous cell death accompanied by ER stress, and promotes ER stress-dependent cell death. In addition, the proteins of the present invention promote cell death (neural cell death) and neurofibrillary tangles. Furthermore, the protein of the present invention binds to Akt1 and inhibits Akt1 activity. Therefore, the protein of the present invention is useful as a marker for early diagnosis, determination of symptom severity, and prediction of disease progression in neurodegeneration, diabetes, cancer, rheumatic diseases and the like. In addition, a compound or a salt thereof that inhibits the activity of the protein of the present invention, a compound or a salt thereof that inhibits the expression of a gene encoding the protein of the present invention, an antisense polynucleotide of the present invention, and an antibody against the protein of the present invention. The contained medicament can be used, for example, as a safe prophylactic / therapeutic agent for neurodegenerative diseases, diabetes and its complications. Further, a compound or a salt thereof that promotes the activity of the protein of the present invention, a compound or a salt thereof that promotes the expression of a gene encoding the protein of the present invention, the protein of the present invention, and the DNA of the present invention include, for example, cancer, It can be used as a safe prophylactic / therapeutic agent for rheumatic diseases and the like.
(1) Screening of drug candidate compounds for disease The expression of the protein of the present invention is increased by nervous cell death accompanied by ER stress, and promotes ER stress-dependent cell death. Further, the protein of the present invention binds to Akt1, a key molecule regulating IGF-1 / insulin signal, which plays an important role in maintaining survival of nerve cells and phosphorylation of tau, and inhibits Akt1 activity. Causes hyperphosphorylation of tau. Accordingly, the protein of the present invention has a neurofibrillary tangle change promoting activity, a nerve cell death promoting activity, and the like.

Therefore, compounds or salts thereof that regulate (inhibit or promote, preferably inhibit) the activity of the protein of the present invention include, for example, neurodegenerative diseases [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic disease] Alzheimer's disease, Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.), diabetes and its complications (eg, It can be used safely as a prophylactic / therapeutic agent for diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesion, arteriosclerosis, hypertension, hyperlipidemia, etc.).

On the other hand, in many cancers, PTEN (one of the tumor suppressor genes that inactivate Akt1) functions are lost due to DNA mutation, and Akt1 is constantly activated. Playing an important role in In addition, since Akt1 activity is enhanced in synovial cells of rheumatoid patients and apoptosis can be induced by suppressing the function of Akt1, activation of Akt1 leads to the pathology of rheumatic diseases accompanied by synovial cell proliferation. Plays an important role in the formation.

Accordingly, compounds or salts thereof that regulate (inhibit or promote, preferably promote) the activity of the protein of the present invention include, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract) Cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, ovarian cancer, blood tumor, etc., rheumatic diseases (eg, rheumatoid arthritis, osteoarthritis, gout, etc.) It can be used as a safe prophylactic / therapeutic agent.

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.

{Specifically, the present invention provides a method for screening a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention, which comprises using the protein of the present invention.

For example, by comparing (i) the activity of the protein of the present invention (neurofibrillary tangle change promoting activity, neuronal cell death promoting activity, etc.) and (ii) the activity of a mixture of the protein of the present invention and a test compound, A compound or its salt that regulates (promotes or inhibits) the activity of the protein of the present invention is screened.

Specifically, for example, (i ′) a case where the protein expression vector of the present invention is introduced into cells and cultured, and (ii ′) a case where the protein expression vector of the present invention is introduced into cells and culture in the presence of a test compound. In this case, the nerve fiber is stained by an immunostaining method (eg, using an anti-tau antibody, etc.), the stained image is obtained by a microscope, and the degree of the change in the nerve fiber is measured, thereby promoting the activity of promoting neurofibrillary tangle change. Are measured and compared, and a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention is screened.

Specifically, for example, (i ″) when the protein expression vector of the present invention is introduced into cells and cultured, and (ii ″) when the protein expression vector of the present invention is introduced into cells and the cells are cultured in the presence of a test compound. When cultured, a cell death inducer (eg, tunicamycin, thapsigargin, 2-deoxyglucose, β-amyloid, okadaic acid, homocysteine, etc.) is added, and axon degeneration using calcein staining, mitochondrial respiration The activity of the protein of the present invention is regulated (promoted or inhibited) by measuring and comparing the activity, the amount of LDH (lactate dehydrogenase) in the culture supernatant, or the amount of DNA cleavage associated with cell death, and measuring and comparing the activity of promoting neuronal cell death. Or a salt thereof to be screened.

Akt1 also phosphorylates Cot (an oncogene found in thyroid cancer cell lines and a member of the mitogen-activated protein kinase kinase kinase family) and IKK (phosphorylates IκB, an inhibitor of NF-κB). , Which is a kinase that activates the transcription of NF-κB) (Mol. Cell. Biol., 22, 5962-5974, 2002).

よ り From this, the following screening methods are also used.

(I ′ ″) The protein expression vector of the present invention is introduced into cells together with a reporter gene containing a NFκB binding sequence in a promoter or enhancer, and if necessary, (a) microbial cell lysate, microbial culture supernatant , A solution containing a ligand such as a lysate of eukaryotic cells, a culture supernatant of eukaryotic cells, (b) the ligand itself or (c) a culture with the addition of a substance having the same binding activity as the natural ligand, and ( ii ′ ″) The protein expression vector of the present invention is introduced into cells together with a reporter gene containing a NFκB binding sequence in a promoter or enhancer, and if necessary, (a) microbial cell lysate, microbial culture supernatant, A solution containing a ligand such as a lysate of eukaryotic cells, a culture supernatant of eukaryotic cells, or a substance having the same binding activity as (b) the ligand itself or (c) a natural ligand is added. The amount of reporter expression when cultured in the presence of a test compound is measured and compared, and a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention is screened.

The reporter expression level can be measured by using the reporter protein activity using luciferase activity, alkaline phosphatase activity or the like as an index.

タ ン パ ク 質 As the above-mentioned protein of the present invention, those produced by culturing cells capable of producing the protein of the present invention and the like are used. Further, a culture solution of the cells, a supernatant thereof, a crushed cell, or the like may be used.

細胞 As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as COS7 cells, CHO cells, and HEK293 cells are preferably used. For the screening, for example, a transformant which secretes the protein of the present invention extracellularly or expresses it intracellularly by culturing by the above-mentioned method is preferably used. 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.

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, the activities in the above (ii), (ii ′), (ii ″) and (ii ′ ″) are the same as those in the above (i), (i ′), (i ″) and (i ′ ″). ), A test compound that reduces by about 20% or more, preferably 30% or more, more preferably about 50% or more, as a compound that inhibits the activity of the protein of the present invention, as described in (ii) and (ii) above. '), (Ii' ') and (ii' '') have an activity of about 20 compared to (i), (i '), (i' ') and (i' ''). Test compounds that increase by at least%, preferably at least 30%, more preferably at least about 50% can be selected as compounds that promote the activity of the protein of the invention.

The compound having an activity of inhibiting the activity of the protein of the present invention is safe and low toxic, for example, a neurodegenerative disease [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.), diabetes And its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.) and useful as therapeutic agents.

Compounds having an activity of promoting the activity of the protein of the present invention are safe and low toxic, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer) in order to enhance the action of the protein of the present invention. , Gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, ovarian cancer, blood tumor, etc.), rheumatic diseases (eg, rheumatoid arthritis, It is useful as a prophylactic / therapeutic agent for osteoarthritis, gout, etc.).

Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts And compounds selected from plasma and the like. As the salt of the compound, those similar to the aforementioned salts of the peptide of the present invention are used.

In addition to the effects of the protein of the present invention described above, the gene encoding the protein of the present invention also regulates the expression of the gene encoding the protein of the present invention by increasing the expression by inducing neuronal cell death accompanied by endoplasmic reticulum stress. The (preferably inhibiting) compound or a salt thereof is used, for example, as a prophylactic / therapeutic agent for neurodegenerative diseases, diabetes and its complications, and regulates (preferably promotes) the expression of a gene encoding the protein of the present invention. Or a salt thereof can be used, for example, as a prophylactic / therapeutic agent for cancer, rheumatic diseases and the like.

Therefore, the polynucleotide (eg, DNA) of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (inhibits or promotes) the expression of a gene encoding the protein of the present invention.

The screening method includes (iii) culturing cells capable of producing the protein of the present invention and (iv) culturing cells capable of producing the protein used in the present invention in the presence of the test compound. A screening method characterized by performing a comparison with the case.

に お い て In the above method, the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases (iii) and (iv) is measured and compared.

細胞 The test compound and cells having the ability to produce the protein of the present invention include the same cells as described above.

The protein amount is measured by a known method, for example, using an antibody recognizing the protein of the present invention, and measuring the protein present in a cell extract or the like according to a method such as Western analysis or ELISA method or a method analogous thereto. can do.

The amount of mRNA can be measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a probe, or a PCR method using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a primer or It can be measured according to a method corresponding thereto.

For example, a test compound which reduces the gene expression level in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii), according to the present invention As a compound that inhibits the expression of a gene encoding the protein, the gene expression level in the case of the above (iv) is about 20% or more, preferably 30% or more, more preferably A test compound that increases about 50% or more can be selected as a compound that promotes the expression of the gene encoding the protein of the present invention.

ス ク リ ー ニ ン グ The screening kit of the present invention contains the protein or partial peptide used in the present invention or a salt thereof, or a cell capable of producing the protein or partial peptide used in the present invention.

Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention are test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts. A compound selected from animal tissue extract, plasma and the like, or a salt thereof, which is a compound or a salt thereof that regulates the expression of the gene of the protein of the present invention.

As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.
Compounds that regulate (preferably inhibit) the expression of the gene encoding the protein of the present invention or salts thereof include, for example, neurodegenerative diseases [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease] Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.], diabetes and its complications (eg, diabetic) It can be used as a prophylactic / therapeutic agent for nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesion, arteriosclerosis, hypertension, hyperlipidemia, etc.). Compounds that regulate (preferably enhance) the expression of the gene encoding the protein of the present invention or salts thereof include, for example, cancer (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer) , Spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, ovarian cancer, blood tumor, etc., rheumatic diseases (eg, rheumatoid arthritis, osteoarthritis, gout, etc.) Can be used as a prophylactic / therapeutic agent.

化合物 When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.

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

As a composition for parenteral administration, for example, injections, suppositories, etc. are used. Injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Dosage forms such as agents. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, 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 is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form suitable for the dose of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, and the like. Usually, each dosage unit dosage form has a dosage of 5 to 500 mg, especially 5 to 100 mg for an injection. Other dosage forms preferably contain 10 to 250 mg of the above compound.

Each of the above-mentioned compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending with the above-mentioned compounds.

The preparations obtained in this way are safe and low toxic, for example, human or warm-blooded animals (eg, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.) ) Can be administered orally or parenterally.

The dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, administration route and the like.For example, for the purpose of treating Alzheimer's disease, the activity of the protein of the present invention or the gene of When a compound or a salt thereof that inhibits expression is orally administered, generally in an adult (with a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day. , More preferably about 1.0 to 20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, the target disease, and the like. For example, for the purpose of treating Alzheimer's disease, the activity of the protein of the present invention or the protein When a compound or a salt thereof that inhibits the expression of the gene of the present invention 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 amount can be administered in terms of the weight per 60 kg.

For example, when a compound or its salt that promotes the activity of the protein of the present invention or the expression of the gene of the protein is orally administered for the purpose of treating rheumatoid arthritis, generally, in adults (with a body weight of 60 kg), About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound or a salt thereof is administered per day. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like. When a compound or a salt thereof that promotes the expression of a protein gene 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 to 30 mg per day. Conveniently, 0.1-20 mg, more preferably about 0.1-10 mg, will be administered by intra-articular injection. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
(2) Quantification of the protein of the present invention, its partial peptide or its salt An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. Since it can be used, it can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay.

That is, the present invention
(I) reacting the antibody of the present invention with a test solution and the labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody. And (ii) simultaneously or sequentially reacting the test solution with the antibody of the present invention insolubilized on a carrier and another antibody of the present invention labeled on a carrier. The present invention provides a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction.

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

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

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

(4) For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In addition, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may.

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

モ ノ ク ロ ー ナ ル 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.

In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. A solid-phase method using a soluble antibody as the first antibody and using a solid-phased antibody as the second antibody is used.

  In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.

  In nephelometry, the amount of insoluble sediment produced as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

  In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.

For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Radioimmunoassay" continued (edited by Kodansha, published in 1979), and "Enzyme immunoassay" edited by Eiji Ishikawa (Medical Shoin, Showa) Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (Third Edition), 3rd edition (Medical Shoin, Showa) Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 74 (Immunochemical Techniques (Part C)) 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies). Academic Press Company)).

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, a neurodegenerative disease [eg, Alzheimer's disease (family Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis Disease), diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.), or future illness It can be diagnosed that there is a high possibility of performing.

Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. Further, for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.
(3) Gene Diagnostics The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog) , Monkeys, chimpanzees, etc.) can detect abnormalities (genetic abnormalities) in DNA or mRNA encoding the protein of the present invention or partial peptides thereof, for example, damage, mutation or reduced expression of the DNA or mRNA, It is useful as a diagnostic agent for a gene such as an increase or an overexpression of the DNA or mRNA.

The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or 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, a neurodegenerative disease [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease) Disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.), diabetes and its It can be diagnosed that there is a high possibility of a complication (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesion, arteriosclerosis, hypertension, hyperlipidemia, etc.).
(4) Pharmaceutical Containing Antisense Polynucleotide The antisense polynucleotide of the present invention, which can complementarily bind to the DNA of the present invention and suppresses the expression of the DNA, has low toxicity, and exhibits the present invention in vivo. For example, neurodegenerative diseases [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome Amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's disease, diabetic neuropathy, multiple sclerosis, etc.], diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, Diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.) It can be used as an anti-therapeutic agent.

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

Also, for example, after inserting the antisense polynucleotide alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, a human or mammal (eg, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, and the like) can be administered orally or parenterally. The antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered using a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered locally into the trachea as an inhalant.

Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of intracellular uptake, the above antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection), and is used for intravenous, subcutaneous, lesion, Etc. may be administered.

The dose of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense polynucleotide of the present invention is administered for the purpose of treating Alzheimer's disease, it is generally used in adults. (60 kg body weight), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.

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

Like the above-mentioned antisense polynucleotide, a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a ribozyme containing a part of the RNA encoding the protein of the present invention, and the like also include the gene of the present invention. Can be suppressed, and the function of the protein used in the present invention or the DNA used in the present invention in a living body can be suppressed, for example, a neurodegenerative disease [eg, Alzheimer's disease (familial Alzheimer's disease) , Juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.) , Diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, Urine diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.) can be used as a preventive or therapeutic agent for such.

A double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).

A ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known ribozyme to a part of RNA encoding the protein of the present invention. As a part of the RNA encoding the protein of the present invention, a portion (RNA fragment) close to a cleavage site on the RNA of the present invention, which can be cleaved by a known ribozyme, can be mentioned.

When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide.
(5) Pharmaceuticals Containing Antibody of the Present Invention The antibodies of the present invention having the activity of neutralizing the activity of the protein of the present invention include, for example, neurodegenerative diseases [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, Sporadic Alzheimer's disease, Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.), diabetes and its complications (Eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.) and the like.

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

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

As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections and the like. Is included. Such injections are prepared according to a known method, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), a nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil, 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 is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form suitable for the dose of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, and the like. Usually, each dosage unit dosage form has a dosage of 5 to 500 mg, especially 5 to 100 mg for an injection. Other dosage forms preferably contain 10 to 250 mg of the above antibody.

Each of the above-described compositions may contain other active ingredients 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 is used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rat, rabbit, sheep, pig, It can be administered orally or parenterally (eg, intravenously) to cattle, cats, dogs, monkeys, and the like. The dose varies depending on the administration subject, target disease, symptoms, administration route and the like.For example, when used for treatment of Alzheimer's disease in adults, the antibody of the present invention is usually used in a dose of 0.1%. About 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 3 times a day It is convenient to administer as an injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.

Further, the antibody of the present invention may be, for example, a neurodegenerative disease (eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, Prion disease, Creutzfeldt-Jakob disease, Huntington's disease, diabetic neuropathy, multiple sclerosis, etc.), diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions) , Arteriosclerosis, hypertension, hyperlipidemia, etc.).
(6) DNA-transferred animal The present invention relates to a DNA encoding an exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (hereinafter abbreviated as the exogenous mutant DNA of the present invention). Is provided.

That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) The animal according to (2), wherein the rodent is a mouse or rat, and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. Things.

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, etc. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by transferring a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. 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 the above-mentioned germ cells with a cell fusion method known per se.

As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. Among them, rodents, which are relatively short in ontogeny and biological cycle in terms of preparation of disease animal model systems, and are easy to breed, especially mice (for example, hybrids such as C57BL / 6 strain and DBA2 strain as pure strains) As a system, _one B6C3F system, _one BDF system, _one B6D2F system, a BALB / c system, an ICR system, or a rat (for example, Wistar, SD, etc.) is preferable.

「As the“ mammal ”in the recombinant vector that can be expressed in mammals, humans and the like can be mentioned in addition to the above-mentioned non-human mammals.

外 The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the DNA of the present invention originally possessed by a non-human mammal.

As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA.

異常 The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of a normal protein of the present invention is used.

(4) The exogenous DNA of the present invention may be derived from mammals of the same species or different species from the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is transferred, DNA derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto is expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-transferring mammals can be created.

Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.

Examples of the promoter that regulates the DNA expression include, for example, (i) a promoter of a DNA derived from a virus (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example, albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acid Protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen types I and II, cyclic AMP-dependent protein kinase βI subunit, dyst Lophine, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin , Α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α A Chin, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, a human and chicken β-actin promoter, and the like are preferable.

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

In addition, a splicing signal of each DNA, an enhancer region, a part of an intron of a eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region, or between the translation region for the purpose of further expressing the desired foreign DNA. It is also possible to connect 3 ′ downstream of the above depending on the purpose.

The normal translation region of the protein of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNAs. From a variety of genomic DNA libraries, or as a starting material, a complementary DNA prepared by known methods from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above-described cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA.

(5) The translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which the translation region is ligated downstream of the promoter and, if desired, upstream of the transcription termination site.

転 移 Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal cells and somatic cells. means. The offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germ cells and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably retained by mating. .

転 移 Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells.

(4) A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, all the offspring can be bred to have the DNA in excess.

The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level and promotes the function of the endogenous normal DNA to eventually cause hyperactivity of the protein of the present invention. Occasionally, it can be used as a disease model animal. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of 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.

Further, 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 preventive / therapeutic agent for a disease associated with the protein of the present invention, for example, a neurodegenerative disease [ Examples: Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's disease, diabetes Neuropathy, multiple sclerosis, etc.), diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.) It can also be used for screening tests for prophylactic and therapeutic agents.

On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be used as a raw material by incorporating it into the aforementioned plasmid. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The progeny of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed the cells so that all offspring have the DNA.

The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of endogenous normal DNA, and finally, the functionally inactive form of the protein of the present invention. It can be refractory and can be used as a model animal for the disease. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.

Also, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention can inhibit the abnormal protein of the present invention (dominant negative action) by the abnormal protein of the present invention in the inactive refractory disease of the protein of the present invention. A model to elucidate.

In addition, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, a prophylactic / therapeutic agent for the protein of the present invention or a functionally inactive refractory disease, for example, neurodegeneration Diseases [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's disease , Diabetic neuropathy, multiple sclerosis, etc.], diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.) ) Can also be used for screening tests for prophylactic and therapeutic agents.

In addition, other possible uses of the above two DNA-transferred animals of the present invention include, for example,
(I) use as a cell source for tissue culture,
(Ii) a peptide specifically expressed or activated by the protein of the present invention, by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or analyzing a peptide tissue expressed by the DNA; Analysis of the relationship with
(Iii) culturing cells of DNA-bearing tissue by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture;
(Iv) Screening of a drug that enhances the function of the cell by using the cell described in (iii) above, and (v) Isolation and purification of the mutant protein of the present invention and production of an antibody thereof are conceivable.

Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, and the like. More detailed pathological findings in each organ of the disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.

In addition, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a proteolytic enzyme such as trypsin to obtain the released DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, it is possible to examine the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and its abnormalities. It is an effective research material for

Further, in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, using the DNA-transferred animal of the present invention, It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease by using a method or the like. In addition, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for diseases associated with the protein of the present invention.
(7) Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention.

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

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA which is artificially mutated to the DNA of the present invention possessed by the non-human mammal to suppress the DNA expression ability, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA does not substantially have the ability to express the protein of the present invention (hereinafter, may be referred to as the knockout DNA of the present invention). ) Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells).

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

方法 The method of artificially mutating the DNA of the present invention can be performed, for example, by deleting part or all of the DNA sequence and inserting or substituting another DNA by genetic engineering techniques. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene or a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl). A reporter gene or the like typified by a transferase gene) to disrupt the exon function, or insert a DNA sequence that terminates gene transcription into an intron between exons (for example, a polyA addition signal); Inability to synthesize complete messenger RNA By introducing a DNA strand having a DNA sequence constructed so as to eventually disrupt the gene (hereinafter abbreviated as a targeting vector) into the chromosome of the animal by, for example, homologous recombination, the obtained ES cell PCR using a DNA sequence on or near the DNA of the present invention as a probe and a DNA sequence on a targeting vector and PCR using a DNA sequence of a nearby region other than the DNA of the present invention used for the preparation of the targeting vector as a primer It can be obtained by analyzing by the method and selecting the knockout ES cells of the present invention.

As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, or according to the known Evans and Kaufma method. It may be a newly established one. For example, in the case of mouse ES cells, currently, 129-line ES cells are generally used. for example the purpose of acquiring the obvious ES cells, the BDF ₁ mice (C57BL / 6 and DBA / 2 for the egg collection number of lack of C57BL / 6 mice and C57BL / 6 were improved by crossing with DBA / 2 Those established using F ₁ ) can also be used favorably. BDF ₁ mice are often egg number, and, in addition to the advantage that the egg is tough, since with C57BL / 6 mice in the background, when the ES cells obtained therefrom, which were generated pathological model mice , C57BL / 6 mice can be advantageously used in that their genetic background can be replaced with C57BL / 6 mice by backcrossing.

In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. However, a large number of blastocysts can be efficiently obtained by collecting embryos at the 8-cell stage and culturing them up to blastocysts. Early embryos can be obtained.

Either male or female ES cells may be used, but male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor.

As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a PCR method can be mentioned. By using this method, the number of ES cells of about 1 colony (about 50 cells) is sufficient, whereas the conventional method required about 10 ⁶ cells for karyotype analysis. The primary selection of ES cells in the early stage can be performed by discriminating between male and female, and the early stage of culture can be greatly reduced by enabling the selection of male cells at an early stage.

{Circle around (2)} The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the normal cells (for example, chromosomes in mice) are knocked out. It is desirable to clone again into cells (number 2n = 40).

胚 The embryonic stem cell line obtained in this way usually has very good growth properties, but must be subcultured carefully since it tends to lose its ontogenic ability. For example, on a suitable feeder cell such as STO fibroblast in the presence of LIF (1-10000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% The cells are cultured by a method such as culturing at about 37 ° C. in carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001 to 0.5% trypsin / 0.1 to 5 mM EDTA, Preferably, a method is used in which cells are made into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on newly prepared feeder cells. Such subculture is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.

ES cells are differentiated into various types of cells, such as parietal muscle, visceral muscle, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proc. Natl. Acad. Sci. USA 78, 7634, 1981; TC Doetschman et al., Journal Of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], and the DNA-deficient cell of the present invention obtained by differentiating the ES cell of the present invention is a cell of the present invention in vitro. Useful in protein cell biology studies.

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

非 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 is, for example, a DNA in which the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.

Cells in which the DNA of the present invention has been knocked out can be used as a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the DNA of the present invention derived from the mouse used for the targeting vector. It can be determined by analysis by PCR using a DNA sequence of a nearby region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. The chimeric embryo is injected into an animal embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.

When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individual thus obtained is usually an individual with a heterozygous expression of the protein of the present invention, which is crossed with an individual with a heterozygous expression of the protein of the present invention, and homozygous expression of the protein of the present invention from their offspring. Defective individuals can be obtained.

In the case of using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method, and these transgenic non-human mammals can be obtained. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.

(4) In the individual knocked out of the DNA of the present invention in this way, animal individuals obtained by mating can confirm that the DNA has been knocked out, and can be reared in a normal rearing environment.

生殖 Furthermore, the acquisition and maintenance of the germ line may be performed in accordance with ordinary methods. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.

非 The non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.

In addition, since the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a model of a disease caused by inactivation of the biological activity of the protein of the present invention. Therefore, it is useful for investigating the causes of these diseases and examining treatment methods.
(7a) Method of screening for a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage of the present invention The non-human mammal deficient in DNA expression of the present invention is deficient or damaged of the DNA of the present invention Can be used for screening for a compound having a therapeutic / preventive effect on diseases caused by the above.

That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring a change in the animal. Diseases such as neurodegenerative diseases [eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion's disease, Creutzfeld-Jakob Disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.), diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, A method for screening a compound or a salt thereof having a therapeutic / preventive effect against hyperlipidemia and the like.

ＤＮＡ The non-human mammal deficient in expressing DNA of the present invention used in the screening method includes the same ones as described above.

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

Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.

(4) As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection, or the like is used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

For example, neurodegenerative diseases (eg, Alzheimer's disease (familial Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion's disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.), diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, high fat) When a compound having a therapeutic / preventive effect is screened for, for example, a test compound is administered to a non-human mammal deficient in expressing DNA of the present invention, and the difference between the test compound non-administration group and the number of neuronal cell deaths, The differences in the amount of various proteins and the like are observed over time in the tissue.

In the screening method, when the test compound is administered to a test animal, if the disease symptom of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound is administered. It can be selected as a compound having a therapeutic / preventive effect on the above diseases.

The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect on a disease caused by deficiency or damage of the protein of the present invention. It can be used as a safe and low toxic drug such as a prophylactic / therapeutic agent. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). ) Is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.

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

Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.

The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the compound is orally administered, it is generally used in an adult (assuming a body weight of 60 kg) Alzheimer's disease patient. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound 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 is usually administered in the form of an injection to an adult (with a body weight of 60 kg) Alzheimer's disease patient. When administered, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound by intravenous injection per day. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.
(7b) A method for screening a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention. The present invention comprises administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and detecting the expression of a reporter gene. The present invention provides a method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for DNA of the present invention.

In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.

(4) Examples of the test compound include the same compounds as described above.

As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.

In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue that expresses the protein of the present invention should be used instead of the protein of the present invention. Β-galactosidase is expressed. Therefore, for example, the present invention can be easily performed by staining with a reagent serving as a substrate for β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal). Of the protein in an animal body can be observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. After reacting for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.

The compound or its salt 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 of the DNA of the present invention.

The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid) and a base (eg, an alkali metal). And particularly preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.

The compound or its salt that promotes or inhibits the promoter activity of the DNA of the present invention or the salt thereof can regulate the expression of the protein of the present invention and regulate the function of the protein, for example, a neurodegenerative disease [eg, Alzheimer's disease (family Alzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson's disease, Down's syndrome, amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jakob disease, Huntington's chorea, diabetic neuropathy, multiple sclerosis Disease), as a preventive or therapeutic agent for diabetes and its complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic foot lesions, arteriosclerosis, hypertension, hyperlipidemia, etc.) Useful.

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

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

Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.

The dose of the compound or a salt thereof varies depending on the target disease, the subject to be administered, the administration route, and the like. For example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally the adult (body weight) is used. For an Alzheimer's disease patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like. For example, the compound of the present invention that inhibits the promoter activity for DNA is usually administered in the form of an injection to an adult ( When administered to an Alzheimer's disease patient (with a body weight of 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound is administered intravenously per day. It is convenient. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.

As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to investigating the cause of various diseases caused or developing preventive and therapeutic agents.

Further, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene-transferred animal). Once created, it will also be possible to specifically synthesize the protein and study its effects in living organisms. Furthermore, if a suitable reporter gene is bound to the above promoter portion, and a cell line that expresses the reporter gene is established, a low-molecular compound having an action of specifically promoting or suppressing the production ability of the protein itself of the present invention in the body. Can be used as a search system.
(8) Agent for Prevention / Treatment of Various Diseases Related to the Protein of the Present Invention The protein of the present invention binds to Akt1 and inhibits Akt1 activity.

Therefore, when the DNA encoding the protein of the present invention is abnormal or defective, or when the expression level of the protein of the present invention is reduced, for example, cancer (eg, colon cancer, breast cancer, lung cancer) , Prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, ovarian cancer, blood tumor, etc., rheumatic diseases ( For example, various diseases such as rheumatoid arthritis, osteoarthritis, and gout develop.

Therefore, the protein of the present invention and the DNA of the present invention can be used, for example, in cancers (eg, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterus) Can be used as a prophylactic / therapeutic agent for cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor, ovarian cancer, blood tumor, etc., and rheumatic diseases (eg, rheumatoid arthritis, osteoarthritis, gout, etc.). .

For example, when there is a patient in whom the activity of the protein of the present invention is not sufficiently or normally exerted because the protein of the present invention is reduced or deficient in the living body, (a) the DNA of the present invention is By expressing the protein of the present invention in vivo, (b) inserting the DNA of the present invention into cells, expressing the protein of the present invention, and then transplanting the cells into a patient. Alternatively, (c) the role of the protein of the present invention in the patient can be sufficiently or normally exerted by administering the protein of the present invention to the patient.

When the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus-associated virus vector, and then used in a conventional manner. It can be administered to warm-blooded animals. The DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.

When the protein of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it is preferable to use a protein purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. preferable.

The protein of the present invention can be used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, orally, or aseptic with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as solutions or suspensions. For example, the protein of the present invention may be mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like in the unit dosage form generally required for the practice of formulations. Can be manufactured by The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained.

Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like.

Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents. For example, it may be used in combination with an alcohol (eg, ethanol or the like), a polyalcohol (eg, propylene glycol, polyethylene glycol or the like), a nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50, or the like). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Also, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with a preservative (for example, benzyl alcohol, phenol etc.), an antioxidant, etc. The prepared injection is usually filled in an appropriate ampoule.

ベ ク タ ー The vector into which the DNA of the present invention is inserted is also formulated in the same manner as described above, and is usually used parenterally.

The preparation obtained in this way is safe and low toxic, and thus, for example, is useful for warm-blooded animals (eg, human, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow, horse, cat, dog, monkey). , Chimpanzees, etc.).

The dose of the protein of the present invention varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the protein of the present invention is orally administered for the purpose of treating rheumatoid arthritis, it is generally adult (60 kg body weight). ), The protein is 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 per day. When administered parenterally, the single dose of the protein varies depending on the administration subject, target disease, and the like. For example, for the purpose of treating rheumatoid arthritis, the protein of the present invention is administered in the form of an injection to an adult (body weight). (As 60 kg), it can be administered by injecting about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the protein per day into the affected area. It is convenient. In the case of other animals, the amount can be administered in terms of the weight per 60 kg.

塩 基 In this 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 art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.

DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threin Crythine : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Ar g: arginine His: histidine Phe: phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Sec: selenocysteine
Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine -4 (R) - carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc : N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBT: 3,4-dihydro-3-hydroxy-4-oxo-
1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide The sequence numbers in the sequence listing in the present specification indicate the following sequences. .
[SEQ ID NO: 1]
1 shows the amino acid sequence of human Neuronal cell death inducible putative kinase (NIPK).
[SEQ ID NO: 2]
1 shows the nucleotide sequence of DNA encoding human NIPK.
[SEQ ID NO: 3]
The base sequence of the primer used in Example 3 and Example 5 is shown.
[SEQ ID NO: 4]
3 shows the nucleotide sequence of a primer used in Example 3.
[SEQ ID NO: 5]
The nucleotide sequence of GenBank Accession No. NM021158 is shown.
[SEQ ID NO: 6]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 5 is shown.
[SEQ ID NO: 7]
This shows a nucleotide sequence in which A at position 251 of GenBank Accession No. NM021158 has been substituted with G.
[SEQ ID NO: 8]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 7 is shown.
[SEQ ID NO: 9]
The nucleotide sequence of GenBank Accession No. AAF30480 is shown.
[SEQ ID NO: 10]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 9 is shown.
[SEQ ID NO: 11]
The nucleotide sequence of GenBank Accession No. AAH76218 is shown.
[SEQ ID NO: 12]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 11 is shown.
[SEQ ID NO: 13]
The nucleotide sequence of GenBank Accession No. AAI58064 is shown.
[SEQ ID NO: 14]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 13 is shown.
[SEQ ID NO: 15]
The nucleotide sequence of GenBank Accession No. AAI59850 is shown.
[SEQ ID NO: 16]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 15 is shown.
[SEQ ID NO: 17]
2 shows the nucleotide sequence of GenBank Accession No. AAS06709.
[SEQ ID NO: 18]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 17 is shown.
[SEQ ID NO: 19]
The nucleotide sequence of GenBank Accession No. AF250311 is shown.
[SEQ ID NO: 20]
The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 19 is shown.
[SEQ ID NO: 21]
7 shows the base sequence of a primer used in Example 4.
[SEQ ID NO: 22]
7 shows the base sequence of a primer used in Example 4.
[SEQ ID NO: 23]
14 shows the base sequence of a primer used in Example 5.
[SEQ ID NO: 24]
14 shows the base sequence of a primer used in Example 5.
[SEQ ID NO: 25]
14 shows the base sequence of a primer used in Example 5.

Hereinafter, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto.
Example 1
In order to clarify a group of genes whose expression fluctuates due to endoplasmic reticulum stress, the following experiment was performed.

After suspending rat primary nerve cells prepared from TP-17 CD rats (Charles River Japan) in a type I collagen-coated 24-well plate (SUMILON) in a B27-containing Neurobasal medium (NB medium: GIBCO), 250,000 cells were suspended. / Well, and cultured for 3 days. (I) tunicamycin (Tnc) is added to the above cultured cells to a final concentration of 100 nM, (ii) thapsigargin (Thap) is added to the above cultured cells to a final concentration of 2 nM, or (iii) the cultured cells are After the medium was exchanged twice with a BMEM-containing DMEM medium containing no glucose, 2-deoxyglucose (2DG) was added to a concentration of 3 mM. Total RNA was extracted according to the experimental manual. As a control, instead of the above drugs (Tnc, Thap and 2DG), cells to which each medium was added (cells to which no drug was added) were used. Using these as materials, gene expression analysis was performed using an oligonucleotide microarray (Rat Genome U34A; Affymetrix).

The experimental method was in accordance with the experimental manual (Expression analysis technical manual) of Affymetrix. As a result of comparing the gene expression profiles of cells stimulated with each drug and cells not added, Neuronal cell death inducible putative kinase (NIPK) gene stimulates each endoplasmic reticulum stress ([i), (ii) and (iii) above). ] (Table 1).

実施例２
小胞体ストレスにより発現亢進したNIPK遺伝子の発現にβアミロイド刺激が影響を与えるか否かを検討する目的で、βアミロイドで刺激した細胞を用いて実施例１と同様に遺伝子発現解析を行なった。

Example 2
Gene expression analysis was performed in the same manner as in Example 1 using cells stimulated with β-amyloid in order to examine whether β-amyloid stimulation affects the expression of the NIPK gene whose expression was enhanced by endoplasmic reticulum stress.

ラ ッ ト Rat primary neurons prepared in the same manner as in Example 1 were suspended in N2-containing DMEM medium (GIBCO), seeded at 250,000 cells / well, and cultured for 4 days. After the culture, β-amyloid suspended in the above medium was added to a final concentration of 25 μM, and after 4, 8, and 24 hours, total RNA was extracted from the cells and subjected to gene analysis as in Example 1. As a control, cells to which the above medium was added instead of β-amyloid were used. NIPK expression was enhanced in cells stimulated with β-amyloid as compared to controls (Table 2).

実施例３
NIPKが細胞死に与える影響を検討する目的で、一過性形質導入を用いてNIPK遺伝子発現細胞を作製し、細胞死誘導系に供した。

Example 3
To investigate the effect of NIPK on cell death, NIPK gene-expressing cells were prepared using transient transduction and used in a cell death induction system.

Thapsigargin was added to human neuroblastoma SK-N-AS cells (purchased from ATCC) to a final concentration of 500 nM, and the cells were cultured for 8 hours. After the culture, total RNA was extracted using ISOGEN (Nippon Gene). A reverse transcription reaction was performed using the obtained total RNA as a template and an RNA PCR kit (TAKARA). For amplification of the human NIPK gene, PCR was performed using synthetic primers (SEQ ID NO: 3 and SEQ ID NO: 4) and Pfu turbo (Stratagene) as an enzyme under the following conditions (1) to (5), Specific PCR products were obtained.
(1) 95 ° C for 1 minute (2) 95 ° C for 20 seconds -68 ° C for 15 seconds -72 ° C for 1 minute three times (3) 95 ° C for 20 seconds -66 ° C for 15 seconds -72 ° C for 1 minute three times (4) 95 ° C 20 seconds-64 ° C 15 seconds-72 ° C 1 minute 35 times (5) 72 ° C 5 minutes The obtained PCR product is cloned into pCDNA3.1- / V5-His TOPO (Invitrogen), and E. coli DH5α is cloned. Transformed. The obtained colony was cultured in an LB medium, and the vector was recovered using a QUAGEN plasmid midi kit (Qiagen). This NIPK gene expression vector was transduced into SK-N-AS cells using Nucleofector (AMAXA). SK-N-AS cells were transduced with pCDNA3.1 (Invitrogen) as a control.

Each transduced cell was seeded at 7,500 cells / well in a type I collagen-coated 96-well plate (IWAKI), and after overnight culture, tunicamycin or thapsigargin was added at various concentrations and cultured for 1 or 2 days to kill cells. Induced. After the cultivation, DNA cleavage accompanying cell death was detected using CELL DEATH DETECTION ELISA ^PLUS kit (Roche). The experimental method followed the experimental manual attached to the kit.

The results are shown in FIGS.

Compared with pCDNA3.1-transduced SK-N-AS cells (control cells), DNA breakage was enhanced in the cells transduced with the human NIPK gene. This clearly shows that NIPK has a cell death promoting effect.
Example 4
For the purpose of examining the effect of NIPK on cell death, rat NIPK gene-expressing cells were prepared using a lentivirus and used in a cell death induction system.

Tunicamycin was added to rat hippocampal neurons to a final concentration of 100 nM and cultured for 8 hours. After the culture, total RNA was extracted from the cells using an RNeasy Mini kit (Qiagen). A reverse transcription reaction was performed using the obtained total RNA as a template and an RNA PCR kit (TAKARA). For amplification of the rat NIPK gene, PCR was performed under the following conditions (1) to (3) using synthetic primers (SEQ ID NO: 21 and SEQ ID NO: 22) and Pfu turbo (Stratagene) as an enzyme. Specific PCR products were obtained.
(1) 94 ° C for 1 minute (2) 94 ° C for 30 seconds -65 ° C for 30 seconds -72 ° C for 30 seconds 25 times (3) 72 ° C for 5 minutes The obtained PCR product is subjected to the ViraPower Lentiviral Directional TOPO Expression Kit (INVITROGEN) Was used to clone into the pLenti6 / V5-TOPO vector, and Escherichia coli DH5α was transformed. The obtained colony was cultured in LB medium, and the vector was recovered using QIAGEN endofree maxi kit (Qiagen). The prepared NIPK lentiviral vector or LacZ lentiviral vector and packaging Mix were simultaneously introduced into HEK293FT cells (Invitrogen) using Nucleofector (AMAXA). The medium was changed 24 hours later, and the culture supernatant was collected 48 and 72 hours after the introduction. The collected culture supernatant was centrifuged at 40,000 × g for 4 hours, and the precipitate was dissolved in a 1/100 amount of Neurobasal medium, left at 4 ° C. overnight, and then heated to 37 ° C. before use.

海 Using TP17 fetuses of SD-IGS rats (purchased from Japan Charles River), hippocampal neurons were isolated according to a standard method, and seeded at 250,000 cells / well on a poly-L-lysine-coated 24-well plate (SUMILON). Four days after the culture, 500 μl of a medium containing a lentivirus (equivalent to 50 ml of the culture supernatant) was added, and 24 hours later, the total amount was replaced with a medium containing 3 nM thapsigargin. Two days after the culture, 1/10 of the medium was added to WST-8, and the medium was cultured at 37 ° C for 90 minutes. Then, 200 µl of the medium was recovered, and the absorbance at 450 nm was measured.

The results are shown in FIG.

Compared to LacZ-transduced rat neurons (control cells), rat NIPK gene-transduced cells showed reduced mitochondrial respiratory activity. This clearly shows that NIPK has a cell death promoting effect.
Example 5
The following experiment was performed for the purpose of confirming the binding between NIPK and Akt1.

Using human NIPK (Example 3) cloned in pCDNA3.1- / V5-His TOPO (Invitrogen) vector as a template, synthetic primers (SEQ ID NO: 3 and SEQ ID NO: 23) and Pfu turbo (Stratagene) as an enzyme Was performed under the following conditions (1) to (3) to obtain a specific PCR product.
(1) 94 ° C for 5 minutes (2) 94 ° C for 30 seconds-60 ° C for 30 seconds-72 ° C for 25 times of 1 minute (3) 72 ° C for 5 minutes The obtained PCR product was used for pCDNA3.1- / V5-His TOPO (Invitrogen) and E. coli DH5α was transformed. The obtained colonies were cultured in an LB medium, and a V5-tagged human NIPK gene expression vector was recovered using a QIAGEN endofree maxi kit (Qiagen).

The Akt1 gene expression vector was prepared by the following method. Using Quick-clone human whole brain cDNA (Clontech) as a template, synthetic primers (SEQ ID NO: 24 and SEQ ID NO: 25) and Pfu turbo (Stratagene) as enzyme, the following conditions (1) to (3) PCR was carried out.
(1) 94 ° C for 5 minutes (2) 35 cycles of 94 ° C for 30 seconds -58 ° C for 30 seconds -72 ° C for 2 minutes (3) 72 ° C for 5 minutes Using 2 µl of the obtained PCR product as a template, the same synthetic primer and enzyme were used. Was performed under the following conditions (1) to (3) to obtain a specific PCR product.
(1) 94 ° C for 5 minutes (2) 94 ° C for 30 seconds -58 ° C for 30 seconds -72 ° C for 2 times 20 times (3) 72 ° C for 5 minutes The obtained PCR product was subjected to pCDNA3.1- / V5-His TOPO (Invitrogen) and E. coli DH5α was transformed. The obtained colony was cultured in LB medium, and the vector was recovered using QIAGEN endofree maxi kit (Qiagen).

V The V5-tagged human NIPK gene expression vector and Akt1 gene expression vector obtained above were simultaneously introduced into COS7 cells using Nucleofector (AMAXA). The cells were seeded on a type I collagen-coated 10 cm petri dish at 4 million cells / dish and cultured at 37 ° C. for 18 hours. After removing the medium and collecting the cells, the cells were suspended in a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NonidetP-40, 1 mM β-mercaptoethanol, protease inhibitor cocktail (ROCHE)]. The cells were disrupted with a homogenizer. After adding rec-protein G sepharose 4B (ZYMED) to the supernatant collected by centrifugation and reacting at 4 ° C for 2 hours, the supernatant was collected, and rec-protein G sepharose 4B and anti-V5 antibody (INVITROGEN) were added. The reaction was performed while rotating at 4 ° C. for 18 hours. After washing four times with 1 ml of lysis buffer, electrophoresis was performed, and Western blotting was performed using an anti-Akt1 antibody (SANTA CRUZ).

The results are shown in FIG.

AAkt1 was detected when VIP-tagged NIPK and Akt1 were simultaneously expressed, whereas Akt1 was not detected when GFP (Wako Pure Chemical) and Akt1 were simultaneously expressed (control group). This confirmed that NIPK and Akt1 bind in cells. It is suggested that the binding between NIPK and Akt1 causes a decrease in Akt1 activity and promotes neuronal cell death through suppression of the neuronal protective action of Akt1. Therefore, treatment of neurodegenerative diseases can be expected by inhibiting NIPK.

FIG. 3 is a diagram showing the amount of DNA cleavage of cells 24 hours after stimulation with tunicamycin as OD405-492. In the figure, the vertical axis indicates the absorbance, and the horizontal axis indicates the concentration of tunicamycin. -○-indicates the amount of DNA cleavage in control cells, and-□-indicates the amount of DNA cleavage in NIPK gene transduced cells. FIG. 4 is a diagram showing the amount of DNA cleavage of cells 48 hours after stimulation with tunicamycin by OD405-492. In the figure, the vertical axis indicates the absorbance, and the horizontal axis indicates the concentration of tunicamycin. -○-indicates the amount of DNA cleavage in control cells, and-□-indicates the amount of DNA cleavage in NIPK gene transduced cells. FIG. 4 is a diagram showing the amount of DNA cleavage of cells 24 hours after stimulation with thapsigargin, represented by OD405-492. In the figure, the vertical axis indicates absorbance, and the horizontal axis indicates the concentration of thapsigargin. -○-indicates the amount of DNA cleavage in control cells, and-□-indicates the amount of DNA cleavage in NIPK gene transduced cells. FIG. 4 is a diagram showing the amount of DNA cleavage of cells 48 hours after stimulation with thapsigargin, represented by OD405-492. In the figure, the vertical axis indicates absorbance, and the horizontal axis indicates the concentration of thapsigargin. -○-indicates the amount of DNA cleavage in control cells, and-□-indicates the amount of DNA cleavage in NIPK gene transduced cells. FIG. 3 is a diagram showing the respiratory activity of cells 48 hours after stimulation with thapsigargin by OD450. In the figure, the vertical axis indicates the absorbance, and the horizontal axis indicates the transgene. FIG. 4 is a diagram showing the binding between NIPK and Akt1 detected by Western blotting using an anti-Akt1 antibody after immunoprecipitation with an anti-V5 antibody.

Claims (40)

A neurodegenerative disease or diabetes comprising a compound having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a compound inhibiting the activity of a salt thereof, or a salt thereof Prophylactic and therapeutic agents. Neurodegenerative disease containing a compound 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 compound inhibiting the expression of a gene of a salt thereof, or a salt thereof Or a preventive or therapeutic agent for diabetes. A nucleotide sequence complementary or substantially complementary to the nucleotide sequence of a polynucleotide encoding a protein or a partial peptide thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or An antisense polynucleotide containing a portion. A medicament comprising the antisense polynucleotide according to claim 3. The medicament according to claim 4, which is an agent for preventing or treating a neurodegenerative disease or diabetes. An antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. A medicament comprising the antibody according to claim 6. The medicament according to claim 7, which is a prophylactic / therapeutic agent for a neurodegenerative disease or diabetes. A diagnostic agent comprising the antibody according to claim 6. The diagnostic agent according to claim 9, which is a diagnostic agent for a neurodegenerative disease or diabetes. A diagnostic agent for a neurodegenerative disease or diabetes 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. Activity of the above protein, its partial peptide, or its salt, characterized by using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, its partial peptide, or its salt. For screening a compound or a salt thereof that inhibits lipase. A protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1; A kit for screening a compound that inhibits the activity or a salt thereof. A compound or a salt thereof obtainable by using the screening method according to claim 12 or the screening kit according to claim 13. A compound that inhibits the expression of the above protein gene, characterized by using 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. Also, a method for screening a salt thereof. Inhibiting the expression of the above protein gene, 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. A kit for screening a compound or a salt thereof. A compound obtainable by using the screening method according to claim 15 or the screening kit according to claim 16, or a salt thereof. A medicament comprising the compound according to claim 14 or 17 or a salt thereof. The medicament according to claim 18, which is an agent for preventing or treating a neurodegenerative disease or diabetes. 18. A method for preventing or treating a neurodegenerative disease or diabetes, comprising administering an effective amount of the compound according to claim 14 or a salt thereof to a mammal. Neurodegeneration characterized by inhibiting the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, or inhibiting the expression of the protein gene Prevention and treatment of disease or diabetes. Use of the compound according to claim 14 or claim 17 or a salt thereof for producing a prophylactic / therapeutic agent for a neurodegenerative disease or diabetes. Cancer or rheumatic disease comprising a compound having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a compound promoting the activity of a salt thereof, or a salt thereof Prophylactic and therapeutic agents. Cancer or rheumatism comprising a compound having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a compound promoting the gene expression of a salt thereof, or a salt thereof Agent for the prevention and treatment of sexual diseases. A medicament comprising a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. The medicament according to claim 25, which is an agent for preventing or treating cancer or rheumatic disease. A pharmaceutical 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. 28. The medicament according to claim 27, which is an agent for preventing or treating cancer or rheumatic disease. A diagnostic agent for cancer or rheumatic 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. Using a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, wherein the activity of the above protein, its partial peptide or a salt thereof is used. For screening a compound or a salt thereof that promotes lipase. A protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1; A screening kit for a compound or salt thereof that promotes activity. A compound or a salt thereof obtainable by using the screening method according to claim 30 or the screening kit according to claim 31. A compound that promotes the expression of the above protein gene, characterized by using a polynucleotide having 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. Also, a method for screening a salt thereof. Promotes the expression of the protein gene, characterized by containing 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. A kit for screening a compound or a salt thereof. A compound or a salt thereof obtainable by using the screening method according to claim 33 or the screening kit according to claim 34. A medicament comprising the compound according to claim 32 or 35 or a salt thereof. 37. The medicament according to claim 36, which is a prophylactic / therapeutic agent for cancer or rheumatic disease. A method for preventing or treating cancer or rheumatic disease, which comprises administering to a mammal an effective amount of the compound according to claim 32 or 35 or a salt thereof. A cancer characterized by promoting the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, or promoting the expression of the protein gene; Prevention and treatment of rheumatic diseases. Use of the compound according to claim 32 or claim 35 or a salt thereof for the manufacture of a prophylactic / therapeutic agent for cancer or rheumatic disease.

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