JP2004323506A - MEDICINAL APPLICATION OF MIP-3alpha SUPPRESSOR AND METHOD FOR SCREENING BRAIN AND NERVE CELL-PROTECTIVE AGENT - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a brain and nerve cell-protecting agent, especially the agent for protecting brain and nerve cell traumas caused by cerebrovascular disorders, craniocerebral injuries or the like. <P>SOLUTION: The brain and nerve cell-protective agent contains a material for reducing expression and/or activity of MIP-3α (liver and activation-regulated chemokine), e.g., an antiMIP-3α antibody or an antisense nucleic acid of MIP-3α. A diagnostic agent for brain and nerve cell disorders contains the antibody or the nucleic acid coding for MIP-3α. A method for screening the material having an action for protecting brain and nerve cells by using MIP-3α and/or CCR6 (MIP-3α receptor), the nucleic acid coding for MIP-3α or CCR6 or the antibody against MIP-3α or CCR6, and a kit for the screening method, and the like, are provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel agent for protecting brain and nerve cells, particularly to a protective agent effective for prevention and treatment of cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage, or head injury. The present invention also relates to a diagnostic agent for brain / nerve cell injury such as cerebrovascular disorder and head injury. Furthermore, the present invention relates to a method for screening a substance having a brain / neuron protective effect and a kit therefor.

  Cerebrovascular disease is the second largest cause of death in Japan, the United States and Europe, and the number one cause of severe sequelae. At present, some patients with cerebral embolism and cerebral thrombosis undergo aggressive treatment (such as tPA), but due to limitations in the treatment time window, only a small percentage of patients are treated. I have. In most cases, only maintenance treatment is performed for the purpose of anti-cerebral edema and suppression of recurrence and spread (antithrombotic drug), and there is no effective drug for the purpose of cure and brain protection.

  It has been well recognized that central nervous system cells are vulnerable to ischemic stress.According to basic experiments using a cerebral ischemia model, nerve cells can be Have been said to suffer irreversible obstacles and lead to death. It is undeniable that this has caused a great sense of despair in the clinical setting of stroke. However, in recent years, vigorous research in the field of neuroscience has been able to solve various stress responses at the level of individual cells, crosstalk between neurons and glial cells, and even programmed cell death during ischemic stress. Various aspects with potential potential have been identified and are expected to lead to more aggressive therapeutic strategies.

  However, to date, a number of development products having various mechanisms of action, such as glutamate antagonists, calcium antagonists, antioxidants, etc., have been tried, but all of them have failed in clinical trials. In Japan, the antioxidant Radicut / RADICUT (registered trademark, Mitsubishi Pharma Corporation) has been approved, but this drug has not been marketed overseas and there is still no worldwide brain-protecting drug. It is the current situation.

  With the enhancement of the intensive care system for stroke patients, there is cerebral hypothermia as a brain protection therapy whose effectiveness has been reviewed in clinical practice. Cerebral hypothermia is a technique for maintaining the temperature of the brain (brain temperature) at a temperature of 32 to 35 ° C., and is gradually receiving attention because of its remarkable cerebral protective effect. However, this therapy requires intensive care facilities and 24-hour intensive management of a plurality of medical staff, so it is difficult to spread it as a general therapy.

  In order to comprehensively analyze gene expression, a microarray method in which cDNA or oligonucleotides are immobilized has been developed, and techniques for finding disease-specific changes in gene expression have become widespread, and their usefulness has been confirmed. For example, Affymetrix's GeneChip system is being widely used for diagnosis of diseases such as cancer and discovery of drug discovery target genes. In fact, discover genes and proteins whose expression is specifically upregulated or specifically suppressed in cerebral ischemia, and create therapeutic and diagnostic agents for cerebrovascular disorders from them. Attempts are being made. For example, it is known that the expression of inflammatory cytokines such as interleukin 1β (IL-1β) and tumor necrosis factor (TNFα) is increased at the infarct site during cerebral ischemia, and receptor antagonism against these cytokines is known. The drug is being studied as an acute treatment for cerebrovascular disease.

MIP-3α (LARC (liver and activation-regulated chemokine), also referred to as Exodus, ST38, CCL20; referred to as “MIP-3α” in this specification) is a CC reported from several laboratories in 1997. It is a kind of chemokine (for example, see Non-Patent Document 1). A rat homolog of this protein is isolated as a gene whose expression is increased in the cerebral cortex of a cerebral ischemia model, and its expression may increase or decrease in an allergic encephalomyelitis model in correlation with the onset of inflammation and the therapeutic effect. It has been reported (Patent Document 1, Non-Patent Document 2).
In addition, the intestinal humoral immunity in mice knocked out of CCR6, which is a MIP-3α receptor, is impaired, suggesting that the MIP-3α / CCR6 signaling system is involved in lymphocyte transport and activation. (Patent Document 2).

WO 98/49309 pamphlet WO 01/17558 pamphlet Hieshima et al., "Journal of Biological Chemistry", USA, 1997, vol. 272, p.5846-5853. Utans-Schneitz et al., "Journal of Neuroimmunol.", Netherlands, 1998, Vol. 92, p. 179-190.

Currently, treatment of cerebrovascular disorders most often requires waiting for a definitive diagnosis, such as X-ray CT or MRI imaging, which limits the treatment time window. Therefore, there is an urgent need to establish a new means for preventing and treating cerebrovascular disorders that does not require any type of disease and does not require a definitive diagnosis.
An object of the present invention is to provide a safe and excellent agent for preventing and treating cerebrovascular disorders. Another object of the present invention is to provide a simple and effective screening method for a substance having a prophylactic or therapeutic effect on cerebrovascular disorders.

The present inventors have verified the characteristics of hypothermia therapy using a rat cerebral ischemia model to achieve the above object, and thought that they will play a major role in hypothermia therapy using a microarray method. Pursued target genes. As a result, in the cerebral ischemia rat model, the expression is significantly increased during reperfusion after transient focal ischemia, and the induction of expression is significantly suppressed in parallel with the cerebral protective effect by hypothermic treatment As the MIP-3α gene.
Furthermore, the present inventors have confirmed that administration of an anti-MIP-3α antibody into the ventricle of a cerebral ischemic rat suppresses the activity of MIP-3α, thereby significantly reducing the infarct volume.
In addition, the present inventors have newly cloned the rat CCR6 gene, and performed PCR based on the nucleotide sequence of the obtained cDNA to express the CCR6 gene in brain tissue in a rat local cerebral ischemia model by hypothermia treatment. As a result of examining the presence or absence of decreased expression, it was found that the expression of the CCR6 gene was significantly increased during reperfusion after transient focal ischemia, and the expression was significantly suppressed in parallel with the cerebral protective effect of hypothermia treatment. Was. The present inventors have further studied based on these findings, and have completed the present invention.

That is, the present invention
[1] It contains a protein having the same or substantially the same amino acid sequence as the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a substance inhibiting a salt thereof. Brain and nerve cell protective agent,
[2] Contains a substance that reduces the activity of a protein or a salt thereof having an amino acid sequence identical or substantially identical to the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 Brain and nerve cell protective agent
[3] A protein or a partial peptide thereof, wherein the substance that reduces the activity contains an amino acid sequence identical or substantially identical to the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. Or the brain / nerve cell protective agent according to the above [2], which is a neutralizing antibody against a salt thereof.
[4] A brain / nerve comprising a substance that inhibits the expression of a gene encoding a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 Cytoprotective agent,
[5] a base sequence complementary to a base sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or The brain / nerve cell protective agent according to the above [4], which is a nucleic acid containing a part thereof.
[6] containing an antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence from amino acid No. 1 in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a partial peptide thereof, or a salt thereof Diagnostic agent for brain and nerve cell injury
[7] a nucleic acid comprising a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a nucleic acid comprising a part thereof Diagnostics for brain and nerve cell injury,
[8] a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, and a receptor thereof; A brain / nerve cell protective agent comprising a substance that inhibits binding,
[9] The cell stimulating activity of a receptor for a protein containing the same or substantially the same amino acid sequence as the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a salt thereof A brain / nerve cell protective agent comprising an inhibitory substance,
[10] of a gene encoding a receptor for a protein or a salt thereof having an amino acid sequence identical or substantially identical to the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 A brain / nerve cell protective agent comprising a substance that inhibits expression,
[11] The receptor according to any of the above [8] to [10], wherein the receptor is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a salt thereof. Brain and nerve cell protective agent,
[12] The use of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, a partial peptide thereof, or a salt thereof A method for screening a substance having a brain / neuron protective action,
[13] A protective effect on brain / nerve cells characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a partial peptide thereof, or a salt thereof. Screening method for substances having,
[14] containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a partial peptide thereof or a salt thereof A kit for screening for a substance having a brain / nerve cell protective action, characterized by the fact that
[15] A brain / nerve cell protective effect comprising a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a partial peptide thereof, or a salt thereof. A kit for screening a substance having
[16] Use of a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4, or 6, or a nucleic acid containing a part thereof Screening method for a substance having a brain / nerve cell protective action,
[17] Use of a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a nucleic acid containing a part thereof A method for screening a substance having a brain / nerve cell protective action,
[18] A nucleic acid containing a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a nucleic acid containing a part thereof A screening kit for a substance having a protective effect on brain and nerve cells,
[19] A nucleic acid comprising a base sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a nucleic acid containing a part thereof Screening kit for a substance having a protective effect on brain and nerve cells
[20] A method for protecting brain or nerve cells, wherein a subject in need thereof is identical or substantially identical to the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. A method comprising administering a substance that inhibits a protein containing the same amino acid sequence or a salt thereof, and [21] a method for producing a brain / nerve cell protective agent, which is represented by SEQ ID NO: 2, 4, or 6 Use of a substance that inhibits a protein or a salt thereof that contains the same or substantially the same amino acid sequence as the amino acid sequence of amino acid number 1 or later in the amino acid sequence,
Provide

  The MIP-3α of the present invention is a diagnostic marker for brain / nerve cell injury, for example, cerebrovascular disorder, and therefore, a substance that inhibits the activity of the protein, for example, a neutralizing antibody against the protein, gene expression of the protein Substances, for example, the antisense nucleic acids of the present invention are useful as brain / nerve cell protectants, particularly as brain / nerve cell protectants during cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage or head trauma. It is.

The present invention relates to a brain / nerve cell protective agent comprising a MIP-3α inhibitor. Here, “MIP-3α” refers to a protein containing the same or substantially the same amino acid sequence as the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. “MIP-3α inhibitor” refers to a substance that directly or indirectly reduces the expression and / or activity of MIP-3α. The term “brain / nerve cell protection” refers to an action of preventing (or at least delaying) brain cells and / or nerve cells that have been or may be damaged by cell injury from leading to cell death. There is no particular limitation on the cause or the like.
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 (hereinafter referred to as “MIP-3α of the present invention” or simply “ "MIP-3α") is a cell of a human or other warm-blooded animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) [eg, hepatocytes, spleen Cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, 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, cartilage Vesicles, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells, or any tissue in which those cells are present [eg, brain, 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, gall bladder, Bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeleton Muscle or the like), or may be a protein synthesized by chemical synthesis or a cell-free translation system. Alternatively, it may be a recombinant protein produced from a transformant into which a polynucleotide having the nucleotide sequence encoding the above amino acid sequence has been introduced.

The amino acid sequence substantially identical to the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 includes the amino acid number in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 At least about 50%, preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, particularly preferably at least about 90%, most preferably at least about 95% Amino acid sequences having the above homology are exemplified.
Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 include, for example, the aforementioned SEQ ID NO: 2, 4 or 6 It contains an amino acid sequence substantially the same as the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented, and contains the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. Proteins having substantially the same activity as proteins are preferred.

Examples of substantially the same activity include signal transduction activity and receptor binding activity. Substantially homogenous indicates that the properties are homogenous (eg, physiologically or pharmacologically). Therefore, it is preferable that the signal transduction activity or the receptor binding activity is equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). The quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
The signal transduction activity is measured according to a method known per se. For example, stimulatory activity on CCR6-expressing cells (eg, increase of intracellular cAMP concentration via CCR6, release of intracellular Ca ²⁺ , production of inositol phosphate, fluctuation of cell membrane potential, phosphorylation or dephosphorylation of intracellular protein) , C-fos activation, pH lowering, etc. promoting or suppressing activities) are measured by a known method. Specifically, a plasmid into which DNA encoding CCR6 has been inserted can be used, for example, in Chinese hamster ovary cells (eg, CHO-K1 cells; Journal of Experimental Medicine (J. Exp. Med.), Volume 108, 945, 1958), and cultured CHO-K1 cells in which CCR6 was highly expressed, in the presence of MIP-3α, and signal transduction activity via CCR6 expressed on the cell membrane (for example, cell Increase or decrease of intracellular cAMP concentration, release of intracellular Ca ²⁺ , production of inositol phosphate, fluctuation of cell membrane potential, phosphorylation or dephosphorylation of intracellular protein, activation of c-fos, decrease of pH, etc. Activity or inhibitory activity) is measured by a known method.
The receptor binding activity can be measured according to a method known per se. For example, (1) contacting a labeled MIP-3α protein with a cell expressing CCR6 or a membrane fraction of the cell, and measuring a binding amount of the labeled MIP-3α protein to the cell or the membrane fraction; 2) The labeled MIP-3α protein is brought into contact with the protein expressed on the cell membrane by culturing a transformant containing DNA encoding the CCR6 protein, and the labeled MIP-3α cell or the membrane fraction is contacted. The receptor binding activity can be measured by measuring the amount of binding per minute.
The DNA encoding CCR6 used in the above-mentioned activity measurement includes a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10 or SEQ ID NO: 14. (See, for example, Baba et al., Journal of Biological Chemistry (J. Biol. Chem.) 272, 14893-14898, 1997).

The MIP-3α of the present invention includes, for example, (i) one or two or more (preferably, An amino acid sequence in which about 1 to 30, more preferably about 1 to 10, and still more preferably a number (1 to 5) amino acids are deleted, (ii) represented by SEQ ID NO: 2, 4, or 6 One or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably, about 1 to 5) amino acids are included in the amino acid sequence of amino acid number 1 or later in the amino acid sequence. (Iii) one or more (preferably about 1 to 30, more preferably 1 to 30 amino acid sequences) About 1 to 10 More preferably, an amino acid sequence in which several (1 to 5) amino acids have been inserted; An amino acid sequence in which two or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably about 1 to 5) amino acids are substituted with another amino acid, or (v) So-called muteins such as proteins containing an amino acid sequence obtained by combining them are also included.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

In the protein in this specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) in accordance with the convention of peptide labeling. MIP-3α of the present invention including a protein containing the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 has a carboxyl group (-COOH) at the C-terminus and a carboxylate at the C-terminus. It may be any of (—COO ⁻ ), amide (—CONH ₂ ), and ester (—COOR).
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 And 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 aralkyl groups, pivaloyloxymethyl groups and the like are used.
When the MIP-3α of the present invention has a carboxyl group (or carboxylate) other than at the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the MIP-3α of 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 (for example, 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.
Preferred specific examples of the MIP-3α of the present invention include, for example, a mature human MIP-3α consisting of the amino acid sequences represented by amino acid numbers 1 to 70 in the amino acid sequence represented by SEQ ID NO: 2, represented by SEQ ID NO: 4. Mature rat MIP-3α comprising the amino acid sequence represented by amino acid numbers 1 to 71 in the amino acid sequence represented, or mature mouse MIP comprising the amino acid sequence represented by amino acid numbers 1 to 70 in the amino acid sequence represented by SEQ ID NO: 6 -3α and the like.

The partial peptide of MIP-3α of the present invention is the above-mentioned peptide having the partial amino acid sequence of MIP-3α of the present invention, and preferably has substantially the same activity as MIP-3α of the present invention. Any one may be used. Substantially the same activity includes, for example, signal transduction activity, receptor binding activity, epitope activity and the like. Substantially the same is as defined above.
For example, a peptide having a partial amino acid sequence of at least 20 or more, preferably 30 or more, more preferably 40 or more, more preferably 50 or more of the constituent amino acid sequences of MIP-3α of the present invention is used.
In the partial peptide of MIP-3α of the present invention, one or more (preferably, about 1 to 10, more preferably, about 1 to 5) amino acids in the amino acid sequence are deleted. Or 1 or 2 or more (preferably, about 1 to 20, more preferably, about 1 to 10, and more preferably, about 1 to 5) amino acids are added to the amino acid sequence, 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 inserted into the amino acid sequence, or the amino acid sequence thereof. One or more (preferably about 1 to 10, more preferably about (1 to 5)) amino acids therein may be substituted with another amino acid.

In the partial peptide of MIP-3α of the present invention, the C-terminal may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Here, as R in the ester, those similar to those described above for MIP-3α of the present invention can be mentioned. When the partial peptide of MIP-3α of the present invention has a carboxyl group (or carboxylate) at a position other than the C-terminus, the carboxyl group amidated or esterified is also included in the partial peptide. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
Further, like the above-described MIP-3α of the present invention, the partial peptide has the amino group of the N-terminal amino acid residue (eg, methionine residue) protected with a protecting group, and the N-terminal has Glutamine residues generated by cleavage in the body are pyroglutamine-oxidized, those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those in which a sugar chain is bonded, such as a so-called glycopeptide Complex peptides and the like are also included.
The partial peptide of MIP-3α of the present invention can also be used as an antigen for producing an antibody.

  MIP-3α or its partial peptide of the present invention may be in a free form or in a salt form. Examples of the salt include a physiologically acceptable salt with an acid or a base, 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) 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 MIP-3α or a salt thereof of the present invention can be produced from a human or other warm-blooded animal cell or tissue described above by a protein purification method known per se. Specifically, after homogenizing human or other warm-blooded animal tissues or cells, extraction with an acid or the like is performed, and the extract is purified by a combination of chromatography such as reverse phase chromatography and ion exchange chromatography. Can be isolated.

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

  The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, and dimethyl sulfoxide. Examples thereof include 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. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.

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

  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. Also, 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.

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

The partial peptide of MIP-3α of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving MIP-3α of 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 of MIP-3α of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide. be able to. 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., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd. (1975)
(iv) Haruaki Yajima and Shunpei Sakakibara, Biochemistry Laboratory Course 1, Protein Chemistry IV, 205, (1977)
(v) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten After the reaction, normal purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. And the partial peptide can be purified and isolated. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide obtained by a salt is a known method or analogous method It can be converted into a free form or another salt by a method.

  Furthermore, the MIP-3α or the partial peptide thereof of the present invention can be obtained by culturing a transformant containing the DNA encoding the MIP-3α of the present invention or the partial peptide thereof, and culturing the transformant containing the MIP-3α or the partial peptide of the present invention. It can also be produced by separating and purifying the partial peptide. Alternatively, MIP-3α or its partial peptide of the present invention is subjected to in vitro translation using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, Escherichia coli lysate, etc., using RNA corresponding to the DNA as a template. Can also be synthesized. Alternatively, the DNA can also be synthesized using the DNA as a template by using a cell-free transcription / translation system containing an RNA polymerase.

The polynucleotide encoding MIP-3α of the present invention or a partial peptide thereof may be any polynucleotide as long as it contains the above-described nucleotide sequence encoding MIP-3α of the present invention or a partial peptide thereof. Preferably it is DNA. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the aforementioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the above-mentioned cells / tissues.

  Examples of the DNA encoding the MIP-3α of the present invention include a DNA containing the nucleotide sequence represented by SEQ ID NO: 1, 3 or 5, or a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1, 3 or 5. It contains a nucleotide sequence that hybridizes under high stringent conditions, and is substantially the same as a protein containing the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 described above. Any DNA may be used as long as it encodes a protein having activity.

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

  The DNA encoding the partial peptide of MIP-3α of the present invention has the same or substantially the same amino acid sequence as a part of the amino acid sequence from amino acid number 1 onward in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6. Any DNA can be used as long as it contains a coding base sequence and encodes a peptide having substantially the same activity (eg, signal transduction activity, receptor binding activity, epitope activity, etc.) as the above-mentioned MIP-3α of the present invention. It may be something. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissues, cDNA library derived from the above-described cells / tissues, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, amplification can be performed directly by RT-PCR using an mRNA fraction prepared from the cells and tissues described above.

Specifically, the DNA encoding the partial peptide of MIP-3α of the present invention includes, for example, (1) a DNA having a partial nucleotide sequence of a DNA having a nucleotide sequence represented by SEQ ID NO: 1, 3, or 5, Or (2) a protein having a nucleotide sequence that hybridizes under high stringent conditions to DNA having the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5, and substantially comprising a protein containing an amino acid sequence encoded by the DNA. DNA encoding a peptide having the same activity (eg, signal transduction activity, receptor binding activity, epitope activity, etc.) is used.
DNAs capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 1, 3 or 5 have the same significance as described above.
The same hybridization method and high stringency conditions as described above are used.

  The DNA encoding the MIP-3α or its partial peptide of the present invention may be amplified by the PCR method using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein or the peptide, or may be DNA incorporated into an appropriate vector. Can be cloned by hybridizing with a DNA fragment encoding a part or the entire region of MIP-3α of the present invention or a labeled synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

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

  The cloned DNA can be used as it is depending on the purpose, or after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation 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 containing the DNA encoding the MIP-3α or the partial peptide thereof of the present invention can be obtained by, for example, (a) cutting out a DNA fragment of interest from the DNA encoding the MIP-3α of the present invention or the partial peptide thereof, B) It can be produced by ligating the DNA fragment downstream of a promoter in an appropriate expression vector.
Examples of expression vectors include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13); Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194); Yeast-derived plasmids (eg, pSH19, pSH15); Animal viruses such as retrovirus, vaccinia virus and baculovirus; pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.
As the promoter, any promoter may be used as long as it is appropriate for the host used for gene expression.
For example, when the host is an animal cell, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter and the like are used. Among them, CMV promoter, SRα promoter and the like are preferable.
When the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter and the like are preferable.
When the host is a bacterium belonging to the genus Bacillus, an SPO1 promoter, an SPO2 promoter, a penP promoter and the like are preferable.
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.

As the expression vector, in addition to the above, those 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, if desired, may be used. it can. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r), neomycin resistant gene (hereinafter sometimes abbreviated as Neo ^r, G418 resistance). In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can be selected using a thymidine-free medium.
If necessary, a signal sequence suitable for the host is replaced with a native signal sequence (for example, the amino acid sequence before amino acid number-1 in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6). May be added to the N-terminal side of MIP-3α of the present invention or a partial peptide thereof. When the host is Escherichia, PhoA signal sequence, OmpA signal sequence, etc .; When the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc .; When the host is yeast When the host is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. are used.

The transformant containing "the DNA encoding MIP-3α of the present invention or its partial peptide" obtained as described above is used to transform a host with an expression vector containing the DNA according to a known method. Can be manufactured. Here, the expression vectors include those described above.
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12 DH1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecular Biology)]. Biology, 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc. Used.
Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984)]. )] Is used.
Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036P, and so on. Used.

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

Transformation can be performed according to known methods depending on the type of host.
The genus Escherichia is described, for example, in Procesings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 (1972) and Gene ( Gene, Vol. 17, 107 (1982).
Bacillus spp. Can be transformed, for example, according to the method described in Molecular & General Genetics, Volume 168, 111 (1979).
Yeasts are described, for example, in Methods in Enzymology, Vol. 194, 182-187 (1991), Procesings of the National Academy of Sciences of the USA (1991). Proc. Natl. Acad. Sci. USA), 75, 1929 (1978).
Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
Animal cells are described in, for example, Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha) and Virology, 52, 456 (1973).

Culture of the transformant can be performed according to a known method depending on the type of the host.
For example, when a transformant whose host is Escherichia or Bacillus is cultured, a liquid medium is preferably used as the culture medium. In addition, the medium preferably contains a carbon source, a nitrogen source, an inorganic substance, and the like necessary for growing the transformant. Here, 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, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, a yeast extract, vitamins, a growth promoting factor, and the like may be added to the medium. The pH of the medium is preferably about 5-8.
As a medium for culturing a transformant whose host is Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Mill) Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as, for example, 3β-indolylacrylic acid may be added to the medium to make the promoter work efficiently.
Culture of the transformant whose host is Escherichia is usually performed at about 15 to 43 ° C. for about 3 to 24 hours. If necessary, ventilation or stirring may be performed.
Culture of a transformant whose host is a bacterium belonging to the genus Bacillus is usually performed at about 30 to 40 ° C. for about 6 to 24 hours. If necessary, ventilation or stirring may be performed.
As a medium for culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, KL et al., Processings of the National Academy of Sciences of Science] Natl. Acad. Sci. USA, 77, 4505 (1980)] and an SD medium containing 0.5% casamino acid [Bitter, GA et al., Processings of the National. Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours. If necessary, ventilation or stirring may be performed.
As a culture medium for culturing an insect cell or a transformant whose host is an insect, for example, the addition of 10% bovine serum inactivated to Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) What added the thing suitably is used. The pH of the medium is preferably between about 6.2 and 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days. Aeration and stirring may be performed as necessary.
Examples of a medium for culturing a transformant whose animal host is an animal cell 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, Vol. 73, 1 (1950)] and the like are used. The pH of the medium is preferably about 6-8. The cultivation is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours. Aeration and stirring may be performed as necessary.
As described above, the MIP-3α of the present invention or a partial peptide thereof can be produced in the cell, cell membrane or extracellular cells of the transformant.

The MIP-3α of the present invention or its partial peptide can be separated and purified from the above culture by, for example, the following method.
When extracting MIP-3α or its partial peptide of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culture, and suspended in an appropriate buffer. A method of obtaining a crude protein (peptide) extract by centrifugation or filtration after disrupting cells or cells by freeze-thawing or the like 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 (peptide) is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected.
The protein (peptide) contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and use of difference in hydrophobicity such as reverse phase high performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.

When the thus obtained protein (peptide) is 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 a method analogous thereto Can be converted to a free form or another salt.
The protein (peptide) produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
The presence of the thus produced MIP-3α or its partial peptide of the present invention can be measured by an enzyme immunoassay using a specific antibody against them or Western blotting.

  Further, the MIP-3α or its partial peptide of the present invention can be prepared by using an RNA corresponding to the DNA encoding the above-described MIP-3α or its partial peptide as a template, as a rabbit reticulocyte lysate, a wheat germ lysate, an E. coli lysate, or the like. Can also be synthesized by in vitro translation using a cell-free protein translation system consisting of Alternatively, it can also be synthesized using a DNA encoding the protein of the present invention or its partial peptide as a template, using a cell-free transcription / translation system further containing an RNA polymerase.

  As shown in the Examples below, MIP-3α significantly increases its gene expression during cerebral ischemia, and significantly decreases in correlation with the therapeutic effect of hypothermia. Furthermore, the administration of a neutralizing antibody against MIP-3α significantly reduces the infarct volume in a cerebral ischemia model. Therefore, the MIP-3α inhibitor of the present invention has a protective effect on brain and nerve cells, particularly cerebrovascular disorder and head. It has a protective effect on brain and nerve cells during trauma. Therefore, the substance is useful for prevention and treatment of diseases in which protecting brain cells and nerve cells from cell injury is effective for prevention and treatment. Such diseases include, for example, cerebrovascular disorders (eg, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, etc.) and head trauma, as well as cerebral disorders during resuscitation after cardiac arrest caused by brain / nerve cell damage. Brain dysfunction before and after brain surgery, hypoxia, hypoglycemia, trauma to the brain or spinal cord, drug poisoning, gas poisoning, diabetes, antitumor agent administration, nervous system disorders due to alcohol, etc .; old age such as Alzheimer's disease Parkinson's disease; Huntington's chorea; Prion disease; Amyotrophic lateral sclerosis; Spinocerebellar degeneration; and AIDS. The MIP-3α inhibitor of the present invention is particularly useful for the prevention and treatment of cerebrovascular disorders (eg, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, etc.) and head trauma.

  Accordingly, a MIP-3α inhibitor (these substances may form a salt, and specific examples of the salt include the same as the above-described salt of MIP-3α of the present invention) is necessary. After mixing with a pharmacologically acceptable carrier to form a pharmaceutical composition, it can be used as a brain / nerve cell protective agent.

  Among the MIP-3α inhibitors, examples of the substance that reduces the activity of MIP-3α include a neutralizing antibody against MIP-3α or a partial peptide thereof, an antagonist of MIP-3α or its receptor, and inhibition of signal transmission. Substances, substances that inhibit the expression of receptors, substances that stimulate the inactivation mechanism of MIP-3α, substances that increase the degradation or metabolism of MIP-3α, and substances that activate them. It is not limited to.

In a preferred embodiment, the substance that reduces the activity of MIP-3α is an antibody against the above-described MIP-3α of the present invention, a partial peptide thereof, or a salt thereof.
The antibody against MIP-3α or its partial peptide or a salt thereof of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize them. The antibody may be the antibody molecule itself, or a single-chain antibody in which a light chain and a heavy chain of a variable region are linked by a linker, or F (ab ′) ₂ , Fab ′, or a Fab fraction of the antibody molecule. (ScFv) or the like.

  The antibody against the MIP-3α of the present invention or a partial peptide thereof or a salt thereof (hereinafter sometimes referred to simply as “MIP-3α of the present invention” inclusive of these in the description of the antibody) is the present invention. Using MIP-3α as an antigen, it can be produced according to a known method for producing an antibody or antiserum.

[Preparation of monoclonal antibody]
(A) Preparation of Monoclonal Antibody-Producing Cell The MIP-3α of the present invention is administered to a non-human warm-blooded animal to a site where the antibody can be produced by administration, 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 2 to 6 weeks, for a total of about 2 to 10 times. Examples of the non-human warm-blooded animal to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained therein. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of 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 an antigen protein (peptide) is adsorbed directly or together with a carrier. A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (an anti-mouse immunoglobulin antibody is used when cells used for cell fusion are mice) or protein A A method of adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a protein (peptide) labeled with a radioactive substance or an enzyme, and detecting a monoclonal antibody bound to the solid phase Is mentioned.
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 the 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 against MIP-3α of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immunizing antigen (protein or peptide) itself or a complex thereof with a carrier protein is formed, and a warm-blooded animal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting the contents 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 carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by crosslinking the carrier. If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin, etc. are used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ~ 5 is used.
Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
The polyclonal antibody can be collected from the blood, ascites, or the like, preferably from the blood of a warm-blooded animal immunized by the above method.
The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.

The brain / nerve cell protective agent containing the antibody of the present invention has low toxicity, and is used as it is as a liquid or as a mixture with a pharmacologically acceptable carrier to obtain a pharmaceutical composition in an appropriate dosage form. It can be administered orally or parenterally (eg, intra-articular administration) to other mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.).
Here, as the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used, and excipients, lubricants, binders, disintegrants in solid preparations, solvents in liquid preparations , A solubilizing agent, a suspending agent, a tonicity agent, a buffer, a soothing agent and the like. If necessary, formulation additives such as preservatives, antioxidants, coloring agents and sweeteners can also be used.

Preferred examples of the excipient include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, dextrin, pullulan And light anhydrous silicic acid, synthetic aluminum silicate and magnesium aluminate metasilicate.
Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Preferred examples of the binder include pregelatinized starch, sucrose, gelatin, acacia, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxy Propylmethylcellulose, polyvinylpyrrolidone and the like.
Preferable examples of the disintegrant include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, sodium carboxymethyl starch, light anhydrous silicic acid, low-substituted hydroxypropylcellulose and the like.
Preferred examples of the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
Preferred examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate. And the like.
Preferred examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol and polyvinyl alcohol. Hydrophilic polymers such as pyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose and hydroxypropylcellulose; polysorbates, and polyoxyethylene hydrogenated castor oil.
Preferred examples of the tonicity agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
Preferred examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate.
Preferred examples of the soothing agent include benzyl alcohol and the like.
Preferable examples of the preservative include p-hydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Suitable examples of the antioxidant include sulfite, ascorbate and the like.
Preferred examples of the coloring agent include water-soluble edible tar dyes (eg, edible dyes such as edible red Nos. 2 and 3, edible yellows 4 and 5, edible blues 1 and 2, water-insoluble lake dyes ( Examples include the water-soluble edible tar dye aluminum salt, and the like, and natural dyes (eg, β-carotene, chlorophyll, and red bean).
Preferred examples of the sweetener include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia and the like.

Examples of the dosage form of the pharmaceutical composition include oral preparations such as tablets, capsules (including soft capsules and microcapsules), granules, powders, syrups, emulsions and suspensions; and injections (eg, subcutaneous injections). Agents, intradermal injections, intravenous injections, intramuscular injections, intraperitoneal injections, intraarticular injections, etc.), external preparations (eg, nasal preparations, transdermal preparations, ointments, etc.), suppositories Parenteral preparations such as (eg, rectal suppositories, vaginal suppositories, etc.), pellets, drops, sustained-release preparations (eg, sustained-release microcapsules, etc.), which are orally or parenterally, respectively. Can be administered safely.
The pharmaceutical composition can be produced by a method commonly used in the technical field of formulation, for example, a method described in the Japanese Pharmacopoeia and the like. Hereinafter, a specific method for producing the preparation will be described in detail. The content of the inhibitor of the transcriptional regulatory factor of the present invention in the pharmaceutical composition varies depending on the dosage form, dosage of the compound, and the like.

  For example, oral preparations include, as an active ingredient, excipients (eg, lactose, sucrose, starch, D-mannitol, etc.), disintegrants (eg, carboxymethylcellulose calcium, etc.), binders (eg, pregelatinized starch, gum arabic). , Carboxymethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.) or a lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000, etc.) and the like, and then compression-molded, and if necessary, taste masking, enteric coating or the like. For the purpose of sustainability, it is produced by coating using a coating base by a method known per se.

Examples of the coating base include a sugar coating base, a water-soluble film coating base, an enteric film coating base, and a sustained release film coating base.
As the sugar-coating base, sucrose is used, and one or more selected from talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, carnauba wax and the like may be used in combination.
Examples of the water-soluble film coating base include cellulosic polymers such as hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, and methylhydroxyethylcellulose; polyvinyl acetal diethylaminoacetate, aminoalkyl methacrylate copolymer E [Eudragit E (trade name), Rohm Pharma Co., Ltd.], synthetic polymers such as polyvinylpyrrolidone, and polysaccharides such as pullulan.
Examples of the enteric film coating base include cellulosic polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, and cellulose acetate phthalate; methacrylic acid copolymer L [Eudragit L (trade name); Acrylic polymers such as Rohm Pharma Co., Ltd., methacrylic acid copolymer LD [Eudragit L-30D55 (trade name), Rohm Pharma Co., Ltd.] and methacrylic acid copolymer S [Eudragit S (trade name), Rohm Pharma Co.]; Natural products such as shellac are exemplified.
Examples of the sustained-release film coating base include cellulosic polymers such as ethyl cellulose; aminoalkyl methacrylate copolymer RS (Eudragit RS (trade name), Rohm Pharma Co., Ltd.); ethyl acrylate / methyl methacrylate copolymer suspension Acrylic polymers such as suspensions (Eudragit NE (trade name), Rohm Pharma Co., Ltd.) and the like.
The above-mentioned coating bases may be used by mixing two or more kinds thereof at an appropriate ratio. Further, at the time of coating, a light-shielding agent such as titanium oxide, iron sesquioxide and the like may be used.

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

  The dosage of the brain / nerve cell protective agent containing the antibody of the present invention varies depending on the administration subject, target disease, symptoms, administration route, and the like, and is used, for example, for the treatment and prevention of cerebrovascular disorders in adults. In such a case, the amount of the antibody of the present invention per dose is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, and more preferably about 0.1 to 5 mg / kg body weight. It is convenient to administer by powder inhalant about 1 to 5 times a day, preferably about 1 to 3 times a day. 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.

  As the substance that inhibits the expression of MIP-3α, preferably, a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence encoding MIP-3α of the present invention or a part thereof is used. The nucleic acid containing the nucleotide sequence complementary to the nucleotide sequence encoding MIP-3α of the present invention or a part thereof (hereinafter, sometimes abbreviated as “antisense nucleic acid of the present invention”) includes the MIP of the present invention. Translation of said protein from RNA which has a base sequence completely complementary to or substantially complementary to -3α or a part of said complementary base sequence and encodes MIP-3α of the present invention What is necessary is just to have the effect | action which suppresses. As the “substantially complementary nucleotide sequence”, a nucleotide sequence encoding MIP-3α of the present invention and a nucleotide sequence capable of hybridizing under physiological conditions of a cell expressing the protein, more specifically, About 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more, of the complementary sequence of the base sequence encoding MIP-3α of the present invention or a partial base sequence thereof. Base sequences having the above homology are exemplified.

  The antisense nucleic acid of the present invention can be designed and synthesized based on the cloned or determined nucleotide sequence information of the nucleic acid encoding MIP-3α of the present invention. Such a nucleic acid can inhibit the replication or expression of the gene encoding MIP-3α of the present invention. That is, the antisense nucleic acid of the present invention can hybridize to RNA transcribed from the gene encoding MIP-3α of the present invention, and inhibits mRNA synthesis (processing) or function (translation into protein). be able to.

The length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the translation of the MIP-3α of the present invention is inhibited by the hybridization of the antisense nucleic acid. May be the entire sequence or partial sequence of the RNA encoding MIP-3α, and may be as short as about 15 bases and as long as the entire sequence of mRNA or early transcript. Considering the ease of synthesis and the problem of antigenicity, oligonucleotides consisting of about 15 to about 30 bases are preferred, but not limited thereto. Specifically, for example, the 5 'end hairpin loop, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, protein coding region of the gene encoding MIP-3α of the present invention , The ORF translation initiation codon, the 3 ′ untranslated region, the 3 ′ palindrome region, and the 3 ′ end hairpin loop may be selected as target regions, but any region within the gene may be selected as a target. For example, it is also preferable to use the intron portion of the gene as the target region.
Further, the antisense nucleic acid of the present invention not only hybridizes with the mRNA or the initial transcript encoding MIP-3α of the present invention to inhibit translation into a protein, but also double-stranded MIP-3α of the present invention. It may bind to a gene encoding -3α to form a triplex, thereby inhibiting RNA transcription.

  Antisense nucleic acids include deoxyribonucleotides containing 2-deoxy-D-ribose, ribonucleotides containing D-ribose, other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or non-nucleotides. Other polymers having a 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 pair of bases as found in DNA or RNA (Including a nucleotide having a configuration permitting attachment of a ring or base). 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, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), Such as proteins (nucleases, nuclease inhibitors, toxic , Antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides) having side chain groups, interacting compounds (for example, 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., where one or more hydroxyl groups have been replaced by halogens, aliphatic groups, etc., or by functional groups such as ethers, amines, etc. It may have been converted.

  The antisense nucleic acid 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. Many such 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.

  Antisense nucleic acids can contain altered or modified sugars, bases, or linkages and can be provided in special forms such as liposomes, microspheres, applied or added by gene therapy. It could be given in 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.

  A ribozyme capable of specifically cleaving mRNA or gene early transcript encoding MIP-3α of the present invention inside the coding region (including the intron portion in the case of the initial transcript) is also an antisense nucleic acid of the present invention. May be included. The term "ribozyme" refers to RNA having an enzymatic activity for cleaving a nucleic acid. Recently, it has been revealed that an oligo DNA having the nucleotide sequence of the enzyme active site also has a nucleic acid cleaving activity. In this document, the term is used as a concept including DNA as long as it has a sequence-specific nucleic acid cleavage activity. The most versatile ribozymes include self-splicing RNAs found in infectious RNAs such as viroids and viruses, and hammerhead and hairpin types are known. The hammerhead type exerts enzymatic activity at about 40 bases, and converts several bases at both ends (about 10 bases in total) adjacent to the hammerhead structure into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the further advantage that it does not attack genomic DNA since it uses only RNA as substrate. When the mRNA encoding MIP-3α of the present invention has a double-stranded structure by itself, the target sequence can be determined by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid capable of specifically binding to RNA helicase. Can be single-stranded [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Further, when the ribozyme is used in the form of an expression vector containing a DNA encoding the ribozyme, a hybrid ribozyme in which a sequence modified from tRNA is further linked in order to promote the transfer of a transcript to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

Double-stranded oligo RNA (small interfering RNA; siRNA) complementary to a partial sequence in the coding region of mRNA or gene early transcription product of MIP-3α of the present invention (including an intron portion in the case of early transcription product) Can also be included in the antisense nucleic acids of the invention. When a short double-stranded RNA is introduced into cells, mRNA complementary to the RNA is degraded, a phenomenon called so-called RNA interference (RNAi), which has long been known in nematodes, insects, plants, and the like. Recently, it has been confirmed that this phenomenon also occurs in mammalian cells [Nature, 411 (6836): 494-498 (2001)], and is attracting attention as an alternative technique to ribozymes.
The antisense oligonucleotide and ribozyme of the present invention determine the target region of mRNA or early transcript based on the cDNA sequence encoding MIP-3α or genomic DNA sequence information of the present invention, and a commercially available automatic DNA / RNA synthesizer (Applied Biosystems, Beckman, etc.), and can be prepared by synthesizing a sequence complementary thereto. The siRNA is obtained by synthesizing the sense strand and the antisense strand with a DNA / RNA automatic synthesizer, denaturing them in an appropriate annealing buffer at, for example, about 90 to about 95 ° C. for about 1 minute, and then denaturing about 30 to 30 ° C. It can be prepared by annealing at about 70 ° C. for about 1 to about 8 hours. Alternatively, a longer double-stranded polynucleotide can be prepared by synthesizing complementary oligonucleotide chains so as to alternately overlap each other, annealing them, and ligating them with a ligase.

The brain / nerve cell protective agent containing the antisense nucleic acid of the present invention can be formulated by a method known per se, as in the case of the antibody of the present invention.
In addition, for example, after inserting the antisense nucleic acid of the present invention alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, a human or mammal (eg, rat, Rabbits, sheep, pigs, cows, cats, dogs, monkeys, and the like). The antisense nucleic acid 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, and administered with 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 antisense nucleic acid of the present invention is formulated alone or together with a carrier such as liposome (injection), and injected into the vein, subcutaneous or joint cavity. It may be administered internally or the like.
The dose of the antisense nucleic acid varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense nucleic acid of the present invention is administered for the purpose of treating acute cerebrovascular disease, generally, In an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense nucleic acid is administered per day.

  Further, the antisense nucleic acid of the present invention can also be used as a diagnostic nucleic acid probe for examining the presence of the nucleic acid encoding MIP-3α of the present invention in a tissue or a cell and the state of expression thereof.

The present invention also provides a brain / nerve cell protective agent containing a substance that inhibits the binding between MIP-3α and its receptor or a substance that inhibits the signal transduction activity of MIP-3α receptor. Examples of such a substance include MIP-3α or an antagonist of its receptor.
The receptor for MIP-3α is not particularly limited as long as it specifically binds to MIP-3α and exhibits a signal information transmitting action, and examples thereof include CCR6 which is a known MIP-3α receptor. . “CCR6 of the present invention” is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10, or a salt thereof. Here, the “protein containing substantially the same amino acid sequence” has the same meaning as that described in detail in the above-mentioned MIP-3α of the present invention.

  Examples of the substance that inhibits the binding between MIP-3α and its receptor include the following MIP-3α of the present invention or a partial peptide thereof or a salt thereof, and / or a MIP-3α receptor (for example, CCR6 of the present invention) Alternatively, it can be obtained by a screening method using a partial peptide thereof or a salt thereof.

[Screening of substances with protective effects on brain and nerve cells]
The present invention provides the MIP-3α of the present invention or a partial peptide thereof or a salt thereof (hereinafter, in the description of the present screening, these may be abbreviated simply as “MIP-3α of the present invention”) and / or MIP-3α receptor (for example, CCR6 of the present invention) or a partial peptide thereof or a salt thereof (hereinafter, in the description of the present screening, these are sometimes abbreviated simply as “CCR6 of the present invention”) A method for screening a compound or a salt thereof that inhibits the activity of MIP-3α (for example, signal transduction activity, receptor binding activity, etc.) of the present invention is provided.
More specifically, for example, (i) measuring and comparing the binding activity between the MIP-3α of the present invention and the CCR6 of the present invention in the presence and absence of a test compound; The MIP of the present invention, characterized by measuring and comparing the signal transduction activity in a cell having the ability to produce the CCR6 of the present invention in the presence of 3α and the presence of the MIP-3α of the present invention and a test compound. A method for screening a compound or a salt thereof that inhibits the activity of -3α is used.
In the above screening method, for example, in the cases (i) and (ii), the signal transduction activity and the receptor binding activity are measured by a method known per se.
Specifically, in the presence and absence of a test compound, for example, a CCR6-expressing cell [a DNA encoding the CCR6 of the present invention, for example, a DNA containing the base sequence represented by SEQ ID NO: 7 or 9, Alternatively, a DNA containing the nucleotide sequence represented by nucleotide numbers 343 to 1440 in the nucleotide sequence represented by SEQ ID NO: 13, or a DNA containing the nucleotide sequence represented by SEQ ID NO: 7 or 9 or SEQ ID NO: 13 A DNA which can hybridize under high stringent conditions to a DNA containing the nucleotide sequence represented by nucleotide numbers 343 to 1440 in the nucleotide sequence shown, wherein the DNA is SEQ ID NO: 8 or 10, or SEQ ID NO: DNA encoding a protein having substantially the same activity as the protein containing the amino acid sequence represented by No. 14 (this And “highly stringent conditions” and “substantially the same activity” have the same meanings as described above) are inserted into the same expression vector as exemplified in the description of MIP-3α of the present invention. To a host cell (eg, CHO-K1 cell, etc.) similar to that exemplified in the description of MIP-3α of the present invention, using the same transformation method. Cell signaling activity through CCR6 expressed on the cell membrane (e.g., increase or decrease of intracellular cAMP concentration, release of intracellular Ca2 ⁺ , production of inositol phosphate, cell membrane The fluctuation of potential, the activity of promoting or suppressing the phosphorylation or dephosphorylation of intracellular proteins, the activation of c-fos, the reduction of pH, etc.) are measured by known methods.
Alternatively, (1) the labeled MIP-3α of the present invention is brought into contact with the CCR6 of the present invention in the presence and absence of a test compound, and the amount of labeled MIP-3α bound to the receptor is measured and compared. (2) The labeled MIP-3α of the present invention is converted to a cell containing the CCR6 of the present invention (eg, immune cells such as lymphocytes known to express CCR6, dendritic cells, astrocytes, etc.) Alternatively, (3) the labeled MIP-3α of the present invention, which is brought into contact with the membrane fraction of the cell and measures the amount of binding of the labeled MIP-3α to the cell or the membrane fraction, is compared with CCR6 encoding Culturing a transformant (eg, the above-described transformed cell) containing the DNA to be brought into contact with the protein expressed on the cell membrane, and then labeling the cell of labeled MIP-3α or By measuring the amount of binding compared to the membrane fraction.

The MIP-3α of the present invention may be externally added as an isolated protein to a system containing the CCR6 of the present invention or a cell capable of producing the same, or the above-described MIP-3α of the present invention may be added. A host (transformant) transformed with a vector containing the encoding DNA may be used. As the host, for example, animal cells such as mouse ATDC5 cells and CHO cells are preferably used. For the screening, for example, a transformant that secretes MIP-3α of the present invention extracellularly by culturing by the above-described method is preferably used. The method for culturing cells capable of expressing MIP-3α 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 signal transduction activity or receptor binding activity in the presence of the test compound is reduced by about 20% or more, preferably 30% or more, more preferably about 50% or more, compared to the absence of the test compound. In such a case, the test compound can be selected as a compound that inhibits the activity of MIP-3α of the present invention.

MIP-3α receptor antagonists include the receptor, preferably CCR6 of the present invention, and other ligands of the receptor, for example, compounds selected as CCR6 agonists in the above screening method (eg, synthetic low molecular weight compounds) Can be similarly screened using
In addition, an inverse agonist of the MIP-3α receptor is also preferable as the substance having a protective effect on the brain and nerve cells of the present invention. Screening can also be performed by applying the above method (i) or (ii).

  The kit used in the above-mentioned screening method of the present invention contains MIP-3α of the present invention and / or CCR6 of the present invention. When screening is performed using the signal transduction activity as an index, the CCR6 of the present invention is provided as cells having the ability to produce it. The MIP-3α of the present invention may be provided as an isolated protein or as a cell capable of producing it. When the receptor binding activity is used as an index, the MIP-3α of the present invention is preferably labeled.

  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, and a compound or a salt thereof that inhibits the receptor binding activity and / or signal transduction activity of MIP-3α of the present invention. As the salt of the compound, those similar to the aforementioned salt of MIP-3α of the present invention are used.

The brain / nerve cell protective agent containing the compound or its salt that inhibits the receptor binding activity and / or signal transduction activity of MIP-3α of the present invention is formulated in the same manner as the above-mentioned antibody of the present invention. be able to.
The dose of the cerebral / nerve cell protective agent varies depending on the administration subject, target disease, symptoms, administration route and the like. For example, when used for treatment / prevention of cerebrovascular disorders in adults, the present invention Of a compound or a salt thereof that inhibits the receptor binding activity and / or signal transduction activity of MIP-3α as a single dose is usually about 0.01 to 20 mg / kg body weight, preferably 0.1 to 10 mg / kg. It is convenient to administer the substance by a powder inhalant about the body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day. 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.

Since the expression of the gene encoding MIP-3α of the present invention increases in cerebral ischemia, substances that inhibit the expression of the gene encoding MIP-3 of the present invention are also substances having a brain / nerve cell protective action. Can be used.
Therefore, the nucleic acid containing the nucleotide sequence encoding MIP-3α of the present invention or a part thereof is useful as a reagent for screening a substance that inhibits the expression of the gene encoding MIP-3α of the present invention.

That is, the present invention also provides a method for screening a substance having a brain / nerve cell protective effect, which comprises using a nucleic acid containing the nucleotide sequence encoding MIP-3α of the present invention or a part thereof.
As the screening method, cells having the ability to produce MIP-3 of the present invention are cultured in the presence and absence of a test compound, and the amount of MIP-3α mRNA is measured and compared under both conditions.
Examples of the test compound and the cells having the ability to produce MIP-3α of the present invention include those described above.
The amount of mRNA can be measured by a known method, for example, Northern hybridization using a nucleic acid containing the nucleotide sequence encoding MIP-3α of the present invention or a part thereof as a probe, or encoding MIP-3α of the present invention as a primer It can be measured according to a PCR method using a nucleic acid containing a base sequence or a part thereof, or a method analogous thereto.
For example, a test compound that inhibits the amount of gene expression in the absence of a test compound by about 20% or more, preferably 30% or more, more preferably about 50% or more, was tested for expression of the gene encoding MIP-3α of the present invention. The compound can be selected as an inhibitory compound, and therefore, a compound having a brain / neuron protective effect.

The expression level of the gene encoding MIP-3α of the present invention can also be measured using an antibody against MIP-3α of the present invention.
That is, after culturing the cells having the ability to produce MIP-3α of the present invention in the presence and absence of a test compound, the amount of MIP-3α protein present in the cell supernatant or cell extract is determined by the present method. Using the antibody against MIP-3α of the present invention, the measurement can be performed according to a known method, for example, a method such as Western analysis or ELISA method or a method analogous thereto.
For example, a test compound that inhibits the amount of protein production in the absence of a test compound by about 20% or more, preferably 30% or more, more preferably about 50% or more, was tested for expression of the gene encoding MIP-3α of the present invention. The compound can be selected as an inhibitory compound, and therefore, a compound having a brain / neuron protective effect.

MIP-3α exerts its physiological activity by binding to its receptor (for example, CCR6) and transmitting signal information to the receptor-expressing cell, so that the expression of the receptor serving as a receptor is inhibited. Activity will decrease. Therefore, a substance that inhibits the expression of the receptor is also a MIP-3α inhibitor in the present invention.
Therefore, the present invention also provides a brain / nerve comprising using an antibody against the nucleic acid containing the nucleotide sequence encoding the CCR6 of the present invention or a part thereof, or the CCR6 of the present invention, a partial peptide thereof, or a salt thereof. A method for screening a substance having a cytoprotective action is provided.
The screening method includes a method of culturing cells having the ability to produce the CCR6 of the present invention in the presence and absence of a test compound, and measuring and comparing the amount of CCR6 mRNA or protein under both conditions. No.
The amount of mRNA can be measured by a known method, for example, Northern hybridization using a nucleotide sequence encoding the CCR6 of the present invention as a probe or a nucleic acid containing a part thereof, or a nucleotide sequence encoding the CCR6 of the present invention as a primer or It can be measured according to a PCR method using a nucleic acid containing a part thereof or a method analogous thereto.
The amount of protein is determined by culturing cells capable of producing the CCR6 of the present invention in the presence and absence of a test compound, isolating a cell membrane fraction or a cell extract, and clarifying an antibody against the CCR6 of the present invention. It can be carried out according to a known method, for example, a method such as Western analysis and ELISA method or a method analogous thereto. An antibody against CCR6 can be prepared using the same method as the above-described antibody against MIP-3α.

  The kit used in the above-described screening method of the present invention produces a nucleic acid containing the nucleotide sequence encoding MIP-3α of the present invention or a part thereof, or an antibody against MIP-3α of the present invention, and produces MIP-3α of the present invention. A cell having the ability, or a nucleic acid containing the nucleotide sequence encoding the CCR6 of the present invention or a part thereof, an antibody against the CCR6 of the present invention, a cell having the ability to produce the CCR6 of the present invention, or the like. .

  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, and a compound or a salt thereof that inhibits expression of MIP-3α of the present invention or CCR6 of the present invention. As the salt of the compound, those similar to the aforementioned salt of MIP-3α of the present invention are used.

The brain / nerve cell protective agent containing the above-mentioned compound of the present invention that inhibits the expression of MIP-3α or the present invention CCR6 or a salt thereof can be formulated as in the case of the above-mentioned antibody of the present invention. .
The dose of the cerebral / nerve cell protective agent varies depending on the administration subject, target disease, symptoms, administration route and the like. For example, when used for treatment / prevention of cerebrovascular disorders in adults, the present invention Of MIP-3α or the compound of the present invention that inhibits the expression of CCR6, or a salt thereof, as a single dose, is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, and more preferably. Is conveniently administered in a powder inhalant at a dose of about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day. 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.

[Quantification of MIP-3α of the present invention or its partial peptide or salt thereof]
Since the antibody against MIP-3α of the present invention can specifically recognize the MIP-3α of the present invention, it can be used for quantification of the MIP-3α of the present invention in a test solution, particularly for quantification by sandwich immunoassay. Can be used.
That is, the present invention
(I) The antibody against MIP-3α of the present invention is allowed to react competitively with the test solution and the labeled MIP-3α of the present invention, and the labeled MIP-3α of the present invention bound to the antibody is reacted. And (ii) an antibody against the MIP-3α of the present invention insolubilized on the test solution and a carrier and a labeled antibody. And reacting the antibody against MIP-3α of the present invention simultaneously or successively, and then measuring the activity of the labeling agent on the insolubilized carrier. Provides a quantification method.
In the quantification method (ii), if one of the antibodies is an antibody that recognizes the N-terminal of MIP-3α of the present invention, the other antibody is an antibody that recognizes another portion of MIP-3α of the present invention. It is desirable that

Further, the MIP-3α of the present invention can be quantified using the monoclonal antibody against MIP-3α 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 MIP-3α of the present invention using the antibody against MIP-3α of the present invention is not particularly limited, and may be an antibody, an antigen or an antigen corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount of the antibody-antigen complex 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. You may. For example, nephelometry, a competitive method, an immunometric method, and a sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance 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 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.

For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
In the sandwich method, a test solution is reacted with an insolubilized monoclonal antibody against MIP-3α of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody against MIP-3α of the present invention (secondary reaction). After the reaction), the amount of MIP-3α of the present invention in the test solution can be determined by measuring the activity of the labeling agent on the insolubilized carrier. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving the measurement sensitivity and the like. You may.
In the method for measuring MIP-3α of the present invention by the sandwich method of the present invention, the monoclonal antibody against MIP-3α of the present invention used in the primary reaction and the secondary reaction differs in the site to which the protein of the present invention binds. Antibodies are preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of MIP-3α of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The monoclonal antibody against MIP-3α of the present invention can also 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, an antigen in a test solution and a labeled antigen are allowed to react competitively with an antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody. As the first antibody, a soluble antibody is used, and an immobilization method using an immobilized antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction against a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.
For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al., "Enzyme Immunoassay" (Medical Shoin, Showa Ed. Ishikawa et al., “Enzyme Immunoassay” (2nd edition) (Issue Shoin, 1982), Eiji Ishikawa et al. “Enzyme Immunoassay” (3rd Edition), Ishikawa, Showa Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 70 (Immunochemical Techniques (Part C)) 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies). )) (The above is from Academic Press) ) And the like can be referred to.
As described above, the MIP-3α of the present invention can be quantified with high sensitivity by using the antibody against MIP-3α of the present invention.
Furthermore, when an increase or decrease in the concentration of the MIP-3α of the present invention is detected by quantifying the concentration of the MIP-3α of the present invention using the antibody against the MIP-3α of the present invention, It can be diagnosed that the subject has suffered cerebral vascular injury such as cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage or brain / neural cell injury due to head trauma, or has a high possibility of undergoing cell injury in the future.
Further, the antibody against MIP-3α of the present invention can be used for detecting the MIP-3α of the present invention present in a subject such as a body fluid or a tissue. In addition, preparation of an antibody column used for purifying MIP-3α of the present invention, detection of MIP-3α of the present invention in each fraction during purification, and behavior of MIP-3α of the present invention in test cells It can be used for analysis and the like.

[Genetic diagnostics]
The DNA encoding the MIP-3α of the present invention (hereinafter, may be simply abbreviated as “DNA of the present invention” in the description of the DNA transgenic animal / knockout animal in the present section (gene diagnostic agent) and the next section) may be, for example, The MIPs of the invention in humans or other warm-blooded animals (eg, rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) by using as probes. Since abnormalities (genetic abnormalities) in DNA or mRNA encoding -3α or a partial peptide thereof can be detected, for example, damage, mutation, or reduced expression of the DNA or mRNA, and increase or expression of the DNA or mRNA It is useful as a diagnostic agent for gene overload.
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), Processings of The National Academy of Sciences of the USA (Proceedings of the National Academy of Sciences of the United States of America), Vol. 86, pp. 2766-2770 (1989)) can be used.
For example, when overexpression is detected by Northern hybridization or DNA mutation is detected by PCR-SSCP method, cerebrovascular disorders such as cerebral infarction, cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, etc. Alternatively, it can be diagnosed that the patient has suffered brain / nervous cell injury due to head injury or the like, or is likely to suffer cell injury in the future.

[DNA transfer animal]
The present invention relates to an exogenous DNA encoding the MIP-3α of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). And a non-human mammal having the same.
That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) The animal according to (2), wherein the rodent is a mouse or rat, and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. Things.
Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. And preferably at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized eggs and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, It can be produced by transferring a target DNA by a microinjection method, a particle gun method, a DEAE-dextran method, or the like. In addition, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like by the DNA transfer method, and can be used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se.
As the non-human mammal, for example, cow, pig, sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat and the like are used. 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 the like, or a rat (for example, Wistar, SD, etc.) is preferable.
"Mammals" in a recombinant vector that can be expressed in mammals include humans in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by non-human mammals, but to the DNA of the present invention once isolated and extracted from the mammal.
As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA and the like are used, and also include abnormal DNA.
The abnormal DNA means a DNA that expresses the abnormal MIP-3α of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal MIP-3α of the present invention is used.
The exogenous DNA of the present invention may be derived from a mammal that is the same or different from the animal of interest. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is transferred, DNA derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto is expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, a vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-transferring mammals can be created.

Examples of the expression vector of MIP-3α of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as phage λ, retroviruses such as Moloney leukemia virus, vaccinia viruses and baculoviruses. Animal viruses and the like are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used.
Examples of the promoter for controlling the DNA expression include, for example, (i) a promoter of a DNA derived from a virus (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, polio virus, 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 acidic 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, dystrophin 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 chains, metallothionein I and IIA, Metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin, α And β-myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, Step Promoters such as reproenkephalin A and vasopressin are used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoters and the like are preferable.
The above vector preferably has a sequence that terminates transcription of a messenger RNA of interest in a DNA-transferred mammal (generally called a terminator). A simian virus SV40 terminator or the like is preferably used.

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

In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and the function of MIP-3α of the present invention is finally enhanced by promoting the function of endogenous normal DNA. The disease may develop and can be used as a disease model animal. For example, using the normal DNA transgenic animal of the present invention, elucidation of the pathological mechanism of the hyperactivity of MIP-3α of the present invention and the diseases associated with MIP-3α of the present invention, and examination of a method for treating these diseases Can be performed.
In addition, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released MIP-3α of the present invention, a preventive / therapeutic agent for a disease associated with MIP-3α of the present invention, for example, It can also be used for screening tests for prophylactic and therapeutic agents for cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage and subarachnoid hemorrhage, head trauma, and various inflammatory diseases.
On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual DNA engineering technique. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The progeny of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all progeny have the DNA.

The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, impairs the function of the MIP-3α of the present invention by inhibiting the function of endogenous normal DNA. It may be active 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 MIP-3α function-inactive refractory disease of the present invention and to examine a method for treating this disease.
Further, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention can be used for inhibiting the function of a normal protein by the abnormal protein of the present invention (dominant negative action) in the inactive refractory disease of MIP-3α of the present invention. ).
In addition, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of free MIP-3α of the present invention, the preventive / therapeutic agent for MIP-3α or a functionally inactive refractory disease of the present invention. For example, it can be used for screening tests for prophylactic and therapeutic agents for cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage, head trauma, and various inflammatory diseases.
In addition, other possible uses of the above two kinds of DNA transgenic animals of the present invention include, for example,
(i) use as a cell source for tissue culture,
(ii) Expression or activation specifically by MIP-3α of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing a peptide tissue expressed by the DNA Analysis of the relationship with the peptide
(iii) culturing cells of tissue having DNA by standard tissue culture techniques and using them to study the function of cells from tissues that are generally difficult to culture,
(iv) screening for an agent that enhances the function of cells by using the cells according to (iii), and
(v) Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered.

Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine the clinical symptoms of a disease associated with MIP-3α of the present invention, including the inactive refractory function of MIP-3α of the present invention, Obtain more detailed pathological findings in each organ of the disease model related to MIP-3α of the present invention, contribute to the development of new treatment methods, and further to the research and treatment of secondary diseases caused by the disease. Can be.
In addition, it is possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain the released DNA-transferred cells, culture them, or systematize the cultured cells. . Furthermore, the MIP-3α-producing cells of the present invention can be identified, examined for their relationship with apoptosis, differentiation or proliferation, or their signal transduction mechanisms, and their abnormalities can be examined. It is an effective research material for elucidating its action.
Further, in order to develop a therapeutic agent for a disease associated with MIP-3α of the present invention, including the inactive refractory function of MIP-3α 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 using a test method, a quantitative method, and the like. Further, 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 a disease associated with MIP-3α of the present invention.

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

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is a DNA which is artificially mutated to the DNA of the present invention possessed by the non-human mammal, thereby suppressing the expression ability of the DNA, or By substantially eliminating the activity of the MIP-3α of the present invention encoded by the DNA, the DNA has substantially no ability to express the MIP-3α of the present invention (hereinafter referred to as the knockout DNA of the present invention). (Sometimes referred to as "ES cells").
As the non-human mammal, the same one as described above is used.
The method of artificially mutating the DNA of the present invention can be carried out, for example, by deleting a part or all of the DNA sequence and inserting or substituting another DNA by a genetic engineering technique. With these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.

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

As a method for determining the sex of ES cells, for example, a method of amplifying and detecting a gene in a sex-determining region on the Y chromosome by a PCR method can be mentioned. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture The primary selection of ES cells in the early stage can be performed by discriminating between male and female, and the early stage of culture can be greatly reduced by enabling the selection of male cells at an early stage.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations at the time of establishment, after knocking out the gene of the ES cells, the cells are knocked out of normal cells (for example, chromosomes in mice). It is desirable to clone again into cells (number 2n = 40).
The embryonic stem cell line obtained in this manner usually has very good growth properties, but it must be carefully subcultured because it tends to lose its ontogenic ability. For example, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% on a suitable feeder cell such as STO fibroblast) in the presence of LIF (1-10000 U / ml). 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 0.001-0.5% trypsin / 0.1-5 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 Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embryology and Experimental Morphology, 87, 27, 1985]. The cells deficient in expression of the DNA of the present invention obtained by differentiating are useful in the cell biology of MIP-3α of the present invention in vitro.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
As the non-human mammal, those similar to the aforementioned can be used.
The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination.
Cells in which the DNA of the present invention has been knocked out can be used as a DNA sequence on a Southern hybridization analysis or targeting vector using the DNA sequence on or near the DNA of the present invention as a probe, and the DNA of the present invention derived from the mouse used for the targeting vector. The determination can be made by analysis by a PCR method using a DNA sequence of a nearby region other than DNA as a primer. When non-human mammalian embryonic stem cells are used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cells are cultured at an appropriate time, for example, at the 8-cell stage of non-human mammalian cells. The chimeric embryo is injected into an animal embryo or a blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention.
When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individual thus obtained is usually an individual having a heterozygous expression of MIP-3α of the present invention, which is crossed with an individual having a heterozygous expression of MIP-3α of the present invention, and the offspring of the present invention An individual lacking in homoexpression of MIP-3α can be obtained.

When an egg cell is used, for example, a transgenic non-human mammal having a targeting vector introduced into a chromosome can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method. Compared to mammals, they can be obtained by selecting those having a mutation in the DNA locus of the present invention by homologous recombination.
In this manner, the individual in which the DNA of the present invention has been knocked out can be bred in an ordinary breeding environment after confirming that the DNA has been knocked out in the animal individual obtained by mating.
Furthermore, the germline can be obtained and maintained in accordance with a conventional method. That is, by crossing male and female animals having the inactivated DNA, a homozygous animal having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by breeding the mother animal in such a manner that there are one normal animal and one homozygote. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention.
In addition, the non-human mammal deficient in expressing the DNA of the present invention lacks various biological activities that can be induced by the MIP-3α of the present invention. Since it can be a model for such diseases, it is useful for investigating the causes of these diseases and examining treatment methods.

(A) 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. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against a disease.
Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same ones as described above.
Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma and the like. These compounds are novel compounds. Or a known compound.
Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The therapeutic / preventive effects of the compounds can be tested.
As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.

For example, when screening for a compound having a therapeutic / preventive effect on cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage, head trauma, and various inflammatory diseases, the DNA expression deficiency of the present invention is insufficient. A test compound is administered to a non-human mammal, and the behavior, infarct volume, and the like of the animal are observed over time, and the symptoms of the above-mentioned disease are observed.
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 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 MIP-3α deficiency or damage of the present invention. It can be used as a drug such as a safe and low toxic prophylactic / therapeutic agent for diseases. Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like are used.
A drug containing the compound obtained by the screening method or a salt thereof can be produced in the same manner as the above-mentioned drug containing MIP-3α of the present invention.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered, it is generally used in an adult (with a body weight of 60 kg) cerebrovascular disorder patient. Administers 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 in the form of an injection and is usually administered to an adult (60 kg) patient with cerebrovascular disease. When administered to a subject, 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 60 kg.

(B) Method of screening for a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention. The present invention provides a method for detecting a reporter gene expression by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention. And a method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for the DNA of the present invention.
In the above-described screening method, the non-human mammal deficient in expressing DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound include the same compounds as described above.
As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable.
In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention is replaced with a reporter gene, since the reporter gene is under the control of the promoter for the DNA of the present invention, the expression of the substance encoded by the reporter gene is traced. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the MIP-3α of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, the tissue of the present invention expressing the MIP-3α of the present invention may Β-galactosidase is expressed instead of MIP-3α. 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 MIP-3α in an animal body can be observed. Specifically, the MIP-3α-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 at 37 ° C. After reacting at about 30 ° C. for about 30 minutes to 1 hour, the β-galactosidase reaction may be stopped by washing the tissue specimen with a 1 mM EDTA / PBS solution, and coloration may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method.
The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt, and the salt of the compound may include a physiologically acceptable acid (eg, an inorganic acid) and a base (eg, an organic acid). 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 MIP-3α of the present invention and the function of the protein. For example, cerebral infarction, cerebral hemorrhage, subarachnoid It is useful as a prophylactic / therapeutic agent for cerebrovascular disorders such as bleeding, head trauma, and various inflammatory diseases.
Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.
A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing MIP-3α or a salt thereof 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 route of administration, and the like. In a stroke 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. When administered to a stroke patient (as 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 by intravenous injection per day. It is convenient to do so. In the case of other animals, the amount can be administered in terms of 60 kg.
As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention, It can greatly contribute to the investigation of the cause of various diseases caused or the development of preventive / therapeutic agents.
Further, using a DNA containing the promoter region of MIP-3α of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (transgenic animal). ) Makes it possible to specifically synthesize the protein and examine its action in the living body. Furthermore, if a suitable reporter gene is linked to the above-mentioned promoter portion and a cell line that expresses the same is established, a low-potential cell capable of specifically promoting or suppressing the ability of the MIP-3α itself of the present invention to produce in vivo is established. It can be used as a search system for molecular compounds.

The MIP-3α inhibitor of the present invention may be used in combination with other active ingredients. Examples of such active ingredients include thrombolytic agents (eg, tissue plasminogen activator, urokinase, etc.), anticoagulants (eg, argatroban, warfarin, etc.), factor 10 inhibitors, thromboxane synthase inhibitors (eg, Eg, ozagrel, etc.), antioxidants (eg, edaravone, etc.), anti-edema agents (eg, glycerol, mannitol, etc.), neurogenesis / regenerative promoters (eg, Akt / PKB activator, GSK-3β inhibitor, etc.) ), Acetylcholinesterase inhibitors (eg, donepezil, rivastigmine, galantamine, zanapezil, etc.), β-amyloid protein production, secretion, accumulation, aggregation and / or deposition inhibitors [β-secretase inhibitors (eg. Compounds described in WO 98/38156) , WO 02/2505, WO 02/2506, WO 02/2512, OM99-2 (WO 01/00663)), γ-secretase inhibitor, β-amyloid protein coagulation Inhibitors (eg, PTI-00703, ALZHEMED (NC-531), PPI-368 (Tokuhei 11-514333), PPI-558 (Tokihyo 2001-500852), SKF-74652 (Biochem. J. 1999), 340 (1), 283-289)), β-amyloid vaccine, β-amyloid-degrading enzyme, etc.), brain function stimulants (eg, aniracetam, nicergoline, etc.), and other therapeutic drugs for Parkinson's disease (eg, dopamine receptor Body agonists (L-dopa, bromocriptene, pergolide, talipexole, prasipexole, cabergoline, adamantine, etc.), monoamine oxidase (MAO) inhibitors (deprenyl, sergiline (selegiline), remacemide, riluzole) ), Anticholinergics (eg, trihexyphenidyl, biperiden, etc.), COMT inhibitors (eg, entacapone, etc.)], muscle atrophy Drugs for treatment of lateral sclerosis (eg, riluzole, neurotrophic factor, etc.), drugs for hyperlipidemia such as cholesterol-lowering drugs [statins (eg, pravastatin sodium, atorvastatin, simvastatin, rosuvastatin, etc.), fibrates (eg, , Clofibrate, etc.), squalene synthase inhibitors], therapeutic agents for abnormal behavior associated with the progression of dementia, wandering (eg, sedatives, anxiolytics, etc.), apoptosis inhibitors (eg, CPI-1189, IDN- 6556, CEP-1347, etc.), neuronal differentiation / regenerative promoters (Leteprinim, Xaliproden (SR-57746-A), SB-216763, etc.), antihypertensives, antidiabetic drugs, antidepressants, anxiolytics Drugs, nonsteroidal anti-inflammatory drugs (eg, meloxicam, teoxicam, indomethacin, ibuprofen, celecoxib, rofecoxib, aspirin, indomethacin, etc.), diseases Decorative antirheumatic drugs (DMARDs), anti-cytokine drugs (TNF inhibitors, MAP kinase inhibitors, etc.), steroid drugs (eg, dexamethasone, hexestrol, cortisone acetate, etc.), sex hormones or derivatives thereof (eg, progesterone, Estradiol, estradiol benzoate, etc.), parathyroid hormone (PTH), calcium receptor antagonists and the like. The other active ingredient and the MIP-3α inhibitor of the present invention are mixed in accordance with a method known per se to give one pharmaceutical composition (eg, tablet, powder, granule, capsule (including soft capsule), liquid, Injections, suppositories, sustained-release preparations, etc.) and may be used in combination. Alternatively, they may be separately formulated and administered to the same subject simultaneously or with a time interval.
Further, the MIP-3α inhibitor of the present invention is not limited to drugs, and can be used in combination with other treatment methods. For example, for cerebrovascular disorders, hypothermia or hypothermia therapy, cerebral thrombosis / cerebral embolus removal surgery can be used in combination, and for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, treatment methods such as neural stem cell transplantation Can be used in combination with, but not limited to.

In the present specification, when bases, amino acids and the like are indicated by abbreviations, they are based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: thyronine : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe Phenylalanine Tyr: tyrosine Trp: tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Sec: selenocysteine (selenocysteine)

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

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
1 shows the nucleotide sequence of human MIP-3α cDNA.
[SEQ ID NO: 2]
2 shows the amino acid sequence of the precursor polypeptide of human MIP-3α.
[SEQ ID NO: 3]
1 shows the nucleotide sequence of rat MIP-3α cDNA.
[SEQ ID NO: 4]
2 shows the amino acid sequence of the precursor polypeptide of rat MIP-3α.
[SEQ ID NO: 5]
1 shows the nucleotide sequence of mouse MIP-3α cDNA.
[SEQ ID NO: 6]
2 shows the amino acid sequence of the precursor polypeptide of mouse MIP-3α.
[SEQ ID NO: 7]
1 shows the nucleotide sequence of human CCR6 cDNA.
[SEQ ID NO: 8]
1 shows the amino acid sequence of human CCR6.
[SEQ ID NO: 9]
1 shows the nucleotide sequence of mouse CCR6 cDNA.
[SEQ ID NO: 10]
2 shows the amino acid sequence of mouse CCR6.
[SEQ ID NO: 11]
2 shows the nucleotide sequence of an oligonucleotide designed to function as a primer for amplifying a fragment of a rat MIP-3α gene transcript.
[SEQ ID NO: 12]
2 shows the nucleotide sequence of an oligonucleotide designed to function as a primer for amplifying a fragment of a rat MIP-3α gene transcript.
[SEQ ID NO: 13]
1 shows the nucleotide sequence of rat kidney-derived CCR6 cDNA.
[SEQ ID NO: 14]
2 shows the amino acid sequence of rat CCR6.
[SEQ ID NO: 15]
1 shows the nucleotide sequence of rat liver-derived CCR6 cDNA.
[SEQ ID NO: 16]
1 shows the nucleotide sequence of an oligonucleotide designed to function as a primer for amplifying rat kidney-derived CCR6 cDNA.
[SEQ ID NO: 17]
1 shows the nucleotide sequence of an oligonucleotide designed to function as a primer for amplifying rat kidney or rat liver-derived CCR6 cDNA.
[SEQ ID NO: 18]
2 shows the nucleotide sequence of an oligonucleotide designed to function as a primer for amplifying rat liver-derived CCR6 cDNA.
[SEQ ID NO: 19]
1 shows the nucleotide sequence of an oligonucleotide designed to function as a primer for amplifying a fragment of the rat CCR6 gene product.
[SEQ ID NO: 20]
1 shows the nucleotide sequence of an oligonucleotide designed to function as a primer for amplifying a fragment of the rat CCR6 gene product.
[SEQ ID NO: 21]
1 shows the nucleotide sequence of an oligonucleotide designed to function as a primer for amplifying a fragment of the rat CCR6 gene product.

  Hereinafter, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 Increased expression of MIP-3α gene in a rat cerebral ischemia model and decreased expression by hypothermia treatment Existence of increased expression of MIP-3α gene in brain tissue and decreased expression by hypothermia treatment in rat regional cerebral ischemia model Was investigated. As a cerebral ischemia model, a middle cerebral artery occlusion model was prepared using 8-week-old male SD rats (Charles River Japan) (Kiyota et al., Experimental Brain Research, Vol. 95). 388-396, 1993). That is, under halothane anesthesia, a silicon-coated obturator was inserted from the right common carotid artery to the base of the middle cerebral artery and occluded for 120 minutes. Thereafter, 0, 2, 4, 6, 8, 24, 48 and 96 hours after the start of reperfusion by obturator removal, 5 rats per group were sacrificed and whole brain was removed, and the infarct center and margin were removed. After sorting, one sample was collected for each group. Similarly, the hypothermia treatment group was subjected to local cerebral ischemia, and at the same time as the start of reperfusion, low-temperature treatment (maintaining the brain temperature at 35 ° C. by cooling the cage with cold air) was started. 2, 4, 6, 8, 24, 48, and 96 hours later, the whole brain was excised and collected as one sample for each group. After each of the above brain tissue samples was pulverized under liquid nitrogen, total RNA was purified from the pulverized wet tissues using Isogen (Nippon Gene) according to the method described in the attached document. After removing the contaminated genomic DNA using a message clean kit (Gene Hunter), single-stranded cDNA was synthesized using a Thermocrypt RT-PCR kit (GIBCO BRL). Based on the obtained cDNA, the expression level of the MIP-3α gene was analyzed by quantitative PCR. That is, a quantitative PCR primer set of the MIP-3α gene was designed using Primer Express (Applied Biosystems), and two types of selected oligonucleotides (SEQ ID NO: 11: 5′-AGAATGGCCTGCAAGCATCT-3 ′; SEQ ID NO: 12: 5′-TGCAGAGGTAAGCCAGCAGTA-3 ′) was synthesized (consigned to Proligo Japan). The MIP-3α gene fragment amplified using the above primer set was used as a reference sequence for quantification. The primer set and the PCR Quantitec Cyber Green PCR Master Kit (Qiagen) were used as the reaction system for quantitative PCR. One used above rat cDNA derived from the reaction per poly A ⁺ RNA 0.8 ng, or from 0 to ¹⁰⁶ copies the standard MIP-3.alpha. Gene as a template, subjected to analysis using an ABI Prism 7000 (Applied Biosystems) Was. The expression level of the MIP-3α gene was determined by calculating the copy number of the MIP-3α cDNA in each sample from the calibration curve calculated from the standard MIP-3α gene. In order to correct the value between each sample, the number of GAPDH copies in the same sample, which was calculated using a Tackman Rodent GAPDH Control Regent VIC probe (Applied Biosystems), Was calculated as the MIP-3α gene copy number. As a result, as shown in FIG. 1, it was shown that the expression level of the MIP-3α gene increased from 2 hours after the start of reperfusion in both the central part and the peripheral part of the infarction. In the central part of the infarction, the expression level of the MIP-3α gene reached the maximum 24 hours after the start of reperfusion and 8 hours in the peripheral part, and thereafter decreased. It was also found that these expressions were suppressed by hypothermia treatment in both the central part and the peripheral part of the infarction. From the above results, it was shown that MIP-3α is a gene whose expression is significantly induced by reperfusion after ischemia, and which is significantly suppressed by hypothermic treatment.

Example 2 Cerebral Protective Effect of Anti-MIP-3α Antibody in Rat Cerebral Ischemia Model Anti-rat MIP-3α monoclonal antibody having neutralizing activity against cerebral infarction volume in a rat local cerebral ischemia model (hereinafter referred to as “anti-MIP-3α antibody”) ) Were examined for their inhibitory effect by intracerebroventricular administration. As a cerebral ischemia model, a middle cerebral artery occlusion model was prepared using 8-week-old male SD rats (Charles River Japan) (Kiyoda et al., Experimental Brain Research, Vol. 95). 388-396, 1993). That is, under halothane anesthesia, a silicon-coated obturator was inserted from the right common carotid artery to the middle cerebral artery origin and occluded for 120 minutes. In experiment 1, the rat was attached to a stereotaxic apparatus under halothane anesthesia immediately before occlusion of the middle cerebral artery, and the ischemic side ventricle (AP: -0.8, ML: -1.6, DV: -4.0). , But from the dura), anti-MIP-3α antibody (IgG1: Genzyme Techno, Cat. No. 43540; 20 μg / 10 μl) or control antibody (IgG1: Genzyme Techno, Cat. No. 43002; 20 μg / 10 μl) ) Was injected. One day after the ischemia treatment, the rat brain was excised, a 2 mm-thick premaxillary slice was prepared, and the cerebral infarct volume was measured by image analysis from the TTC-stained image. It was significantly (P <0.05) smaller compared to the group (FIG. 2A). In Experiment 2, immediately after middle cerebral artery occlusion reperfusion, a control antibody or anti-MIP-3α antibody was injected into the ventricle by the same method as above, and the cerebral infarct volume was measured two days later. The cerebral infarct volume was significantly smaller in the group (P <0.05) (FIG. 2B). The above results indicate that suppressing the function of MIP-3α has a cerebral infarction inhibitory effect.

Example 3 Increased production of MIP-3α protein in a rat cerebral ischemia model and decreased production by hypothermia treatment Existence of increased production of MIP-3α protein in brain tissue and decreased production by hypothermia treatment in a rat regional cerebral ischemia model Was examined. As a cerebral ischemia model, a middle cerebral artery occlusion model was prepared using 8-week-old SD male rats (Charles River Japan) (Kiyoda et al., Experimental Brain Research, 95). 388-396, 1993). That is, under halothane anesthesia, a silicon-coated obturator was inserted from the right common carotid artery to the base of the middle cerebral artery and occluded for 120 minutes. Then, at 0, 4, 8, 24, 48 and 96 hours after the start of reperfusion by obturator removal, 5 rats per group were sacrificed, the whole brain was removed, and the infarct center and margin were collected. Each group was collected as one sample. In the hypothermia treatment group, similarly, after local cerebral ischemia, low-temperature treatment (maintaining the brain temperature at 35 ° C. by cooling the cage with cold air) was started simultaneously with the start of reperfusion, and the same as in the ischemia group After 4, 8, 24, 48, and 96 hours, the whole brain was excised and collected as one sample for each group. After each of the above brain tissue samples was pulverized under liquid nitrogen, a protease inhibitor cocktail (CellLytic-MT Mammalian Tissue Lysis / Extraction Reagent; Sigma) was added to Cellulic-MT Mammalian Tissue Lysis / Extraction Reagent (Sigma). (P8340; Sigma) was added in a volume of 1/100, and 1 ml was added to each 50 mg of ground brain tissue and solubilized by Polytron. Thereafter, each solubilized tissue fluid was centrifuged at 12,000 rpm for 5 minutes at 4 ° C., and the supernatant liquid from which the insoluble fraction was removed was used as a sample solution. This sample solution was subjected to a rat MIP-3α sandwich ELISA described below, and is shown in FIG. 3 as the amount of MIP-3α protein per 1 mg of brain tissue protein. The amount of protein in the sample solution was quantified using a BCA protein assay kit (Pierce) according to the attached manual. The rat MIP-3α sandwich ELISA was carried out using a commercially available anti-rat MIP-3α antibody (43540; Genzyme Techne) and a biotin-labeled anti-rat MIP-3α polyclonal antibody (44540; Genzyme Techne). That is, the anti-rat MIP-3α antibody (43540) was diluted with a phosphate buffer (PBS) to a concentration of 2 μg / ml, and the diluted 100 μl was dispensed to a 96-well ELISA plate. After leaving at room temperature for 2 hours, the plate washed three times with a washing buffer (PBS containing 0.05% Tween 20, pH 7.2) was added to a block buffer (1% fetal bovine serum, 5% sucrose, 300 μl per well of PBS containing 0.05% sodium azide) was added, and the mixture was left at room temperature for 1 hour. Thereafter, the wells were washed three times with a washing buffer, 100 μl of each diluted sample solution and rat MIP-3α standard solution were added, and the mixture was left at room temperature for 2 hours. After washing three times with a washing buffer, 100 μl of a biotin-labeled anti-rat MIP-3α polyclonal antibody (44540) diluted to 50 ng / ml with PBS was added, and the mixture was allowed to stand at room temperature for 2 hours. After washing, streptavidin-horseradish peroxidase was bound to a biotin-labeled anti-rat MIP-3α polyclonal antibody according to the manual attached to a protein detector ELISA kit (KPL), followed by reaction. The substrate was added to each well and reacted, and the absorbance at 405 nm was measured. The result is shown in FIG.
It was found that the amount of MIP-3α protein in brain tissue reached the maximum value 24 hours after the start of reperfusion in both the central part and the peripheral part of the infarction. It was also shown that their production was significantly suppressed by hypothermia treatment. From the above results, it became clear that the production of MIP-3α protein was significantly induced by reperfusion after ischemia, and was significantly suppressed by hypothermia treatment.

Example 4 Cloning of cDNA Encoding CC Chemokine Receptor 6 (CCR6) and Determination of Nucleotide Sequence cDNAs encoding rat kidney-derived CCR6 and rat liver-derived CCR6 were cloned and their nucleotide sequences were determined. The cloning and the determination of its nucleotide sequence are described below.
First, PCR was performed using rat kidney Marathon-Ready cDNA (Clontech) as a template and two types of primers, Primer 1 (see below) and Primer 2 (see below). The composition of the reaction solution in this reaction was as follows: 5 μl of the above cDNA was used as a template, 4 μl of Advantage 2 Polymerase Mix (Clontech), 0.4 μM each of primer 1 and primer 2, 200 μM dNTP mixture, 10 × cDNA PCR Reaction Buffer (Clontech) was added to make a liquid volume of 200 μl. The PCR reaction was performed by repeating a heat treatment at 94 ° C. for 1 minute and then a cycle of 94 ° C. for 30 seconds, 67 ° C. for 30 seconds, and 68 ° C. for 2 minutes 35 times. After confirming the amplified fragment by agarose gel electrophoresis, the PCR reaction product was subcloned into a plasmid vector pCR 2.1-TOPO (Invitrogen) according to the prescription of TOPO TA Cloning Kit (Invitrogen). Escherichia coli TOP10 was transformed with this plasmid, and clones into which cDNA had been introduced were selected on LB agar medium containing kanamycin. As a result of analyzing the nucleotide sequence of each clone, a cDNA sequence encoding rat CCR6 (SEQ ID NO: 13) was obtained, and the plasmid DNA was named rCCR6-kidney. Further, Escherichia coli DH5α (Invitrogen) was transformed with the plasmid DNA, and the transformant was named DH5α / rCCR6-kidney.
Next, PCR was performed using rat liver Marathon-Ready cDNA (Clontech) as a template and two kinds of primers, Primer 2 (see below) and Primer 3 (see below). The composition of the reaction solution in the reaction was as follows: 5 μl of the above cDNA was used as a template, 1 μl of Pyrobest DNA Polymerase (Takara Bio Inc.), 0.4 μM each of primer 2 and primer 3, 200 μM dNTP mixture, 10 × PyrobestII buffer (Takara Bio). (Bio Inc.) was added to make a liquid volume of 200 μl. The PCR reaction was performed by repeating a cycle of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, and 72 ° C. for 2 minutes 40 times after heat treatment at 94 ° C. for 30 seconds. After confirming the amplified fragment by agarose gel electrophoresis, the PCR reaction product was subcloned into a plasmid vector pCR-BluntII-TOPO (Invitrogen) according to the instructions of Zero Blunt TOPO PCR Cloning Kit (Invitrogen). Escherichia coli TOP10 was transformed with this plasmid, and clones into which cDNA had been introduced were selected on LB agar medium containing kanamycin. As a result of analyzing the nucleotide sequence of each clone, a cDNA sequence encoding rat CCR6 (SEQ ID NO: 15) was obtained, and the plasmid DNA was named rCCR6-liver. Further, Escherichia coli DH5α (Invitrogen) was transformed with the plasmid DNA, and the transformant was named DH5α / rCCR6-liver.
The rat kidney-derived CCR6 cDNA and the rat liver-derived CCR6 cDNA completely matched the nucleotide sequence of the protein coding region, and encoded a protein having an amino acid sequence of 366 amino acids (SEQ ID NO: 14). The results of the hydrophobicity plot analysis of the amino acid sequence suggested that this protein had seven transmembrane domains.
Primer 1: 5'-TGTATTGAAGACAGAACACTTGTGG-3 '(SEQ ID NO: 16)
Primer 2: 5'-TCACATGTAATAGCAAGTTTCACAAAGG-3 '(SEQ ID NO: 17)
Primer 3: 5'-GCATCTCACTACCCGTCTCTC-3 '(SEQ ID NO: 18)

Example 5 Increased expression of CCR6 gene in rat cerebral ischemia model and decreased expression by hypothermia treatment The presence or absence of increased CCR6 gene expression in brain tissue and decreased expression by hypothermia treatment in a rat regional cerebral ischemia model was examined. As a cerebral ischemia model, a middle cerebral artery occlusion model was prepared using 8-week-old male SD rats (Charles River Japan) (Experimental Brain Research), 95 388-396, 1993). That is, under halothane anesthesia, a silicon-coated obturator was inserted from the right common carotid artery to the base of the middle cerebral artery and occluded for 120 minutes. Thereafter, 0, 2, 4, 6, 8, 24, 48 and 96 hours after the start of reperfusion by obturator removal, 5 rats per group were sacrificed and whole brain was removed, and the infarct center and margin were removed. After sorting, one sample was collected for each group. The hypothermia treatment group was similarly subjected to local cerebral ischemia, and at the same time as the start of reperfusion, low-temperature treatment (maintaining a brain temperature of 35 ° C. by cooling the cage with cold air) was started. 2, 4, 6, 8, 24, 48, and 96 hours later, the whole brain was excised and collected as one sample for each group. After each of the above brain tissue samples was pulverized under liquid nitrogen, total RNA was purified from the pulverized wet tissues using Isogen (Nippon Gene) according to the method described in the attached document. After removing the contaminated genomic DNA using Message Clean Kit (Gene Hunter), single-stranded cDNA was synthesized using Thermoscript RT-PCR kit (GIBCO BRL). Using the obtained cDNA as a template, the expression level of the CCR6 gene was analyzed by quantitative PCR. That is, two types of oligonucleotides (SEQ ID NO: 19: 5'-GGACGATGCGTTGTCATTTTC-3 '; SEQ ID NO: 20: 5'-CCGCAGCTGCAGCGCCGAGAAA-3', based on the rat CCR6 gene sequence cloned in Example 4 (previous paragraph) Was also synthesized by Proligo Japan) to amplify the rat CCR6 gene fragment and used it as a reference sequence for quantification. In the reaction system of quantitative PCR, a primer set for quantitative PCR (SEQ ID NO: 19: 5′-GGACGATGCGTTGTCATTTTC-3 ′, SEQ ID NO: 21: 5 ′) designed using Primer Express (Applied Biosystems) was used. GTGCCCGGGTTTACTCAGAA-3 ', both of which were commissioned by Proligo Japan) and PCR Quantititech Cyber Green PCR Master Kit (Qiagen). Using one each rat cDNA samples corresponding to the reaction per poly ^A + RNA 0.8 ng amount, or a standard CCR6 gene fragment 0-10 ⁶ copies as templates, an analysis using ABI Prism 7000 (Applied Biosystems) Done. The CCR6 gene expression level was normalized by the GAPDH gene expression level in the same sample. That is, the number of CCR6 cDNA copies in each sample was calculated from the calibration curve created from the standard CCR6 gene, and at the same time, the GAPDH cDNA copy in the same sample was analyzed using the Tackman Rodent GAPDH / Control Reagent VIC probe (Applied Biosystems). The number was calculated as the number of CCR6 cDNA copies per one copy of GAPDH cDNA. As a result, as shown in FIG. 4, after the start of reperfusion, the expression level of the CCR6 gene was shown to increase from 4 hours in the central part of the infarction and from 6 hours in the peripheral part. The expression level of the CCR6 gene reached the maximum 48 hours after the start of reperfusion in both the central part and the peripheral part of the infarction, and the expression level decreased thereafter. Furthermore, it was found that these expressions were suppressed by hypothermia treatment in the central part of the infarction after 8 hours and in the peripheral part after 24 hours. From the above results, it was shown that the expression of the CCR6 gene was significantly induced with reperfusion after ischemia, and was significantly suppressed by hypothermia treatment.

  The MIP-3α of the present invention is a diagnostic marker for brain / nerve cell injury, for example, cerebrovascular disorder, and therefore, a substance that inhibits the activity of the protein, for example, a neutralizing antibody against the protein, gene expression of the protein Substances, for example, the antisense nucleic acids of the present invention are useful as brain / nerve cell protectants, particularly as brain / nerve cell protectants during cerebrovascular disorders such as cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage or head trauma. It is.

FIG. 4 is a graph showing the time course of MIP-3α gene expression after reperfusion in the central part (A) and the peripheral part (B) of the infarcted lesion in a rat cerebral ischemia model, and the effect of hypothermia treatment on it. In the figure, C is a case where cDNA as a control, that is, cDNA from untreated rat brain was used as a template. It is a figure which shows the effect which the anti-rat MIP-3 (alpha) monoclonal antibody which has neutralizing activity has on the cerebral infarction volume in a rat cerebral ischemia model. A is the infarct volume in the brain extracted one day after the ischemic treatment from the rat to which the antibody was administered immediately before the ischemic treatment, and B is the cerebral infarct volume after 2 days in the rat to which the antibody was administered immediately after the ischemic reperfusion. Show. In the figure, * indicates p <0.05 with respect to the control antibody group by the Student t-test. Time course of MIP-3α protein production after reperfusion in the central infarct (NA) and margin (NB) of a rat cerebral ischemia model, and the effect of hypothermic treatment on it [center of infarct (HA) ), Edge portion (HB)]. In the figure, N indicates a control, that is, a solubilized fraction derived from untreated rat brain. FIG. 2 is a graph showing the time course of the expression of the CCR6 gene after reperfusion in the central part (A) and the peripheral part (B) of the infarct lesion in a rat cerebral ischemia model, and the effect of hypothermia treatment on it. In the figure, C is a case where cDNA as a control, that is, cDNA from untreated rat brain was used as a template.

Claims (21)

A brain containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a substance inhibiting a salt thereof; Neuroprotective agent. It comprises a substance having the same or substantially the same amino acid sequence as the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a substance which reduces the activity of a salt thereof. Brain and nerve cell protective agent. A protein or a partial peptide thereof or a salt thereof, wherein the substance whose activity is reduced has the same or substantially the same amino acid sequence as the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6; 3. The agent for protecting brain and nerve cells according to claim 2, which is a neutralizing antibody against the following. A brain / nerve cell protective agent comprising a substance that inhibits the expression of a gene encoding a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4, or 6 . A base sequence complementary to a base sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a part thereof, wherein the substance inhibiting the expression is The brain / nerve cell protective agent according to claim 4, which is a nucleic acid containing It comprises an antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence from amino acid No. 1 in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a partial peptide thereof or a salt thereof. Diagnostic agent for brain and nerve cell injury. Brain / nerve comprising a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a nucleic acid comprising a part thereof Diagnostic agent for cell injury. Inhibits binding of a protein or a salt thereof having an amino acid sequence identical or substantially identical to the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 to a receptor for the protein or a salt thereof Protective agent for brain and nerve cells, containing a substance that acts. A substance that inhibits a cell stimulating activity of a receptor for a protein having the same or substantially the same amino acid sequence as the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a salt thereof A brain / nerve cell protective agent comprising: Inhibits the expression of a gene encoding a receptor for a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence of amino acid No. 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 Protective agent for brain and nerve cells, containing a substance that acts. The brain / nerve cell protection according to any one of claims 8 to 10, wherein the receptor is a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a salt thereof. Agent. It is characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence after amino acid number 1 in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a partial peptide thereof or a salt thereof. A method for screening a substance having a brain / nerve cell protective action. Use of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10 or a partial peptide thereof or a salt thereof, the substance having a brain / neural cell protective action. Screening method. It is characterized by containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a partial peptide thereof or a salt thereof. Screening kit for a substance having a protective effect on brain and nerve cells. A substance having a protective effect on brain and nerve cells, comprising a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a partial peptide thereof, or a salt thereof. Screening kit. Brain characterized by using a nucleotide sequence encoding a polypeptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a nucleic acid containing a part thereof -A method for screening a substance having a neuronal protective action. Brain / nerve characterized by using a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a nucleic acid containing a part thereof A method for screening a substance having a cytoprotective action. It is characterized by containing a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, 4 or 6, or a nucleic acid containing a part thereof A screening kit for a substance having a brain / nerve cell protective action. A brain comprising a nucleotide sequence encoding a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 8 or 10, or a nucleic acid containing a part thereof. A kit for screening a substance having a neuroprotective effect. A method for protecting brain or nerve cells, comprising the steps of: providing a subject in need thereof with an amino acid identical or substantially identical to the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6; A method comprising administering a substance that inhibits a protein containing a sequence or a salt thereof. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence of amino acid number 1 or later in the amino acid sequence represented by SEQ ID NO: 2, 4 or 6 for producing a brain / nerve cell protective agent, or a protein thereof. Use of salt-suppressing substances.

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