JP2006050908A - Mib GENE-INDUCING beta-AMYLOID - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find out a new gene of which expression in astroglia cells is induced by a β-amyloid, identify a polypeptide encoded by the gene, and use the gene and polypeptide as means for diagnosing and treating neuropathy associated with the β-amyloid. <P>SOLUTION: This new gene of which expression is induced by a β-amyloid stimulation, is obtained by a cDNA subtraction method, and a new polynucleotide, a polypeptide encoded by the polynucleotide, a recombinant vector containing the polypeptide, a transformant having the recombinant vector, an antibody against the polypeptide, a method for producing the polypeptide, a method for identifying a compound regulating the expression of the gene and/or protein by using the above materials, the compound obtained from the method, a medicinal composition for the neuropathy associated with the β-amyloid by using them, a method of diagnosis, a diagnosis kit, a method of treatment, etc., are provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel gene Mib (a membrane protein induced by beta-amyloid) whose expression is induced by β-amyloid protein which is considered to be one of the causes of Alzheimer's disease. The present invention uses a polynucleotide containing the base sequence of Mib, a polypeptide encoded by the polynucleotide, a recombinant vector containing the polynucleotide, a transformant transformed with the recombinant vector, and the transformant. A method for producing a polypeptide, an antibody against the polypeptide, a method for identifying a compound that inhibits and / or suppresses the expression of Mib using the polypeptide, a compound obtained by the identification method, the above-mentioned polynucleotide, etc. Diagnostic methods such as astroglial cell migration, astroglial cell extension, and / or diseases associated with astroglial inflammatory factor production (eg, Alzheimer's disease), detection of Mib expression Migration of astroglia cells, extension of astroglial processes, and / or Disease (e.g., Alzheimer's disease) associated with the production of inflammatory factor astroglial diagnostic kits, such as, and pharmaceutical compositions such as comprising said polynucleotides and the like.

  β-amyloid is a constituent factor of senile plaques observed in the brain of Alzheimer's disease patients, and is considered to be an important factor in the early stage of Alzheimer's disease. β-amyloid is processed from β-amyloid precursors with certain proteases to form insoluble β-sheet structured aggregates. β-amyloid is thought to act directly or indirectly on nerve cells and promote the progression of Alzheimer's disease. As one of indirect actions of β-amyloid, activation of astroglial cells is known. When activated by β-amyloid, astroglial cells are known to extend their processes into senile plaques and produce several functional factors including inflammatory factors (eg, non-patented). See references 1-3). The activation of astroglial cells, which were thought to play a role in the repair of nerve cells damaged by senile plaques and the elimination of β-amyloid, actually, the astroglial cells secrete inflammatory factors. Therefore, it is known that the damage of nerve cells is worsened. In addition, it has been reported that astroglia cells that promote neurite outgrowth in the absence of β amyloid promote the destruction of neurons by β amyloid in the presence of β amyloid (for example, Non-Patent Document 4). checking).

  As a gene whose expression is induced by β-amyloid, the present inventor has prostaglandin E2 synthase (see Patent Document 1), aggrecanase-1 (ADAMTS-4: a disintegrin and metalloproteinase with thrombospondin motifs-4) (patent) We have already found Lib (see Document 2) and Lib (see Patent Document 3). However, analysis of the mechanism by which β-amyloid exacerbates Alzheimer's disease through activation of astroglial cells and factors that induce or suppress gene and / or protein expression in astroglia cells by β-amyloid are still insufficient.

JP 2001-258575 A JP 2001-352991 A JP 2003-164290 A Mark, R.A. E. et al. , Human Pathol. 26, pp 816-823 (1995) McGeer, P.M. L. et al. Brain Res. Rev. 21, pp 195-218 (1995) Edwardston, M.M. et al. , Neuroscience 54, pp 15-36 (1993). M.M. R. Domenici, et al. , Journal of Physiology-Paris 96, pp 313-316 (2002).

  The problem to be solved by the present invention is to find a gene whose expression is induced by β-amyloid in astroglial cells, and to use the found gene as astroglial cell migration, astroglial cell extension, and / or astrophile. It is to be used for diagnosis and / or treatment of diseases (eg, Alzheimer's disease) associated with the production of inflammatory factors of glial cells. Another subject of the present invention is the diagnosis and / or diagnosis of diseases associated with astroglial cell migration, astroglial cell extension, and / or production of astroglial inflammatory factors (eg Alzheimer's disease) The object is to provide a polypeptide encoded by a polynucleotide comprising the base sequence of the gene and an antibody against the polypeptide, which can be used for therapy. Another object of the present invention is to provide a method for identifying a compound that inhibits and / or suppresses the expression of the polynucleotide and / or the polypeptide, and a compound identified by the identification method. Further, another subject of the present invention is a disease (for example, Alzheimer's disease) associated with migration of astroglial cells, extension of astroglial processes, and / or production of inflammatory factors of astroglial cells using them. Diagnostic kits, diagnostic methods, pharmaceutical compositions, and therapeutic methods.

  In order to solve the problem, the present inventor performed cDNA subtraction treatment using β-amyloid-treated and untreated rat fetus-derived astroglia cells. The present invention was completed by identifying a gene whose expression is induced by β-amyloid treatment and determining a protein having an amino acid sequence encoded by the base sequence of the gene. Furthermore, an ortholog of the gene in humans was found and sequenced.

  Furthermore, the gene of the present invention is expressed in nerve cells in human brains not affected by Alzheimer's disease, decreased in nerve cells and expressed in astroglial cells in human brains affected by Alzheimer's disease. It was increased. This suggests that the gene of the present invention is a gene associated with a disease associated with astroglial cells (for example, Alzheimer's disease).

That is, the present invention provides the following:
[1] Any of the following polynucleotides or their complementary strands:
(1) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3 in the sequence listing; or (2) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing;
[2] A polynucleotide selected from the following group or a complementary strand thereof:
(1) having at least 70% base sequence homology with the polynucleotide according to (1) or (2) of [1], and migration of astroglial cells, extension of astroglial processes, and A polynucleotide encoding a polypeptide having a function associated with the production of inflammatory factors of astroglial cells;
(2) Hybridization under stringent conditions with the polynucleotide according to (1) or (2) of [1], and migration of astroglial cells, extension of astroglial processes, and / or astroglia A polynucleotide encoding a polypeptide having a function associated with the production of inflammatory factors in cells; and (3) one or several in the nucleotide sequence of the polynucleotide according to (1) or (2) of [1] Which encodes a polypeptide having a function associated with astroglial cell migration, astroglial cell extension, and / or astroglial inflammatory factor production A polynucleotide;
[3] A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing;
[4] Any of the following polypeptides:
(1) It has at least 70% amino acid sequence homology with the polypeptide according to [3], and astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammation A polypeptide having a function associated with factor production; or (2) in the amino acid sequence of the polypeptide according to [3], wherein one or several amino acids are deleted, substituted, or added, and the astroglial cell A polypeptide having a function associated with migration, elongation of astroglial processes, and / or production of astroglial inflammatory factors;
[5] A recombinant vector comprising the polynucleotide according to [1] or [2];
[6] Plasmid pBS SK (-)-rMib (FERM BP-08654);
[7] A transformant transformed with the recombinant vector according to [5] or the plasmid according to [6];
[8] A method for producing a polypeptide, comprising a step of culturing the transformant according to [7];
[9] An antibody that immunologically recognizes the polypeptide of [3] or [4];
[10] A method for identifying a compound that inhibits and / or suppresses the production of astroglial cell migration, astroglial cell extension, and / or production of inflammatory factors of astroglial cells, comprising the following steps:
(1) adding β-amyloid and a candidate compound to cells; and (2) mib expression of astroglia cells when mib expression is inhibited and / or suppressed in the cells. Identifying as a compound that inhibits and / or suppresses process elongation and / or production of inflammatory factors in astroglial cells;
[11] The polynucleotide according to [1] or [2], the polypeptide according to [3] or [4], the recombinant vector according to [5], the plasmid according to [6], [7] The method according to [10], wherein at least one of the transformant according to [9] or the antibody according to [9] is used;
[12] A compound identified by the identification method according to [10] or [11];
[13] The test tissue derived from brain tissue is tissue derived from a patient suffering from a disease associated with astroglial cell migration, astroglial cell extension, and / or production of astroglial inflammatory factors. A method for determining whether or not there is a method, comprising measuring the expression level of the polynucleotide according to [1] or [2] in the test tissue;
[14] A method for determining whether a test tissue derived from brain tissue is a tissue derived from an Alzheimer's disease patient, wherein the expression of the polynucleotide according to [1] or [2] in the test tissue A determination method characterized by measuring a quantity;
[15] The polynucleotide according to [1] or [2], the polypeptide according to [3] or [4], the recombinant vector according to [5], the plasmid according to [6], [7] Using at least one of the transformant according to [9], the antibody according to [9], or the compound according to [12], astroglial cell migration, astroglial cell extension, And / or a kit for diagnosis of a disease associated with the production of inflammatory factors of astroglial cells;
[16] The polynucleotide of [1] or [2], the polypeptide of [3] or [4], the recombinant vector of [5], the plasmid of [6], [7] A diagnostic kit for Alzheimer's disease, characterized by using at least one of the transformant according to [9], the antibody according to [9], or the compound according to [12];
[17] The polynucleotide of [1] or [2], the polypeptide of [3] or [4], the recombinant vector of [5], the plasmid of [6], [7] A pharmaceutical composition comprising at least one of the transformant according to [9], the antibody according to [9], or the compound according to [12];
[18] The polynucleotide according to [1] or [2], the polypeptide according to [3] or [4], the recombinant vector according to [5], the plasmid according to [6], [7] Astroglial cell migration, astroglial cell extension, and / or astroglia, comprising at least one of the transformant according to [9], the antibody according to [9], or the compound according to [12] An agent for ameliorating and / or treating a disease associated with the production of cellular inflammatory factors;
[19] The polynucleotide according to [1] or [2], the polypeptide according to [3] or [4], the recombinant vector according to [5], the plasmid according to [6], [7] An agent for improving and / or treating Alzheimer's disease, comprising at least one of the transformant according to [9], the antibody according to [9], or the compound according to [12];
About.

  The present invention provides a gene whose expression is induced by β-amyloid treatment. The provision of a pharmaceutical composition and / or diagnostic means utilizing the expression and / or function of the gene of the present invention can be used in clinical and basic medical fields of diseases related to β-amyloid such as Alzheimer's disease.

(Blastic amyloid treatment of astroglial cells)
Astroglia cells can be prepared from the brains of various animals. Examples of the animal to be used include rodents such as rats, mice, guinea pigs, and rabbits that can easily collect brain tissue, and preferably rats. As the brain tissue to be used, a brain derived from a fetus is preferably used. A method known per se is used for the preparation of astroglial cells from the embryo-derived brain. The method is disclosed in an experimental medicine separate volume neurobiochemistry manual (Junichi Ito et al. Yotsuya (1989)).

  Β-amyloid that acts on astroglial cells is commercially available and can be obtained from Anaspec. Β amyloid can also be prepared by genetic engineering techniques. Prior to treatment, β-amyloid may be aggregated by centrifugation or the like. The addition concentration and treatment time of β-amyloid are appropriately selected within the range showing physiological activity. The concentration is preferably 25 to 50 μM, and the treatment time is preferably 10 to 20 hours. In addition, β-amyloid-untreated astroglial cells can be prepared by seeding astroglia cells in a culture vessel and adding an equal amount of the buffer used for dilution of β-amyloid.

(Preparation of expressed gene)
The gene expressed in β-amyloid-treated and untreated astroglia cells can be collected by a commonly used RNA extraction method. The expressed gene may be used as mRNA or in the form of cDNA. For the conversion from mRNA to cDNA, a method using reverse transcriptase or the like can be applied. In order to obtain total RNA, for example, the AGPC method (acid guanidinium-phenol-chloroform method; Chomczynski et al., Anal. Biochem. 162, pp156-159 (1986)) can be applied to extract mRNA from total RNA. May be obtained by a purification method of poly A (+) RNA using an oligo dT column or the like. RNA samples obtained by a plurality of experiments can be mixed and used. In order to analyze the quality of each lot, the expression of several genes known to be expressed in astroglial cells may be monitored. For example, nerve growth factor (NGF), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), p75, etc. are selected as monitor genes, and a sample in which these expressions are equivalent may be used. .

(Subtraction process)
Examples of a method for obtaining a gene whose expression is induced by β-amyloid treatment include a subtraction treatment or a differential display method. In the subtraction process, two sets of gene groups are used: a subtracted gene group (tester) and a subtracted gene group (driver). For example, in order to obtain a gene whose expression is induced by β-amyloid, a gene obtained from a β-amyloid-treated cell as a tester (β-amyloid-treated gene) and a gene obtained from an untreated cell as a driver (untreated gene) Subtraction using) is recommended. Various known means can be used as the subtraction processing method (Japanese Patent Laid-Open No. 3-117488). Preferably, the cDNA subtraction method is used, and as the cDNA subtraction method, the RDA method (Representative Difference Analysis), the PCR-selection (PCR-select) cDNA method and the like are particularly preferable.

  In the subtraction treatment, the efficiency of the subtraction treatment can be confirmed by adding an appropriate DNA fragment to the tester side as a positive control for subtraction and confirming its concentration. In addition, if the blots before and after subtraction are subjected to Southern blot hybridization using a gene whose expression is known to be induced by β-amyloid treatment as a probe, the success or failure of the subtraction treatment should be confirmed as well. Can do. Examples of genes whose expression is known to be induced by β-amyloid treatment include NGF, iNOS (Inductive Nitric Oxide Synthase), and RANTES (Regulated on Activation Normal T-cell Expressed and Secreted).

  The PCR product after the subtraction treatment is inserted into an appropriate vector to prepare a plasmid library or the like. Two sets are prepared by amplifying the vector insertion part by PCR and dot blotting the genes on the membrane. A PCR product after subtraction treatment using a β-amyloid-treated gene as a tester and an untreated gene as a driver on the dot-blotted membrane [hereinafter, the subtraction treatment is represented by the formula of (tester)-(driver). ] And (untreated gene)-(β amyloid-treated gene) are subjected to hybridization with the PCR product probe after subtraction, respectively. A clone that is specifically enriched in the PCR product after subtraction of (β-amyloid-treated gene)-(untreated gene) compared to the PCR product after subtraction of (untreated gene)-(β-amyloid-treated gene) May be a gene whose expression is induced by β-amyloid treatment. The sequence of the vector insertion part can be determined by the DNA sequencing method.

(Acquisition of rat gene)
The gene provided in the present invention has been obtained by obtaining a part of the cDNA as a gene encoding a novel protein by cDNA subtraction treatment using rat fetal astroglial cells. This cDNA fragment cloned from rat fetal astroglial cells using the cDNA subtraction method as a gene induced by the addition of β-amyloid did not show homology to any gene in the database, and was confirmed to be a novel gene. . Screening was performed using the prepared β-amyloid-treated rat fetal astroglial cell-derived cDNA library as a probe to obtain a cDNA of about 7 kilobase pairs (bp). A sequencing reaction was performed using a fluorescent dye (Cy5; manufactured by Pharmacia) labeled M13 universal primer, a reverse primer, and an internal sequence primer, and a cDNA sequence was determined using an ALF Express fluorescent sequencer (Pharmacia). This sequence analysis revealed that this rat gene encodes a protein having a base sequence (SEQ ID NO: 1) of 6,996 bp and an amino acid sequence (SEQ ID NO: 2) of 2,106 amino acids. In addition, the ORF region (nucleotide 371 to nucleotide 6681) of this rat gene was registered (unpublished) in DDBJ with accession number AB161229.

  When this rat protein is analyzed by the prediction program PSORT, the signal sequence (amino acids 1 to 27) is followed by 23 transmembrane regions (amino acids 33 to 49, amino acids 82 to 98, amino acids 143 to 159, amino acids 165 to 181, Amino acids 196-212, amino acids 248-264, amino acids 361-377, amino acids 387-403, amino acids 409-425, amino acids 486-502, amino acids 548-564, amino acids 568-584, amino acids 599-615, amino acids 717-733, Amino acids 739 to 755, amino acids 769 to 785, amino acids 1247 to 1263, amino acids 1264 to 1280, amino acids 1538 to 1554, amino acids 1576 to 1592, amino acids 1610 to 1626, amino acids 1753 to 1769, amino 2020-2036) was had. Since it has many transmembrane regions, it was named a membrane protein induced by beta-amyloid (Mib), and this rat gene was named rat Mib (hereinafter also referred to as rMib). When the tissue distribution of the rat Mib of the present invention was examined, expression of a transcript of about 9 kbp was observed in the lung and kidney.

  In this specification, “Mib” is a membrane protein induced by beta-amyloid, and is used when referring to a Mib gene or a Mib protein or both unless otherwise specified.

(Acquisition of human ortholog)
The NCBI human genome database was searched to find the human ortholog of rat Mib (hereinafter also referred to as human Mib or hMib). A portion of the human counterpart was found on the NCBI genome database. Primers and the like were designed from a part of this corresponding sequence, and cloned from human small intestine-derived poly A (+) RNA (manufactured by CLONTECH) by RT-PCR. A sequencing reaction is performed using a fluorescent dye (Bigdye; manufactured by ABI) labeling terminator, an M13 universal primer, a reverse primer, and an internal sequence primer, and a sequence is determined using a fluorescent capillary sequencer ABI PRISM 3100 (manufactured by ABI). did. This human gene encoded a protein having a base sequence (SEQ ID NO: 3) of 6,795 bp and an amino acid sequence (SEQ ID NO: 4) of 2090 amino acids. The ORF region (nucleotide 265 to nucleotide 6534) of this human Mib gene was registered (unpublished) in DDBJ with accession number AB161230. When this human protein is analyzed by the prediction program PSORT, the signal sequence (amino acids 1-27) is followed by 24 transmembrane regions (amino acids 33-49, amino acids 83-99, amino acids 148-164, amino acids 166-182, Amino acids 197-213, amino acids 249-265, amino acids 378-394, amino acids 399-415, amino acids 421-437, amino acids 498-514, amino acids 560-576, amino acids 580-596, amino acids 611-627, amino acids 729-745, Amino acids 751 to 767, amino acids 781 to 797, amino acids 1258 to 1274, amino acids 1275 to 1291, amino acids 1300 to 1316, amino acids 1532 to 1548, amino acids 1569 to 1585, amino acids 1604 to 1620, amino acids 747-1763, had the amino acid 2004-2020). Human Mib showed 83% homology with rat Mib at the amino acid level and 69% at the nucleotide level (FIGS. 2-4). When the tissue distribution of human Mib was examined, strong expression of transcripts of about 11.6 kbp and about 9.0 kbp was observed in the heart, placenta, lung, liver, kidney and pancreas.

  The present inventor has identified the Mib gene as a gene whose expression is induced by β-amyloid treatment by the cDNA subtraction method. β-amyloid is a factor thought to promote the progression of Alzheimer's disease. The Mib gene was hardly expressed in rat brain in the absence of β-amyloid stimulation, and was also expressed lower in human brain than other organs. The present inventor believes that, from these expression kinetics, the Mib gene is expressed in the brain by β-amyloid stimulation and thereby exerts its function.

  It is expected to suppress Alzheimer's disease by suppressing the expression and / or function of the Mib gene. For example, even if β-amyloid deposits exist in the brain, suppression of the expression of the Mib gene suppresses the expression, delay, improvement, and / or treatment of Alzheimer's disease by suppressing the expression of the Mib gene. I think it will be possible.

(Mib function)
In general, cells do not migrate in the adult steady state brain, but when nerve cells are damaged, such as by brain disease, glial cells may migrate to the site of injury to repair and / or protect this damage. Are known. In the vicinity of senile plaques in which β-amyloid of Alzheimer's disease is aggregated, it is known that nerve cells are damaged and neuronal cells are lost due to neurofibrillary tangles. At this time, the astroglial cells move to the damaged site so as to surround the senile plaque so as not to further spread the influence of the insoluble protein aggregate (ie, senile plaque) that has become a foreign substance in the living body. It is known to elongate the protrusion toward the head. Accumulation of astroglial cells at the lesion site and extension of the process should be a biological defense mechanism originally intended to repair nerve cells damaged by senile plaques and eliminate foreign substances. It is known that astroglial cells secrete inflammatory factors, further exacerbating fragile neuronal damage. It is also known that astroglial cells that promote neurite outgrowth in the absence of β-amyloid promote neuronal damage by β-amyloid in the presence of β-amyloid.

  On the other hand, the present inventors have found a rat Mib gene whose expression is induced in the brain by treatment with β-amyloid. This gene was presumed to be localized in the cell membrane from its structure. The human Mib gene is expressed in nerve cells in human brains not affected by Alzheimer's disease, but is decreased in neurons around senile plaques in human brains affected by Alzheimer's disease. And found that expression is enhanced.

  Characteristics of astroglial cells when β-amyloid is aggregated as described above, expression of Mib is induced by β-amyloid, and expression of Mib is specific to astroglial cells adjacent to senile plaques in Alzheimer's disease patients Judging from the fact that Mib is presumed to be localized in the cell membrane, Mib is expressed in the cell membrane of astroglial cells by β-amyloid stimulation, migratory astroglial cells, and processes of astroglial processes. The inventor believes that it is associated with elongation and / or production of inflammatory factors in astroglial cells and as a result is involved in the progression of Alzheimer's disease. In this case, Mib is directly associated with migration of astroglial cells, elongation of astroglial processes, and / or production of inflammatory factors of astroglial cells, or a ligand or the like binds to Mib expressed on the cell membrane. It is believed to be associated with signal transduction downstream, migration of astroglial cells, extension of astroglial processes, and / or production and / or secretion of inflammatory factors from astroglial cells.

  As a method for measuring the migration of astroglial cells, for example, a method using a membrane having pores coated with a reconstituted extracellular matrix (ECM) can be mentioned. Specifically, astroglial cells or the like in which the Mib gene of the present invention is expressed or astroglial cells or the like in which the expression level of the Mib gene is reduced by the siRNA method are prepared, and the cells are seeded on the membrane. After leaving still, the cells remaining on the surface of the upper chamber without moving are removed with a cotton swab, and the moved cells are fixed to the back of the chamber. The migration of astroglial cells can be measured by staining the cells and counting with a microscope or the like. In this experiment, astroglial cells or the like in which the Mib gene is not expressed or astroglial cells not subjected to the siRNA method can be used as control cells. Examples of the membrane having pores coated with the reconstructed ECM include a BioCoat matrix 24 well chamber (manufactured by BD Falcon).

  Examples of the method for measuring the elongation of the astroglial process include astroglial cells in which the Mib gene of the present invention is expressed by gene transfer or astroglial cells in which the expression level of the Mib gene is reduced by the siRNA method. There is a method in which protrusion extension is induced and the length or the number of protrusions is directly measured with a microscope. In this experiment, astroglial cells or the like in which the Mib gene is not expressed or astroglial cells not subjected to the siRNA method can be used as control cells.

  As a method for measuring the production and / or secretion of inflammatory factors in astroglial cells, for example, in astroglial cells in which the Mib gene of the present invention is expressed, inflammatory factors are encoded by Northern blot or RT-PCR. Examples thereof include a method for measuring the expression level of mRNA or a method for measuring the amount of inflammatory factor protein by ELISA. Alternatively, a method for producing astroglia cells and the like in which the expression level of the Mib gene is reduced by the siRNA method, and measuring the expression level or protein amount of the inflammatory factor in the cells using Northern blot, RT-PCR, or ELISA Is mentioned. In these methods, astroglial cells or the like that do not express the Mib gene, or astroglial cells that are not subjected to the siRNA method can be used as control cells. Examples of the inflammatory factor considered to be related to the Mib gene of the present invention and the amount of production and / or secretion thereof include, for example, nerve growth factor (NGF), inducible nitric oxide synthase (iNOS), and regulated on activation normal T-cell. Expressed and Secreted (RANTES), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), S100β and the like.

(Polynucleotide and its complementary strand)
The polynucleotide of the present invention or a complementary strand thereof is a polynucleotide consisting of the base sequence described in SEQ ID NO: 1 or 3 in the sequence listing or a partial base sequence thereof, or a complementary strand thereof, or the sequence described in SEQ ID NO: 1 or 3 in the sequence listing. A polynucleotide comprising a base sequence or a partial base sequence thereof or a complementary strand thereof, a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing or a partial amino acid sequence thereof, or a complementary strand thereof, or a sequence thereof Examples thereof include a polynucleotide encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a partial amino acid sequence thereof, or a complementary chain thereof. The polynucleotide of the present invention is preferably a polynucleotide having a function associated with migration of astroglial cells, extension of astroglial processes, and / or production of inflammatory factors of astroglial cells. In addition, the polynucleotide of the present invention is preferably a polynucleotide in which the translated protein has cell membrane localization.

  Examples of the partial base sequence include a base sequence encoding an amino acid fragment of a characteristic structural part of Mib. Examples of such a partial base sequence include a signal sequence portion or a transmembrane region of the amino acid sequence described in SEQ ID NO: 2 in the sequence listing, that is, amino acids 1 to 27, or amino acids 33 to 49, amino acids 82 to 98, amino acids. 143-159, amino acids 165-181, amino acids 196-212, amino acids 248-264, amino acids 361-377, amino acids 387-403, amino acids 409-425, amino acids 486-502, amino acids 548-564, amino acids 568-584, amino acids 599 to 615, amino acids 717 to 733, amino acids 739 to 755, amino acids 769 to 785, amino acids 1247 to 1263, amino acids 1264 to 1280, amino acids 1538 to 1554, amino acids 1576 to 1592, amino acids 1610 to 1626, a Amino Acids 1753-1769, or the base sequence can be mentioned that encode any of the amino acid fragment of an amino acid 2020-2036. Alternatively, as such a partial base sequence, for example, a signal sequence or a transmembrane region of the amino acid sequence described in SEQ ID NO: 4 of the sequence listing, that is, amino acids 1 to 27, or amino acids 33 to 49, amino acids 83 to 99, Amino acids 148-164, Amino acids 166-182, Amino acids 197-213, Amino acids 249-265, Amino acids 378-394, Amino acids 399-415, Amino acids 421-437, Amino acids 498-514, Amino acids 560-576, Amino acids 580-596, Amino acids 611-627, Amino acids 729-745, Amino acids 751-767, Amino acids 781-797, Amino acids 1258-1274, Amino acids 1275-1291, Amino acids 1300-1316, Amino acids 1532-1548, Amino acids 1569-158 , Amino 1604 to 1620, amino acid 1747 to 1,763, the base sequence can be mentioned that encode any of the amino acid fragment of an amino acid 2004 to 2020.

  In addition, the polynucleotide of the present invention or a complementary strand thereof is a polynucleotide having at least about 70% base sequence homology with the above-described polynucleotide or a complementary strand thereof, and substantially the same as the full-length Mib. The thing which has the function of the same quality is mentioned. Mib functions preferably include functions associated with astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammatory factor production. The polynucleotide of the present invention is preferably a polynucleotide in which the translated protein has cell membrane localization. The selected polynucleotide is preferably about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% or more, more preferably about 80%, about 85% , About 90%, about 95%, or about 99% or more, more preferably about 90%, about 95%, or about 99% or more. A technique for determining the homology of a base sequence is known per se, and for example, a method for directly determining a base sequence can be used.

  The polynucleotide of the present invention or a complementary strand thereof is a polynucleotide that hybridizes with the above-mentioned polynucleotide under stringent conditions or a complementary strand thereof, and has a function substantially the same as that of the full-length Mib. The thing which has. Mib functions preferably include functions associated with astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammatory factor production. The polynucleotide of the present invention is preferably a polynucleotide in which the translated protein has cell membrane localization. A “polynucleotide that hybridizes to a polynucleotide under stringent conditions” can be obtained, for example, by the method described in Molecular Cloning 2nd edition (J. Sambrook et al. (1989)). Here, “hybridization under stringent conditions” means, for example, 0.1 × SSC after heating in a solution of 6 × SSC, 0.5% SDS and 50% formamide at 42 ° C. This shows that a positive hybridization signal that can be discriminated even under a condition of washing at 68 ° C. in a 0.5% SDS solution is observed.

  The polynucleotide of the present invention or a complementary strand thereof is a polynucleotide or its complementary strand in which one or several nucleotides are deleted, substituted, or added in the base sequence of the above-mentioned polynucleotide, and has a full length. Those having substantially the same function as Mib. Mib functions preferably include functions associated with astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammatory factor production. The polynucleotide of the present invention is preferably a polynucleotide in which the translated protein has cell membrane localization. Nucleotide deletion, substitution, or addition means are known per se, and examples include a method for producing a deletion mutant using exonuclease, a site-directed mutagenesis method, and the like.

  All of the polynucleotides of the present invention provide genes useful for the production of the polypeptides of the present invention. In addition, the polynucleotide of the present invention can be used as a probe or primer for mRNA detection or as an antisense oligomer that regulates gene expression. For example, when the polynucleotide of the present invention is used as an antisense, the expression of the polynucleotide of the present invention is specifically inhibited. Furthermore, the polynucleotide of the present invention can also be used as a reagent for nucleic acids. The polynucleotide of the present invention may be single-stranded or double-stranded, and in the case of single-stranded, it may be a sense strand or an antisense strand. The polynucleotide of the present invention may be DNA or RNA.

(Polypeptide)
Examples of the polypeptide of the present invention include a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing or a partial amino acid sequence thereof. Moreover, as a polypeptide of this invention, the polypeptide containing the amino acid sequence of the sequence number 2 or 4 of a sequence table, or its partial amino acid sequence is mentioned. The polypeptide of the present invention is preferably a polypeptide having a function related to migration of astroglial cells, elongation of astroglial processes, and / or production of inflammatory factors of astroglial cells. The polypeptide of the present invention is preferably a polypeptide in which the translated protein has cell membrane localization.

  Examples of the partial amino acid sequence include amino acid fragments of the characteristic structural portion of Mib. Examples of such a partial amino acid sequence include, for example, a signal sequence portion or a transmembrane region of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, that is, amino acids 1 to 27, or amino acids 33 to 49, amino acids 82 to 98, amino acids. 143-159, amino acids 165-181, amino acids 196-212, amino acids 248-264, amino acids 361-377, amino acids 387-403, amino acids 409-425, amino acids 486-502, amino acids 548-564, amino acids 568-584, amino acids 599-615, amino acids 717-733, amino acids 739-755, amino acids 769-785, amino acids 1247-1263, amino acids 1264-1280, amino acids 1538-1554, amino acids 1576-1592, amino acids 1610-1626 Amino 1753-1769, or include any of the amino fragment of an amino acid 2020 to 2036). Alternatively, as such a partial amino acid sequence, for example, a signal sequence or a transmembrane region of the amino acid sequence described in SEQ ID NO: 4 in the sequence listing, that is, amino acids 1 to 27, or amino acids 33 to 49, amino acids 83 to 99, Amino acids 148-164, Amino acids 166-182, Amino acids 197-213, Amino acids 249-265, Amino acids 378-394, Amino acids 399-415, Amino acids 421-437, Amino acids 498-514, Amino acids 560-576, Amino acids 580-596, Amino acids 611-627, Amino acids 729-745, Amino acids 751-767, Amino acids 781-797, Amino acids 1258-1274, Amino acids 1275-1291, Amino acids 1300-1316, Amino acids 1532-1548, Amino acids 1569-1 85, amino acids 1604-1620, amino acids 1747 to 1,763, and any amino acid fragment of amino acids 2004 to 2020 is.

  In addition, the polypeptide of the present invention includes a polypeptide having at least about 70% amino acid sequence homology with the above-mentioned polypeptide and having substantially the same function as Mib. . Mib functions preferably include functions associated with astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammatory factor production. The polypeptide of the present invention is preferably a polypeptide having cell membrane localization. The selected polypeptide is preferably about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% or more, more preferably about 80%, about 85% About 90%, about 95%, or about 99% or more, more preferably about 90%, about 95%, or about 99% or more of amino acid sequence homology.

  The polypeptide of the present invention is a polypeptide in which one or several amino acids are deleted, substituted, or added in the amino acid sequence of the above-mentioned polypeptide, and has substantially the same function as Mib. A polypeptide having Mib functions preferably include functions associated with astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammatory factor production. The polypeptide of the present invention is preferably a polypeptide having cell membrane localization.

  The polypeptide of the present invention can be altered to the extent that no significant change in function is involved, such as modification of its constituent amino group or carboxyl group. The polypeptide of the present invention is a natural polypeptide, a recombinant polypeptide, or a synthetic polypeptide, preferably a recombinant polypeptide.

  The polypeptides of the present invention can themselves be used in pharmaceutical compositions for regulating in vivo functions. Moreover, the polypeptide of the present invention can be used for screening for obtaining compounds capable of regulating their functions, such as inhibitors, antagonists, activators, and the like, and obtaining antibodies against them. Furthermore, the polypeptide of the present invention can also be used as a reagent.

(Recombinant vector)
A recombinant vector can be obtained by incorporating the polynucleotide of the present invention into an appropriate vector. As a vector, in addition to a naturally occurring one extracted, a part of a polynucleotide other than a part necessary for propagation may be missing. For example, there are vectors derived from Col E1 and vectors derived from lambda phage. As a method for incorporating the polynucleotide of the present invention into the vector, a method known per se can be applied. For example, a method is used in which an appropriate restriction enzyme is selected, the polynucleotide is cleaved at a specific site, then mixed with a similarly treated vector, and religated by ligase. In pBS SK (−)-rMib (FIG. 5) shown in Examples described later, pBluescript SK (−) (manufactured by Stratagene) was used as a vector, but it is not limited thereto. Plasmid pBSSK (-)-rMib was deposited on March 5, 2004 as FERM BP-08654 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary, and on April 19, 2004 Received a certificate of survival certificate.

(Transformant)
In addition to the cell-free protein expression system as described above, the polypeptide of the present invention and its derivative can be obtained by gene recombination technology using a host known per se such as E. coli, yeast, Bacillus subtilis, insect cells, animal cells and the like. Can be provided.

  For the transformation, a method known per se can be applied. For example, the host is transformed using a plasmid, chromosome, virus or the like as a replicon. As a more preferable system, an integration method into a chromosome can be mentioned in consideration of gene stability. For convenience, an autonomous replication system using an extranuclear gene is used. The vector is selected depending on the type of host, and has as its constituent elements a gene sequence for expression and a gene sequence carrying information on its replication and control. The constituent elements are selected depending on whether the host is a prokaryotic cell or a eukaryotic cell, and a promoter, a ribosome binding site, a terminator, a signal sequence, an enhancer and the like are used in combination by a method known per se.

(Method for producing polypeptide)
The transformant can be used for the production of the polypeptide of the present invention by selecting and culturing optimal conditions known for the culture conditions of each host known per se. The culture may be performed using the physiological activity of the polypeptide to be expressed and produced as an index, or may be performed by subculture or batch using the amount of transformant in the medium as an index.

  A transformant for producing the polypeptide of the present invention may be, for example, a competent cell using a vector (for example, pBS SK (−)-rMib) incorporating the gene of the present invention. It can be obtained by transforming E. coli DH5α. The polypeptide of the present invention can be obtained by performing amplification of a vector (for example, pBSSK (−)-rMib) incorporating the gene of the present invention using these transformants. As a method for purifying and / or recovering a polypeptide consisting of a polypeptide from a medium and its derivative, a combination of molecular sieve, ion column chromatography, affinity chromatography, etc., or fractionation of ammonium sulfate, alcohol, etc. based on a difference in solubility. For example, but not limited to. More preferably, examples of the method for recovering the polypeptide include, but are not limited to, a method in which an antibody against the amino acid sequence is prepared based on the information on the amino acid sequence and specifically adsorbed and recovered by a polyclonal antibody or a monoclonal antibody.

(antibody)
An antibody is prepared by selecting antigenic determinants of a polypeptide comprising the polypeptide of the present invention and its derivative. The antigenic determinant is composed of at least 5-15, preferably at least 7-12, more preferably at least 8-10 amino acids. This amino acid sequence does not necessarily need to be homologous to the polypeptide encoded by the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3; It may be an exposed part. If the exposed part is a discontinuous part, it is also effective that the exposed part has a continuous amino acid sequence. The antibody is not particularly limited as long as it immunologically recognizes a polypeptide comprising the protein and / or polypeptide of the present invention and a polypeptide derived therefrom. The presence or absence of this recognition can be determined by a known antigen-antibody binding reaction.

  In order to produce antibodies, a polypeptide comprising the polypeptide of the present invention and a derivative thereof can be singly or conjugated to a carrier in the presence or absence of an adjuvant to a humoral response to animals and / or Induction of immunity such as cellular response. The carrier is not particularly limited as long as it does not adversely affect the host itself, and examples thereof include cellulose, polymerized amino acid, albumin and the like. As the animal to be immunized, mouse, rat, rabbit, goat, horse and the like are preferably used, but are not limited thereto. The polyclonal antibody is obtained by a method for recovering antibody from serum known per se. Preferable means includes, for example, immunoaffinity chromatography.

  Production of a monoclonal antibody can be performed by collecting antibody-producing cells from an animal that has been subjected to the above-described immunization means, and introducing a means for transformation into a per se known permanent proliferative cell.

  Such a polyclonal antibody or monoclonal antibody can directly bind to the protein and / or polypeptide of the present invention and control its function and / or activity. Such polyclonal or monoclonal antibodies are useful for diagnosis, prevention, suppression, delay, amelioration and / or treatment of diseases involving the function and / or activity of the protein and / or polypeptide of the present invention.

(Use of the above polynucleotide, polypeptide, recombinant vector, transformant, antibody)
One of the features of the present invention is associated with migration of astroglia cells, extension of astroglial processes, and / or production of inflammatory factors in astroglia cells by detecting Mib expression in the brain It relates to the diagnosis of diseases such as Alzheimer's disease. That is, when the expression of Mib in a brain sample is detected and the expression of Mib is high, the sample can be determined as a brain sample derived from a subject suffering from Alzheimer's disease. The detection of Mib expression may be performed by detecting the Mib gene or by detecting the Mib protein. For the detection of Mib expression, the above-mentioned polynucleotides, polypeptides, recombinant vectors, transformants, and antibodies can be used. Specifically, the Mib gene expression detection method includes Northern blotting or RT-PCR using a probe designed from the above-mentioned polynucleotide. Examples of a method for detecting the expression of the Mib protein include Western blotting using the above-described antibody.

  Another of the features of the invention is that astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammation by suppressing and / or inhibiting Mib expression by β-amyloid It relates to methods for inhibiting, delaying, ameliorating and / or treating the onset and / or progression of diseases associated with the production of sex factors, such as Alzheimer's disease. That is, it was found for the first time that expression of Mib is induced by β-amyloid, and it was found that it is related to the relationship with the brain, particularly the onset and / or progression of Alzheimer's disease. Based on this novel finding, even if β-amyloid is present, the onset and / or progression of Alzheimer's disease can be suppressed, delayed, ameliorated, and / or inhibited by suppressing and / or inhibiting the induction of expression of Mib. It can be treated.

  Inhibiting and / or inhibiting Mib expression includes, for example, inhibiting mRNA expression of a structural gene encoding Mib. Such an inhibitor can be obtained, for example, by screening a candidate compound using β amyloid and using an mRNA expression system of a structural gene encoding Mib. A compound capable of suppressing and / or inhibiting the expression of Mib mRNA by β-amyloid obtained by such screening can be provided as a prophylactic / therapeutic agent for Alzheimer's disease.

(Identification of compounds)
The polynucleotides, polypeptides, recombinant vectors, transformants and antibodies thus prepared can be used for astroglial cell migration, astroglial cell extension, and / or astroglial cells, alone or in combination of multiple means. It provides an effective means for identifying compounds that inhibit and / or suppress the production of other inflammatory factors.

  Such an identification method is not particularly limited as long as it can compare the expression change of Mib in cells to which β-amyloid and a candidate compound are added. Specifically, for example, (1) β amyloid is added to one cell, β amyloid and a candidate compound are added to the other cell, and (2) the expression of Mib in the cell to which β amyloid is added, and β amyloid And the expression of Mib in cells to which the candidate compound was added, and (3) the expression of Mib in the cells to which β-amyloid and the candidate compound were added compared to the expression of Mib in the cells to which β-amyloid was added In addition, the candidate compound can be identified as a compound that inhibits and / or suppresses astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammatory factor production. The addition of the candidate compound may be before β amyloid addition, at the same time as β amyloid addition, or after β amyloid addition. The expression of Mib to be compared may be mRNA expression or protein expression as long as the expression levels can be compared. The amount of Mib mRNA can be detected by, for example, Northern blotting or RT-PCR using a polynucleotide containing a part of the base sequence represented by SEQ ID NO: 1 or 3 in the sequence listing as a probe. The amount of the Mib protein can be detected by, for example, Western blotting or ELISA using an antibody against a polypeptide containing a part of the amino acid sequence represented by SEQ ID NO: 2 or 4 in the sequence listing. A polynucleotide containing a part of the base sequence represented by SEQ ID NO: 1 or 3 in the sequence listing is incorporated into a recombinant vector. A transformant is produced by transforming E. coli cells, and a polypeptide is produced using this transformant, or a polypeptide is synthesized from the amino acid sequence represented by SEQ ID NO: 2 or 4 in the sequence listing. An antibody against the Mib protein is prepared using such a polypeptide. Specific antibody production methods have been described in detail above. Also, when the expression of Mib in cells added with β-amyloid and a candidate compound is reduced by at least 20% compared to the expression of Mib in cells added with β-amyloid, preferably at least 30%, more preferably at least 40% And / or more preferably at least 50% reduced and / or suppressed the astroglial cell migration, astroglial process extension, and / or astroglial inflammatory factor production when the candidate compound is reduced by at least 50% It can be identified as a compound.

  In addition, for example, selection of antagonists by drug design based on the three-dimensional structure of polypeptides, selection of expression regulators at the gene level using a protein expression system, selection of antibody recognition substances using antibodies, etc. are known per se. It can be used in a pharmaceutical screening system.

  The compound obtained by this identification method is useful as a pharmaceutical composition and a therapeutic agent for diseases described later.

(Compound, pharmaceutical composition, diagnostic means)
Compounds obtained by the above identification methods treat diseases associated with astroglial cell migration, astroglial cell extension, and / or astroglial inflammatory factor production (eg, Alzheimer's disease) Can be used as a candidate compound. Candidate compounds include proteins, polypeptides, non-antigenic polypeptides, and low molecular compounds, but are not limited thereto, and are preferably low molecular compounds.

  The candidate compounds thus selected are selected in consideration of biological usefulness, toxicity, etc., so that astroglial cell migration, astroglial process extension, and / or astroglial inflammatory factor It can be prepared as a pharmaceutical composition for use in the treatment of diseases associated with production (eg, Alzheimer's disease). Further, a polynucleotide of the present invention or a complementary strand thereof, a polypeptide of the present invention, a recombinant vector containing the polynucleotide or the complementary strand thereof, a transformant transformed using these recombinant vectors, and the polypeptide The immunologically recognizing antibody itself has functions such as inhibition, antagonism and activation of the expression of the gene and / or protein of the present invention, and the expression and / or function of the gene and / or protein of the present invention. It can be used as a pharmaceutical means used for the treatment and / or prevention of a disease involving selenium.

  Examples of diseases associated with migration of astroglial cells, elongation of astroglial cell processes, and / or production of inflammatory factors of astroglial cells include neurological diseases such as Alzheimer's disease. The expression of Mib of the present invention is induced by β-amyloid treatment, and the expression is kept low in the absence of β-amyloid stimulation. Since Mib is expressed in the brain by β-amyloid stimulation and thereby exerts its function, an inhibitory effect on Alzheimer's disease by suppressing Mib expression and / or function is expected. In addition, since cell membrane localization is predicted with a high probability, it is highly likely that Mib is related to a disease mechanism or a drug discovery target. Therefore, the present inventor believes that inhibition of Mib expression and / or function inhibition can be used for treatment of diseases, particularly Alzheimer's disease.

  The above candidate compound, or the polynucleotide of the present invention or a complementary strand thereof, the polypeptide of the present invention, a recombinant vector containing the polynucleotide or the complementary strand thereof, a transformant transformed using these recombinant vectors, or An antibody that immunologically recognizes the polypeptide may be formulated in combination with an appropriate pharmaceutical carrier to migrate astroglial cells, extend astroglial processes, and / or produce inflammatory factors of astroglial cells. It can be used as an agent for preventing and / or treating related diseases (eg, Alzheimer's disease). Such a formulation uses a therapeutically effective amount of the above candidate compound, or a polynucleotide of the present invention or a complementary strand thereof, a polypeptide of the present invention, a recombinant vector comprising the above polynucleotide or the complementary strand thereof, and these recombinant vectors. A transformed transformant or an antibody that immunologically recognizes the above polypeptide and a pharmaceutically acceptable carrier and / or excipient are included. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and mixtures thereof. What is necessary is just to select a prescription suitable for an administration route.

  The route of administration may be systemic administration or local administration. One preferred embodiment of systemic administration includes injection, for example, intravenous injection. Other injection routes such as subcutaneous, intramuscular, or intraperitoneal can also be used. As another mode of administration, oral administration such as enteric formulation or capsule formulation is also possible. In addition, transmucosal or transdermal administration using penetrants such as bile salts or fusidic acid or other surfactants may be used. For topical administration, forms such as salves, pasta and gels can be used.

  The required dose range is the above-mentioned candidate compound, or the polynucleotide of the present invention or a complementary strand thereof, the polypeptide encoded by them, the recombinant vector containing the polynucleotide or the complementary strand thereof, and the characteristics obtained using these recombinant vectors. Depending on the effectiveness of the transformed transformant or antibody immunologically recognizing the polypeptide, route of administration, nature of the formulation, subject's symptoms and the judgment of the attending physician, the appropriate dose may be, for example, subject weight The range is 0.01 to 1000 μg per kg, preferably 0.05 to 500 μg per kg body weight, more preferably 0.1 to 100 μg per kg body weight.

  In formulating a compound, a polypeptide, polypeptide, protein, polynucleotide, antibody, etc., a formulating means known per se according to each control may be introduced. Specifically, for example, preparation methods such as powders, pills, tablets, capsules, aqueous solution preparations, ethanol solution preparations, liposome preparations, delipidated emulsions, and inclusion bodies such as cyclodextrins can be used.

  Powders, pills, capsules and tablets are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, cidroxypropyl It can be produced using a binder such as cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin.

  The suspension can be produced using water, sugars such as sucrose, sorbitol and fructose, glycols such as polyethylene glycol, oils and the like.

  The solution for injection can be prepared using a carrier made of a salt solution, a glucose solution, or a mixture of salt water and a glucose solution.

  Liposomeization is performed, for example, by adding a solution in which the substance is dissolved in a solvent (such as ethanol) to a solution in which the phospholipid is dissolved in an organic solvent (such as chloroform), and then distilling off the solvent. And soaking, sonicating and centrifuging, and then collecting the supernatant by filtration.

  The fat emulsifier is, for example, mixed with the substance, oil components (vegetable oil such as soybean oil, sesame oil, olive oil, etc.), emulsifier (phospholipid etc.), etc., heated to make a solution, and then added with the required amount of water to emulsify. It can be carried out by emulsification and / or homogenization using a machine (homogenizer). It can also be lyophilized. In addition, when emulsifying fat, an emulsification aid may be added. Examples of the emulsification aid include glycerin and saccharides (eg, glucose, sorbitol, fructose, etc.).

  In cyclodextrin inclusion, for example, a solution in which cyclodextrin is heated and dissolved in water or the like is added to a solution in which the substance is dissolved in a solvent (ethanol), and then cooled and the deposited precipitate is filtered and sterilized. This can be done by drying. At this time, the cyclodextrin to be used may be appropriately selected from cyclodextrins (α type, β type, γ type) having different pore diameters according to the size of the substance.

  A polynucleotide of the present invention or a complementary strand thereof, a polypeptide of the present invention, a recombinant vector comprising the polynucleotide or the complementary strand thereof, a transformant transformed with these recombinant vectors, and an immunology of the polypeptide Antibody that recognizes is useful as a diagnostic tool for diseases associated with astroglial cell migration, astroglial cell extension, and / or production of astroglial inflammatory factors (eg, Alzheimer's disease) . In particular, it is useful as a diagnostic tool for diagnostic markers and / or reagents for Alzheimer's disease. In the present specification, means means a method and / or medium used for achieving the object. That is, for example, the diagnostic means includes a method for diagnosis, a reagent used for diagnosis, a kit, and the like.

  Diagnosis is performed by determining the abundance of the polynucleotide and / or polypeptide of the present invention in a sample. As the sample, blood, sputum fluid, brain biopsy sample and the like are preferable, and cerebrospinal fluid is particularly preferable. As a measurement method used for diagnosis, a known antigen-antibody reaction, an enzyme reaction system, a PCR reaction system, or the like can be used.

  Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following Examples.

(Culture of glial cells)
Astroglia cells were obtained from rat cerebral cortex. The cerebrum of Wister rat fetus (Charles River, 17 days old) was collected, and the cells were dispersed by trypsin treatment. The obtained cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal calf serum (FCS) and antibiotic solution for 1 week, and then further subcultured in DMEM containing 10% FCS. It was confirmed by a fluorescent antibody method that 90% or more of the cells were positive for glial fibrillary acidic protein.

(Preparation of β-amyloid and β-amyloid treatment of astroglia cells)
β-amyloid (anaspec, β-amyloid 1-40) was dissolved in sterilized water, stored at −70 ° C. and melted at the time of use. Aggregation of β-amyloid was performed by dissolving 1 mM β-amyloid in Eagle buffer, holding it in a CO ₂ incubator for 1 week, and then centrifuging at 10,000 × g for 10 minutes. The pellet was resuspended in DMEM containing B27 supplement (Gibco BRL), adjusted to an appropriate concentration, and used. The astroglia cells after the secondary culture were washed with a phosphate buffer (PBS), converted to a serum-free medium containing a B27 supplement, cultured for 2 days, and further cultured for 15 hours with addition of 25 μM aggregated β-amyloid. .

(CDNA subtraction and library screening)
Total RNA was extracted nine times by AGPC from β-amyloid-treated and untreated astroglial cells (6 cm plastic dishes, 20 each). Poly A (+) RNA was prepared using oligo dT-latex (Roche).

β-amyloid-treated and untreated astroglial cell-derived poly A (+) RNA 2 μg each (β-amyloid-treated gene) − (untreated gene) and (untreated gene) − (β-amyloid-treated gene) The cDNA subtraction treatment was performed using a PCR-select cDNA subtraction kit (manufactured by CLONTECH) according to the method of Diachenko et al. (Proc. Natl. Acad. USA 93, pp 6025-6030 (1996)). As a positive control for the subtraction treatment, φΧ174 / Hae III digested DNA fragment was added to the tester after Rsa I digestion so that the weight ratio in the tester was about 2.5 × 10 ⁻⁵ .

  In order to verify the efficiency of the subtraction treatment performed, Southern blot hybridization was performed on the PCR product after subtraction and the PCR product before subtraction using the φ 対 照 174 / Hae III digested DNA fragment as a positive control as a probe.

  Ligation of the (β-amyloid-treated gene)-(untreated gene) PCR product vector [pBluescript SK + (Stratagene)] to prepare a plasmid library, and DH5α competent cell (Gibco BRL) Transformation was performed.

Two sets of 960-clone vector insertion portions of the plasmid library were amplified by PCR and dot-blotted 60-100 ng of each gene on a membrane (Hybond-N +; manufactured by Amersham). A probe of the PCR product after subtraction of (β amyloid-treated gene)-(untreated gene) and (untreated gene)-(β-amyloid-treated gene) was applied to this dot blot membrane with 0.5 M Na ₂ HPO _4. (PH 6.8) Hybridization was carried out at 68 ° C. for 16 hours in a 7% SDS solution containing 10 μg / ml salmon sperm DNA. The membrane was washed at 68 ° C. for 30 minutes using 0.1 × SSC containing 0.1% SDS. Compared with the PCR product after subtraction of (untreated gene)-(β-amyloid-treated gene), clones that are specifically enriched in the PCR product after subtraction of (β-amyloid-treated gene)-(untreated gene) Selected.

(Analysis by Northern blot hybridization)
Northern blot hybridization using β amyloid-treated and untreated astroglial cell-derived poly A (+) RNA was carried out with each vector insertion part of the selected clone group labeled with ³² P as a probe. By the above method, each poly A (+) RNA sample was prepared from β-amyloid-treated and untreated astroglial cells. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA and β-actin cDNA (manufactured by CLONTECH), which are normally expressed genes, were used as internal standard probes. Detection was performed using BAS2000 (Fuji Film). The G3PDH gene and β-actin gene showed no difference in the expression level when treated with β amyloid and when not treated, whereas the gene of the present invention was maintained at a low level when not treated with β amyloid However, it increased about 7.0 times by the beta amyloid treatment (FIG. 1).

(Base sequence analysis and homology search)
The gene of the present invention has a higher order structure, and it was difficult to identify the sequence under normal sequence reaction conditions. The inventor has examined the denaturation temperature, denaturation time, and annealing temperature of the sequencing reaction in detail, and has made possible the sequence analysis of this gene. The specific sequence reaction conditions were such that the denaturation temperature during the amplification reaction was increased to 96 ° C., and the denaturation time was extended to 20-30 seconds. The annealing temperature was set as high as possible for each primer in order to avoid re-formation of the secondary structure of the template DNA at a low temperature. This clone, the expression of which is induced by β-amyloid, is subjected to a sequencing reaction using a fluorescent dye (Cy5; Pharmacia) -labeled M13 universal primer, reverse primer, and internal sequence primer, and an ALF Express fluorescence sequencer (manufactured by Pharmacia) is used. Thus, the base sequence of the vector insertion part was analyzed. The obtained sequence was subjected to a homology search in the NCBI database. One clone of the obtained full-length cDNA had no gene showing homology and was considered novel. The total length of cDNA obtained by screening a phage library derived from β-amyloid-added rat astroglia cells was 6,996 bp (SEQ ID NO: 1) and a novel gene encoding 2,106 amino acids (SEQ ID NO: 2). When this protein is analyzed by the prediction program PSORT, the signal sequence (amino acids 1-27) is followed by 23 transmembrane regions (amino acids 33-49, amino acids 82-98, amino acids 143-159, amino acids 165-181, amino acids 196-212, amino acids 248-264, amino acids 361-377, amino acids 387-403, amino acids 409-425, amino acids 486-502, amino acids 548-564, amino acids 568-584, amino acids 599-615, amino acids 717-733, amino acids 739-755, amino acids 769-785, amino acids 1247-1263, amino acids 1264-1280, amino acids 1538-1554, amino acids 1576-1592, amino acids 1610-1626, amino acids 1753-1769, amino acids 20 0-2036) was had. Since it has many transmembrane regions, this gene was named Mib (A membrane protein induced by beta-amyloid). This is the first time that rat Mib is a novel sequence and has been transcribed as mRNA.

(Acquisition of human ortholog)
When searching the NCBI human genome database to find the human orthologue, a fragment of the corresponding human gene was found on the NCBI genome database. Based on this information, RT-PCR method using a superscript II (GIBCO) from human small intestine-derived poly A (+) RNA (1 μg, manufactured by CLONTECH) as a primer oligo dT (12-18) The full-length sequence was cloned and identified at Similar to rat Mib, human Mib also has a higher-order structure, and it is difficult to identify the sequence under normal sequence reaction conditions. The conditions for the sequence reaction were examined in detail. The specific sequence reaction conditions were such that the denaturation temperature during the amplification reaction was increased to 96 ° C., and the denaturation time was extended to 20-30 seconds. The annealing temperature was set as high as possible for each primer in order to avoid re-formation of the secondary structure of the template DNA at a low temperature. The base sequence of the vector insertion part was sequenced using BigDye-labeled terminator (ABI), M13 universal primer, reverse primer, and internal sequence primer, and fluorescent capillary sequencer ABI PRISM 3100 (ABI) was used. Analyzed. In order to avoid sequencing mistakes due to PCR errors of this gene having a long cDNA full length, the sequences of 5 independent clones were determined, and these were comprehensively determined to determine the sequence of this gene. Further, in order to completely identify the 5 ′ end and 3 ′ end of the cDNA, 5′-lace method and 3′-lace method (GeneRacer kit: manufactured by Invitrogen) were performed. For PCR, KOD plus (manufactured by TOYOBO) was used as a DNA polymerase, and the PCR product was incorporated into pCR-Blunt II-TOPO (manufactured by Invitrogen) and sequenced.

  This human cDNA has a total length of 6,795 bp (SEQ ID NO: 3) and encodes 2,090 amino acids (SEQ ID NO: 4). Following the signal sequence (amino acids 1 to 27), 24 transmembrane regions (amino acids) 33-49, amino acids 83-99, amino acids 148-164, amino acids 166-182, amino acids 197-213, amino acids 249-265, amino acids 378-394, amino acids 399-415, amino acids 421-437, amino acids 498-514, amino acids 560-576, amino acids 580-596, amino acids 611-627, amino acids 729-745, amino acids 751-767, amino acids 781-797, amino acids 1258-1274, amino acids 1275-1291, amino acids 1300-1316, amino acids 1532-1548, amino acids 569-1585, amino acids 1604 to 1620, amino acids 1747 to 1763, had an amino acid 2004 to 2020). This is the first time that human Mib is a novel gene and is transcribed as mRNA. Human Mib showed 83% homology at the amino acid level and 69% homology at the nucleotide level compared to rat Mib. Comparison of the amino acid sequences of rMib and hMib is shown in FIGS.

(Tissue expression distribution of rat Mib and human Mib)
The Northern blot analysis method using poly A (+) RNA extracted from rat and human tissues is the same as the method described in JP-A No. 2001-258575, JP-A No. 2001-35291, or JP-A No. 2003-164290. Carried out. Specifically, a 295 bp gene fragment of nucleotides 6645 to 6939 of SEQ ID NO: 1 was used as a probe for rat Mib, and a 360 bp gene fragment of nucleotides 3616 to 3975 of SEQ ID NO: 3 was used as a probe for human Mib. The rat Ab or human Mib cDNA was hybridized to a Northern blot (both manufactured by CLONTECH) of poly A (+) RNA extracted from each tissue of rat or human after ³² P labeling. The inventor has shown that rat Mib is expressed as an approximately 9 kbp transcript in the lung and kidney, and that human Mib is approximately 11.6 kbp or approximately 9.0 kbp in the heart, placenta, lung, liver, kidney, and pancreas. It was confirmed that it was expressed as a transcript.

(Preparation of pBS SK (-)-rMib (FERM BP-08654))
pBS SK (−)-rMib was obtained in a form in which the entire length of rat Mib cDNA (about 7.0 kbp) was incorporated between the PstI site and the XhoI site of pBluescript SK (−) (Stratagene). Specifically, the phage obtained by screening a β amyloid-added rat astroglial cell-derived phage library was obtained by an in vivo excision method using a helper phage.

FIG. 1 is a diagram showing the results of Northern blotting confirming that the gene of the present invention is induced by β-amyloid treatment. In the figure, lane N shows an untreated rat astroglial cell-derived poly (A) + RNA sample, and lane A shows a rat astroglial cell-derived poly (A) + RNA sample treated with 25 μM β-amyloid for 15 hours. Show. (A) shows the results of using the Mib gene, and (B) shows the results of using the G3PDH gene as an internal standard as a probe. The G3PDH gene, which is an internal standard, shows no difference in the expression level between β amyloid treatment and untreated, whereas the gene of the present invention is maintained at a low level when β amyloid is not treated. Increased by about 7.0 times by β-amyloid treatment. FIG. 2 is a diagram showing a comparison of amino acid sequences of human Mib (upper) and rat Mib (lower). In the figure, “*” indicates that the amino acids are the same, and “.” Indicates that the amino acids have similar properties. FIG. 3 is a continuation of FIG. It is a figure which shows the comparison of the amino acid sequence of human Mib (upper part) and rat Mib (lower part). In the figure, “*” indicates that the amino acids are the same, and “.” Indicates that the amino acids have similar properties. FIG. 4 is a continuation of FIG. It is a figure which shows the comparison of the amino acid sequence of human Mib (upper part) and rat Mib (lower part). In the figure, “*” indicates that the amino acids are the same, and “.” Indicates that the amino acids have similar properties. FIG. 5 is a diagram showing the construction of pBS SK (−)-rMib (FERM BP-08654). In the figure, rMib (about 7.0 kbp) indicates the inserted portion of the rat Mib gene. rMib is inserted between the Pst I restriction enzyme site and the Xho I restriction enzyme site of the multicloning site of the vector. f1 (−) ori represents a phagemid replication initiation sequence, Amp represents an ampicillin resistance gene site, and ColE1 ori represents a plasmid replication initiation sequence.

  Sequence number 1 of a sequence table: The base sequence of a rat Mib gene.

  Sequence number 2 of a sequence table: The amino acid sequence of rat Mib protein. A signal sequence is present at amino acids 1-27. There are 23 transmembrane regions in the following parts: amino acids 33-49, amino acids 82-98, amino acids 143-159, amino acids 165-181, amino acids 196-212, amino acids 248-264, amino acids 361-377, amino acids 387-403, amino acids 409-425, amino acids 486-502, amino acids 548-564, amino acids 568-584, amino acids 599-615, amino acids 717-733, amino acids 739-755, amino acids 769-785, amino acids 1247-1263, amino acids 1264-1280, amino acids 1538-1554, amino acids 1576-1592, amino acids 1610-1626, amino acids 1753-1769, amino acids 2020-2036.

  Sequence number 3 of a sequence table: The base sequence of a human Mib gene.

  Sequence number 4 of a sequence table: The amino acid sequence of human Mib protein. A signal sequence is present at amino acids 1-27. There are 24 transmembrane regions in the following parts: amino acids 33-49, amino acids 83-99, amino acids 148-164, amino acids 166-182, amino acids 197-213, amino acids 249-265, amino acids 378-394, amino acids 399 to 415, amino acids 421 to 437, amino acids 498 to 514, amino acids 560 to 576, amino acids 580 to 596, amino acids 611 to 627, amino acids 729 to 745, amino acids 751 to 767, amino acids 781 to 797, amino acids 1258 to 1274, amino acids 1275-1291, amino acids 1300-1316, amino acids 1532-1548, amino acids 1569-1585, amino acids 1604-1620, amino acids 1747-1763, amino acids 2004-2020.

Claims (19)

Any of the following polynucleotides or their complementary strands:
(1) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3 in the sequence listing; or (2) a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing. A polynucleotide selected from the following group or a complementary strand thereof:
(1) having at least 70% base sequence homology with the polynucleotide according to (1) or (2) of claim 1, and migration of astroglial cells, extension of astroglial processes, and A polynucleotide encoding a polypeptide having a function associated with the production of inflammatory factors of astroglial cells;
(2) hybridization with the polynucleotide according to (1) or (2) of claim 1 under stringent conditions, and migration of astroglia cells, extension of astroglial processes, and / or astroglia A polynucleotide encoding a polypeptide having a function associated with the production of inflammatory factors of cells; and (3) one or several in the nucleotide sequence of the polynucleotide according to (1) or (2) of claim 1 Which encodes a polypeptide having a function associated with astroglial cell migration, astroglial cell extension, and / or astroglial inflammatory factor production Polynucleotide. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4 in the sequence listing. Any of the following polypeptides:
(1) A polypeptide having at least 70% amino acid sequence homology with the polypeptide according to claim 3 and having astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammation A polypeptide having a function associated with the production of factors; or (2) in the amino acid sequence of the polypeptide according to claim 3, wherein one or several amino acids are deleted, substituted or added, and A polypeptide having a function associated with migration, astroglial cell extension, and / or production of astroglial inflammatory factors. A recombinant vector comprising the polynucleotide according to claim 1 or 2. Plasmid pBS SK (-)-rMib (FERM BP-08654). A transformant transformed with the recombinant vector according to claim 5 or the plasmid according to claim 6. A method for producing a polypeptide, comprising a step of culturing the transformant according to claim 7. An antibody that immunologically recognizes the polypeptide according to claim 3 or 4. A method for identifying a compound that inhibits and / or inhibits astroglial cell migration, astroglial cell extension, and / or astroglial cell inflammatory factor production, comprising the following steps:
(1) adding β-amyloid and a candidate compound to a cell; and (2) mib expression of astroglia cells when mib expression is inhibited and / or suppressed in the cells. Identifying as a compound that inhibits and / or suppresses process elongation and / or production of inflammatory factors in astroglial cells. A polynucleotide according to claim 1 or 2, a polypeptide according to claim 3 or 4, a recombinant vector according to claim 5, a plasmid according to claim 6, a transformant according to claim 7, 11. The method according to claim 10, wherein at least one of the antibodies according to claim 9 is used. The compound identified by the identification method of Claim 10 or Claim 11. Whether the test tissue derived from brain tissue is from a patient suffering from a disease associated with astroglial cell migration, astroglial cell extension, and / or production of astroglial inflammatory factors A method for determining whether or not the expression level of the polynucleotide according to claim 1 or 2 in the test tissue is measured. A method for determining whether a test tissue derived from a brain tissue is a tissue derived from an Alzheimer's disease patient, wherein the expression level of the polynucleotide according to claim 1 or 2 in the test tissue is measured. The determination method characterized by this. A polynucleotide according to claim 1 or 2, a polypeptide according to claim 3 or 4, a recombinant vector according to claim 5, a plasmid according to claim 6, a transformant according to claim 7, Use of at least one of the antibody according to claim 9 or the compound according to claim 12, migration of astroglial cells, extension of astroglial processes, and / or astroglial cells A diagnostic kit for diseases associated with the production of inflammatory factors. A polynucleotide according to claim 1 or 2, a polypeptide according to claim 3 or 4, a recombinant vector according to claim 5, a plasmid according to claim 6, a transformant according to claim 7, A diagnostic kit for Alzheimer's disease, wherein at least one of the antibody according to claim 9 or the compound according to claim 12 is used. A polynucleotide according to claim 1 or 2, a polypeptide according to claim 3 or 4, a recombinant vector according to claim 5, a plasmid according to claim 6, a transformant according to claim 7, A pharmaceutical composition comprising at least one of the antibody according to claim 9 or the compound according to claim 12. A polynucleotide according to claim 1 or 2, a polypeptide according to claim 3 or 4, a recombinant vector according to claim 5, a plasmid according to claim 6, a transformant according to claim 7, Production of astroglial cell migration, astroglial process extension, and / or astroglial cell inflammatory factor comprising at least one of the antibody of claim 9 or the compound of claim 12. An agent for improving and / or treating a disease associated with the disease. A polynucleotide according to claim 1 or 2, a polypeptide according to claim 3 or 4, a recombinant vector according to claim 5, a plasmid according to claim 6, a transformant according to claim 7, An agent for improving and / or treating Alzheimer's disease comprising at least one of the antibody according to claim 9 or the compound according to claim 12.

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