JP2001258575A - Gene capable of being induced by beta-amyloid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of obtaining a new gene of which the expression is induced or depressed by being treated with a β-amyloid, so as to control neurological diseases, such as Alzheimer's disease. SOLUTION: This method for obtaining a new gene of which the expression is induced or depressed by the β-amyloid treatment comprises applying a cDNA (complementary deoxyribonucleic acid) subtraction technique. And the new DNA obtained by the above method, a peptide encoded by the DNA, a recombinant vector, containing the DNA, a transformant having the recombinant vector an antibody against the peptide, a method of producing the polypeptide, a method of screening such a compound that controls the gene expression of a prostaglandin E synthetic enzyme and/or its enzyme activity by using the above DNA or the like, and compounds screened by the screening method are provided. Further, medicinal compositions and diagnostic measures used for the neurological diseases relating to the β-amyloid are provided by utilizing the above compounds and methods.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for obtaining a gene whose expression is induced or suppressed by protein amyloid involved in Alzheimer's disease, and a novel gene obtained by the method. More specifically, DNA having a DNA sequence of a novel gene, said DNA
, A recombinant vector containing the DNA, a transformant transformed with the recombinant vector, a method for producing a polypeptide using the transformant, an antibody against the polypeptide, and the like. For screening compounds, compounds selected by the screening method, inhibitors of the activity and / or expression of the polypeptide acting on the polypeptide or the DNA, pharmaceutical compositions related thereto, and related The present invention relates to a means for diagnosing a disease.

[0002]

2. Description of the Related Art β-amyloid is a component of senile plaques observed in the brain of Alzheimer's disease patients, and is considered to be the most important factor in the early onset stage of Alzheimer's disease. β-amyloid is produced by processing of a β-amyloid precursor by a certain protease to form an insoluble β-sheet structure aggregate. β-amyloid directs nerve cells,
By acting indirectly, it is thought to promote the progression of Alzheimer's disease.

[0003] Activation of astroglial cells is known as an indirect action of β-amyloid. Astroglial cells produce several functional factors when activated by β-amyloid. It is thought that the cooperative action of astroglial cells and β-amyloid exacerbates Alzheimer's disease. It is also known that most of activated astroglial cells are localized in senile plaques in lesions in the brain of Alzheimer's disease patients.

[0004] However, analysis of the mechanism by which β-amyloid activates astroglial cells and the factor by which β-amyloid induces or suppresses expression in astroglial cells is not yet sufficiently analyzed.

[0005]

The problem to be solved by the present invention is to find a novel gene whose expression is induced in astroglial cells by β-amyloid treatment, and to find the found gene such as Alzheimer's disease. It is to be used as a means for diagnosing and controlling neurological diseases. Another object is to provide a polypeptide encoded by the novel gene and provide an antibody against the novel polypeptide. Another object of the present invention is to screen for a compound that regulates the action and / or expression of the novel polypeptide using the above-mentioned compounds. The present invention also provides a means for diagnosing a disease and a therapeutic agent utilizing the same.

[0006]

In order to solve the problem, the present inventors have
A β-amyloid treatment and a cDNA subtraction treatment using an untreated rat fetal brain-derived astroglial cell-expressed gene were performed, a novel gene whose expression was induced by the β-amyloid treatment was identified, its nucleotide sequence and the novel gene The amino acid sequence encoded by was determined, and the present invention was completed.

That is, the present invention provides a method for obtaining a gene whose expression is induced or suppressed by β-amyloid by subtraction treatment using β-amyloid-treated astroglial cells and untreated cells; And cDNA using untreated cells
A method of obtaining a gene whose expression is induced or suppressed by β-amyloid by performing a subtraction treatment; Expression or induction of β-amyloid by performing a cDNA subtraction treatment using β-amyloid-treated rat astroglial cells and untreated cells A method for obtaining a gene to be suppressed; a DNA group obtained by the above-described gene obtaining method; a DNA selected from the following or a complementary strand thereof; a DNA represented by the DNA sequence shown in SEQ ID NO: 1 in the sequence listing; The DN of SEQ ID NO: 1
DNA containing the A sequence, DNA encoding a polypeptide having at least about 70% DNA sequence homology with said DNA and having prostaglandin E synthase activity, and DNA from said DNA. D
Deletion or substitution of one or several DNAs in the NA sequence,
DNA encoding a polypeptide having a mutation such as addition or induced mutation, and having a prostaglandin E synthase activity; hybridizing with the above DNA or its complementary strand under stringent conditions; A DNA encoding a polypeptide having a synthase activity; a polypeptide encoded by the DNA of the present invention; a recombinant vector containing the DNA of the present invention or its complementary strand; a trait transformed with the recombinant vector of the present invention E. coli E. coli coli 437 (F
ERM BP-7074); coli 4
Plasmid p437 contained in No. 37 (FERM BP-7074); a method for producing the polypeptide of the present invention, which comprises a step of culturing the transformant of the present invention; an antibody that immunologically recognizes the polypeptide of the present invention; Activity of prostaglandin E synthase and / or prostaglandin E
A method for screening a compound that regulates the expression of a synthase gene, comprising using at least one of the DNA of the present invention or its complementary strand, polypeptide, recombinant vector, transformant, or antibody. A screening method characterized by the above; a compound selected by the above-mentioned screening method; a prostaglandin E synthase inhibitor selected by the above-mentioned screening method; a DNA of the present invention or its complementary strand, polypeptide, recombinant vector, transformant , An antibody, a compound, or an inhibitor; a pharmaceutical composition comprising at least one of the following: a DNA of the present invention or a complementary chain thereof, a polypeptide, a recombinant vector, a transformant, an antibody, Characterized by containing at least one of a compound and an inhibitor. A pharmaceutical composition for use in the prevention / treatment of a disease associated with prostaglandin E synthase; at least one of the DNA or its complementary strand, polypeptide, recombinant vector, transformant, antibody, compound or inhibitor of the present invention; A preventive / therapeutic agent for neuronal cell death comprising any one of the above; a DNA of the present invention or a complementary chain thereof, a polypeptide, a recombinant vector, a transformant,
A prophylactic / therapeutic agent for Alzheimer's disease, which comprises at least one of an antibody, a compound and an inhibitor; the DNA of the present invention or a complementary strand thereof, a polypeptide, a recombinant vector, a transformant, Antibodies, compounds,
Or a means for diagnosing a disease characterized by containing at least one of inhibitors; a means for diagnosing neuronal cell death comprising a step of measuring the gene expression level and / or enzyme activity of prostaglandin E synthase A means for diagnosing Alzheimer's disease, comprising the step of measuring the gene expression level and / or enzyme activity of prostaglandin E synthase;
A prophylactic / therapeutic agent for neuronal cell death containing a prostaglandin E synthase inhibitor; and an Alzheimer's disease preventive / therapeutic agent containing a prostaglandin E synthase inhibitor.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (β-Amyloid Treatment of Astroglial Cells) Astroglial cells can be prepared from brains of various animals. A fetal brain is preferably used, and as an animal to be used, a rodent small animal such as a rat, a mouse, a guinea pig, or a rabbit, which is relatively easy to collect a fetal brain, is preferable, and a rat is particularly preferably used. Astroglial cells can be prepared from the fetal brain by a method known per se, which method is disclosed in Experimental Biomedical Separate Neurobiochemistry Manual (Jinichi Ito et al. Yodosha (1989)).

Β-amyloid acting on astroglial cells is commercially available and can be obtained from Anaspec, etc., but can also be prepared by genetic engineering techniques. β-amyloid treatment is performed by inoculating various animal brain-derived astroglial cells into a suitable culture vessel, culturing for about 2 days in a serum-free medium, and then adding appropriately diluted β-amyloid using a suitable buffer. Do. The concentration of β-amyloid added and the treatment time are appropriately selected within a range where the physiological activity can be observed, but the concentration is preferably 25 to 50 μM, and the treatment time is preferably 10 to 20 hours. In parallel with β-amyloid treatment, astroglial cells can be seeded in another culture vessel under the same conditions, and β-amyloid-untreated astroglial cells can be prepared by adding an equal amount of the buffer used for diluting β-amyloid. .

(Preparation of expressed gene) The expressed gene is mR
It may be used as it is or as a cDNA. m
For conversion from RNA to cDNA, a method using reverse transcriptase or the like can be applied.

Genes expressed in β-amyloid-treated and untreated astroglial cells can be collected by a commonly used RNA collection method. For collecting total RNA, for example, AG
PC method (Chomczynski et al., A
nal. Biochem. 162, pp156-1
59 (1986)) is applicable and the mR from total RNA
For collection of NA, polyA using an oligo dT column or the like was used.
(+) RNA purification and the like. In some cases, RNA samples obtained by mixing a plurality of times are mixed and used. For the purpose of analyzing the quality of the harvested lot, it is advisable to monitor the expression of some genes known to be expressed in astroglia. For example, nerve growth factor (NGF), tumor necrosis factor-α (TNF-α), interleukin-6 (I
L-6), p75 and the like may be used as monitor genes, and only samples having the same expression may be used.

(Subtraction treatment) In order to obtain a gene whose expression is induced or suppressed by β-amyloid treatment, subtraction treatment can be applied. In the subtraction processing, two sets of gene groups, ie, a gene group to be subtracted (tester) and a gene group to be subtracted (driver) are used. For example, to obtain a gene whose expression is induced by β-amyloid, β
Subtraction using a gene obtained from amyloid-treated cells (β-amyloid-treated gene) and a gene obtained from untreated cells (unprocessed gene) as a driver to obtain a gene whose expression is suppressed by β-amyloid It is preferable to perform subtraction using an unprocessed gene as a tester and a β-amyloid-treated gene as a driver. As a subtraction processing method, various known means can be used (Japanese Patent Laid-Open No.
117488), preferably a cDNA subtraction method. As the cDNA subtraction method, RDA method, PCR-select cDNA
Method is particularly preferred.

In the subtraction process, an appropriate DNA fragment is added to the tester side as a positive control for subtraction, and the concentration of the fragment is confirmed, whereby the efficiency of the subtraction process can be confirmed. In addition, using a gene known to be induced to be expressed by β-amyloid treatment as a probe, PCR products before and after subtraction were used for Southern blot analysis.
When hybridization is performed, the success or failure of the subtraction process can be similarly confirmed. Genes whose expression is currently known to be induced by β-amyloid treatment include NGF and inducible Nitric Oxi.
de Synthase (iNOS), Regulat
ed on Activation Normal T
-Cell Expressed and Secret
ted (RANTES) and the like.

[0014] The PCR product after the subtraction treatment is inserted into an appropriate vector to prepare a plasmid library or the like. The vector insertion portion is amplified by PCR, and four sets of those genes obtained by dot-blotting 60 to 100 ng of each gene on a membrane are prepared. For this dot membrane, a PCR product before subtraction treatment using a β-amyloid-treated gene as a tester and an untreated gene as a driver (hereinafter, the subtraction treatment is represented by the formula (tester) − (driver)) and (untreated) Gene) -PCR product before subtraction of (β-amyloid-treated gene), and (β-amyloid-treated gene)-(unprocessed gene) and (unprocessed gene)-
PC after subtraction of (β-amyloid-treated gene)
Differential hybridization is performed for each of the four probes of the R product. (Unprocessed gene)
PC after subtraction of (β-amyloid-treated gene)
A clone that is specifically enriched in the PCR product after subtraction of (β-amyloid-treated gene)-(untreated gene) as compared with the R product may be determined as a gene whose expression is induced by β-amyloid treatment. . The sequence of the vector insertion portion can be determined by a DNA sequencing method known per se.

(Acquisition of Gene) The gene provided in the present invention is a cDNA obtained as a gene encoding a novel protein by cDNA subtraction treatment using astroglia derived from rat fetal brain. β-amyloid-treated and β-amyloid-untreated astroglial expression genes were prepared, subjected to (β-amyloid-treated gene)-(unprocessed gene) subtraction treatment, and found as clones whose expression was induced by β-amyloid treatment. Was. Using a fluorescent dye (Cy5; manufactured by Pharmacia) -labeled M13 universal and reverse primers, a sequencing reaction was performed, and ALF Ex
When the base sequence of the insert was determined using a press fluorescence sequencer (manufactured by Pharmacia) or the like, the cDNA of the present invention encoded a protein consisting of 153 amino acids. Gene database (DDBJ / EMB)
L / GenBank), the gene of the present invention was found to be microsomal g, a gene recently reported to encode human prostaglandin E synthase.
lutatione S-transferase1
-Like1 (MGST1-L1) (Jakobso
net et al. Proc. Natl. Aca
d. Sci. USA, 96, pp7220-7
225 (1999)) and 82.5% for nucleotides and 79.7% for amino acids. In addition, rat tissue blots (tissue bl)
ots) showed that this gene showed high expression in rat testis and kidney, which was similar to the distribution pattern of MGST1-L1 in human tissues. Therefore, this gene is predicted to encode rat prostaglandin E synthase.

The present gene was also found to be significantly induced by β-amyloid treatment by Northern blotting using β-amyloid-treated and untreated rat astroglial cell-expressed genes.

(DNA and its complementary strand) DN of the present invention
A and its complementary strand correspond to DN shown in SEQ ID NO: 1 in the sequence listing.
A DNA consisting of the A sequence and its complementary strand, a DNA having the partial sequence shown in SEQ ID NO: 1 and its complementary strand, and a DNA that hybridizes with these DNAs under stringent conditions. “DNA that hybridizes to DNA under stringent conditions” is, for example, Molecular Clon.
ing second edition (J. Sambrook et al.
(1989)). Here, “hybridize under stringent conditions” means, for example, 6 × SSC, 0.5
After heating at 42 ° C. in a solution of 50% formamide at 42 ° C. and washing at 68 ° C. in a solution of 0.1 × SSC and 0.5% SDS, a signal of a positive hybridization was still obtained. Indicates what is observed.

The DNA of the present invention is at least about 70% of the DNA represented by SEQ ID NO: 1 in the sequence listing.
Encoding a polypeptide having homology on the DNA sequence of E. coli and having prostaglandin E synthase activity
A is selected. The selected DNA preferably has a homology of about 70% or more, more preferably about 80% or more, and even more preferably about 90% or more. Techniques for determining DNA sequence homology are known per se, and for example, a method for directly determining a DNA sequence can be used. Confirmation of prostaglandin E synthase activity of the encoded polypeptide can be carried out by expressing the polypeptide using the DNA of the present invention. A known protein expression system, for example, a cell-free protein expression system can be used for expression of the polypeptide. As a cell-free protein expression system, for example, ribosome-based techniques derived from embryos, rabbit reticulocytes, etc. can be used (Nature, 179, p.
p160-161 (1957)).

Furthermore, the DNA of the present invention is obtained by
It means a DNA having a mutation such as deletion, substitution, addition or the like of one or several DNAs in the A sequence or an induced mutation, and encoding a polypeptide having prostaglandin E synthase activity. Means for deletion, substitution, addition or insertion of DNA are known per se, and examples thereof include a method for producing a deletion mutant using exonuclease, and a site-directed mutagenesis method.

Each of these DNAs provides a gene useful for producing the novel polypeptide of the present invention, and a nucleic acid such as a gene encoding the gene or an mRNA encoding the gene.
It can be used as a probe or primer for detection, or as an antisense oligomer that regulates gene expression. For example, when the DNA of the present invention is used as an antisense, the expression of the novel polypeptide of the present invention is specifically inhibited. Further, the D of the present invention
NA can also be used as a reagent for nucleic acids.

(Polypeptide) The polypeptide of the present invention comprises a DNA represented by the DNA sequence of SEQ ID NO: 1 in the Sequence Listing and a DNA represented by the DNA sequence of SEQ ID NO: 1 in the Sequence Listing.
It is a polypeptide encoded by DNA containing NA. Furthermore, the polypeptide is encoded by DNA having at least about 70% homology on the DNA sequence with the DNA represented by SEQ ID NO: 1 in the sequence listing, and has prostaglandin E synthase activity. In addition, these polypeptides can be modified to a degree that does not involve a significant change in function, such as by modifying constituent amino groups or carboxyl groups.

The polypeptides of the present invention may themselves be:
They can be used in pharmaceutical compositions to modulate their own in vivo functions. Further, the polypeptide of the present invention can be used for screening for obtaining compounds capable of regulating their functions, for example, inhibitors, antagonists, activators, etc., and for obtaining antibodies against them. Further, the polypeptide of the present invention can be used as a reagent.

(Recombinant Vector) A recombinant vector can be obtained by incorporating the DNA of the present invention into an appropriate vector DNA. As the vector DNA, a naturally occurring DNA may be extracted, or may be a DNA in which a part of DNA other than a part necessary for growth is partially deleted. For example, there are vectors derived from Col E1 and vectors derived from lambda phage. As a method for incorporating the DNA of the present invention into the vector DNA, a method known per se can be applied. For example, a method is used in which a DNA is cleaved at a specific site by selecting and treating an appropriate restriction enzyme, then mixed with a DNA to be used as a vector similarly treated, and religated with ligase. In p437 shown in Examples described later, pBluesc was used as vector DNA.
Rip SK (manufactured by STRATAGENE) was used, but is not limited to this.

(Transformant) In addition to the above-described cell-free protein expression system, the present invention can be carried out by a gene recombination technique using a host known per se, such as Escherichia coli, yeast, Bacillus subtilis, insect cells, and animal cells. Can be provided.

Transformation can be performed by any means known per se. 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 the stability of the gene. For convenience, an autonomous replication system using an extranuclear gene is used. The vector is selected depending on the type of the host, and includes a gene sequence to be expressed and a gene sequence carrying information on its replication and control as components. Components 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.

The transformant can be used for the production of the polypeptide of the present invention by culturing under conditions that are optimal for the culture conditions of each host known per se. The cultivation may be performed using the physiological activity of the novel polypeptide to be expressed and produced, particularly the prostaglandin E synthase activity as an indicator, or by subculture or batch using the amount of transformant in the medium as an indicator.

The transformants E. coli shown in the examples described later. co
li 437 is the plasmid DNA D
This is a novel microorganism obtained by using NA and introducing it into Escherichia coli and carrying out a transformation reaction, and is deposited as FERM BP-7074 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

(Recovery of New Polypeptide and Its Derivative) The recovery of the novel polypeptide and the polypeptide consisting of its derivative from the culture medium is performed by molecular sieve and ion column chromatography using the activity of prostaglandin E synthase as an index. , Affinity chromatography or the like, or a fractionation means such as ammonium sulfate or alcohol based on the difference in solubility. More preferably, a method is used in which an antibody against the amino acid sequence is prepared based on the information on the amino acid sequence, and the antibody is specifically adsorbed and collected using a polyclonal antibody or a monoclonal antibody.

(Antibody) An antibody is prepared by selecting an antigenic determinant of a polypeptide comprising the novel polypeptide of the present invention and a derivative thereof. The antigenic determinant has at least 5
And more preferably at least 8 to 10 amino acids. This amino acid sequence does not necessarily need to be homologous to the polypeptide sequence encoded by the DNA of SEQ ID NO: 1 in the sequence listing, and may be any exposed site on the three-dimensional structure of the protein. Then, it is also effective that the exposed site has a continuous amino acid sequence. The antibody is not particularly limited as long as it immunologically recognizes a polypeptide consisting of a novel polypeptide and a derivative thereof. The presence or absence of this recognition is determined by a known antigen-antibody binding reaction.

In order to produce an antibody, a polypeptide comprising the novel polypeptide of the present invention and a derivative thereof is prepared by
In the presence or absence of an adjuvant, immunity induction such as a humoral response and / or a cellular response is performed on an animal alone or in combination with a carrier. The carrier is not particularly limited as long as it does not cause harmful effects on the host, and examples thereof include cellulose, polymerized amino acids, and albumin. As animals to be immunized, mice, rats, rabbits, goats, horses, and the like are preferably used. The polyclonal antibody is obtained by a known antibody recovery method from serum. A preferred means is an immunoaffinity chromatography method.

To produce a monoclonal antibody,
Antibody-producing cells are recovered from the animal to which the above-mentioned immunization has been applied, and a method for transforming perpetually proliferating cells known per se is introduced.

The polyclonal or monoclonal antibody can directly bind to the polypeptide of the present invention and control its activity, and is useful for the treatment and prevention of diseases associated with the activity of the polypeptide of the present invention.

(Screening) D thus prepared
NA and its complementary chain, polypeptide, recombinant vector, transformant, and antibody can be used alone or in combination with a plurality of means to inhibit prostaglandin E synthase activity or prostaglandin E synthase gene expression. Alternatively, it provides an effective means for screening an activator. That is, screening of a compound that inhibits or enhances the expression of prostaglandin E synthase by using at least one of the DNA of the present invention or its complementary strand, a recombinant vector, a transformant, or an antibody is performed. In addition, by using at least one of the polypeptide or the antibody of the present invention, a compound that inhibits or enhances prostaglandin E synthase activity can be screened. For example, drug screening known per se includes screening of antagonists by drug design based on the tertiary structure of a polypeptide, screening of an expression regulator at the gene level using a protein expression system, screening of an antibody recognizing substance using an antibody, and the like. Available in the system. Further, the compounds selected by this screening are useful as pharmaceutical compositions described later and therapeutic agents for diseases.

(Prostaglandin E synthase) Prostaglandin is a group of physiologically active substances synthesized in animal tissues from eicosapolyenoic acid such as arachidonic acid. A to J are distinguished in accordance with the difference between the oxygen atom and the double bond attached to. In addition, there are 1 to 3 groups according to the number of double bonds in the side chain, and both are combined and classified and represented as prostaglandin E ₁ and prostaglandin H ₂ . 2
In the group eicosapolyenoic acid precursor is arachidonic acid biosynthesis begins by oxygen bimolecular arachidonic acid by cyclooxygenase is added, prostaglandin G ₂ occurs. Making prostaglandin H ₂ the 15-hydroperoxide is a hydroxyl group. Prostaglandin E ₂ is produced by isomerization of prostaglandin H ₂ , and the enzyme that catalyzes the isomerization reaction is prostaglandin E synthase.
Human prostaglandin E synthase is 15-16 kDa
Recently, a protein and a gene have been identified (Jakobsson et al. Proc. N).
atl. Acad. Sci. USA, 96,
pp 7220-7225 (1999)).

The prostaglandin E synthase activity can be measured by a method known per se. For example, Proc. Natl. Acad. Sci. US
A, 96, pp 7220-7225 (1999).

Prostaglandin E produced by the catalytic action of prostaglandin E synthase is a physiologically active substance having activities such as a blood pressure lowering action and a vasodilatory action. Its production is inhibited by non-steroidal anti-inflammatory agents such as aspirin which inhibits cyclooxygenase which is an enzyme that catalyzes the upstream process of prostaglandin E synthase. Has been reported to be effective against
ndersen et al. Neurolog
y, 45, pp 1441-1445 (199
5)). Furthermore, recently, in a test using cerebrospinal fluid from a patient with Alzheimer's disease, it has been reported that the amount of prostaglandin E is increased in the brain of an Alzheimer's disease patient (Montine et al. Neuro).
ology, 53, pp1495-1498 (1
999)). These suggest that Alzheimer's disease may be involved in overproduction of prostaglandin E. On the other hand, the novel gene of the present invention was identified as a gene whose expression was induced by β-amyloid treatment, and from the result of DNA sequence homology search, it was considered to encode rat prostaglandin E synthase. From this fact, the overproduction of prostaglandin E in Alzheimer's disease is not affected by β-amyloid treatment.
It is conceivable that overexpression of a synthase may be involved, and inhibiting the activity and expression of prostaglandin E synthase indicates a possibility of leading to treatment of neurological diseases such as Alzheimer's disease.

(Compounds, Pharmaceutical Compositions, and Diagnostic Means) The compounds selected by the above screening method and the compounds that inhibit or enhance the activity and / or gene expression of prostaglandin E synthase are prostaglandin E It can be used as a candidate compound such as an inhibitor, an antagonist, or an activator that regulates synthase activity and prostaglandin E synthase expression. Examples of the candidate compounds such as the above inhibitors, antagonists, activators and the like include proteins, polypeptides, polypeptides having no antigenicity, and low molecular weight compounds, preferably low molecular weight compounds.

[0038] The candidate compounds thus selected are selected for their biological usefulness in view of toxicity and the like, so as to be used in the treatment of diseases associated with the activity and / or expression of prostaglandin E synthase. It can be prepared as a product. In addition, the novel DNA of the present invention or its complementary strand, the polypeptide encoded by them, the above DNA
Or a recombinant vector containing the complementary chain thereof, a transformant transformed with the recombinant vector, and an antibody that immunologically recognizes the polypeptide are themselves prostaglandin E synthase activities. It has functions such as inhibition, antagonism, and activation of expression, and can be used as a pharmaceutical means for treating diseases related to the activity and expression of prostaglandin E synthase.

Diseases related to the activity and expression of prostaglandin E synthase include diseases involving nerve cell death, for example, neurological diseases such as Alzheimer's disease. In preparation, a known means of preparing a compound, such as a compound, a polypeptide, a protein, a polynucleotide, or an antibody, corresponding to each control may be introduced.

The novel DNA of the present invention or its complementary chain, the polypeptide encoded by them, the recombinant vector containing the above DNA or its complementary chain, the transformant transformed by using these recombinant vectors, and the above polypeptide Antibody that immunologically recognizes prostaglandin E
It is useful as a diagnostic means for neuronal cell death, diseases and the like related to the activity and expression of the synthase. In particular, it is useful as a diagnostic tool such as a diagnostic marker and / or reagent for Alzheimer's disease. Diagnosis is to determine the abundance of the nucleic acid sequence of prostaglandin E synthase by utilizing the interaction / reactivity with the DNA of the present invention, and / or to determine the presence of prostaglandin E synthase in the sample. Do this by determining the amount. As a sample, blood, a sputum, a brain biopsy sample and the like are suitable, and a cerebral sputum is particularly preferable. As a measurement method used for diagnosis, an antigen-antibody reaction, an enzyme reaction system, a PCR reaction system, or the like known per se may be used. Measuring the amount and / or activity of prostaglandin E in a sample is also useful as a means for indirectly measuring the activity and expression of prostaglandin E synthase. Prostaglandin E was quantified by a radioimmunoassay using an antibody against prostaglandin E (Fumio Hirata et al., Med. Te.
chnol. , 6, pp1242 (1978))
And other methods known per se. Here, the means means a method and / or a medium used for achieving the object. That is, for example, the diagnosis means includes a method for diagnosis, a reagent kit used for diagnosis, and the like.

[0041]

Example (Poly A from astroglial cells)
(+) Preparation of RNA) Astroglia cells were
It was prepared from r-system rat fetal brain according to the Experimental Medicine Separate Neurobiochemistry Manual (Jinichi Ito et al. Yodosha (1989)). After subculture of rat fetal brain-derived astroglial cells, serum-free for 2 days, β-amyloid (A) was added to a 2.5 μM / 6 cm plastic dish.
Naspec Corp., β-amyloid 1-40) or an equivalent amount of a solvent was added, and the mixture was cultured for 15 hours.

Β-amyloid-treated and untreated astroglial cells (20 cm each of 6 cm plastic dish)
Total RNA was extracted 9 times from each of them) by the AGPC method.

For the purpose of analyzing the quality of total RNA in each lot of β-amyloid-treated and untreated lots, NGF and TNF-genes known to be expressed in astroglial cells were used.
RT-PCR was performed using the α, IL-6 and p75 genes. Further, for NGF, Northern blotting was performed using the RT-PCR product as a probe. Based on these results, a lot to be used for the subtraction treatment is selected, and the total RNAs of the selected lots are mixed, and the resulting mixture is subjected to the po dT-latex method.
ly A (+) RNA was prepared.

(Subtraction treatment) Poly derived from astroglial cells not treated with β-amyloid and untreated
Using 2 μg of each A (+) RNA as a starting material, cDNA subtraction of (β-amyloid-treated gene)-(unprocessed gene) and (unprocessed gene)-(β-amyloid-treated gene) was performed by the method of Diatchenko et al.
oc. Natl. Acad. USA, 93,
pp6025-6030 (1996)). As a positive control for the subtraction treatment, φΧ174 / Ha was added to the tester after Rsa I digestion.
e III was added digested DNA fragments to be about 2.5 × 10 ^-5 at a weight ratio in the tester.

To verify the efficiency of the subtraction processing performed, the positive control φ 対 照 174 / Hae II
Using the I-digested DNA fragment and the NGF gene as probes, the PCR product after the subtraction and the PCR product before the subtraction were subjected to Southern blot hybridization.

The PCR product after subtraction of (β-amyloid-treated gene)-(unprocessed gene) and (unprocessed gene)-(β-amyloid-treated gene) was 900 bp.
The size was fractionated into the above (Group I), 500 bp to 900 bp (Group II), and 250 bp to 500 bp (Group III).

A PCR product belonging to Group III of (β-amyloid-treated gene)-(unprocessed gene) was ligated to a vector (pBluescript II) to prepare a plasmid library.

The vector insertion portion of the 480 clones of the plasmid library was amplified by the PCR method, and those genes were each separated by 60 to 100 ng into a membrane (Hybon).
d-N +) (manufactured by Amersham). A PCR product after subtraction of (β-amyloid-treated gene)-(unprocessed gene) and (unprocessed gene)-(β-amyloid-treated gene) and (β
Differential hybridization was performed using a total of four probes of PCR products before subtraction of (amyloid-treated gene)-(untreated gene) and (untreated gene)-(β-amyloid-treated gene).
(Unprocessed gene) -A clone that is specifically enriched in the PCR product after subtraction of (β-amyloid-treated gene)-(unprocessed gene) compared to the PCR product after subtraction of (β-amyloid-treated gene) Selected.

(Analysis by Northern Blot Hybridization) Poly A (+) derived from β-amyloid-treated and untreated astroglial cells was used as a probe with each vector-inserted portion of the clones selected by differential hybridization screening.
Northern blot hybridization using RNA was performed. Among clones selected in the differential hybridization screening, clones that may be duplicated were checked for their presence or absence by Southern blot hybridization, and clones found to be duplicated were identified in advance by Northern blot hybridization. Excluded from analysis by hybridization.

(Base sequence analysis and homology search) β
The clone group whose expression is induced by amyloid is Cy5
A sequencing reaction was carried out using labeled M13 universal and reverse primers, and ALF Express
The nucleotide sequence of the vector insertion portion was analyzed using a fluorescent sequencer (Pharmacia). For the resulting sequence,
A homology search was performed on the NCBI database. One of the obtained full-length cDNA clones is a novel gene, which is 79.7% in base sequence and 82.5% in amino acids with the gene MGST1-L1, which was recently reported to encode human prostaglandin E synthase. % Homology (FIG. 1).

(Confirmation of Novel Gene Induction by Northern Blot Method) By the above-mentioned method, each of poly A was obtained from β-amyloid-treated and untreated astroglia.
(+) RNA samples were prepared. After the above-described subtraction treatment of the novel gene, Northern blotting was performed using the cDNA fragment as a probe. Glyceraldehyde triphosphate dehydrogenase (G3PDH) cDNA which is a constantly expressed gene (manufactured by CLONTECH)
Was used as an internal standard probe. Detection is BAS200
0 (manufactured by Fuji Film Co., Ltd.). The G3PDH gene has no difference in expression level between the β-amyloid treatment and the non-treatment, whereas the novel gene of the present invention has a low expression level maintained at a low level when the β-amyloid is not treated. Amyloid treatment increased about 22-fold (FIG. 2).

(Tissue distribution of novel gene expression) A random-primed cDNA probe derived from the novel gene of the present invention was hybridized to a Northern blot (manufactured by CLONTECH) of poly A (+) RNA extracted from each rat tissue. . Strong expression of the novel gene of the present invention was observed in kidney and testis. Weak expression was found in brain, lung, and skeletal muscle (FIG. 3). The expression pattern of this gene in each tissue is determined by the expression pattern of human prostaglandin E synthase gene MGST1-L1 (Jakobsson et al. Proc.
tl. Acad. Sci. USA, 96, p.
p7220-7225 (1999)), and the results support that this gene encodes the same type of prostaglandin E synthase as previously reported in rats.

(E. coli 437 (FPRM)
Production of BP-7074) ZAP vector (STR
B in the multiple cloning site of ATAGENE
a cD containing the open reading frame (ORF) 462 base pairs of the gene of the present invention at the amH1-XhoI site
About 1.3 kB of full length NA was inserted. This recombinant vector is used in a ZAP-cDNA synthesis system (STRATAGEN).
PBluescript SK (S
(Trade name, manufactured by TRATAGENE) to obtain p437 (FIG. 4). E. coli competent cells (Max Effici)
encyDH5α competent cells;
To 50 μl of GIBCO BRL), 25 pg of the obtained plasmid was added for transformation. 450
μl of SOC (20 g / l tryptone, 5 g / l
Yeast extract, 0.5 g / l NaCl,
2.5 mM KCl, 10 mM MgCl ₂ , 20 mM
Glucose), and after shaking culture at 37 ° C. for 1 hour, LB-ampicillin plate (10 g / l NaC
1, 10 g / l tryptone, 5 g / l yeast extract, 25 μg / ml ampicillin) to obtain ampicillin-resistant clones.

[0054]

As described above, the present invention provides a novel prostaglandin E synthase whose expression is induced by β-amyloid treatment. Providing a novel pharmaceutical composition and diagnostic means utilizing this property provides great utility in clinical and basic medical fields for β-amyloid-related diseases such as Alzheimer's disease.

[Sequence List] SEQUENCE LISTING <110> Daiichi Pharmaceutical Co., Ltd. <120> Novel gene induced by beta-amyloid <130> N00032201A <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 462 < 212> DNA <213> rat <220> <221> CDS <222> (1) .. (462) <400> 1 atg act tcc ctg ggt ttg gtg atg gag aac agc cag gtg ctc ccc gcc 48 Met Thr Ser Leu Gly Leu Val Met Glu Asn Ser Gln Val Leu Pro Ala 1 5 10 15 ttt ctg ctc tgc agc aca ctg ctg gtc atc aag atg tac gcg gtg gct 96 Phe Leu Leu Cys Ser Thr Leu Leu Val Ile Lys Met Tyr Ala Val Ala 20 25 30 gtc atc aca ggc caa gtc agg ctg cgg aag aag gct ttt gcc aac ccc 144 Val Ile Thr Gly Gln Val Arg Leu Arg Lys Lys Ala Phe Ala Asn Pro 35 40 45 gag gac gcg ttg aaa cgt gga ggt ctc cag tact agg agt gac cca 192 Glu Asp Ala Leu Lys Arg Gly Gly Leu Gln Tyr Cys Arg Ser Asp Pro 50 55 60 gat gtg gag cgc tgc ctc aga gcc cac cgc aac gac atg gag acg atc 240 Asp Val Glu Arg Cys Leu Arg Ala His Arg Asn Asp Met Glu Thr Ile 65 70 75 80 tac ccc ttc ctc ttc ctt ggt ttc gtc tac tca ttc ctg g ga ccc aac 288 Tyr Pro Phe Leu Phe Leu Gly Phe Val Tyr Ser Phe Leu Gly Pro Asn 85 90 95 cct ctc atc gcc tgg ata cat ttc ctc gtg gtc ctc aca ggc cgt gtg 336 Pro Leu Ile Ala Trp Ile His Phe Leu Val Val Leu Thr Gly Arg Val 100 105 110 gta cac acc gtg gcc tac ctg ggc aaa atg aac cca cgc att cgc tcc 384 Val His Thr Val Ala Tyr Leu Gly Lys Met Asn Pro Arg Ile Arg Ser 115 120 125 ggg gcc tat gtt ctg gcc cag ttc gcc tgt ttc tcc atg gcc cta cag 432 Gly Ala Tyr Val Leu Ala Gln Phe Ala Cys Phe Ser Met Ala Leu Gln 130 135 140 atc ctc tgg gaa gtg gcc cac cat ctg tga 462 Ile Leu Trp Glu Val Ala His Leu 145 150 <210> 2 <211> 153 <212> PRT <213> rat <400> 2 Met Thr Ser Leu Gly Leu Val Met Glu Asn Ser Gln Val Leu Pro Ala 1 5 10 15 Phe Leu Leu Cys Ser Thr Leu Leu Val Ile Lys Met Tyr Ala Val Ala 20 25 30 Val Ile Thr Gly Gln Val Arg Leu Arg Lys Lys Ala Phe Ala Asn Pro 35 40 45 Glu Asp Ala Leu Lys Arg Gly Gly Leu Gln Tyr Cys Arg Ser Asp Pro 50 55 60 Asp Val Glu Arg Cys Leu Arg Ala His Arg Asn Asp Met G lu Thr Ile 65 70 75 80 Tyr Pro Phe Leu Phe Leu Gly Phe Val Tyr Ser Phe Leu Gly Pro Asn 85 90 95 Pro Leu Ile Ala Trp Ile His Phe Leu Val Val Leu Thr Gly Arg Val 100 105 110 Val His Thr Val Ala Tyr Leu Gly Lys Met Asn Pro Arg Ile Arg Ser 115 120 125 Gly Ala Tyr Val Leu Ala Gln Phe Ala Cys Phe Ser Met Ala Leu Gln 130 135 140 Ile Leu Trp Glu Val Ala His His Leu 145 150

[Brief description of the drawings]

FIG. 1 is a diagram illustrating a comparison between the amino acid sequence of the polypeptide encoded by the gene of the present invention and the amino acid sequence of human prostaglandin E synthase encoded by the human gene MGST1-L1. The upper amino acid sequence is the deduced amino acid sequence of the polypeptide encoded by the gene of the present invention, and the lower amino acid sequence is the amino acid sequence of human prostaglandin E synthase. In the figure, “*” indicates that the amino acids are the same, and “.” Indicates that they are different.

FIG. 2 shows the results of Northern blotting, which confirmed that the expression of the gene of the present invention was induced by β-amyloid treatment. In the figure, lane U is untreated rat astroglial cell-derived poly (A) + RNA sample, and lane A is rat astroglial cell-derived poly (A) + RN after treatment with 25 μM β-amyloid for 15 hours.
Sample A is shown. The upper row shows m of the gene of the present invention.
The result using an RNA-derived cDNA fragment as a probe is shown. The lower part is G3PDH used as an internal standard.
1 shows the results of using cDNA (CLONTECH) as a probe.

FIG. 3 shows the results of analysis of the tissue distribution of the gene of the present invention by rat tissue blots. CDNA derived from mRNA of the gene of the present invention
The fragment was used as a probe.

FIG. 4 is a diagram showing the construction of a plasmid p437 of the present invention. The portion represented as "PGE synthase" in the figure is the inserted portion of the gene of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61K 48/00 4B065 45/00 A61P 25/00 4C084 48/00 25/28 4C085 A61P 25 / 00 43/00 111 4C086 25/28 C07K 16/40 4C087 43/00 111 C12N 1/15 4H045 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/90 1/21 C12Q 1/533 5/10 1/68 A 9/90 G01N 33/15 Z C12Q 1/533 33/50 Z 1/68 33/53 D G01N 33/15 C12P 21/08 33/50 (C12N 1/21 33/53 C12R 1:19) // C12P 21/08 C12N 15/00 ZNAA (C12N 1/21 A61K 37/02 C12R 1:19) C12N 5/00 A F term (reference) 2G045 AA34 AA35 AA40 BB20 CB01 CB21 DA12 DA13 DA14 DA36 DA77 FB 02 FB03 4B024 AA01 AA11 BA07 BA53 CA04 CA09 DA02 DA06 DA07 DA11 DA12 EA04 GA11 HA03 HA12 4B050 CC03 CC10 DD07 LL01 LL03 4B063 QA01 QA05 QA19 QQ03 QQ39 QQ44 QR19 QR33 QR48 QR51 QR56 QR59 CA14 DA80 QR20 AG20 4B065 AA19X AA26X AA72X AA90X AA90Y AA91Y AB01 AB04 BA02 BA08 CA25 CA44 CA46 4C084 AA01 AA02 AA13 BA21 CA53 DC32. AA20 AA30 BA10 CA45 DA75 DA76 DA89 EA21 EA50 FA72 FA73 FA74 HA05

Claims (25)

[Claims] 1. A method for obtaining a gene whose expression is induced or suppressed by β-amyloid by performing a subtraction treatment using β-amyloid-treated astroglial cells and untreated cells. 2. A method for obtaining a gene whose expression is induced or suppressed by β-amyloid by subjecting cDNA subtraction treatment to β-amyloid-treated astroglial cells and untreated cells. 3. A method for obtaining a gene whose expression is induced or suppressed by β-amyloid by subjecting cDNA subtraction to β-amyloid-treated rat astroglial cells and untreated cells. 4. A DNA group obtained by the method according to any one of claims 1 to 3. 5. A DNA selected from the following group or a complementary strand thereof: D represented by the DNA sequence of SEQ ID NO: 1 in the sequence listing
NA, a DNA containing the DNA sequence of SEQ ID NO: 1 in the sequence listing
NA, a DNA having at least about 70% homology on the DNA sequence with said DNA and encoding a polypeptide having prostaglandin E synthase activity, and one to several nucleotides in the DNA sequence of said DNA. DNA encoding a polypeptide having mutations such as deletion, substitution, addition or the like or induced mutations of individual DNAs and having prostaglandin E synthase activity. 6. A DNA which hybridizes with the DNA of claim 5 or a complementary strand thereof under stringent conditions and encodes a polypeptide having a prostaglandin E synthase activity. 7. A polypeptide encoded by the DNA according to claim 5 or 6. 8. A recombinant vector containing the DNA according to claim 5 or its complementary strand. [9] A transformant transformed with the recombinant vector according to [8]. 10. An E. coli E. coli. coli 437 (FERM)
BP-7074). 11. E. coli E. coli. coli 437 (FERM)
BP-7074). 12. A method for producing the polypeptide according to claim 7, comprising a step of culturing the transformant according to claim 9. 13. An antibody that immunologically recognizes the polypeptide according to claim 7. 14. A method for screening a compound that regulates the activity of prostaglandin E synthase and / or the expression of a prostaglandin E synthase gene, wherein the DNA or its complementary strand according to claim 5 or 6 is provided. The polypeptide according to claim 7, the recombinant vector according to claim 8, the transformant according to claim 9, or the transformant according to claim 13.
7. A screening method, wherein at least one of the antibodies described in 1) is used. 15. A compound selected by the screening method according to claim 14. 16. A prostaglandin E synthase inhibitor selected by the screening method according to claim 14. 17. The DNA according to claim 5 or 6, or a complementary strand thereof, the polypeptide according to claim 7, and the polypeptide according to claim 7.
, The transformant according to claim 9, the antibody according to claim 13, the compound according to claim 15, or the inhibitor according to claim 16. A pharmaceutical composition characterized by containing: 18. The DNA according to claim 5 or 6, or a complementary strand thereof, the polypeptide according to claim 7, and the polypeptide according to claim 7.
, The transformant according to claim 9, the antibody according to claim 13, the compound according to claim 15, or the inhibitor according to claim 16. A pharmaceutical composition for use in the prevention / treatment of a disease associated with prostaglandin E synthase, characterized by comprising: (19) a DNA according to (5) or (6), a complementary strand thereof, a polypeptide according to (7), (8);
, A transformant according to claim 9, an antibody according to claim 13, a compound according to claim 15, or an inhibitor according to claim 16. An agent for preventing / treating neuronal cell death. 20. The DNA according to claim 5 or 6, or a complementary strand thereof, the polypeptide according to claim 7, and 8).
, A transformant according to claim 9, an antibody according to claim 13, a compound according to claim 15, or an inhibitor according to claim 16. An agent for preventing / treating Alzheimer's disease. 21. The DNA according to claim 5 or 6, or a complementary strand thereof, the polypeptide according to claim 7, and the polypeptide according to claim 7.
, A transformant according to claim 9, an antibody according to claim 13, a compound according to claim 15, or an inhibitor according to claim 16. Means for diagnosing a disease. 22. A means for diagnosing nerve cell death, comprising a step of measuring the gene expression level and / or enzyme activity of prostaglandin E synthase. 23. A means for diagnosing Alzheimer's disease, comprising the step of measuring the gene expression level and / or enzyme activity of prostaglandin E synthase. 24. A preventive / therapeutic agent for nerve cell death, comprising a prostaglandin E synthase inhibitor. (25) A prophylactic / therapeutic agent for Alzheimer's disease, comprising a prostaglandin E synthase inhibitor.