JP2003164290A - beta-AMYLOID-INDUCING Lib GENE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new gene in which expression is induced by β-amyloid stimulation and control neurological disorders such as Alzheimer's disease. <P>SOLUTION: This invention provides a new DNA having a DNA sequence of the new gene, in which expression is induced by β-amyloid stimulation, obtained by a cDNA subtraction method, a polypeptide encoded by the DNA, a recombinant vector containing the DNA, a transformant having the recombinant vector, an antibody against the polypeptide, a method for producing the polypeptide, a method for identifying a compound for regulating expression and activity of the gene by utilizing the DNA, the polypeptide, the vector, the transformant or the antibody, a compound obtained by the method and further provides a medicinal composition used for neurological disorders related to β-amyloid, a diagnostic means and a kit for diagnosis by utilizing the DNA, the polypeptide, the recombinant vector, the transformant or the antibody. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel gene whose expression is induced by a protein β-amyloid involved in Alzheimer's disease. More specifically, DN of a novel gene
A DNA having an A sequence, a polypeptide encoded by the DNA, a recombinant vector containing the DNA, a transformant transformed with the recombinant vector, a method for producing a polypeptide using the transformant, Antibodies against polypeptides, methods for identifying compounds using these, compounds obtained by the methods, the polypeptides or the DN
The present invention relates to an inhibitor of the activity and / or expression of the polypeptide which acts on A, a pharmaceutical composition related thereto, and a diagnostic means for diseases related thereto.

[0002]

BACKGROUND ART β-amyloid is a constituent factor of senile plaques observed in the brains 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 a β-amyloid precursor by a certain protease, and forms an insoluble aggregate having a β-sheet structure. β-amyloid is direct to nerve cells,
It is believed to act indirectly to promote the progression of Alzheimer's disease. As an indirect action of β-amyloid, activation of astroglial cells is known. Astroglia cells are known to produce several functional factors when activated by β-amyloid (Mark, RE et al., Human Pa).
thol. 26, pp816-823 (1995), M
cGeer, P.C. L. et al. , Brain R
es. Rev. 21, pp195-218 (199
5), Eddleston, M .; et al. , Neu
roscience 54, pp15-36 (199
3)). The present inventors have proposed that as a gene whose expression is induced by β-amyloid, prostaglandin E2 synthase (JP 2001-258575 A), ADAMTS-4.
(A Disintegrinand Metallo
proteinase with Thrombosp
ondin Motifts-4) has already been found (Japanese Patent Application No. 2000-181599). It is considered that Alzheimer's disease is exacerbated by the cooperative action of astroglial cells and β-amyloid.In a lesion of the brain of an Alzheimer's disease patient, activated astroglial cells are localized around senile plaques and their projections extend to senile plaques. It is also known to be.

However, analysis of the mechanism by which β-amyloid exacerbates Alzheimer's disease through activation of astroglial cells and the factors whose expression is induced or suppressed in astroglial cells by β-amyloid are still insufficient.

[0004]

The problem to be solved by the present invention is to find a novel gene whose expression is induced by β-amyloid in astroglial cells, and to find the found gene in nerves such as Alzheimer's lesions. It is to be used as a means for diagnosing and controlling diseases. Furthermore, it is to provide a polypeptide encoded by the novel gene and to provide an antibody against the novel polypeptide. Another object of the present invention is to provide a method for identifying a compound that regulates the action and / or expression of the novel polypeptide, which utilizes the above, and further,
Providing a compound identified by the identification method,
Further, the present invention provides a means for diagnosing a disease, a kit for diagnosing a disease, and a therapeutic drug for a disease using these.

In order to solve the problem, the present inventor has
β-amyloid treatment and untreated rat fetal brain-derived astroglial cells were subjected to cDNA subtraction treatment using the expressed gene to identify a novel gene whose expression is induced by β-amyloid treatment. The present invention was completed by determining the encoding amino acid sequence. Furthermore, an ortholog of the novel gene in human was found and the sequence was determined.

That is, the present invention includes (1) a DNA selected from the following group or a complementary strand thereof: D represented by the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing.
NA, D represented by the DNA sequence set forth in SEQ ID NO: 2 in the Sequence Listing
NA, DNA containing the DNA sequence set forth in SEQ ID NO: 1 or 2 of the Sequence Listing, and at least about 70% DN with said DNA
A DNA encoding a peptide having homology on the A sequence and having a cell-cell adhesion regulating function and / or a cell-extracellular matrix adhesion regulating function, and a DNA sequence of the DNA described above, Have mutations such as deletions, substitutions and additions of one to several DNAs,
And cell-cell adhesion control function and / or cell-cell
A DNA encoding a peptide having a function of regulating adhesion between extracellular matrices.

(2) Hybridization with the DNA described in (1) or a complementary strand thereof under stringent conditions, and a function of regulating adhesion between cells and / or a function of regulating adhesion between cells and extracellular matrix. DNA encoding a peptide having:

(3) The DNA according to (1) or (2)
A polypeptide encoded by.

(4) The DNA according to (1) or (2)
Alternatively, a recombinant vector containing the complementary strand thereof.

(5) pCMV-Tag4-rLib (F
ERM BP-7811).

(6) A transformant transformed with the recombinant vector according to (4) or the plasmid according to (5). (7) A method for producing the polypeptide according to claim 3, comprising a step of culturing the transformant according to (6).

(8) An antibody which immunologically recognizes the polypeptide according to (3).

(9) A method for identifying a compound having a cell-cell adhesion regulation function and / or a cell-extracellular matrix adhesion regulation function, which comprises (1) or (2)
DNA or complementary strand thereof, described in (3), recombinant vector described in (4), or (5)
A method comprising using at least one of the plasmid according to (6), the transformant according to (6) and the antibody according to (8).

(10) A compound identified by the identification method described in (9).

(11) An adhesion regulator between cells and / or cells and extracellular matrix identified by the identification method described in (9).

(12) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). Medicine consisting of two.

(13) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). A pharmaceutical composition containing one.

(14) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). A pharmaceutical composition for use in the prevention / treatment of a disease involving adhesion between cells / cells and / or cells / extracellular matrix containing the same.

(15) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). And a preventive / therapeutic agent for neuronal cell death.

(16) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). An agent for preventing / treating Alzheimer's disease, which comprises

(17) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). A diagnostic method for a disease containing one.

(18) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). A method of diagnosing neuronal cell death containing

(19) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). A diagnostic method for Alzheimer's disease containing:

(20) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). A kit for diagnosing a disease containing one.

(21) The DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). A kit for diagnosing neuronal cell death, which comprises:

(22) DN according to (1) or (2)
A or its complementary strand, the polypeptide according to (3),
At least one of the recombinant vector described in (4) or the plasmid described in (5), the transformant described in (6), the antibody described in (8), and the compound described in (10). A kit for diagnosing Alzheimer's disease, which comprises: Is to provide.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION (β-Amyloid Treatment of Astroglia Cells) Astroglia cells can be prepared from the brains of various animals. Fetal brain is preferably used, and as the animal to be used, rodents such as rat, mouse, guinea pig, and rabbit, which are relatively easy to collect fetal brain, are preferable, and rat is particularly preferably used. A method known per se is used for the preparation of astroglial cells from the fetal brain, and the method is described in a separate volume of Experimental Medicine, Neurobiochemistry Manual (Niichi Ito et al., Yodosha (1
989)) and the like.

Β-amyloid that acts on astroglial cells is commercially available and can be obtained from Anaspec or the like, but can also be prepared by a genetic engineering method. The β-amyloid may be aggregated by centrifugation or the like before being treated. The concentration of β-amyloid added and the treatment time are appropriately selected within a range exhibiting physiological activity, but the concentration is preferably 25 to 50 μM, and the treatment time is preferably 10 to 20 hours. β-amyloid-untreated astroglial cells can be prepared by seeding astroglial cells in other culture vessels under the same conditions in parallel with β-amyloid treatment and adding the same amount of the buffer used for β-amyloid dilution. .

(Preparation of Expression Gene) The expression gene of β-amyloid-treated and untreated astroglial cells can be collected by a commonly used RNA collection method. The expressed gene may be used as it is as mRNA or as cDNA. A method using reverse transcriptase or the like can be applied to the conversion of mRNA into cDNA. For collecting total RNA, for example,
Acidic guanidinium-phenol-chloroform method (A
GPC method; Chomczynski et al. , An
al. Biochem. 162, pp156-159
(1986)) and polyA (+) using oligo dT column etc. for collecting mRNA from total RNA.
Examples include purification of RNA. The RNA sample can be used by mixing a plurality of RNA samples. For the purpose of analyzing the quality of the collected lot, it is recommended to monitor the expression of several genes known to be expressed in astroglia. For example, nerve growth factor (NGF), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-
6), p75, etc. are used as monitor genes, and it is advisable to use a sample whose expression is equivalent.

(Subtraction treatment) To obtain a gene whose expression is induced by β-amyloid treatment,
Subtraction processing can be applied. In the subtraction process, two sets of gene groups are used, a gene group on the subtraction side (tester) and a gene group on the subtraction side (driver). For example, in order to obtain a gene whose expression is induced by β-amyloid, a gene obtained from β-amyloid-treated cells as a tester (β-amyloid-treated gene) and a gene obtained from untreated cells as a driver (untreated gene) ) Should be used for subtraction. As a method of subtraction processing, various known means can be used (JP-A-3-117488).
Preferred is the cDNA subtraction method.
As the cDNA subtraction method, RDA method, PC
The R-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 and confirming its concentration as a positive control for subtraction. In addition, using a gene whose expression is known to be induced by β-amyloid treatment as a probe, Southern blot analysis of PCR products before and after subtraction was performed.
When hybridization is performed, the success or failure of the subtraction process can be similarly confirmed. Currently, as genes whose expression is known to be induced by β-amyloid treatment, NGF and inducible nitric
Oxide Synthase (iNOS), Regu
Later on Activation Norma
l T-cell Expressed and Se
creted (RANTES) and the like.

The PCR product after the subtraction treatment is inserted into an appropriate vector to prepare a plasmid library or the like. The vector inserts are amplified by PCR, and two sets of those genes are dot-blotted on a membrane. PC after subtraction treatment using β-amyloid treated gene as a tester and untreated gene as a driver for this dot membrane
R product [Hereafter, subtraction processing (tester)-
Expressed by the formula of (driver). ] And (unprocessed gene)-
P after subtraction of (β amyloid processing gene)
Hybridization is performed with each CR product probe. (Untreated gene)-(β amyloid-treated gene) Compared with the PCR product after subtraction,
A clone in which specific enrichment is found in the PCR product after the (β-amyloid-treated gene)-(untreated gene) subtraction may be determined as a gene whose expression is induced by β-amyloid treatment. The sequence of the vector insert portion can be determined by a DNA sequencing method known per se.

(Acquisition of rat gene) The gene provided in the present invention is obtained as a gene encoding a novel protein by cDNA subtraction treatment using rat fetal brain-derived astroglia. Nucleotide database (DDBJ /
As a result of a search of EMBL / GenBank), it was confirmed to be novel. Therefore, when the prepared β-amyloid-treated rat astroglial cell-derived cDNA library was screened using the cDNA fragment as a probe, a 5.6 Kb cDNA was obtained. Obtained. This cDNA has almost the same number of nucleotides as the band observed in the Northern blot, and 5'-rapida
mplicification of cDNA ends
Since the 5'-side sequence could not be obtained by the (5'-race) method, it was considered to include the full-length mRNA.
A sequence reaction was performed using a fluorescent dye (Cy5; Pharmacia) labeled M13 universal and reverse primers, and the sequence of the ORF region was determined with an ALF Express fluorescence sequencer (Pharmacia) and the like, this gene encodes ORF1734bp, 578 amino acids. New LRR superfamily (B
uchanan, S .; G. et al. , Prog
Biophys Mol. Biol. 65, pp 1-44
(1996) Shishido, E .; et al. , S
science 280, pp2118-2121 (19
It has been revealed that it is a protein belonging to (98)) (Fig. 1).

The protein encoded by this gene is a signal sequence (1-19 amino acids), Leucine Rich.
Repeat (LRR) N-flanking region (25
-53 amino acids), 15 times LRR region (54-413 amino acids), LRR C-flanking region (423-4)
75 amino acids), a transmembrane region (535-557 amino acids), and an intracellular region (558-578 amino acids). Each LRR has a consensus residue of 24 amino acids (Fig. 2). This protein was named Lib. Fl
When Lig added with an ag tag was expressed in Cos7 cells, it was larger than 62 kDa calculated, which was considered to be the effect of glycosylation. By β-amyloid treatment, Lib showed a 13-fold increase in expression in rat astroglial cells (FIG. 3).

(Acquisition of human ortholog) When the human genome database was searched to find the human ortholog of Lib, the corresponding human gene was found on the NCBI genome database. It was cloned from human brain-derived poly A (+) RNA (CLONTECH) by RT-PCR, the sequence was determined, and DDBJ / EMBL was used.
/ GenBank with accession number AB0710
Registered at 37 (unpublished). Human Lib showed 84% homology with rat at the amino acid level and 81% at the nucleotide level (Fig. 2). The present inventors showed for the first time that human Lib exists in the chromosome 3q29 region and that this gene is transcribed as mRNA. When the tissue distribution of human Lib was examined, strong expression was observed in the placenta, and it was below the detection level in the brain (Fig. 4).

(Function of Lib) LRR is a repeat of a short sequence consisting of 20 to 29 amino acids, and its most typical length is 24 amino acids. The LRR domain functions in specific protein-protein interactions including adhesion, target recognition, and receptor-ligand binding. Lib
Is a novel molecule belonging to the LRR superfamily. Furthermore, it is characteristic of the extracellular domains of certain adhesion factors and receptors, and both cysteine-rich N- and C-terminal LRR flanking regions (which are thought to form disulfide bonds as binding motifs) have. Judging from the characteristics of these LRRs and the expression of Lib remarkably by β-amyloid treatment, Lib expression-induced in astroglial cells by β-amyloid was detected between neurons and astroglial cells.
Alternatively, it is speculated that the interaction between glial cells is strengthened, and as a result, the present inventors believe that this is involved in the progression of Alzheimer's disease.

Among the members of the LRR family, a protein (Carp that acts as a target recognition molecule during the growth of nerve axons into specific muscles of the Drosophila development stage) (Carp).
RICIOUS) (Shishido, E. et a.
l. , Science 280, pp2118-21
(1998), Taniguchi, H .; et a
l. J. Neurobiol. 42, pp104-1
6 (2000)), and a protein (Glycoprotein V) (Hickkey, which is a constituent molecule of the GPIb-V-IX complex involved in platelet adhesion and activation).
M. J. et al. , Proc. Natl. Aca
d. Sci. USA 90, pp8327-8331
(1993), Lanza, F .; et al. J.
Biol. Chem. 268, pp 20801-208
07 (1993), Modderman, P .; W.
et al. J. Biol. Chem. 267, pp
364-369 (1992), Lopez, J .; A.
et al. , Blood Coagul. Fibri
nolysis 5, pp97-119 (1994))
Exists. Lib shows a domain structure similar to that of the above-mentioned protein and has almost the same number of repeats. Not all functions of the above proteins have been clarified, but Lib
Similarly, the inventor believes that it is a molecule involved in cell-cell interaction and cell-extracellular matrix interaction at the developmental stage or nerve injury.

Lib is a novel inflammation-related factor because it is an inducible gene, not a constitutively expressed gene, and it also showed increased expression in cytokines. TNF-α, IL-1β
Has been detected in Alzheimer's diseased brain (McGee
r, P. L. , Et al. , Brain Re
s. Rev. 21, pp195-218 (199
5)), and IFN-γ has also been pointed out to be involved in exacerbation of Alzheimer's disease (Blasko, I. et a.
l. J. Neuroimmunol. 116, pp
1-4 (2001), Ii, M. et al. et al. , Br
ain Res. 720, pp93-100 (199
6)). As observed in Alzheimer's disease and other neurological diseases, glial cells migrate to the affected area during inflammation and surround it, and extend a protrusion inside. Lib is suppressed to a low expression at a steady state, but expression is induced in response to inflammatory stimuli (Fig. 6), and cells found in glia
It is considered to be a molecule involved in dynamic changes between cells or between cells and extracellular matrix.

(DNA and complementary strand thereof) DN of the present invention
A and its complementary strand are a DNA consisting of the DNA sequence shown in SEQ ID NO: 1 or 2 of the sequence listing and its complementary strand, a DNA having the partial sequence shown in SEQ ID NO: 1 or 2 of the sequence listing.
And its complementary strands and DNAs that hybridize with these DNAs under stringent conditions. "DNA that hybridizes to DNA under stringent conditions" refers to, for example, Molec.
ural Cloning 2nd Edition (J. Sambrook
k et al. (1989)). Here, “hybridize under stringent conditions” means, for example, 6 ×
After warming at 42 ° C. in a solution of SSC, 0.5% SDS and 50% formamide, 0.1 × SSC, 0.5
This shows that a positive hybridization signal which can be discriminated is observed even under the condition of washing at 68 ° C. in a solution of% SDS.

The DNA of the present invention has at least about 70% homology in the DNA sequence with the DNA represented by the DNA sequence of SEQ ID NO: 1 or 2 in the sequence listing, and has a cell-cell adhesion regulating function. And / or DNA encoding a polypeptide having a function of regulating adhesion between cells and extracellular matrix.

The selected DNA is preferably about 7
It has a homology of 0% or more, more preferably about 80% or more, and further preferably about 90% or more. Techniques for determining homology of DNA sequences are known per se, for example DNA
Methods that directly determine the sequence can be used. Confirmation of the cell-cell adhesion regulation function of the encoded polypeptide and / or the cell-extracellular matrix adhesion regulation function can be carried out by expressing the polypeptide in an appropriate cell using the DNA of the present invention. it can. A known protein expression system, for example, a cell-free protein expression system can be used to express the polypeptide. As the cell-free protein expression system, for example, a technique of a ribosome system derived from embryo, rabbit reticulocyte and the like can be used (Nature, 179, pp1.
60-161 (1957)). The ability of the cells expressing the polypeptide to adhere to each other, or the cells expressing the polypeptide to the extracellular matrix may be measured by visual or physical means.

Further, the DNA of the present invention is DN of the above DNA.
Encodes a polypeptide having a mutation such as a deletion, substitution, or addition of one to several DNAs in the A sequence and having a cell-cell adhesion regulating function and / or a cell-extracellular matrix adhesion regulating function Means DNA that Means for deleting, substituting, adding or inserting DNA is known per se, and examples thereof include a method for producing a deletion mutant using exonuclease, and a method for site-directed mutagenesis.

All of these DNAs provide a gene useful for producing the novel polypeptide of the present invention, and a nucleic acid such as a gene encoding the gene or mRNA.
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. Furthermore, D of the present invention
NA can also be used as a reagent for nucleic acids.

(Polypeptide) The polypeptide of the present invention is D represented by the DNA sequence of SEQ ID NO: 1 or 2 in the sequence listing.
NA and the DNA described in SEQ ID NO: 1 or 2 of the sequence listing
It is a polypeptide encoded by a DNA containing the DNA represented by the sequence. Furthermore, it is encoded by a DNA having at least about 70% homology in the DNA sequence with the DNA represented by the DNA sequence set forth in SEQ ID NO: 1 or 2 of the Sequence Listing, and the cell-cell adhesion regulating function and / or cell -A polypeptide having a function of regulating adhesion between extracellular matrices. In addition, it is a polypeptide encoded by a DNA that hybridizes with the above DNA or a complementary strand thereof under stringent conditions, and has a function of controlling adhesion between cells and / or cells and extracellular matrix. Further, it is encoded by a DNA having mutations such as deletion, substitution, addition of one or several DNAs in the DNA sequence of the above DNA, and has a cell-cell adhesion regulating function and / or a cell-extracellular matrix. It is a polypeptide having an adhesion regulating function of. In addition, these polypeptides are
Modifications such as modification of the constituent amino group or carboxyl group are possible without causing a significant change in function.

The polypeptides of the present invention are, by themselves,
It can be used in pharmaceutical compositions for regulating their own in vivo function. Further, the polypeptide of the present invention can be used for screening for obtaining compounds capable of regulating their functions, such as inhibitors, antagonists, activators, etc., and for obtaining antibodies against them. Furthermore, 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. The vector DNA may be one obtained by extracting a naturally occurring one, or one in which a part of the DNA other than the part necessary for growth is partially deleted. For example, there are a vector derived from Col E1 and a vector 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 an appropriate restriction enzyme is selected and treated to cut the DNA at a specific site, which is then mixed with DNA similarly used as a vector and religated with ligase. In pCMV-Tag4-rLib (FIG. 5) shown in Examples described later, pCMV-Tag4 (STRATAGE) was used as vector DNA.
(Manufactured by NE Co.) was used, but it is not limited to this. Also, pCMV-Tag4-rLib has been deposited at the Patent Organism Depositary Center as FERM BP-7811.

(Transformant) In addition to the cell-free protein expression system as described above, the present invention can be carried out by a gene recombination technique utilizing a host known per se, such as Escherichia coli, yeast, Bacillus subtilis, insect cells, and animal cells. It is possible to provide a novel polypeptide consisting of and a derivative thereof.

For the transformation, means known per se can be applied. For example, a replicon such as a plasmid, a chromosome or a virus is used to transform a host. As a more preferable system, if the stability of the gene is taken into consideration, the method of integrating into the chromosome can be mentioned. For convenience, an autonomous replication system using an extranuclear gene is used. The vector is selected depending on the type of host, and has a gene sequence for expression and a gene sequence carrying information on its replication and control as its constituent elements. The component is 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 in the production of the polypeptide of the present invention by culturing by selecting the optimum conditions for the culture conditions of each known host. Culturing may be carried out by subculture or batch using the amount of transformants in the medium as an index, although the physiological activity of the novel polypeptide produced and produced may be used as an index.

The above pCMV-Tag4-rLib is used.
Using the competent cell E. The transformant obtained by transforming E. coli DH5α was used to obtain pC
Amplification of MV-Tag4-rLib was performed.

(Recovery of Novel Polypeptide and Its Derivative) For recovering the polypeptide consisting of the novel polypeptide and its derivative from the medium, molecular sieve, ion column chromatography, affinity chromatography, etc. may be combined or the solubility difference may be detected. It can also be purified and recovered by a fractionation means based on ammonium sulfate, alcohol, etc. 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 is specifically adsorbed and recovered by 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 its derivative. The antigenic determinant is at least 5
, More preferably at least 8-10 amino acids. This amino acid sequence does not necessarily have to be homologous to the polypeptide sequence encoded by the DNA of SEQ ID NO: 1 in the sequence listing, but may be an exposed site on the outside in the three-dimensional structure of the protein, and the exposed site is a discontinuous site. 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 novel polypeptide and a polypeptide consisting of a derivative thereof. The presence or absence of this recognition is determined by a known antigen-antibody binding reaction.

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

To produce a monoclonal antibody,
It is carried out by recovering antibody-producing cells from the animal to which the above-mentioned immunization means has been applied and introducing a means for transforming permanently proliferating cells known per se.

A polyclonal antibody or a monoclonal antibody is capable of directly binding to the polypeptide of the present invention and controlling the activity thereof, and is useful for treating / preventing diseases associated with the activity of the polypeptide of the present invention.

(Identification of compounds) DN thus prepared
A and its complementary strand, polypeptide, recombinant vector,
Transformants and antibodies, alone or in combination of multiple means, provide effective means for identifying compounds that modulate the expression or activity of the polypeptides of the invention. That is, the identification of a compound that inhibits or enhances the expression of the polypeptide of the present invention by using at least one of the DNA of the present invention or a complementary strand thereof, a recombinant vector, a transformant, or an antibody, By using at least one of the polypeptide of the present invention or the antibody, a compound that inhibits or enhances the activity of the polypeptide of the present invention can be identified. For example, screening of antagonists by drug design based on the three-dimensional structure of a polypeptide, screening of expression regulators at the gene level using a protein expression system, screening of antibody recognizing substances using antibodies, etc. It is available in the system. In addition, the compound obtained by the present identification method is useful as a pharmaceutical composition described later and a therapeutic agent for diseases.

(Compound, pharmaceutical composition, diagnostic means) The compound obtained by the above-mentioned identification method is an inhibitor, antagonist or activator which regulates adhesion between cells and / or cells and extracellular matrix. And the like can be used as a candidate compound. 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.

The candidate compounds thus selected are used for treating diseases associated with cell-cell and / or cell-extracellular matrix adhesion by selecting biological utility in consideration of toxicity and the like. It can be prepared as a pharmaceutical composition to be used. Further, the novel DNA of the present invention or its complementary chain, the polypeptide encoded by these, the recombinant vector containing the above DNA or its complementary chain, the transformant transformed with these recombinant vectors, and the above polypeptide Antibodies immunologically recognizing the above have functions such as inhibition, antagonism, and activation of the activity, expression, etc. of the polypeptide of the present invention themselves, and between cells / cells and / or between cells / extracellular matrix. It can be used as a medicinal means for treating / preventing diseases involving adhesion.

Diseases associated with adhesion between cells / cells and / or between cells / extracellular matrix include diseases associated with nerve cell death, for example, neurological diseases such as Alzheimer's disease. Judging from the characteristics of LRR, which is a characteristic structure of Lib, and the expression of Lib by β-amyloid treatment is remarkably induced, Lib whose expression was induced in astroglial cells by β-amyloid, Alternatively, it is speculated that the interaction between glial cells is strengthened, and as a result, the present inventors believe that this is involved in the progression of Alzheimer's disease. Therefore, the inhibition of Lib expression or function is
The inventor believes that it can be applied to the treatment of Alzheimer's disease.

For formulation, a compound, a polypeptide, a protein, a polynucleotide, an antibody or the like may be introduced into a formulation means known per se according to each control.

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 with these recombinant vectors, and the above polypeptide The antibody that immunologically recognizes
Alternatively, it is useful as a diagnostic means for nerve cell death, diseases and the like involving adhesion between cells and extracellular matrix. In particular,
It is useful as a diagnostic means such as a diagnostic marker and / or reagent for Alzheimer's disease. Diagnosis utilizes interaction / reactivity with the DNA of the present invention to determine the abundance of the polypeptide of the present invention and / or the abundance of the polypeptide of the present invention in a sample. Done by. As the sample, blood, liquor, brain biopsy sample and the like are preferable, and cerebral spinal fluid is particularly preferable. The measuring method used for diagnosis is an antigen-antibody reaction, an enzyme reaction system, PCR
A reaction system or the like may be used. The means referred to here means a method and / or medium used for achieving the purpose. That is, for example, the diagnostic means includes a diagnostic method, a reagent used for diagnostics, a kit, and the like.

[0061]

Example (Glial cell culture) Astroglia cells were obtained from rat cerebral cortex. The cerebrum of a Wister rat fetus (Charles River, 17 Days) was collected,
The cells were dispersed by trypsin treatment. The obtained cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 20% fetal calf serum (FCS) and an antibiotic solution for 1 week, and then further subcultured in DMEM containing 10% FCS. It was confirmed by the fluorescent antibody method that 90% or more of the cells were positive for glial fibrillary acidic protein. C6 rat astroglioma cells are American Type C
Obtained from the Ultra Collection (ATCC) and maintained in DMEM with 10% FCS.

(Preparation of β-amyloid and β-amyloid treatment of astroglial cells) β-amyloid (Anasp
ec, β-amyloid 1-40) dissolved in sterile water-
It was stored at 70 ° C and thawed before use. Aggregation of β-amyloid was performed by dissolving 1 mM β-amyloid in Earle's buffer and holding it in a CO ₂ incubator for 1 week.
It was carried out by centrifugation at 000 × g for 10 minutes.
Add pellets to B27 supplement (Gibco BRL)
It was resuspended in DMEM containing and adjusted to an appropriate concentration and used. After the secondary culture, the astroglial cells were washed with a phosphate buffer solution (PBS), converted to a serum-free medium containing B27 supplement, cultured for 2 days, and then added with 25 μM of aggregated β-amyloid and further cultured for 15 hours. .

(CDNA subtraction and library screening) From β-amyloid-treated and untreated astroglial cells (6 cm plastic dishes, 20 sheets each), total RNA was collected 9 times by the AGPC method.
Was extracted. Poly A (+) RNA is oligo dT-
It was prepared using latex (manufactured by Roche).

2 μg each of poly A (+) RNA derived from β-amyloid-treated and untreated astroglial cells was used as a starting material (β-amyloid-treated gene)-(untreated gene)-(untreated gene)-(β-amyloid-treated Gene) cDNA subtraction process, Diat
chenko et al. (Proc. Natl. Aca
d. USA 93, pp 6025-6030 (199
6)) in accordance with PCR-select cDNAs
It was performed using the subtraction kit (CLONTECH). As a positive control for subtraction treatment, φΧ1 was added to the tester after digestion with Rsa I.
The 74 / Hae III digested DNA fragment was added to the tester at a weight ratio of about 2.5 × 10 ⁻⁵ .

In order to verify the efficiency of the subtraction process performed, a positive control φ174 / Hae II
PCR product after subtraction and P before subtraction using I digested DNA fragment as a probe
Southern blot hybridization was performed on the CR products.

(Β-amyloid processing gene)-(unprocessed gene) PCR product vector [pBluescript
t SK + (Stratagene)] to prepare a plasmid library and DH5
Transformation of α-competent cells (Gibco BRL) was performed.

The vector insertion portion of the 960 clone of the plasmid library was amplified by the PCR method, and 60 to 100 ng of each of these genes was inserted into a membrane (Hybon).
d-N +; manufactured by Amersham) were dot-blotted to prepare two sets. On this dot-blot membrane, a PCR product probe after subtraction of (β-amyloid-treated gene)-(untreated gene) and (untreated gene)-(β-amyloid-treated gene) was used, and 0.5MNa ₂ HPO ₄ ( pH 6.8), 10 μg
/ Ml salmon sperm DNA in 7% SDS solution at 68 ° C, 1
Hybridization was carried out for 6 hours each.
Membrane 6 in 0.1 x SSC with 0.1% SDS
It was washed at 8 ° C. for 30 minutes. (Unprocessed gene)-(β
PCR after subtraction of amyloid processing gene)
A clone was selected in which specific enrichment was observed in the PCR product after subtraction of (β-amyloid-treated gene)-(untreated gene) compared to the product.

(Analysis by Northern Blot Hybridization) Each vector insertion portion of the selected clone group was labeled with ³² P and then used as a probe, and β-amyloid-treated and untreated astroglial cell-derived poly were used.
Northern blot hybridization with A (+) RNA was performed. By the above method, respective poly A (+) RNA samples were prepared from β-amyloid-treated and untreated astroglia. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA, β-actin cDNA, which are genes with constant expression
(Manufactured by CLONTECH) was used as an internal standard probe. The detection was performed using BAS2000 (manufactured by Fuji Film Co., Ltd.). G3PDH gene and β-actin gene are
While no difference in expression level was observed between β-amyloid-treated and untreated, the novel gene of the present invention showed a low expression level when β-amyloid-untreated, but the expression level was approximately There was a 13-fold increase (Fig. 3).

(Nucleotide sequence analysis and homology search) β
The clones whose expression was induced by amyloid were subjected to a sequencing reaction using Cy5-labeled M13 universal and reverse primers, and the nucleotide sequence of the vector insertion portion was analyzed with an ALF Express fluorescence sequencer (Pharmacia). NCB for the sequences obtained
A homology search was performed on the I database. One of the obtained full-length cDNAs was considered to be novel because it had no gene showing homology. When the cDNA fragment was screened from the prepared β-amyloid-treated rat astroglial cDNA library, a 5.6 Kb cDNA was obtained. This cd
Since NA has almost the same number of nucleotides as the band observed in the Northern blot, and the 5'side sequence was not obtained by the 5'race method, the mRNA of the novel gene was
It was considered to include the full length. When the sequence of the ORF region was determined, it was revealed that this gene is a protein belonging to a novel LRR superfamily, which encodes ORF1734bp and 578 amino acids (Fig. 1). This protein has a signal sequence (1-19 amino acids), LRR
N-flanking region (25-53 amino acids), 15th LRR region (54-413 amino acids), LRR C-flanking region (423-475 amino acids), transmembrane region (535-557 amino acids), intracellular region ( 558-
578 amino acids). Each LRR has a consensus residue of 24 amino acids. This protein was named Lib (Fig. 1). Lib with a Flag tag added to C
When it was expressed in os7 cells, it was larger than the calculated 62 kDa, which was considered to be the effect of glycosylation.

(Tissue distribution of novel gene expression and cloning of human ortholog) In order to find the human ortholog, the human genome database was searched. Since the human corresponding gene was found on the NCBI genome database, human fetal brain-derived poly A (+) RNA (1
μg, CLONTECH) to SuperScript
II (GIBCO) using oligo dT
(12-18) was cloned by the RT-PCR method. Reverse transcript is 5'-TGTGCTCTC
TCCCTTGCACAG-3 '(5' flanking region) and 5'-ATCACGGGAAAGCTCCAT
Amplification by PCR was performed using GGAC-3 ′ (3 ′ flanking region) as a primer. The PCR product was integrated into pCR2.1 (Invitrogen),
I performed a sequence.

In order to avoid a sequencing error due to a PCR error, the sequences of five independent clones were determined, and they were comprehensively determined to determine the sequence of this gene. Human Lib
Showed 84% homology with the rat at the amino acid level and 81% at the nucleotide level (Fig. 2).

The present inventors have for the first time shown that human Lib exists in the chromosome 3q29 region and that this gene is transcribed as mRNA. Pol extracted from each human tissue to investigate the tissue distribution of human orthologs
Northern blot of y A (+) RNA (CLONTE
CH) was hybridized with human Lib cDNA by ³² P labeling. Strong expression in placenta,
It was below the detection level in the brain (Fig. 4).

(Induction of Expression of Gene of the Present Invention by Inflammatory Cytokine) Rat C6 cells were transferred to rat TNF-α (4
00U / ml, Pepro Tech, UK), rat IL-1β (100U / ml, Pepro Tech,
UK) and rat interferon (IFN) -γ
(200 U / ml, PeproTech, UK). Total RNA is RNAeasy min
It was purified using i column (Qiagen).
The expression level of the gene of the present invention is TaqMan Gold.
RT-PCR kit (Applied Biosy
Quantitative RT using
-Analysis by PCR (ABI PRISM7700).

TNF-α, IL-1β and IFN-γ
The peak of 6 hours was reached by adding the mixed solution of about 1.
An 8-fold (fold increase relative to 0 hour) gene expression was observed. TNF-α and IL-1β each showed a slight increase in expression, but the simultaneous addition of them induced more expression, and the addition of IFN-γ showed a further increase (1.8-fold). (Fig. 6).

(Analysis of intracellular distribution of gene of the present invention) A plasmid in which Ds-Red was fused to the C-terminus of rat Lib gene was transiently expressed in rat astrocyte-ma C6, and then it was examined under a phase contrast microscope. When the intracellular distribution of Lib was observed, it was confirmed that the fusion protein was expressed on the cell surface. (Plasmid pCMV-Tag4-rLib (FPRM
BP-))) A rat rat astrocyte-derived cDNA library screened with rat lat cDNA was used as a template, and
The ORF site of t Lib was amplified using PCR in a form in which restriction enzyme sites (5 'side BglII, 3'side BamHI) were added to both ends of the ORF.

The amplified fragment thus obtained was treated with the above two restriction enzymes and treated with BamHI to obtain pCMV-Tag4 (S
TRAGAGEN) and pCMV-T
ag4-rLib was obtained (BglII and BamHI can be ligated in a convertible manner) (FIG. 5).

[0077]

INDUSTRIAL APPLICABILITY The present invention provides a novel gene whose expression is induced by β-amyloid treatment. This gene has a characteristic LRR region and is considered to be involved in cell-cell and / or cell-extracellular matrix adhesion. The provision of a novel pharmaceutical composition and diagnostic means utilizing this characteristic provides great utility in clinical and basic medical fields of diseases associated with β-amyloid such as Alzheimer's disease.

[Sequence list]                               SEQUENCE LISTING       <110> Daiichi Pharmaceutical co.ltd <120> Novel gene induced by beta-amyloid <130> N01113001A <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 1737 <212> DNA <213> rat <220> <221> CDS <222> (1) .. (1737) <400> 1 atg ccg ctg aaa cat tac ctc ctc ttg ctg gtg ggc tgc cag gcc tgg 48 Met Pro Leu Lys His Tyr Leu Leu Leu Leu Val Gly Cys Gln Ala Trp   1 5 10 15 gct cta ggt ttg gcc tac tat ggc tgc ccc agt gaa tgc acc tgt tcc 96 Ala Leu Gly Leu Ala Tyr Tyr Gly Cys Pro Ser Glu Cys Thr Cys Ser              20 25 30 cgg gcc tcc cag gtg gag tgc aca gga gcg cgg att gtg gcg atg cct 144 Arg Ala Ser Gln Val Glu Cys Thr Gly Ala Arg Ile Val Ala Met Pro          35 40 45 acc ccc ctg ccc tgg aat gct atg agc ctg cag gtc gtt aac acg cac 192 Thr Pro Leu Pro Trp Asn Ala Met Ser Leu Gln Val Val Asn Thr His      50 55 60 att act gag ctc cct gag aac ctg ttc ctc aac att tca gcg ctc att 240 Ile Thr Glu Leu Pro Glu Asn Leu Phe Leu Asn Ile Ser Ala Leu Ile  65 70 75 80 gcc cta aag atg gag aag aat gag ctg tca acc atc atg ccg ggt gcc 288 Ala Leu Lys Met Glu Lys Asn Glu Leu Ser Thr Ile Met Pro Gly Ala                  85 90 95 ttc cgc aat ctg ggc tct ctg cgc tac ctc agc ctc gcc aac aac aaa 336 Phe Arg Asn Leu Gly Ser Leu Arg Tyr Leu Ser Leu Ala Asn Asn Lys             100 105 110 ctg agg atg ctg ccc atc agg gtc ttc cag gac gta aac aat ctg gag 384 Leu Arg Met Leu Pro Ile Arg Val Phe Gln Asp Val Asn Asn Leu Glu         115 120 125 tcc ctt ctg ctc tcc aat aac cag ctg gtg cag atc cag cca gcc cag 432 Ser Leu Leu Leu Ser Asn Asn Gln Leu Val Gln Ile Gln Pro Ala Gln     130 135 140 ttc tcc cag ttt agt aat ctt agg gaa ctc cag ttg cat ggc aac aat 480 Phe Ser Gln Phe Ser Asn Leu Arg Glu Leu Gln Leu His Gly Asn Asn 145 150 155 160 ctg gaa tcc att ccc gaa gaa gcc ttc gac cac cta gta gga ctc acc 528 Leu Glu Ser Ile Pro Glu Glu Ala Phe Asp His Leu Val Gly Leu Thr                 165 170 175 aaa ctc aac ctg ggc agg aac agc ttc acc cac cta tcg cct agg ctc 576 Lys Leu Asn Leu Gly Arg Asn Ser Phe Thr His Leu Ser Pro Arg Leu             180 185 190 ttt cag cat ctg ggc aac ctc cag gtg ctc cgg cta cac gag aac agg 624 Phe Gln His Leu Gly Asn Leu Gln Val Leu Arg Leu His Glu Asn Arg         195 200 205 ctt tca gac atc ccc atg ggc acg ttc gat gca ctc ggc aac ctc cag 672 Leu Ser Asp Ile Pro Met Gly Thr Phe Asp Ala Leu Gly Asn Leu Gln     210 215 220 gag ctt gcc ctc caa gag aac cag att ggc acc ctc tcc cct ggc ttg 720 Glu Leu Ala Leu Gln Glu Asn Gln Ile Gly Thr Leu Ser Pro Gly Leu 225 230 235 240 ttc cac aat aac cgt aac ctc cag aga ctg tat ctg tcc aac aac cac 768 Phe His Asn Asn Arg Asn Leu Gln Arg Leu Tyr Leu Ser Asn Asn His                 245 250 255 atc tcc cag ctg ccc cct ggc atc ttc atg cag ctg ccc cag ctt aac 816 Ile Ser Gln Leu Pro Pro Gly Ile Phe Met Gln Leu Pro Gln Leu Asn             260 265 270 aag ctc aca ctc ttt ggg aat tcc ctg agg gag ctt tcc ccg ggg gtc 864 Lys Leu Thr Leu Phe Gly Asn Ser Leu Arg Glu Leu Ser Pro Gly Val         275 280 285 ttc ggg ccc atg ccc aac ctg agg gag ctt tgg ctc tac aac aac cac 912 Phe Gly Pro Met Pro Asn Leu Arg Glu Leu Trp Leu Tyr Asn Asn His     290 295 300 atc acc tcc ctc gcc gac aac acc ttc agc cac ctg aac cag ctg cag 960 Ile Thr Ser Leu Ala Asp Asn Thr Phe Ser His Leu Asn Gln Leu Gln 305 310 315 320 gtc ctg att ctc agt cac aac cag ctc act tac atc tcc cca gga gcc 1008 Val Leu Ile Leu Ser His Asn Gln Leu Thr Tyr Ile Ser Pro Gly Ala                 325 330 335 ttc aat ggg ctg acg aac ctc cga gaa ctg tcc ctc cac acc aac gct 1056 Phe Asn Gly Leu Thr Asn Leu Arg Glu Leu Ser Leu His Thr Asn Ala             340 345 350 cta cag gac ctg gac agc aac gtc ttc cga tcg ctg gcc aac ctg cag 1104 Leu Gln Asp Leu Asp Ser Asn Val Phe Arg Ser Leu Ala Asn Leu Gln         355 360 365 aac atc tcg ctc cag agt aac cgc ctc cga cag ctt ccg ggc agc atc 1152 Asn Ile Ser Leu Gln Ser Asn Arg Leu Arg Gln Leu Pro Gly Ser Ile     370 375 380 ttt gcc aac gtc aac ggc ctc aca acc atc caa ctg cag aac aac aat 1200 Phe Ala Asn Val Asn Gly Leu Thr Thr Ile Gln Leu Gln Asn Asn Asn 385 390 395 400 cta gaa aac ttg cct ttg ggc atc ttt gat cac ttg gtg aac ctg tgt 1248 Leu Glu Asn Leu Pro Leu Gly Ile Phe Asp His Leu Val Asn Leu Cys                 405 410 415 gag ctt cgc ctc tac gac aac ccc tgg aga tgt gac tca gat atc ctc 1296 Glu Leu Arg Leu Tyr Asp Asn Pro Trp Arg Cys Asp Ser Asp Ile Leu             420 425 430 cca ctt cat aac tgg ctc ctt ctc aac aga gcc agg ctg ggg aca gac 1344 Pro Leu His Asn Trp Leu Leu Leu Asn Arg Ala Arg Leu Gly Thr Asp         435 440 445 act ctt cca gtg tgt tcc agt ccc gcc aac gtc cga ggc cag tcc ctc 1392 Thr Leu Pro Val Cys Ser Ser Pro Ala Asn Val Arg Gly Gln Ser Leu     450 455 460 gtc atc atc aat atc aat ttc cct gga ccc agt gtc cag ggc cca gag 1440 Val Ile Ile Asn Ile Asn Phe Pro Gly Pro Ser Val Gln Gly Pro Glu 465 470 475 480 acc cct gaa gta cct agt tac cca gat acg ccc agc tat ccc gac acc 1488 Thr Pro Glu Val Pro Ser Tyr Pro Asp Thr Pro Ser Tyr Pro Asp Thr                 485 490 495 aca tct gtt tcc tct acc acg gag atc acc agc gcc gtc gac gac tac 1536 Thr Ser Val Ser Ser Thr Thr Glu Ile Thr Ser Ala Val Asp Asp Tyr             500 505 510 acc gat cta acc acc att gaa gcc acc gat gac cga aat acg tgg ggc 1584 Thr Asp Leu Thr Thr Ile Glu Ala Thr Asp Asp Arg Asn Thr Trp Gly         515 520 525 atg act gag gcc cag agc ggg ctg gcc att gct gcc atc gtg atc ggc 1632 Met Thr Glu Ala Gln Ser Gly Leu Ala Ile Ala Ala Ile Val Ile Gly     530 535 540 atc ata gct ttg gcc tgt tcc cta gcc gcc tgc atc tgc tgc tgc tgc 1680 Ile Ile Ala Leu Ala Cys Ser Leu Ala Ala Cys Ile Cys Cys Cys Cys 545 550 555 560 tgc aaa aag agg agc caa gca gtc ctg atg cag atg aag gct ccc aat 1728 Cys Lys Lys Arg Ser Gln Ala Val Leu Met Gln Met Lys Ala Pro Asn                 565 570 575 gag tgc tag 1737 Glu Cys <210> 2 <211> 1746 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (1746) <400> 2 atg cca ctg aag cat tat ctc ctt ttg ctg gtg ggc tgc caa gcc tgg 48 Met Pro Leu Lys His Tyr Leu Leu Leu Leu Val Gly Cys Gln Ala Trp   1 5 10 15 ggt gca ggg ttg gcc tac cat ggc tgc cct agc gag tgt acc tgc tcc 96 Gly Ala Gly Leu Ala Tyr His Gly Cys Pro Ser Glu Cys Thr Cys Ser              20 25 30 agg gcc tcc cag gtg gag tgc acc ggg gca cgc att gtg gcg gtg ccc 144 Arg Ala Ser Gln Val Glu Cys Thr Gly Ala Arg Ile Val Ala Val Pro          35 40 45 acc cct ctg ccc tgg aac gcc atg agc ctg cag atc ctc aac acg cac 192 Thr Pro Leu Pro Trp Asn Ala Met Ser Leu Gln Ile Leu Asn Thr His      50 55 60 atc act gaa ctc aat gag tcc ccg ttc ctc aat atc tca gcc ctc atc 240 Ile Thr Glu Leu Asn Glu Ser Pro Phe Leu Asn Ile Ser Ala Leu Ile  65 70 75 80 gcc ctg agg att gag aag aat gag ctg tcg cgc atc acg cct ggg gcc 288 Ala Leu Arg Ile Glu Lys Asn Glu Leu Ser Arg Ile Thr Pro Gly Ala                  85 90 95 ttc cga aac ctg ggc tcg ctg cgc tat ctc agc ctc gcc aac aac aag 336 Phe Arg Asn Leu Gly Ser Leu Arg Tyr Leu Ser Leu Ala Asn Asn Lys             100 105 110 ctg cag gtt ctg ccc atc ggc ctc ttc cag ggc ctg gac agc ctt gag 384 Leu Gln Val Leu Pro Ile Gly Leu Phe Gln Gly Leu Asp Ser Leu Glu         115 120 125 tct ctc ctt ctg tcc agt aac cag ctg ttg cag atc cag ccg gcc cac 432 Ser Leu Leu Leu Ser Ser Asn Gln Leu Leu Gln Ile Gln Pro Ala His     130 135 140 ttc tcc cag tgc agc aac ctc aag gag ctg cag ttg cac ggc aac cac 480 Phe Ser Gln Cys Ser Asn Leu Lys Glu Leu Gln Leu His Gly Asn His 145 150 155 160 ctg gaa tac atc cct gac gga gcc ttc gac cac ctg gta gga ctc acg 528 Leu Glu Tyr Ile Pro Asp Gly Ala Phe Asp His Leu Val Gly Leu Thr                 165 170 175 aag ctc aat ctg ggc aag aat agc ctc acc cac atc tca ccc agg gtc 576 Lys Leu Asn Leu Gly Lys Asn Ser Leu Thr His Ile Ser Pro Arg Val             180 185 190 ttc cag cac ctg ggc aat ctc cag gtc ctc cgg ctg tat gag aac agg 624 Phe Gln His Leu Gly Asn Leu Gln Val Leu Arg Leu Tyr Glu Asn Arg         195 200 205 ctc acg gat atc ccc atg ggc act ttt gat ggg ctt gtt aac ctg cag 672 Leu Thr Asp Ile Pro Met Gly Thr Phe Asp Gly Leu Val Asn Leu Gln     210 215 220 gaa ctg gct cta cag cag aac cag att gga ctg ctc tcc cct ggt ctc 720 Glu Leu Ala Leu Gln Gln Asn Gln Ile Gly Leu Leu Ser Pro Gly Leu 225 230 235 240 ttc cac aac aac cac aac ctc cag aga ctc tac ctg tcc aac aac cac 768 Phe His Asn Asn His Asn Leu Gln Arg Leu Tyr Leu Ser Asn Asn His                 245 250 255 atc tcc cag ctg cca ccc agc atc ttc atg cag ctg ccc cag ctc aac 816 Ile Ser Gln Leu Pro Pro Ser Ile Phe Met Gln Leu Pro Gln Leu Asn             260 265 270 cgt ctt act ctc ttt ggg aat tcc ctg aag gag ctc tct ctg ggg atc 864 Arg Leu Thr Leu Phe Gly Asn Ser Leu Lys Glu Leu Ser Leu Gly Ile         275 280 285 ttc ggg ccc atg ccc aac ctg cgg gag ctt tgg ctc tat gac aac cac 912 Phe Gly Pro Met Pro Asn Leu Arg Glu Leu Trp Leu Tyr Asp Asn His     290 295 300 atc tct tct cta ccc gac aat gtc ttc agc aac ctc cgc cag ttg cag 960 Ile Ser Ser Leu Pro Asp Asn Val Phe Ser Asn Leu Arg Gln Leu Gln 305 310 315 320 gtc ctg att ctt agc cgc aat cag atc agc ttc atc tcc ccg ggt gcc 1008 Val Leu Ile Leu Ser Arg Asn Gln Ile Ser Phe Ile Ser Pro Gly Ala                 325 330 335 ttc aac ggg cta acg gag ctt cgg gag ctg tcc ctc cac acc aac gca 1056 Phe Asn Gly Leu Thr Glu Leu Arg Glu Leu Ser Leu His Thr Asn Ala             340 345 350 ctg cag gac ctg gac ggg aat gtc ttc cgc atg ttg gcc aac ctg cag 1104 Leu Gln Asp Leu Asp Gly Asn Val Phe Arg Met Leu Ala Asn Leu Gln         355 360 365 aac atc tcc ctg cag aac aat cgc ctc aga cag ctc cca ggg aat atc 1152 Asn Ile Ser Leu Gln Asn Asn Arg Leu Arg Gln Leu Pro Gly Asn Ile     370 375 380 ttc gcc aac gtc aat ggc ctc atg gcc atc cag ctg cag aac aac cag 1200 Phe Ala Asn Val Asn Gly Leu Met Ala Ile Gln Leu Gln Asn Asn Gln 385 390 395 400 ctg gag aac ttg ccc ctc ggc atc ttc gat cac ctg ggg aaa ctg tgt 1248 Leu Glu Asn Leu Pro Leu Gly Ile Phe Asp His Leu Gly Lys Leu Cys                 405 410 415 gag ctg cgg ctg tat gac aat ccc tgg agg tgt gac tca gac atc ctt 1296 Glu Leu Arg Leu Tyr Asp Asn Pro Trp Arg Cys Asp Ser Asp Ile Leu             420 425 430 ccg ctc cgc aac tgg ctc ctg ctc aac cag cct agg tta ggg acg gac 1344 Pro Leu Arg Asn Trp Leu Leu Leu Asn Gln Pro Arg Leu Gly Thr Asp         435 440 445 act gta cct gtg tgt ttc agc cca gcc aat gtc cga ggc cag tcc ctc 1392 Thr Val Pro Val Cys Phe Ser Pro Ala Asn Val Arg Gly Gln Ser Leu     450 455 460 att atc atc aat gtc aac gtt gct gtt cca agc gtc cat gtc cct gag 1440 Ile Ile Ile Asn Val Asn Val Ala Val Pro Ser Val His Val Pro Glu 465 470 475 480 gtg cct agt tac cca gaa aca cca tgg tac cca gac aca ccc agt tac 1488 Val Pro Ser Tyr Pro Glu Thr Pro Trp Tyr Pro Asp Thr Pro Ser Tyr                 485 490 495 cct gac acc aca tcc gtc tct tct acc act gag cta acc agc cct gtg 1536 Pro Asp Thr Thr Ser Val Ser Ser Thr Thr Glu Leu Thr Ser Pro Val             500 505 510 gaa gac tac act gat ctg act acc att cag gtc act gat gac cgc agc 1584 Glu Asp Tyr Thr Asp Leu Thr Thr Ile Gln Val Thr Asp Asp Arg Ser         515 520 525 gtt tgg ggc atg acc cag gcc cag agc ggg ctg gcc att gcc gcc att 1632 Val Trp Gly Met Thr Gln Ala Gln Ser Gly Leu Ala Ile Ala Ala Ile     530 535 540 gta att ggc att gtc gcc ctg gcc tgc tcc ctg gct gcc tgc gtc ggc 1680 Val Ile Gly Ile Val Ala Leu Ala Cys Ser Leu Ala Ala Cys Val Gly 545 550 555 560 tgt tgc tgc tgc aag aag agg agc caa gct gtc ctg atg cag atg aag 1728 Cys Cys Cys Cys Lys Lys Arg Ser Gln Ala Val Leu Met Gln Met Lys                 565 570 575 gca ccc aat gag tgt taa 1746 Ala Pro Asn Glu Cys           580

[Brief description of drawings]

FIG. 1 cDNA of the gene of the present invention (rat Lib)
FIG. 3 is a diagram showing the primary structure of a polypeptide encoded by the gene. In the figure, Nc, Ec, Xb, Xh, and Hi are restriction enzymes NcoI, EcoRI, XbaI, and X, respectively.
The recognition sites for hoI and HindIII are shown. The lower figure shows each region of the Lib protein. In the figure, Signa
l Peptide is a signal peptide, N-plan
ks is the N-flanking region, Fifteen Leu
Cine-rich repeats represent 15 leucine rich repeats, C-flanks represent C-flanking regions, and Transmembrane regions represent transmembrane regions.

FIG. 2 Rat Lib (upper row) and Human Lib (lower row)
It is a figure explaining the comparison of the amino acid sequence of. "*" In the figure
Indicates that the amino acids are the same, and “.” Indicates that the amino acids have similar properties.

FIG. 3 is a diagram showing 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 N is an untreated rat astroglial cell-derived poly (A) + RNA sample, Lane A is a rat astroglial cell-derived poly (A) + RN after treatment with 25 μM β-amyloid for 15 hours.
A sample is shown respectively. A is the Lib gene, B is G
The results of using 3PDH gene (g) and β-actin gene (a) as probes are shown.

FIG. 4 is a view showing the results of analysis of the tissue distribution of the gene of the present invention by human tissue blots. Human Lib cDNA was used as a probe.

FIG. 5 shows the construction of pCMV-Tag4-rLib. In the figure, BglII0.6 and BamHI
2.3 shows the recognition sites for the restriction enzymes BglII and BamHI. Pcmv is the cytomegalovirus promoter region, Tsv40 is the SV40 terminator region, P
sv40 is the SV40 promoter region, ori is the replication initiation sequence, Neo r / Kan r is the neomycin and kanamycin resistance gene site, and Ttk is the thymidine kinase terminator region. rLib is rat Lib
The insertion site of the gene is shown.

FIG. 6 is a diagram showing changes in the expression level of Lib mRNA in rat C6 cells by cytokine treatment. A is T when the expression level of Lib mRNA at 0 hours is 1.
It is a figure which shows the relative expression level of Lib mRNA in each time after a mixed solution treatment of NF-α, IL-1β and IFN-γ. B is TNF-α alone (α), IL-1β alone (β), IFN-γ (γ), TNF-α and IL-1β.
Mixed solution (α + β), TNF-α, IL-1β and IFN
LibmR 6 hours after treatment with a mixture of -γ (α + β + γ)
It is a figure showing NA expression level as a relative value on the basis of G3PDH mRNA expression level.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 39/395 A61K 48/00 4C085 45/00 A61P 25/28 4C087 48/00 29/00 4H045 A61P 25 / 28 43/00 105 29/00 C07K 14/47 43/00 105 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1 / 21 C12Q 1/02 5/10 G01N 33/53 M C12P 21/02 Z C12Q 1/02 33/566 G01N 33/53 C12N 15/00 ZNAA 5/00 A 33/566 A61K 37/02 F term (reference) 4B024 AA01 AA11 BA80 CA04 CA11 HA11 HA17 4B063 QA18 QQ05 QQ42 QQ79 QR32 QR48 QR74 4B064 AG01 CA19 CC24 DA01 DA13 4B065 AA91X AA91Y AA93Y AB01 BA01 CA24 CA44 CA46 4C084 AA02 AA08 CA22 CA17 A17 BA17 A01 A17 A17 A01 A17 A17 BA17 A01 A17 BA17 A18 A17 A17 A17 A18 A17 BA18 A17 A17 A17 A17 BA17 A01 5 DB01 DB70 NA14 ZA16 ZB11 ZB21 4C085 AA12 AA13 BB07 BB11 CC02 CC05 CC07 CC08 CC21 CC28 DD23 DD62 DD63 EE01 4C087 AA02 AA03 BB45 BC34 BC83 CA12 CA16 CA20 NA14 ZA16 ZB11 ZB21 4H045 A50 A20 A20A20A20A20

Claims (22)

[Claims] 1. A DNA selected from the following group or its complementary strand; D represented by the DNA sequence set forth in SEQ ID NO: 1 in the Sequence Listing.
NA, D represented by the DNA sequence set forth in SEQ ID NO: 2 in the Sequence Listing
NA, DNA containing the DNA sequence set forth in SEQ ID NO: 1 or 2 of the Sequence Listing, and at least about 70% DN with said DNA
A DNA encoding a peptide having homology on the A sequence and having a cell-cell adhesion regulating function and / or a cell-extracellular matrix adhesion regulating function, and a DNA sequence of the DNA described above, Have mutations such as deletions, substitutions and additions of one to several DNAs,
And cell-cell adhesion control function and / or cell-cell
A DNA encoding a peptide having a function of regulating adhesion between extracellular matrices. 2. A hybridizing with the DNA according to claim 1 or a complementary strand thereof under stringent conditions and having an adhesion regulating function between cells and cells and / or an adhesion regulating function between cells and extracellular matrix. A DNA encoding the peptide having. 3. A polypeptide encoded by the DNA according to claim 1 or 2. 4. A recombinant vector containing the DNA according to claim 1 or 2, or a complementary strand thereof. 5. A pCMV-Tag4-rLib (FERM
BP-7811). 6. A transformant transformed with the recombinant vector according to claim 4 or the plasmid according to claim 5. 7. The method for producing the polypeptide according to claim 3, which comprises a step of culturing the transformant according to claim 6. 8. An antibody which immunologically recognizes the polypeptide according to claim 3. 9. A method for identifying a compound having a cell-cell adhesion regulating function and / or a cell-extracellular matrix adhesion regulating function, which comprises the DNA according to claim 1 or 2, or a complementary strand thereof. At least one of the polypeptide according to claim 3, the recombinant vector according to claim 4, or the plasmid according to claim 5, the transformant according to claim 6, and the antibody according to claim 8. A method characterized by using two. 10. A compound identified by the identification method according to claim 9. 11. A cell-cell and / or cell-extracellular matrix adhesion regulator identified by the identification method according to claim 9. 12. The DNA according to claim 1 or 2, or its complementary strand, the polypeptide according to claim 3, and 4.
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. Medicine. 13. The DNA according to claim 1 or 2, or its complementary strand, the polypeptide according to claim 3, and 4.
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. A pharmaceutical composition comprising: 14. The DNA according to claim 1 or 2, or its complementary strand, the polypeptide according to claim 3, and 4.
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. A pharmaceutical composition for use in the prevention / treatment of a disease involving adhesion between cells / cells and / or cells / extracellular matrix. 15. The DNA according to claim 1 or 2, or a complementary strand thereof, the polypeptide according to claim 3, and 4.
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. A preventive / therapeutic agent for nerve cell death. 16. The DNA according to claim 1 or 2, or a complementary strand thereof, the polypeptide according to claim 3, claim 4
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. A preventive / therapeutic agent for Alzheimer's disease. 17. The DNA according to claim 1 or 2, or a complementary strand thereof, the polypeptide according to claim 3, and 4.
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. A diagnostic tool for diseases. 18. The DNA according to claim 1 or 2, or a complementary strand thereof, the polypeptide according to claim 3, claim 4
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. Diagnostic method for nerve cell death. 19. The DNA according to claim 1 or 2, or a complementary strand thereof, the polypeptide according to claim 3, claim 4
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. A diagnostic tool for Alzheimer's disease. 20. The DNA according to claim 1 or 2, or a complementary strand thereof, the polypeptide according to claim 3, claim 4
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. Kit for diagnosing diseases. 21. The DNA according to claim 1 or 2, or a complementary strand thereof, the polypeptide according to claim 3, claim 4
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. A diagnostic kit for neuronal cell death. 22. The DNA according to claim 1 or 2, or a complementary strand thereof, the polypeptide according to claim 3, claim 4
At least one of the recombinant vector according to claim 5 or the plasmid according to claim 5, the transformant according to claim 6, the antibody according to claim 8, and the compound according to claim 10. A diagnostic kit for Alzheimer's disease.