JP2002320489A - Scavenger receptor and its application - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new scavenger receptor protein useful in searching or assessing pharmaceutical preparations for preventing or treating arteriosclerosis, to provide a DNA encoding the protein, and to provide applications of the protein. SOLUTION: This new scavenger receptor protein comprises either one amino acid sequence shown in the amino acids No.1 to 254 in the sequence No.2 in the sequence table (See the specification), in the amino acids No.1 to 246 in the sequence No.4 in the sequence table (See the specification), or in the amino acids No.1 to 250 in the sequence No.6 in the sequence table (See the specification).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel scavenger receptor protein useful for the development of a medicament for preventing or treating arteriosclerosis, and a DN encoding the protein.
A and uses of the protein.

[0002]

BACKGROUND OF THE INVENTION The concept of macrophage scavenger receptor was described by Brown and Goldstein of the University of Texas in 1979.
It was proposed in 1965 from a study on the mechanism of atherosclerosis. Br
own and colleagues have studied low-density lipoprotein (LDL) receptors, which are responsible for cholesterol uptake in cells. Was found to be missing. It has been shown that even if the LDL receptor is deficient, cholesterol derived from LDL is taken up by macrophages on the blood vessel wall via the receptor, and atherosclerosis is generated.

[0003] The uptake of acetylated LDL into macrophages is competed by a wide range of negatively charged large substances such as nucleic acids such as poly I and poly G, sugar chains such as fucoidan, and polymers such as maleated albumin and polyvinyl sulfate. For this reason, this receptor is widely known as a scavenger receptor having a broad ligand recognition function.

[0004] The scavenger receptor cDNA is 199
First cloned in 2000 [Kodama, T et al. N
343, 531-535, 1990]. This scavenger receptor is a membrane glycoprotein having a molecular weight of about 220,000 and consisting of a trimer of subunits having a molecular weight of about 70,000. Thereafter, as a scavenger receptor, type I (about 450 amino acids) having a cysteine-rich domain, and about 10
Type II, which has a short C-terminal region by 0 amino acids, has become known. These scavenger receptors penetrate the cell membrane only once, with an intracellular region of about 50 amino acids,
Type I is composed of about 380 amino acids, and type II is composed of about 280 amino acids. The extracellular region is further composed of a spacer sequence, an N-linked sugar chain sequence, a collagen-like sequence, and a sequence specific to each type. In addition, these known scavenger receptors cannot exert their function as a receptor with a monomer, and need to form a trimer to exhibit activity.

[0005] About 50 N-terminals of the human scavenger receptor
Amino acids are the intracellular domain of the receptor, which are located at the 20th (21st in the mouse), 26th (27th in the mouse), and 48th (49th in the mouse) Ser- Xaa-Lys, Arg-Xaa-Xaa-Thr,
There is an amino acid sequence that can be phosphorylated, Ser-Xaa-Lys (Xaa may be any amino acid) (Takeshi Doi et al., Experimental Medicine 10, 19-26, 1992).

[0006] The type I scavenger receptor consists of a domain having six cysteines, and a series of membrane proteins have a homology with this domain of the scavenger receptor, and the SRCR protein (scavenger receptor cystein r).
ich domain like protein). This S
RCR proteins include sea urchin superact receptor and lymphocytes CD5 and CD6.

[0007] The scavenger receptor has a collagen-like domain consisting of 22 amino acids and consisting of four lysines in a cluster at the C-terminal side. This domain forms a ligand binding site and can bind to collagen. It is presumed to play an important role in the treatment of foreign matter and waste products. The scavenger receptor recognizes various nucleic acids, sugar acids, denatured proteins, polymers, and various other substances, and takes these substances into cells to remove them. However, it does not mean that anything is taken in, but it is extremely efficient in treating substances that bind to biological components, negatively charged foreign substances, and wastes. For example, although almost no normal LDL incorporation is observed, LD with increased negative charge due to oxidation or the like is increased.
L is very well incorporated. Scavenger receptors are receptors that bind and take up very well with denatured biological components such as oxidized LDL.

The accumulation of lipid-deposited foam cells derived from macrophages and smooth muscle cells (hereinafter “SMC”)
It is one of the characteristic initial changes in the intima of the ongoing atherosclerotic plaque. It is thought that the macrophage and SMC transformation into foam cells involves the macrophage scavenger receptor, and the results of many in vitro and in vivo studies support this model of atherogenesis. ing. For example, after incubating the modified LDL in vitro and then adding Chinese hamster ovary cells (hereinafter referred to as “CHO cells”) forcibly expressing a bovine scavenger receptor, the CHO cells resemble macrophages in plaques. It can be converted into lipid-depositing cells. Also, in atherosclerotic plaques, mRNA encoding the macrophage scavenger receptor, the receptor protein and modified L
DL is detected (for a description of the importance of scavenger receptors in the symptoms of atherosclerosis see Krieger et al, J. Biol. Chem. 268 (7): 4569-45
72 (1993) and references cited therein).

The scavenger receptor is present not only in tissue macrophages but also in a state where macrophages are differentiated from monocytes and invade with various pathological conditions. The most frequently studied is expression in foam cells in the early stage of atherosclerosis (Masaru Naito et al., Experimental Medicine, 10, 31-35, 1992). According to the high-lipid diet model of rabbits and pigs, which are most commonly observed in the course of atherosclerosis, LDL-like particles adhere to the matrix of the vascular wall several days after starting a high-cholesterol diet. Thereafter, a large number of monocytes are deposited in such a manner as to adhere to endothelial cells, enter the intima, become macrophages, and form lesions called fatty streaks. This fatty gland is an aggregate of hundreds or thousands of macrophages, where the scavenger receptor is also expressed in the highest amount. As the lesion further develops and smooth muscle cells enter the intima and become proliferative, macrophages will only remain in small numbers at the lip and deep in the lesion, and scavenger receptor expression will be significantly reduced.

When macrophages take in foreign substances via scavenger receptors, proteins and nucleic acids are rapidly decomposed and released. However, when macrophages take up lipoproteins via scavenger receptors, they cannot degrade the cholesterol ring in macrophages, so they are transferred to a plasma acceptor such as HDL, or ACAT in macrophages.
Cholesterol esterification enzyme, called cholesterol esterase, causes lipid droplets of cholesterol ester to accumulate in macrophages, and the macrophages change into foam cells.

Also recently, one of a new family of scavenger receptors, called Marco Scavenger Receptors (MMarco SR), has been cloned from a mouse macrophage cDNA library (Eloma et al., Cell, 80:
603-609 (1995)). The Marco Scavenger receptor is also involved in binding Gram-positive and negative bacteria.

Incidentally, in addition to these known scavenger receptors, the existence of unknown scavenger receptors has been suggested (Clinica Chimica Acta 286 (1999) 191-20).
5) However, to date, the structure and characteristics of such scavenger receptors have not been clarified.

[0013]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel scavenger receptor protein useful for search or evaluation of a drug for preventing or treating arteriosclerosis, a DNA encoding the protein, and a use of the protein. To provide.

[0014]

Means for Solving the Problems The present invention provides (1) a protein according to any one of the following 1) to 3): 1) amino acids 1 to 254 of SEQ ID NO: 2 in the sequence listing;
A protein consisting of an amino acid sequence represented by any one of amino acids 1 to 246 of SEQ ID NO: 4 in the sequence listing and amino acids 1 to 250 of SEQ ID NO: 6 in the sequence listing; 2) an amino acid number of SEQ ID NO: 2 in the sequence listing 1 to 254,
In the amino acid sequence represented by any one of amino acids 1 to 246 of SEQ ID NO: 4 in the sequence listing and amino acids 1 to 250 of SEQ ID NO: 6 in the sequence listing, one or more amino acids are added or deleted. A protein comprising an inserted and / or substituted amino acid sequence and having scavenger receptor activity; 3) amino acids 1 to 254 of SEQ ID NO: 2 in the sequence listing;
An amino acid sequence having at least 50% homology with any one of amino acid numbers 1 to 246 of SEQ ID NO: 4 in the sequence listing and amino acid numbers 1 to 250 of SEQ ID NO: 6 in the sequence listing A) a protein having a scavenger receptor activity; coli XL1 Blue
pME18S-hSR-PSOX SANK 7130
0 (FERM BP-7260); coliXL1
Blue pME18S-mSR-PSOX SAN
K 71400 (FERM BP-7261) and E.C. coli XL1 Blue pME18S-pS
R-PSOX SANK 71500 (FERM BP
-7262), a protein consisting of an amino acid sequence encoded by DNA inserted into the multiple cloning site of pME18S, (2) amino acid numbers 1 to 254 of SEQ ID NO: 2 in the sequence listing. A protein having the amino acid sequence shown, (3) amino acids 1 to 2 of SEQ ID NO: 4 in the sequence listing
A protein consisting of the amino acid sequence shown in 46;
(4) Amino acid numbers 1 to 250 of SEQ ID NO: 6 in the sequence listing
A protein consisting of the amino acid sequence represented by (5),
(1) DNA encoding the protein according to any one of (4), (6) DNA according to any one of the following a) to (d): a) SEQ ID NO: 1 in the sequence listing Nucleotides 97 to 8
58, nucleotides 1 to 7 of SEQ ID NO: 3 in the sequence listing
38 and nucleotide number 13 of SEQ ID NO: 5 in the sequence listing
A) a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 762 to 762; b) nucleotide numbers 97 to 8 of SEQ ID NO: 1 in the sequence listing.
58, nucleotides 1 to 7 of SEQ ID NO: 3 in the sequence listing
38 and nucleotide number 13 of SEQ ID NO: 5 in the sequence listing
To 762, comprising a nucleotide sequence that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 762 to 762; and c) a DNA encoding a protein having scavenger receptor activity; Nucleotides 97 to 8
58, nucleotides 1 to 7 of SEQ ID NO: 3 in the sequence listing
38 and nucleotide number 13 of SEQ ID NO: 5 in the sequence listing
Consisting of a nucleotide sequence having a nucleotide sequence identity of at least 67% with a DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOS: to 762, and encoding a protein having scavenger receptor activity.
A; d) Transformed E. coli. coli XL1 Blue
pME18S-hSR-PSOX SANK 7130
0 (FERM BP-7260); coliXL1
Blue pME18S-mSR-PSOX SAN
K 71400 (FERM BP-7261) and E.C. coli XL1 Blue pME18S-pS
R-PSOX SANK 71500 (FERM BP
-7262), the DNA inserted into the multiple cloning site of pME18S, the DNA described in (7), (5) or (6).
(8) an animal cell expression vector, (9) the recombinant vector according to (7), (9) a transformed E. coli E. coli. coli XL1 Bl
ue pME18S-hSR-PSOX SANK 7
1300 (FERM BP-7260); coli
XL1 Blue pME18S-mSR-PSOX
SANK 71400 (FERM BP-7261)
And E. coli XL1 Blue pME18S
-PSR-PSOX SANK 71500 (FERM
BP-7262), which is a plasmid carried by any one of (7) and (8), wherein the recombinant vector is any one of (10), (7) and (9). A host cell carrying the recombinant vector of (1)
1) Transformed E. coli coli XL1 Blue
pME18S-hSR-PSOX SANK 7130
0 (FERM BP-7260); coli XL
1 Blue pME18S-mSR-PSOX SA
NK 71400 (FERM BP-7261) and E.C. coli XL1 Blue pME18S-pS
R-PSOX SANK 71500 (FERM BP
-7262), wherein the host cell is transformed with the recombinant vector for expressing an animal cell according to (12) or (8) or (9), which is selected from the group consisting of: In a system for incorporating or binding oxidized LDL to the host cell by adding oxidized LDL to the host cell, culturing is performed with or without the addition of a test substance at the time of adding oxidized LDL. (13) The method according to (8) or (9), which comprises examining whether or not the test substance inhibits the uptake or binding of oxidized LDL into cells. By culturing host cells transformed with the recombinant vector for animal cell expression on a plastic plate coated with phosphatidylserine, the host cells are transferred to the plate. In the system for attachment, the culture is performed with or without the addition of a test substance to the host cells during or before culturing on the plate, and then the adhesion of the host cells to the plate is tested. (14) a method for testing a preventive or therapeutic agent for arteriosclerosis, which comprises examining whether or not a substance inhibits the disease; (14) consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing A nucleic acid comprising an antisense sequence of a nucleotide sequence, (15) a pharmaceutical composition comprising the nucleic acid according to (14) as an active ingredient, (16)
An antibody which specifically binds to the protein according to any one of (1) to (4).

We are known to not express the class A scavenger receptor, CD36.
Since THP-1 cells differentiated in a macrophage-like manner by PMA bind to phosphatidylserine, which is considered to be a physiological ligand of scavenger receptor, a cDNA encoding a novel scavenger receptor is cloned from the cells. The scavenger receptor was also cloned from mice and pigs. Furthermore, the present inventors have found that the expression of this cDNA in cultured cells revealed that the transformed cells acquired the ability to take in oxidized LDL, which is a substrate of the scavenger receptor. The present inventors have found that an agent for preventing or treating arteriosclerosis can be searched or evaluated using the transformed cells, and the present invention has been completed.

In the present invention, the term "scavenger receptor activity" refers to an activity of binding oxidized LDL on a cell membrane and taking it into cells.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION A DNA encoding the protein of the present invention is obtained by subjecting a cDNA library obtained by performing a reverse transcription reaction to a template using mRNA extracted and purified from animal cells expressing the DNA, as a scavenger receptor activity. Can be obtained by screening a clone having

Animal cells that serve as a source of this mRNA are as follows:
Human monocyte-derived cell line THP-1 (ATCC TIB-2
02) to phorbol 12-myristate 12-acetate:
MA)), spleen and thymus of BALB / c mice, or whole organ tissues of porcine fetus, etc., but various mammals (including humans) that produce the protein of the present invention are preferred. Cells or tissues of origin or cultured cell lines can also be used.

For the extraction of mRNA, guanidine thiocyanate / cesium chloride ultracentrifugation, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method, guanidine acid thiocyanate / phenol / chloroform method can be used, but commercially available mRNA separation method can be used. Kits can also be used.

It is known that most of the mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3 'end. The paramagnetic particles on which mRNA is adsorbed and streptavidin is further immobilized,
The mRNA can be purified by capturing the mRNA using the bond between streptavidin and washing, and then eluting the mRNA. Alternatively, a method in which mRNA is adsorbed on an oligo (dT) cellulose column and then eluted and purified may be employed. Further, mRNA can be further fractionated by sucrose density gradient centrifugation or the like.

Further, after using the mRNA obtained by the above method as a template and synthesizing a single-stranded cDNA using a reverse transcriptase,
A double-stranded cDNA can be synthesized from the single-stranded cDNA. The method includes the S1 nuclease method (Ef
stratiadis, A. et al. (1976) Cell 7, 279-288), Lan
d method (Land, H. et al. (1981) Nucleic Acids Res. 9,
2251-2266), O. Joon Yoo method (Yoo, OJ et al. (198
3) Proc. Natl. Acad. Sci. USA 79, 1049-1053), but the Okayama-Berg method (Okay
ama, H. and Berg, P. (1982) Mol. Cell. Biol. 2, 16
1-170) is preferable.

Next, the obtained cDNA fragment was inserted into a lambda phage vector and allowed to self-replicate, thereby obtaining cDN.
The recombinant phage having the A fragment can be stably retained and amplified. For example, lambda phage λZAPII
(Stratagene), the host E. coli XL
1-Blue MRF 'strain and JM109 strain were made into plaques, and 5-bromo-4-chloro-
3-Indolyl-β-D-galactopyranoside (5-Brom
o-4-chloro-3-indolyl-β-D-galactopyranoside (X-
gal)) Recombinants can be selected based on the presence or absence of color development due to metabolism. As a vector, a plasmid vector can be used in addition to a lambda phage vector.

Also, various commercially available cDNA libraries (for example, Clontech) can be used.

As a method for selecting a clone having a cDNA encoding a protein having a desired scavenger receptor activity from the library obtained as described above, for example, any of the following various methods can be adopted.

(1) Screening method using synthetic oligonucleotide probe: When the whole or a part of the amino acid sequence of the target protein has been elucidated (if the sequence is a plurality of continuous specific sequences, ), And synthesizes an oligonucleotide encoding the amino acid sequence (for amino acids with degenerate codons, using frequently used codons or combining possible codons may be synthesized a plurality of nucleotide sequences, also in the latter case, it is also possible to reduce the types by including inosine), those labeled with this ³² P, ³⁵ S or biotin as a probe, a set The recombinant phage DNA is hybridized with a denatured and fixed nitrocellulose filter or nylon filter, By searching the resulting positive clones,
Select this.

(2) Screening method using a probe prepared by the polymerase chain reaction: When the whole or part of the amino acid sequence of the target protein has been elucidated, a sense corresponding to a part of the amino acid sequence on the N-terminal side An oligonucleotide of the antisense strand corresponding to the chain and a part of the C-terminal side is synthesized, and a polymerase chain reaction (hereinafter referred to as “PCR”) is performed to amplify a DNA fragment encoding the target protein. As the template DNA used here, cDNA synthesized by reverse transcription reaction from mRNA of a cell producing the protein of the present invention, or genomic DNA can be used. The DNA fragment thus prepared is labeled with ³² P, ³⁵ S, biotin, or the like, and a cDNA library or a genomic library is screened by plaque hybridization or colony hybridization using the probe as a probe. Select the desired clone.

(3) Method for Screening by Producing a Protein Having Scavenger Receptor Activity in Other Animal Cell Lines: Plasmid obtained by inserting the cDNA obtained as described above into an expression vector (a self-replicating transcription promoter The plasmid can be used to transform an animal cell host with the cDN.
The protein encoded by A is produced, and the scavenger receptor activity of the transformed cell is measured, or using an antibody that specifically binds to the protein of the present invention, and a secondary antibody against the antibody, By detecting the presence of the protein of the present invention, a strain having a cDNA encoding the protein of the present invention is selected. In addition, a commonly used cell line such as COS or CHO can be used as an animal cell host. However, in order to facilitate detection of the protein of the present invention as a foreign gene product, the host itself is used under certain culture conditions. It is preferable that the cells do not produce the protein.

The DNA encoding the protein of the present invention can be collected from the target transformant obtained as described above by a known method (Maniatis, T. et al. (1982): "Mole
cular Cloning A Laboratory Manual "Cold Spring Ha
rbor Laboratory, NY). For example, it can be carried out by separating a fraction corresponding to plasmid DNA from cells and cutting out a cDNA region from the plasmid DNA.

When obtaining the gene encoding the protein of the present invention, the DNA / RNA
An amplification method can also be suitably used. Especially when the full-length cDNA cannot be obtained from the library, Rapid Amplif
ication of cDNA Ends (RACE: Experimental Medicine, (1994),
See 12 (6), 35-38. Using primers based on partial sequences and mRNA from mammals, or using commercially available kits and cDNA libraries).
Can be obtained up to both ends. Primers used when employing such a PCR method can be appropriately set based on the sequence information of the gene of the present invention, and can be synthesized according to a conventional method.

The nucleotide sequence of the thus obtained DNA of the present invention can be determined, for example, by the chemical modification method of Maxam-Gilbert (Maxam, AM and Gilbert, W. (19).
80): "Methods in Enzymology" 65, 499-559) and M13
Dideoxynucleotide chain termination using phage (Me
ssing, J. and Vieira, J. (1982) Gene 19, 269-276)
It can be performed by such as. Further, an automatic DNA sequence analyzer using a fluorescent dye instead of the radioisotope (for example, Model 373A manufactured by PerkinElmer Japan Applied Biosystems) can be used.

The transformed E. coli strain E. coli harboring a plasmid into which cDNA encoding the most preferred protein of the present invention has been inserted. coli XL1 Bl
uepME18S-hSR-PSOX SANK 71
300; coli XL1 Blue pME18
S-mSR-PSOX SANK 71400 and E.C. coli XL1 Blue pME18S-pS
All of the R-PSOX SANK 71500 have been internationally deposited on August 1, 2000 at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology, and have respective accession numbers FE.
RM BP-7260, FERM BP-7261, and FERM BP-7262. Therefore, the gene encoding the protein of the present invention can be obtained from the strain.

By incorporating the fragment containing the gene encoding the protein of the present invention, cloned as described above, into a vector DNA, other prokaryotic or eukaryotic host cells can be transformed. it can. Further, by introducing an appropriate promoter and a sequence involved in expression into these vectors, the gene can be expressed in each host.

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis.
And the like. To transform the gene of interest into these host cells, the host cells are transformed with a plasmid vector containing replicons or origins of replication from species compatible with the host and regulatory sequences. Further, the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.

For example, K12 strain and the like are often used as Escherichia coli, and pBR322 and pUC-type plasmids are generally used as vectors, but are not limited thereto, and any known various strains and vectors can be used. .

As the promoter, in Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (t
ac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter and the like, and any promoter can be used for producing the protein of the present invention.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura,
K. et al. (1984) J. Biochem. 95, 87-93) and the like, but are not limited thereto.

By connecting a DNA sequence encoding the signal peptide sequence of α-amylase of Bacillus subtilis as a promoter, extracellular secretory expression becomes possible.

Eukaryotic host cells include cells of vertebrates, insects, yeasts, and the like.
For example, COS cells (Gluzman, Y. (1.
981) Cell 23, 175-182, ATCC CRL-165
0) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) lacking dihydrofolate reductase (Urlaub, G. and Chasin, LA (1980) Proc.
Natl. Acad. Sci. USA 77, 4126-4220) and the like are often used, but are not limited thereto.

As a vertebrate cell expression promoter, those having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like which are usually located upstream of the gene to be expressed can be used. May have a replication origin. As an example of the expression vector, pSV2dhfr having the early promoter of SV40 (Subramani, S. et al. (198
1) Mol. Cell. Biol. 1,854-864).
It is not limited to this.

As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcription promoter, a transcription termination signal, Those having an RNA splice site can be used. The expression vector is diethylaminoethyl (D
EAE)-Dextran method (Luthman, H. and Magnusso)
n, G. (1983) Nucleic Acids Res, 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL and
van der Eb, AJ (1973) Virology 52, 456-457),
And electric pulse drilling (Neumann, E. et al. (1982)
EMBO J. 1, 841-845) can be incorporated into COS cells, and thus desired transformed cells can be obtained. When CHO cells are used as host cells, a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, J. et al. (1)
989): "Molecular Cloning ALaboratory Manual" Cold
Spring Harbor Laboratory, NY) and pSV2-neo
(Southern, PJ and Berg, P. (1982) J. Mol. App
l. Genet. 1, 327-341) and the like, and selecting a G418-resistant colony, a transformed cell stably producing the protein of the present invention can be obtained.

When insect cells are used as host cells, cell lines derived from ovarian cells (Sf-9 or Sf-21) of Spodoptera frugiperda of the family Lepidoptera, Noctuidae, and Trichopl
High Five cells derived from usia ni egg cells (Wickham, TJ e
tal, (1992) Biotechnol. Prog. I: 391-396) is often used as a host cell, and a polyhedrin protein promoter of autographa nucleopolyhedrovirus (AcNPV) was used as a baculovirus transfer vector. pVL1392 / 1393 is often used (Kidd, IM and VC Emery (1993) The use
of baculoviruses as expression vectors.Applied Bi
ochemistry and Biotechnology 42, 137-159). In addition, a vector using a promoter of baculovirus P10 or an isobasic protein can also be used. Furthermore, the envelope surface protein GP67 of AcNPV
The recombinant protein can be expressed as a secreted protein by connecting the secretory signal sequence of N. to the N-terminal side of the target protein (Zhe-mei Wang, et al.
(1998) Biol. Chem., 379, 167-174).

As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known. Among them, yeast of the genus Saccharomyces, for example, baker's yeast Saccharomyces cerevi
siae and petroleum yeast Pichia pastoris are preferred. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, a promoter for an alcohol dehydrogenase gene (Bennetzen,
L. and Hall, BD (1982) J. Biol. Chem. 257, 301
8-3025) and the promoter of the acid phosphatase gene (Miyanohara, A. et al. (1983) Proc. Natl. Acad. S
ci. USA 80, 1-5) can be preferably used. Also,
When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and a cleavage site of an endogenous protease or a known protease possessed by the host cell at the N-terminal side. For example, in a system in which human mast cell tryptase, a trypsin-type serine protease, is expressed in petroleum yeast, a secretory signal sequence of α-factor of yeast and KEX2 possessed by petroleum yeast are located on the N-terminal side.
It is known that active tryptase is secreted into the medium by connecting and expressing the protease cleavage sites (Andrew, L. Niles, et al. (1998) Biotech
nol. Appl. Biochem. 28, 125-131).

The transformant obtained as described above is
It can be cultured according to a conventional method.
Alternatively, the protein of the present invention is produced extracellularly. The medium used for the culture can be appropriately selected from various ones commonly used depending on the host cell used. For example, in the case of the above COS cells, RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) A medium obtained by adding a serum component such as fetal bovine serum to a medium such as the above can be used as necessary.

The thus-expressed protein of the present invention can be subjected to various separation procedures utilizing its physicochemical properties, chemical properties and the like (“Biochemical Data Book II”, 1st edition,
1175-1259, Tokyo Chemical Doujin (1980); Biochemistry, v
ol. 25, No. 25, p8274-8277 (1986); Eur. J. Biochem.,
163, pp. 313-321 (1987)). Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, freeze-thaw method, ultrasonic crushing, ultrafiltration, gel filtration, Examples include various liquid chromatography such as adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, and combinations thereof.

Further, by connecting a histidine tag consisting of 6 residues to the recombinant protein to be expressed, the protein can be efficiently purified on a nickel affinity column.

By combining the above-mentioned methods, the protein of the present invention can be easily produced in large quantities with high yield and high purity.

The amino acid sequence of the protein of the present invention purified as described above can be obtained by using an automatic protein amino acid sequencer (for example, Perkin Elmer Japan, Inc.).
It can be confirmed using Applied Biosystems' model 492).

In order for the protein obtained by the genetic engineering technique from the DNA of the present invention to exhibit scavenger receptor activity, the amino acid sequence represented by amino acid numbers 1 to 254 of SEQ ID NO: 2 in the sequence listing is not necessarily required. Amino acid numbers 1 to 246 of SEQ ID NO: 4 in the sequence listing
It is not necessary to consist of the amino acid sequence shown in SEQ ID NO: 6 or a sequence completely matching the amino acid sequence shown in amino acid Nos. 1 to 250 of SEQ ID NO: 6, for example, even if its partial sequence is a scavenger A protein consisting of such a partial sequence is also included in the protein of the present invention as long as it exhibits receptor activity. Further, the DNA encoding the protein is also included in the present invention.

[0049] In general, eukaryotic genes are known as polymorphisms (poly-
morphism) (eg, Nishi, T. et a
l. (1985) J. Biochem. 97, 153-159), this polymorphism may result in the substitution of one or more amino acids, or the substitution of a nucleotide sequence but no amino acids. It may not change. The amino acid sequence represented by amino acid numbers 1 to 254 of SEQ ID NO: 2 in the sequence listing, and the amino acid numbers 1 to 246 of SEQ ID NO: 4 in the sequence listing
At one or more sites in the amino acid sequence of the protein of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 1 to 250, Proteins having deletion, addition, insertion and / or substitution of amino acid residues often have scavenger receptor activity [a protein having an amino acid sequence in which a natural amino acid sequence has been substituted is a natural protein and As an example having the same activity, for example, interleukin 2
It is known that a protein obtained by converting a nucleotide sequence corresponding to cysteine of the (IL-2) gene into a nucleotide sequence corresponding to serine retains IL-2 activity. (Wang, A. et al. (1984) Science 22
4, 1431-1433)]. All such proteins are included in the present invention as long as they have scavenger receptor activity.
In addition, all DNAs encoding these proteins and having the same nucleotide sequence are also included in the present invention.

The various DNAs of the present invention can be prepared, for example, by the phosphite-triester method (Hunk
apiller, M. et al. (1984) Nature 310, 105-111) and the like, and can be produced by chemical synthesis of nucleic acid.

The codon corresponding to the desired amino acid may be arbitrarily selected, and can be determined by a conventional method in consideration of, for example, the codon usage of the host to be used. (Granth
am, R. et al. (1981) Nucleic Acids Res. 9, 143-17
Four). Furthermore, the codons in these nucleotide sequences are partially modified by site-specific mutagenesis (Mark, Mark,
DF et al. (1984) Proc. Natl. Acad. Sci. USA 81,
5662-5666). In addition, in order to prepare a modified form in which one or more amino acid residues are deleted, a method in which DNA is trimmed from the end using exonuclease Bal31 or the like (Toshimi Kishimoto et al. Lecture 1: Gene Research Method II, "335-354", cassette mutation method (Toshimitsu Kishimoto, "New Chemistry Experiment Lecture 2, Nucleic Acid III Recombinant DNA Technology", 242-251), etc.

Among the above-mentioned proteins having a scavenger receptor activity, preferred are those having the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.
A protein consisting of 254 amino acids having a methionine residue at the N-terminus, a protein consisting of 246 amino acids having the methionine residue at the amino acid number 1 of the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing as an N-terminus, A protein consisting of 250 amino acids having an N-terminal methionine residue at amino acid number 1 in the amino acid sequence shown in SEQ ID NO: 6 in the column list can be exemplified.

In addition, a certain DNA is a nucleotide sequence represented by nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing, a nucleotide sequence represented by nucleotide numbers 1 to 738 of SEQ ID NO: 3 in the sequence listing, or SEQ ID NO: 5 of the sequence listing. Whether the DNA hybridizes with the DNA consisting of the nucleotide sequence of nucleotide numbers 13 to 762 is determined by, for example, random primer method (Anal. Biochem., 132: 6013 (1983)) or nick translation method. (Maniatis, T. et al. (1982) in "M
olecular Cloning A Laboratory Manual "Cold Spring
Harbor Laboratory, NY.), Etc., and [α- ³² P] d
Hybridization can be performed by using a probe labeled with CTP or the like to examine the results. The DNA used for hybridization is adsorbed on a known method, for example, a nitrocellulose membrane or a nylon membrane, and solidified by heating or irradiation with ultraviolet rays. Then, the film is
XSSC, dipped in a pre-hybridization solution containing 5% Denhardt solution and 0.1% sodium dodecyl sulfate (hereinafter referred to as “SDS”),
Incubate at 55 ° C for at least 4 hours. Thereafter, the previously prepared labeled probe was added to the same prehybridization solution so as to have a final specific activity of 1 × 10 ⁶ cpm / ml.
Incubate at 60 ° C overnight. The operation of washing the membrane at 57 ° C. for 5 minutes is repeated 5 times, and after washing at 57 ° C. for 20 minutes, autoradiography is performed to determine whether hybridization has occurred. Using this method, cDNA libraries derived from various animal cells can be
Nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing
A cDNA that hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 from nucleotide numbers 1 to 738 in the sequence listing or the nucleotide sequence shown in nucleotide numbers 13 to 762 in SEQ ID NO: 5 in the sequencing listing. Can be isolated.
(Maniatis, T. etal. (1982) in "Molecular Cloning
A Laboratory Mannual "Cold Spring Harbor Laborator
y, NY.).

As the DNA of the present invention obtained by using genetic engineering techniques such as nucleotide sequence modification and nucleic acid hybridization as described above, nucleotides 97 to 97 of SEQ ID NO: 1 in the sequence listing are preferable. To 858, the nucleotide sequence of nucleotide numbers 1 to 738 of SEQ ID NO: 3 in the Sequence Listing or the nucleotide sequence of nucleotides 13 to 762 of SEQ ID NO: 5 in the Sequence Listing and 67%
A DNA consisting of a nucleotide sequence having the above nucleotide sequence identity and encoding a protein having scavenger receptor activity can be mentioned. More preferably, the nucleotide sequence represented by nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing, the nucleotide sequence represented by nucleotide numbers 1 to 738 of SEQ ID NO: 3 in the sequence listing, or the nucleotide number represented by SEQ ID NO: 5 of the sequence listing A DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 13 to 762 can be exemplified, and most preferably a DNA consisting of the nucleotide sequence represented by nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing can be given.

The fact that the protein of the present invention has a scavenger receptor activity is, for example, that oxidized LDL (radioactive isotope) (Labeled with an element or the like), and then cultured.

However, the method for detecting the scavenger receptor activity of the protein of the present invention is not limited to these methods.
Other well-known methods in the technical field of the present invention can also be used.

In the above scavenger receptor activity detection method, a test sample such as a compound synthesized or an extract from a culture of a microorganism may be allowed to coexist when oxidized LDL is added.
By examining whether the test sample inhibits the scavenger receptor activity of the protein of the present invention, scavenger receptor inhibitors can be evaluated or screened. In particular, for substances having a strong effect of specifically inhibiting the scavenger receptor activity of the protein of the present invention,
As a scavenger receptor-specific inhibitor, it can be expected to have drug efficacy in preventing or treating arteriosclerosis.

As an example of an antibody that specifically binds to the protein of the present invention, a monoclonal antibody that specifically binds to the protein of the present invention can be mentioned. The method for obtaining the monoclonal antibody is as described below. is there.

In producing a monoclonal antibody,
Generally, the following work steps are required. That is,
(A) Purification of a biopolymer used as an antigen, (b) Immunization by injecting an antigen into an animal, and then collecting blood and assaying the antibody titer to determine the timing of splenectomy. (C) preparation of myeloma cells (hereinafter referred to as "myeloma"); (d) cell fusion of antibody-producing cells with myeloma; and (e) selection of a hybridoma group producing the desired antibody. (F) splitting (cloning) into single cell clones, (g) culturing hybridomas to produce monoclonal antibodies in large quantities, or breeding hybridoma-transplanted animals, (h) Investigation of the biological activity of the monoclonal antibody produced as described above and its recognition specificity, or assay of its properties as a labeling reagent.

Hereinafter, a method for preparing a monoclonal antibody will be described in detail along the above steps, but the method for preparing the antibody is not limited thereto. For example, antibody-producing cells other than spleen cells and myeloma can be used.

(A) Purification of antigen As the antigen, the protein of the present invention or a part thereof prepared by the method described above can be used. Furthermore, since the primary structure of the protein has been elucidated by the present invention, for example, a partial peptide of the protein can be chemically synthesized using a method well known to those skilled in the art and used as an antigen.

(B) Preparation of antibody-producing cells The antigen obtained in the step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or alum, to immunize a laboratory animal as an immunogen. I do. A mouse is most preferably used as an experimental animal, but is not limited thereto.

The method of immunogen administration for mouse immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.

The immunization can be performed once or repeatedly at appropriate intervals (preferably at intervals of one to five weeks). Thereafter, the antibody titer against the antigen in the serum of the immunized animal is measured, and the effect of the subsequent operation can be enhanced by using the animal whose antibody titer is sufficiently high as a source of antibody-producing cells. Generally, 3 to 3 days after the last immunization
It is preferable to use antibody-producing cells derived from an animal 5 days later for cell fusion later.

The antibody titers used herein include radioisotope immunoassay (hereinafter referred to as "RIA"), solid-phase enzyme immunoassay (hereinafter referred to as "ELISA"), fluorescent antibody assay, passive assay and the like. Various known techniques such as a hemagglutination method can be mentioned, but from the viewpoints of detection sensitivity, speed, accuracy, and possibility of automation of the operation, the RIA method or the ELISA method is more preferable.

In the present invention, the measurement of the antibody titer
According to the LISA method, it can be performed according to the procedure described below. First, the purified or partially purified antigen is adsorbed on a solid surface such as a 96-well plate for ELISA, and the solid surface on which the antigen is not adsorbed is covered with a protein unrelated to the antigen, for example, BSA. The sample is contacted with a serially diluted sample (for example, mouse serum) as the first antibody, and the monoclonal antibody in the sample is bound to the antigen. Further, an antibody against an enzyme-labeled mouse antibody is added as a second antibody, and the antibody is bound to the mouse antibody.After washing, a substrate of the enzyme is added, and a change in absorbance due to color development based on the decomposition of the substrate is measured. calculate.

(C) Step of Preparing Myeloma Myeloma is generally a cell line obtained from a mouse, for example, an 8-azaguanine-resistant mouse (BALB)
/ C) myeloma strain P3X63Ag8U.1 (P3-U1) [Yelton,
DE et al. Current Topics in Microbiology and Imm
unology, 81, 1-7 (1978)], P3 / NSI / 1-Ag4-1 (NS-1)
[Kohler, G. et al. European J. Immunology, 6, 511
-519 (1976)], Sp2 / O-Ag14 (SP-2) [Shulman, M.
et al. Nature, 276, 269-270 (1978)], P3X63Ag8.653
(653) [Kearney, JF et al. J. Immunology, 123,
1548-1550 (1979)], P3X63Ag8 (X63) [Horibata, Ka
nd Harris, AW Nature, 256, 495-497 (1975)]. These cell lines are prepared in a suitable medium, for example, 8-azaguanine medium [RPMI-1640].
A medium in which 8-azaguanine is added to a medium in which glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as “FCS”) are added to the medium],
Iscove's Modified Dulb
ecco's Medium (hereinafter referred to as “IMDM”) or D
The cells are subcultured in MEM, and subcultured in a normal medium [eg, ASF104 medium containing 10% FCS (manufactured by Ajinomoto Co.)] 3 to 4 days before cell fusion.
Secure a cell count of × 10 ⁷ or more.

(D) Cell fusion Antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, and may be obtained from any site of an individual. In general, spleen, lymph node, peripheral blood, Alternatively, it can be obtained from a combination of these as appropriate, and spleen cells are most commonly used.

After the final immunization, a site where antibody-producing cells are present, for example, a spleen is removed from a mouse having a predetermined antibody titer, and spleen cells as antibody-producing cells are prepared. Currently, the most commonly used means for fusing the spleen cells with the myeloma obtained in step (c) is a method using polyethylene glycol, which has relatively low cytotoxicity and simple fusion operation. This method includes, for example, the following procedure.

The spleen cells and myeloma were thoroughly washed with a serum-free medium (for example, RPMI 1640) or phosphate buffered saline (hereinafter referred to as “PBS”), and the ratio of spleen cells to myeloma was 5: 1 to 1: 1. Mix to about 10: 1 and centrifuge. The supernatant was removed, and the precipitated cell group was thoroughly loosened. Then, 1 ml of 50% (w
/ V) polyethylene glycol (molecular weight 1000 to 40)
00) is dropped. Then 10ml
And slowly centrifuge. The supernatant was discarded again, and the precipitated cells were placed in a normal medium (hereinafter referred to as "HAT") containing an appropriate amount of hypoxanthine / aminopterin / thymidine (hereinafter referred to as "HAT") solution and mouse interleukin-2 (hereinafter referred to as "IL-2"). Culture plate (hereinafter referred to as “plate”)
And incubate at 37 ° C for about 2 weeks in the presence of 5% carbon dioxide. HAT medium is supplemented as needed on the way.

(E) Selection of Hybridoma Group When the myeloma cell is an 8-azaguanine resistant strain, that is, a hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) -deficient strain, the myeloma cell not fused is used. , And fusion cells of myeloma cells cannot survive in a HAT-containing medium. On the other hand, a fused cell of antibody-producing cells or a hybridoma of an antibody-producing cell and a myeloma cell can survive, but a fused cell of antibody-producing cells has a life span. Therefore, by continuing the culture in the HAT-containing medium, only the hybridoma of the antibody-producing cell and the myeloma cell survives, and as a result, the hybridoma can be selected.

For the hybridomas that have grown into colonies, the medium is replaced with a medium obtained by removing aminopterin from the HAT medium (hereinafter referred to as “HT medium”). Thereafter, a part of the culture supernatant is collected, and the antibody titer is measured by, for example, an ELISA method.

Although the method using an 8-azaguanine-resistant cell line has been described above, other cell lines can be used according to the selection method of the hybridoma, and in that case, the composition of the medium to be used also changes.

(F) Cloning A hybridoma that has been found to produce a specific antibody by measuring the antibody titer in the same manner as described in step (b) is transferred to another plate and cloned. The cloning method includes a limiting dilution method in which one hybridoma is diluted and cultured so that one hybridoma is contained in one well of the plate, a soft agar method in which a colony is collected by culturing in a soft agar medium, and a micromanipulator. Method of removing and culturing cells from a cell sorter
Examples include "Sota clone" for separating individual cells, and the limiting dilution method is simple and often used.

For the wells in which the antibody titer has been recognized, for example, cloning by the limiting dilution method is repeated 2 to 4 times, and those with a stable antibody titer are selected as the monoclonal antibody-producing hybridoma strain of the present invention.

(G) Preparation of Monoclonal Antibody by Hybridoma Culture After completion of cloning, the culture medium was HT
The medium is replaced with a normal medium and cultured. Large-scale culture is performed by rotary culture using a large culture bottle or spinner culture. By purifying the supernatant in this large-scale culture using a method known to those skilled in the art such as gel filtration, a monoclonal antibody that specifically binds to the protein of the present invention can be obtained. In addition, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the hybridoma in the abdominal cavity of a mouse of the same strain (for example, BALB / c described above) or a Nu / Nu mouse. . As a simple purification method, a commercially available monoclonal antibody purification kit (for example, MAbTrap GII kit; manufactured by Pharmacia) or the like can also be used.

The monoclonal antibody thus obtained is
It has high antigen specificity for the protein of the present invention.

(H) Assay of Monoclonal Antibody The isotype and subclass of the obtained monoclonal antibody can be determined as follows. First,
The identification methods include the Ouchterlony method, E
The LISA method or the RIA method can be used. The Otterlony method is simple but requires a concentration operation when the concentration of the monoclonal antibody is low. Meanwhile, ELISA
If the method or RIA method is used, the culture supernatant is allowed to react with the antigen-adsorbed solid phase as it is, and antibodies corresponding to various immunoglobulin isotypes and subclasses are used as secondary antibodies. Can be identified. As a simpler method, a commercially available identification kit (eg, Mouse Typer Kit; manufactured by Bio-Rad)
Etc. can also be used.

Further, the protein was quantified by the Foreign Lowry method and a method of calculating from the absorbance at 280 nm [1.4 (OD ₂₈₀ ) = 1 m of immunoglobulin.
g / ml].

The thus obtained monoclonal antibody of the present invention can be used for detection, separation and purification of the protein of the present invention utilizing its specificity.

A nucleotide sequence complementary to the partial sequence of the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing can be used for so-called antisense therapy. The antisense molecule is a stable DNA such as DNA consisting of usually 15 to 30 mer, or a phosphorothioate, methylphosphonate or morpholino derivative thereof, which is complementary to a part of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.
Stable R such as NA derivative, 2'-O-alkyl RNA
It can be used as an NA derivative. Such antisense molecules can be introduced into cells by methods well known in the art of the present invention, such as by microinjection, liposome encapsulation, or expression using a vector having an antisense sequence. Such an antisense therapy can be performed by using nucleotide No. 9 of SEQ ID No.
By reducing the activity of the protein encoded by the nucleotide sequence shown from 7 to 858, for example, a therapeutic or preventive effect on arteriosclerosis can be expected.

A composition useful as a medicine containing the antisense oligonucleotide can be produced by a known method such as mixing a pharmaceutically acceptable carrier. Examples of such carriers and manufacturing methods can be found in Remington's Pharmaceut
ical Sciences. Then, an amount sufficient for the treatment of arteriosclerosis in which the expression of the gene containing the nucleotide sequence represented by nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing and the activity of the gene product are recognized is administered to each individual. . The effective amount depends on each person's condition, weight,
It may vary according to various factors such as gender and age, and differences in administration methods such as subcutaneous, topical, oral and intramuscular. For example, it may be changed to 0.02 to 0.2 mg / kg / hour for 2 hours for intravenous injection and 1 to 200 mg / m ² / day for subcutaneous administration.

[0083]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. The molecular biology techniques commonly used in the examples are described in standard laboratory manuals (eg, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Part 2; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). Also, unless otherwise noted, ligation was performed using standard buffers, incubation temperatures and times, approximately equimolar amounts of ligated DNA fragments, and approximately 10 units of T4 / 0.5 μg DNA.
This was performed using DNA ligase.

Embodiment 1 Inheritance of SR-PSOX gene
The human mononuclear cell line THP-1 stimulated with the child cloning PMA is a plastic plate coated with phosphatidylserine (hereinafter referred to as “PS”), which is considered to be a physiological ligand of the scavenger receptor (hereinafter referred to as “PS”). (PS coated plate). Known class A scavenger receptors (hereinafter "SR-A") have been reported to recognize oxidized LDL and acetylated LDL (Koda).
ma T., et al. Nature 1990 Feb 8; 343 (6258): 531-5)
However, since THP-1 cells activated with PMA do not express CD36, which is known as a class A scavenger receptor, THP-1 cells stimulated with PMA
It was suggested that it might be expressing a novel scavenger receptor for oxidized LDL and PS. Therefore, cloning of this novel scavenger receptor was attempted by the following method.

First, in order to isolate the cDNA of the receptor on THP-1 cells stimulated by PMA, a cDNA library of THP-1 cells was prepared as follows,
After transfection into the S-7 cell line, cDNA-transfected COS-7 cells that bind to the PS-coated plate were selected, and DNA was isolated from the cells.

Specifically, PMA was added to THP-1 cells (ATCC TIB-202) cultured in RPMI1640 medium containing 10% fetal bovine serum (hereinafter referred to as “FBS”) to a final concentration of 160 nM. For 72 hours (hereinafter, the same operation was performed for all THP-1 PMA stimulations). After preparing total RNA from TMA-stimulated THP-1 cells using a total RNA extraction and purification reagent (RNAzol: manufactured by Biotex Laboratories), mRNA was prepared using an mRNA isolation kit (manufactured by Pharmacia). did. Using this mRNA as a template, a cDNA was synthesized using a cDNA synthesis kit (Timesaver cDNA synthesis kit: Amersham Pharmacia).
And the expression vector pME18s (Sakamaki, K.,
et al. (1992) EMBO J. 11, 3541-9)
A library was created. Methods for introducing into an expression vector, transforming / selecting into Escherichia coli, and recovering plasmids are described in Sambrook et al., Molecular Cloning: A Laborator.
y Manual, Part 2; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). This cDN
The A library had 1 × 10 ⁶ independent clones.

COS-7 cells (ATCC CRL-16)
51) was cultured in Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM") containing 10% FBS until the cells became semi-confluent, and the cells were recovered by trypsin treatment, and K was adjusted to 2 × 10 ⁷ cells / ml. -PBS
(8.1 mM dipotassium phosphate, 1.46 mM monopotassium phosphate, 30.8 mM sodium chloride and 1 mM
20.7 mM potassium chloride), and the cDNA library obtained above was introduced by electroporation.

The preparation of the PS coated plate and the examination of the binding were carried out as follows. That is, after dissolving phosphatidylserine (sodium salt, bovine brain origin: manufactured by Avanti Polar Lipids) at a concentration of 8 μg / ml in cold methanol, using a sonicator (Ultrasonic: manufactured by Yamato Scientific Co., Ltd.). After sonication for 5 minutes at 4 ° C., the solution was placed on a plastic plate (φ3
(5 mm untreated petri dish: manufactured by Iwaki Glass Co., Ltd.) and dried in a clean bench at room temperature for 5 hours. As a control, a mixture prepared by pouring methanol alone into a plastic plate was used in the same manner. Thereafter, 10% FBS and 0.1% FBS were added to reduce non-specific binding to the plate.
Pour a DMEM medium containing 5% gelatin into a PS-coated plate, incubate (block) at 37 ° C. for 30 minutes, and then use a PBS solution containing 5 mM EDTA and 0.5% gelatin (hereinafter referred to as “solution A”) for 2 minutes. Washed twice. The transformed COS cells were sown on the PS-coated plate thus obtained, and allowed to stand in solution A for 5 minutes. Then, non-adhered cells were separated from the plate by pipetting.

COS remaining on PS coat plate
-7 cells, 0.6% SDS and 10 mM EDT
A vector (episomal DNA) containing the cDNA introduced into the cells with the A solution was extracted and isolated. The resulting episomal DNA was introduced into competent E. coli (Electromax DH10B: Gibco BRL) by electroporation, and the transformed E. coli was cultured to purify the amplified plasmid. The purified plasmid was again introduced into COS-7 cells and selected by binding to a PS-coated plate.

In this way, after the introduction into COS-7 cells and the selection of binding to the PS-coated plate were repeated five times, a single clone showing remarkable binding to the PS-coated plate was isolated. The gene inserted in the plasmid retained in was designated SR-PS.
The nucleotide sequence of this gene was determined by nucleotide sequencing using the dideoxy chain termination method [Sanger F., Nicklen S., Coulson AR, Proc. Natl.
Acad. Sci. USA, 74: 5463-5467 (1977)].

As a result, the coding region of this cDNA clone starts with a start codon (atg) and stops with a stop codon (tg).
It contained an open reading frame ending in a). In addition, an expression sequence tag (hereinafter referred to as “EST”) using the determined nucleotide sequence
One EST as a result of homology search of database
Clone (GenBank accession number AA290712)
Was found to be homologous to SR-PS. This EST clone SR-PSOX (s cavenger r ecept
or for p hophatidyl s erine and ox idized lipoprotei
n: It was named as SEQ ID NO: 1) in the sequence listing. However, SR-
Ala at amino acid number 181 in the amino acid sequence encoded by the PSOX gene (SEQ ID NO: 2 in the sequence listing) is SR-P
In S, it was Val. The SR-PSOX gene was located on human chromosome 17 and encoded a novel type I membrane protein consisting of 254 amino acids.

The transformed E. coli E. coli harboring the expression plasmid pME18S-hSR-PSOX having the SR-PSOX gene was used. coli XL1 Blue pME
18S-hSR-PSOX SANK 71300 is
On August 1, 2000, it was deposited internationally with the Institute of Biotechnology and Industrial Technology, and the deposit number FERM BP-7
260 was added.

Embodiment 2 FIG . Mouse and pig SR-PS
Cloning of OX homologous gene The mouse and pig homologous genes of the human SR-PSOX gene were isolated by the following RT-PCR method. That is, first, total RNA was prepared from the spleen and thymus of a BALB / c mouse by using an RNA extraction reagent (RNAzol: manufactured by Biotex Laboratories) as described in the attached instruction manual. Using this total RNA as a starting material, a cDNA synthesis kit (first-strand cDNA synthesis kit: manufactured by Amersham Pharmacia)
Was used as described in the attached instructions to prepare cDNA. Similarly, a fetal pig (derived from all organ tissues) c
A DNA library was obtained for cloning of porcine homologous genes.

Next, the nucleotide sequence shown below:
tcagaattcc caacaagctc cgcagaa -3 '(upstream primer for mouse: SEQ ID NO: 7 in Sequence Listing); 5'-tcaggatcct gta
ggcgcta gggtcttg -3 '(downstream primer for mouse: SEQ ID NO: 8 in Sequence Listing); 5'-tcagaattcg aggccgccgg gagaag
atg -3 '(upstream primer for pig: SEQ ID NO: 9 in Sequence Listing); and 5'-tcactcgagc tttcatcctt agctcaggtg-
Oligonucleotides consisting of 3 '(downstream primer for pig: SEQ ID NO: 10 in the sequence listing) were synthesized, and PCR was carried out using these as primers under the following conditions. Reaction solution composition: cDNA library 10 μl Upstream primer—10 pmol Downstream primer—10 pmol 10 × PCR buffer 10 μl dNTPs (2.5 mM each) 4 μl Taq polymerase (promega) 5 units 100 μl with sterile water. Temperature condition: After holding at 94 ° C for 2 minutes, then at 94 ° C for 3 minutes
A temperature cycle of 0 second, 52 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times, and then the temperature was further kept at 72 ° C. for 10 minutes. In addition, GeneAmp PCR System 9600 (manufactured by PerkinElmer Japan) was used for controlling the reaction temperature of all PCRs in the examples.

A part of the above PCR reaction solution was
Electrophoresis was performed on agarose (FMC Bioproducts) gel. As a result of estimating the chain length of the band of the PCR product by comparison with the band of the molecular weight marker electrophoresed on the same gel, the size of the mouse and pig SR-PSOX genes was about 800 bp in both cases.

Next, each PCR product was incorporated into a plasmid vector using an original TA cloning kit (manufactured by Invitrogen). That is, first, each PCR reaction solution (the target PC
R product (about 10 ng equivalent) and 50 ng of pCRII vector (attached to the kit) were added to a ligase reaction buffer (6 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride,
Add 4 units of T4 DNA ligase (attached to the kit) in 5 mM sodium chloride, 7 mM β-mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1 mM spermidine, 0.1 mg / ml bovine serum albumin and add at 14 ° C. It was kept warm for 15 hours. next,
Competent Escherichia coli TOP10F '(attached to the kit) 50 containing 2 µl of 0.5 M β-mercaptoethanol
2 μl of the above ligase reaction solution was added and mixed to μl, and the mixture was left on ice for 30 minutes, then at 42 ° C. for 30 seconds, and again on ice for 2 minutes. Add SOC medium (2% tryptone, 0.5% yeast extract, 0.05%
Sodium chloride, 2.5 mM potassium chloride, 1 mM
500 μl of magnesium chloride and 20 mM glucose) were added, and the mixture was cultured for 1 hour with reciprocal shaking culture (37 ° C., 110 rpm). The culture was added to an L-broth agar medium containing 100 μg / ml ampicillin (1% tryptone, 0.5%
Yeast extract, 0.5% sodium chloride, 0.1% glucose, 0.6% were spread on a plate of Bacto-Agar (manufactured by Difco) and cultured at 37 ° C. overnight. A single ampicillin-resistant colony that appeared on the plate was selected, and the colony was scraped with a platinum loop and cultured overnight at 37 ° C. in 5 ml of L-broth medium containing 100 μg / ml ampicillin. The culture was centrifuged to collect the precipitated cells, and plasmid DNA was prepared by an alkaline method. The nucleotide sequence of the cDNA cloning portion in the thus obtained plasmid was determined by nucleotide sequencing using the dideoxy chain termination method [Sanger F., Nicklen S., C
oulson AR, Proc. Natl. Acad. Sci. USA, 74: 5463-
5467 (1977)].

As a result, the obtained mouse and pig SR-PSOX homologous genes had an open reading frame of 738 bp and 750 bp, respectively (SEQ ID NOS: 3 and 5 in the sequence listing). The homology between these nucleotide sequences was analyzed using analysis software (Macvector: manufactured by Oxford Molecular Group). Amino acid sequences respectively encoded by the obtained mouse and pig SR-PSOX homologous genes (SEQ ID NO: 4 and SEQ ID NO: 6 in Sequence Listing)
Had 44% and 51% homology to amino acids encoded by the human SR-PSOX gene, respectively. In the nucleotide sequence of the open reading frame, mouse and pig SR-PSO
The X homologous gene is located between the human SR-PSOX gene and
They had 67% and 70% identity, respectively.

The mouse SR-PS thus obtained
Expression plasmid pME18S-mS having OX gene
Transformed E. coli carrying R-PSOX coli
XL1 Blue pME18S-mSR-PSOX
Expression plasmid pME18S-pSR-PSO with SANK 71400 and porcine SR-PSOX gene
X. coli XL1 B
lue pME18S-pSR-PSOX SANK
71500 was deposited on August 1, 2000 with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, under the accession numbers FERMBP-7261 and FERM BP-7, respectively.
262 is attached.

Embodiment 3 FIG . Scavenger Receptor Activity 1) Binding to PS Coated Plate In order to confirm the binding activity of the SR-PSOX gene product to PS, CO containing the SR-PSOX gene was introduced.
The binding of S-7 cells to PS-coated plates was examined. That is, pM was added to COS-7 cells by the method described in Example 1.
E18S-hSR-PSOX was introduced and this transformed C
OS-7 cells were seeded on a PS-coated plate and allowed to stand for 30 minutes, and then the plate was washed three times with solution A.

Further, in order to examine the influence on the binding to PS, various additives (PS liposome, phosphatidylcholine (PC) liposome, oxidized LDL (preparation method is described in Example 3 below), poly I, dextran sulfate, etc. )
Is added, the cells and the additives are pre-incubated for 15 minutes before performing the binding assay, then the cells are plated on a PS-coated plate and allowed to stand for 30 minutes.
The plate was washed three times with. The number of cells attached to each plate was measured with an optical microscope. All experiments are 3
The number of adherent cells per well was calculated as an average value.

As a result, the COS-7 cells into which the SR-PSOX gene had been introduced did not bind to the PS-uncoated plate but specifically bound to the PS-coated plate. COS-7 cells transfected with mouse SR-PSOX similarly bound to the PS-coated plate. Its binding was inhibited by 100 μM PS liposomes, but not by 100 μM phosphatidylcholine (PC) liposomes. Furthermore, SR-PS
The binding of OX-introduced COS-7 cells to PS-coated plates is also inhibited by specific ligands for scavenger receptors such as oxidized LDL, poly I, dextran sulfate, chondroitin sulfate, LDL, acetyl LDL, etc. Was not inhibited (FIG. 1).

2) Uptake of oxidized LDL into cells Next, uptake of oxidized LDL into COS-7 cells into which the SR-PSOX gene was introduced was examined by the method described below.

First, healthy human peripheral blood plasma (specific gravity: 1.00)
To 6), potassium bromide was added so as to have a specific gravity of 1.019, and then ultracentrifugation (using an ultracentrifuge manufactured by Beckman) at 4 ° C. and 58,000 rpm for 20 hours was performed to remove the upper layer. Next, potassium bromide was added to the obtained lower layer liquid so as to have a specific gravity of 1.063, and again at 4 ° C. and 58,000 rpm.
and ultracentrifuged for 20 hours. The upper layer after centrifugation was recovered as human LDL (Lecturer on Experimental Chemistry 4 Lipid I Neutral lipid lipoprotein (edited by The Biochemical Society of Japan, Tokyo Chemical Dojin))
7. Fractionation and purification of lipoprotein 181 pages, 1993 and Havel
RJ, et al., J. Clin. Invest. 34: 1345-1353 (195
5)).

Next, the obtained human LDL (hereinafter referred to as “native LDL”) was incubated at 37 ° C. for 24 hours in a 7.5 μM copper sulfate solution, whereby
Oxidized LDL was obtained. In addition, the native LDL and acetic anhydride are used in a conventional method [Goldstein JL, Ho YK, Basu SK, Brown
MS, Proc. Natl. Acad. Sci. UAS, 76: 333-337 (197
9)] to give an acetylated form of LDL. The production of oxidized LDL and acetylated LDL
It was confirmed by a change in mobility using agarose gel electrophoresis. The method described in the literature [Voyta JC, Via DP,
Butterfield C.E., Zetter BR, J. Cell Biol., 99: 2
034-2040 (1984)], oxidized LDL was converted to 1,1′-dioctadecyl-3,3,3 ′, 3′-tetramethylindocarbocyanine perchlorate (hereinafter referred to as “DiI”: Molecular Probes, Inc.). Label). Furthermore, human LDL was converted to the iodine monochloride method [MacFarlane AS,
Nature, 182: 53 (1958) ] by was prepared which was radiolabeled with ¹²⁵ I.

According to the method described in Example 2 above, human SR-PS
CO into which OX or mouse SR-PSOX gene has been introduced
For S cells, DiI-labeled oxidized LDL (5 μg / m
l) 500 μg / ml unlabeled oxidized LDL, native LDL or acetylated LDL, 100 μg / ml
The reaction was carried out at 37 ° C. for 2 hours in the presence or absence of poly I or 100 μg / ml of dextran sulfate. Next, the cells were washed three times with DMEM, and the uptake of Dil-labeled lipoprotein into the cells was examined under a fluorescence microscope.

As a result, in COS-7 cells into which human SR-PSOX and mouse SR-PSOX genes had been introduced, significant uptake of DiI-oxidized LDL into cells was observed. This incorporation results in excess unlabeled oxidized LDL,
PolyI and dextran sulfate were inhibited by the coexistence, but were not inhibited by the coexistence of acetylated LDL and native LDL. From the above results, SR-PSO
X has been shown to be a specific receptor for oxidized LDL.

3) Binding ability and degradation activity of oxidized LDL Next, COS-7 into which the human SR-PSOX gene was introduced
Using the cells, the binding ability and proteolytic activity of ¹²⁵ I-labeled oxidized LDL were examined as follows.

COS- into which SR-PSOX gene was introduced
The binding of ¹²⁵ I-labeled oxidized LDL to 7 cells was measured by reacting at 4 ° C. in a 12-well cell culture plate as follows. That is, after washing the cells three times with PBS, the cells are washed with 1 ml of a cold DMEM medium containing 10 mM Hepes-sodium hydroxide (pH 7.4) and 10% FBS (hereinafter referred to as “medium C”) at 4 ° C. for 30 minutes. Pre-processed. After removing the medium, the cells were added with 0.5 ml of medium C containing various concentrations of ¹²⁵ I-labeled oxidized LDL, and incubated at 4 ° C. for 2 hours. The medium was removed, and the cells were washed with a Tris washing solution (50 mM Tris-HCl, 1 mg / ml bovine serum albumin (hereinafter referred to as “BSA”)).
After rapid washing with 50 mM sodium chloride, pH 7.4), the plate was washed twice with a BSA-free Tris washing solution for 10 minutes each. Add 0.5 ml of 0.2N to the washed cells.
The cells were lysed by adding an aqueous sodium hydroxide solution and shaking for 3 to 4 hours, and the radioactivity eluted from the cells was measured with a gamma counter.

Further, the decomposition activity of ¹²⁵ I-labeled oxidized LDL is
It was measured as follows. That is, ¹²⁵ I-labeled oxidized LDL dissolved in medium C was first added to the cells and
For 4-6 hours. Then, trichloroacetic acid was added to the medium, and the medium was allowed to stand at 4 ° C. for 30 minutes.
The supernatant was collected by centrifugation at 5000 rpm for 30 minutes.
Add potassium iodide and hydrogen peroxide to this supernatant and mix.
After addition of chloroform and centrifugation, the upper layer was recovered and its radioactivity was measured with a gamma counter. Each value when cells were not added to the above reaction solution was subtracted as background.

As a result, as shown in FIG.
COS-7 cells transfected with the SOX gene bound ¹²⁵ I-labeled oxidized LDL, and this binding was suppressed by excess unlabeled oxidized LDL, but not acetylated LDL or native LDL.

In addition, C-containing the SR-PSOX gene
OS-7 cells degraded ¹²⁵ I-labeled oxidized LDL,
This degradation was also suppressed by excess unlabeled oxidized LDL and not acetylated LDL or native LDL (FIG. 3).

It should be noted that CHO cells (CHO cells) stably expressing the known scavenger receptor hSR-A gene
-SR-A), a similar experiment was performed, and as a result, acetylated LDL was converted to DiI-oxidized L
It was confirmed that DL incorporation was suppressed, and that acetylated LDL had no abnormality.

The above results indicate that SR-PSOX is a specific receptor for oxidized LDL, and that acetylated LDL and native LDL are not ligands having high affinity for SR-PSOX.

In the experimental system described in any one of 1) to 3) above, a test substance is added by the same operation as in the case of adding various additives, and a change in scavenger receptor activity is observed. Thus, a test for a scavenger receptor inhibitor useful as an agent for preventing or treating arteriosclerosis can be performed.

[0115]

As described above, the present invention provides a scavenger receptor protein having a novel structure and properties, a DNA encoding the protein, and uses of the protein. The protein of the present invention is useful for searching or evaluating a drug for preventing or treating a novel arteriosclerosis.

[Brief description of the drawings]

FIG. 1. COS- transfected with human SR-PSOX gene
Results of an experiment to examine the ability of 7 cells to adhere to a PS-coated plate (the number of adherent cells was 100 when no additive was added).
(%)

FIG. 2: COS- transfected with human SR-PSOX gene
The figure showing the result of the experiment which examined the binding capacity of oxidized LDL in 7 cells (the amount of binding per 1 mg of oxidized LDL added to the experimental system)

FIG. 3 shows COS- into which human SR-PSOX gene has been introduced.
The figure showing the result of the experiment which investigates the resolution of oxidized LDL in 7 cells (decomposition amount per 1 mg of oxidized LDL added to the experimental system)

[Sequence List Free Text] SEQ ID NO: 7: PCR primer for amplifying cDNA encoding a novel mouse scavenger receptor SEQ ID NO: 8: PCR primer for amplifying cDNA encoding a novel mouse scavenger receptor SEQ ID NO: 9 PCR primer for amplifying cDNA encoding porcine novel scavenger receptor SEQ ID NO: 10: PCR primer for amplifying cDNA encoding porcine novel scavenger receptor

[Sequence List] SEQUENCE LISTING <110> Sankyo Company, Limited <120> Scavenger Receptor <130> 2001121SS <140> <141> <150> JP2000-241421 <151> 2000-08-09 <160> 10 <170> PatentIn Ver. 2.0 <210> 1 <211> 1845 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (97) .. (858) <400> 1 aaacgattga gacaccagct ggacgtcacg cgccggagca tgtctgggag tcagagcgag 60 gtggctccat ccccgcagag tccgcggacg cccgag atg gga cgg gac ttg cgg 114 Met Gly Arg Asp Leu Arg 1 5 ccc ggg tcc cgc gtg ctc ctg ctc ctg ctt ctg ctc ctg ctg gtg tac 162 Pro Gly Leu Valu Leu Leu Valu Tyr 10 15 20 ctg act cag cca ggc aat ggc aac gag ggc agc gtc act gga agt tgt 210 Leu Thr Gln Pro Gly Asn Gly Asn Glu Gly Ser Val Thr Gly Ser Cys 25 30 35 tat tgt ggt aaa aga att tct tcc gac tcc ccg cca tcg gtt cag ttc 258 Tyr Cys Gly Lys Arg Ile Ser Ser Asp Ser Pro Pro Ser Val Gln Phe 40 45 50 atg aat cgt ctc cgg aaa cac ctg aga gct tac cat cgg tgt cta tac 306 Met Asn Arg Leu Arg Lys His Leu Arg Ala Tyr His Arg Cys Leu Tyr 55 60 65 70 tac acg agg ttc cag ctc ctt tcc tgg agc gtg tgt ggg ggc aac aag 354 Tyr Thr Arg Phe Gln Leu Leu Ser Trp Ser Val Cys Gly Gly Asn Lys 75 80 85 gac cca tgg gtt cag gaa ttg atg agc tgt gat ctc aaa gaa tgt 402 Asp Pro Trp Val Gln Glu Leu Met Ser Cys Leu Asp Leu Lys Glu Cys 90 95 100 gga cat gct tac tcg ggg att gtg gcc cac cag aag cat tta ctt cct 450 Gly His Ala Tyr Ser Gly Ile Val Ala His Gln Lys His Leu Leu Pro 105 110 115 acc agc ccc cca att tct cag gcc tca gag ggg gca tct tca gat atc 498 Thr Ser Pro Pro Ile Ser Gln Ala Ser Glu Gly Ala Ser Ser Asp Ile 120 125 130 cac acc cct gcc cag atg ctc ctg tcc acc ttg cag tcc act cag cgc 546 His Thr Pro Ala Gln Met Leu Leu Ser Thr Leu Gln Ser Thr Gln Arg 135 140 145 150 ccc acc ctc cca gta gga tca ctg tcc tcg gac aaa gag ctc act cgt 594 Pro Thr Leu Pro Val Gly Ser Leu Ser Ser Asp Lys Glu Leu Thr Arg 155 160 165 ccc aat gaa acc acc att cac act gcg ggc cac agt ctg gca gct ggg 642 Pro Asn Glu Thr Thr Ile His Thr Ala Gly His Ser Leu Ala Ala Gly 170 175 180 cct gag gct ggg gag aac cag aag cag ccg gaa aaa aat gct ggt ccc 690 Pro Glu Ala Gly Glu Asn Gln Lys Gln Pro Glu Lys Asn Ala Gly Pro 185 190 195 aca gcc agg aca tca gcc aca gtg ccg ctg tgc ctc ctg gcc atc 738 Thr Ala Arg Thr Ser Ala Thr Val Pro Val Leu Cys Leu Leu Ala Ile 200 205 210 atc ttc atc ctc acc gca gcc ctt tcc tat gtg ctg tgc aag agg agg 786 Ile Phe Ile Leu Thr Ala Leu Ser Tyr Val Leu Cys Lys Arg Arg 215 220 225 230 agg ggg cag tca ccg cag tcc tct cca gat ctg ccg gtt cat tat ata 834 Arg Gly Gln Ser Pro Gln Ser Ser Pro Asp Leu Pro Val His Tyr Ile 235 240 245 cct gtg gca cct gac tct aat acc tgagccaaga atggaagctt gtgaggagac 888 Pro Val Ala Pro Asp Ser Asn Thr 250 ggactctatg ttgcccaggc tgttatggaa ctcctgagtc aagtgatcct cccaccttgg 948 cctctgaagg tgtgaggatt ataggcgtca cctaccacat ccagcctaca cgtatttgtt 1008 aatatctaac ataggactaa ccagccactg ccctctctta ggcccctcat ttaaaaacgg 1068 ttatactata aaatctgctt ttcacactgg gtgataataa cttggacaaa ttctatgtgt 1128 attttgtttt gttt tgcttt gctttgtttt gagacggagt ctcgctctgt catccaggct 1188 ggagtgcagt ggcatgatct cggctcactg caacccccat ctcccaggtt caagcgattc 1248 tcctgcctcc tcctgagtag ctgggactac aggtgctcac caccacaccc ggctaatttt 1308 ttgtattttt agtagagacc ggggtttcac catgttgacc aggctggtct cgaactcctg 1368 acctggtgat ctgcccaccc aggcctccca aagtgctggg attaaaggtg tgagccacca 1428 tgcctggccc tatgtgtgtt ttttaactac taaaaattat ttttgtaatg attgagtctt 1488 ctttatggaa acaactggcc tcagcccttg cgcccttact gtgattcctg gcttcatttt 1548 ttgctgatgg ttccccctcg tcccaaatct ctctcccagt acaccagttg ttcctccccc 1608 acctcagccc tctcctgcat cctcctgtac ccgcaacgaa ggcctgggct ttcccaccct 1668 ccctccttag caggtgccgt gctgggacac catacgggtt ggtttcacct cctcagtccc 1728 ttgcctaccc cagtgagagt ctgatcttgt ttttattgtt attgctttta ttattattgc 1788 ttttattatc attaaaactc tagttcttgt tttgtcaaaa aaaaaaaaaa aaaaaaa 1845 <210> 2 <211> 254 <212> PRT <213> Homo sapiens <400> 2 Met Gly Arg Asp Leu Arg Pro Gly Ser Arg Val Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Val Tyr Leu Thr Gln P ro Gly Asn Gly Asn Glu Gly 20 25 30 Ser Val Thr Gly Ser Cys Tyr Cys Gly Lys Arg Ile Ser Ser Asp Ser 35 40 45 Pro Pro Ser Val Gln Phe Met Asn Arg Leu Arg Lys His Leu Arg Ala 50 55 60 Tyr His Arg Cys Leu Tyr Tyr Thr Arg Phe Gln Leu Leu Ser Trp Ser 65 70 75 80 Val Cys Gly Gly Asn Lys Asp Pro Trp Val Gln Glu Leu Met Ser Cys 85 90 95 Leu Asp Leu Lys Glu Cys Gly His Ala Tyr Ser Gly Ile Val Ala His 100 105 110 Gln Lys His Leu Leu Pro Thr Ser Pro Pro Ile Ser Gln Ala Ser Glu 115 120 125 Gly Ala Ser Ser Asp Ile His Thr Pro Ala Gln Met Leu Leu Ser Thr 130 135 140 Leu Gln Ser Thr Gln Arg Pro Thr Leu Pro Val Gly Ser Leu Ser Ser 145 150 155 160 Asp Lys Glu Leu Thr Arg Pro Asn Glu Thr Thr Ile His Thr Ala Gly 165 170 175 His Ser Leu Ala Ala Gly Pro Glu Ala Gly Glu Asn Gln Lys Gln Pro 180 185 190 Glu Lys Asn Ala Gly Pro Thr Ala Arg Thr Ser Ala Thr Val Pro Val 195 200 205 Leu Cys Leu Leu Ala Ile Ile Phe Ile Leu Thr Ala Ala Leu Ser Tyr 210 215 220 Val Leu Cys Lys Arg Arg Arg Gly Gln Ser Pro Gln Ser Ser Pro Asp 225 230 235 240 Leu Pro Val His Tyr Ile Pro Val Ala Pro Asp Ser Asn Thr 245 250 <210> 3 <211> 780 <212> DNA <213> Mus musculus <220> <221> CDS <222> ( 1) .. (738) <400> 3 atg agg cgg ggc ttt gga ccc ttg tct ctt gcg ttc ttc ctt ttc ttg 48 Met Arg Arg Gly Phe Gly Pro Leu Ser Leu Ala Phe Phe Leu Phe Leu 1 5 10 15 ttg gcg ctg ctg acc ctg cca ggc gat ggc aac cag ggc agt gtc gct 96 Leu Ala Leu Leu Thr Leu Pro Gly Asp Gly Asn Gln Gly Ser Val Ala 20 25 30 gga agt tgt tct tgt gat cgt acc att tct tct ggc acc cag atacc 144 Gly Ser Cys Ser Cys Asp Arg Thr Ile Ser Ser Gly Thr Gln Ile Pro 35 40 45 cag ggt act ttg gat cac atc cga aaa tac ctg aaa gca ttt cat cgt 192 Gln Gly Thr Leu Asp His Ile Arg Lys Tyr Leu Lys Ala Phe His Arg 50 55 60 tgt cca ttc ttt atc agg ttc cag ttg cag tcc aaa agc gtg tgt ggg 240 Cys Pro Phe Phe Ile Arg Phe Gln Leu Gln Ser Lys Ser Val Cys Gly 65 70 75 80 gga agc caa gac cag tgg gtc cgt gaa cta gtg gac tgc ttt gag cgc 288 Gly Ser Gln Asp Gln Trp Val Arg Glu Leu Val Asp Cys Phe Glu Arg 85 90 95 aaa gag tgt gga act ggt cat ggg aag agt ttt cac cac caa aaa cat 336 Lys Glu Cys Gly Thr Gly His Gly Lys Ser Phe His His Gln Lys His 100 105 110 ttg cct caa gcc agt acc cag acc cct gag gcc gca gag ggg aca cct 384 Leu Pro Gln Ala Ser Thr Gln Thr Pro Glu Ala Ala Glu Gly Thr Pro 115 120 125 tcg gac acg agc acc cct gca cat agt cag agc act cag cac tcc act 432 Ser Asp Thr Ser Thr Pro Ala His Ser Gln Ser Thr Gln His Ser Thr 130 135 140 ctt cca tca gga gca ctg tcc tta aac aaa gag cac acc caa ccc tgg 480 Leu Pro Ser Gly Ala Leu Ser Leu Asn Lys Glu His Thr Gln Pro Trp 145 150 155 160 gag atg acc act ctc cct tca ggc tat ggt ctg gaa gct agg cct gag 528 Glu Met Thr Thr Leu Pro Ser Gly Tyr Gly Leu Glu Ala Arg Pro Glu 165 170 175 gct gag gca aat gag aaa cag caa gat gac aga cag caa gaa gca cca 576 Ala Glu Ala Asn Glu Lys Gln Gln Asp Asp Arg Gln Gln Glu Ala Pro 180 185 190 gga gct gga gct agc aca cca gct tgg gta ccg gtg ctg tcc ctc ctg 624 Gly Ala Gly Ala Ser Pro A Val Pro Val Leu Ser Leu Leu 195 200 205 gcc att gtc ttc ttc ctc act gca gcc atg gcc tat gtg ctg tgc aac 672 Ala Ile Val Phe Phe Leu Thr Ala Ala Met Ala Tyr Val Leu Cys Asn 210 215 220 agg aga gcg aca ca cag aac tct gca ggt ttg cag ctc tgg tac act 720 Arg Arg Ala Thr Gln Gln Asn Ser Ala Gly Leu Gln Leu Trp Tyr Thr 225 230 235 240 cct gtt gaa cca aga ccc tagcgcctac agcaagagtg gaagctcatg 768 Pro Val Glu Prog Glug ga 780 <210> 4 <211> 246 <212> PRT <213> Mus musculus <400> 4 Met Arg Arg Gly Phe Gly Pro Leu Ser Leu Ala Phe Phe Leu Phe Leu 1 5 10 15 Leu Ala Leu Leu Thr Leu Pro Gly Asp Gly Asn Gln Gly Ser Val Ala 20 25 30 Gly Ser Cys Ser Cys Asp Arg Thr Ile Ser Ser Gly Thr Gln Ile Pro 35 40 45 Gln Gly Thr Leu Asp His Ile Arg Lys Tyr Leu Lys Ala Phe His Arg 50 55 60 Cys Pro Phe Phe Ile Arg Phe Gln Leu Gln Ser Lys Ser Val Cys Gly 65 70 75 80 Gly Ser Gln Asp Gln Trp Val Arg Glu Leu Val Asp Cys Phe Glu Arg 85 90 95 Lys Glu Cys Gly Thr Gly His Gly Lys Ser Phe His His Gln Lys His 10 0 105 110 Leu Pro Gln Ala Ser Thr Gln Thr Pro Glu Ala Ala Glu Gly Thr Pro 115 120 125 Ser Asp Thr Ser Thr Pro Ala His Ser Gln Ser Thr Gln His Ser Thr 130 135 140 Leu Pro Ser Gly Ala Leu Ser Leu Asn Lys Glu His Thr Gln Pro Trp 145 150 155 160 Glu Met Thr Thr Leu Pro Ser Gly Tyr Gly Leu Glu Ala Arg Pro Glu 165 170 175 Ala Glu Ala Asn Glu Lys Gln Gln Asp Asp Arg Gln Gln Glu Ala Pro 180 185 190 Gly Ala Gly Ala Ser Thr Pro Ala Trp Val Pro Val Leu Ser Leu Leu 195 200 205 Ala Ile Val Phe Phe Leu Thr Ala Ala Met Ala Tyr Val Leu Cys Asn 210 215 220 Arg Arg Ala Thr Gln Gln Asn Ser Ala Gly Leu Gln Leu Trp Tyr Thr 225 230 235 240 Pro Val Glu Pro Arg Pro 245 <210> 5 <211> 780 <212> DNA <213> Sus scrofa <220> <221> CDS <222> (13) .. (762) < 400> 5 cgccgggaga ag atg ccg ctc tgg gaa ctc tgg ttc ttc ttg ctc gca ctc 51 Met Pro Leu Trp Glu Leu Trp Phe Phe Leu Leu Ala Leu 1 5 10 ttt cta gcg tgg ctg act cca cca ggc atg atg 99 Phe Leu Ala Trp Leu Thr Pro Pro Gly Asn Gly Asn Glu Gly Ser Met 15 20 25 gct gga agt tgt cct tgc aat aga agg att tct tcc cac tcc cct ccg 147 Ala Gly Ser Cys Pro Cys Asn Arg Arg Ile Ser Ser His Ser Pro Pro 30 35 40 45 acc gat cac gac atg aga cat ctc cgg aaa tac ctg aac cac tat caa 195 Thr Asp His Asp Met Arg His Leu Arg Lys Tyr Leu Asn His Tyr Gln 50 55 60 cac tgt act tcc tac gtc agg ttc cag cta ccc cgt ggg agt gta tgt 243 His Cys Thr Ser Tyr Val Arg Phe Gln Leu Pro Arg Gly Ser Val Cys 65 70 75 gga ggc agc agt gac cag tgg gtg ctg aaa ctg atg ggc tgc ttt gat 291 Gly Gly Ser Ser Asp Gln Trp Val Leu Lys Leu Met Gly Cys Phe Asp 80 85 90 cgc gga gaa tgt gga cgt gct cat gcc aga acg gtg gcc cac cag cag 339 Arg Gly Glu Cys Gly Arg Ala His Ala Arg Thr Val Ala His Gln Gln 95 100 105 cat tta gct cct cag aac acc cgg gtt cct gaa ctc cca gaa agg gca 387 His Leu Ala Pro Gln Asn Thr Arg Val Pro Glu Leu Pro Glu Arg Ala 110 115 120 125 cct cca gac agt agc acc cct gcc cag acg aat ctg cca tct acc ctg 435 Pro Pro Asp Ser Ser Thr Pro Ala Gln Thr Asn Leu Pro Ser Thr Leu 1 30 135 140 cag ccg acc cag aag ccc acg ctt cca gaa ggc atg cca tcc ttg gcc 483 Gln Pro Thr Gln Lys Pro Thr Leu Pro Glu Gly Met Pro Ser Leu Ala 145 150 155 aaa aag ctc atc ccc acc agt gaa acc gac aca tcc act gtg ggc cac 531 Lys Lys Leu Ile Pro Thr Ser Glu Thr Asp Thr Ser Thr Val Gly His 160 165 170 agt ctg ggg gct aag tct gag gcc agg gag aac cag gag cag cta gga 579 Ser Leu Gly Ala Lys Ser Glu Ala Arg Glu Asn Gln Glu Gln Leu Gly 175 180 185 aaa aat gtg ggt gct aca gcg gga aca tca gcc ctg gtg cca gtg ctg 627 Lys Asn Val Gly Ala Thr Ala Gly Thr Ser Ala Leu Val Pro Val Leu 190 195 200 205 tcc ctc ttg gtc atc att ttc ctt ctc acc gga gtc cta ctt tac gtg 675 Ser Leu Leu Val Ile Ile Phe Leu Leu Thr Gly Val Leu Leu Tyr Val 210 215 220 atg tgc aag aag agg cag gag cag tca cgt cag tac cct ccaat 723 Met Cys Lys Lys Arg Gln Glu Gln Ser Arg Gln Tyr Pro Pro Asp Pro 225 230 235 cag ctt cat tat gtt cct gtg gca tca aat atc aac acc tgagctaagg 772 Gln Leu His Tyr Val Pro Val Ala Ser Asn Ile Asn Thr 24 0 245 250 atgaaagc 780 <210> 6 <211> 250 <212> PRT <213> Sus scrofa <400> 6 Met Pro Leu Trp Glu Leu Trp Phe Phe Leu Leu Ala Leu Phe Leu Ala 1 5 10 15 Trp Leu Thr Pro Pro Gly Asn Gly Asn Glu Gly Ser Met Ala Gly Ser 20 25 30 Cys Pro Cys Asn Arg Arg Ile Ser Ser His Ser Pro Pro Thr Asp His 35 40 45 Asp Met Arg His Leu Arg Lys Tyr Leu Asn His Tyr Gln His Cys Thr 50 55 60 Ser Tyr Val Arg Phe Gln Leu Pro Arg Gly Ser Val Cys Gly Gly Ser 65 70 75 80 Ser Asp Gln Trp Val Leu Lys Leu Met Gly Cys Phe Asp Arg Gly Glu 85 90 95 Cys Gly Arg Ala His Ala Arg Thr Val Ala His Gln Gln His Leu Ala 100 105 110 Pro Gln Asn Thr Arg Val Pro Glu Leu Pro Glu Arg Ala Pro Pro Asp 115 120 125 Ser Ser Thr Pro Ala Gln Thr Asn Leu Pro Ser Thr Leu Gln Pro Thr 130 135 140 Gln Lys Pro Thr Leu Pro Glu Gly Met Pro Ser Leu Ala Lys Lys Leu 145 150 155 160 Ile Pro Thr Ser Glu Thr Asp Thr Ser Thr Val Gly His Ser Leu Gly 165 170 175 Ala Lys Ser Glu Ala Arg Glu Asn Gln Glu Gln Leu Gly Lys Asn Val 180 185 190 Gly Ala Thr Ala Gly T hr Ser Ala Leu Val Pro Val Leu Ser Leu Leu 195 200 205 Val Ile Ile Phe Leu Leu Thr Gly Val Leu Leu Tyr Val Met Cys Lys 210 215 220 Lys Arg Gln Glu Gln Ser Arg Gln Tyr Pro Pro Asp Pro Gln Leu His 225 230 235 240 Tyr Val Pro Val Ala Ser Asn Ile Asn Thr 245 250 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a cDNA encoding a novel murine scavenger receptor <400> 7 tcagaattcc caacaagctc cgcagaa 27 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a cDNA encoding a novel murine scavenger receptor <400> 8 tcaggatcct gtaggcgcta gggtcttg 28 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a cDNA encoding a novel porcine scavenger receptor <400> 9 tcagaattcg aggccgccgg gagaagatg 29 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Seq uence: PCR primer to amplify a cDNA encoding a novel porcine scavenger receptor <400> 10 tcactcgagc tttcatcctt agctcaggtg 30

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C07K 16/28 C12N 1/21 4H045 C12N 1/21 C12Q 1/02 C12Q 1/02 C12N 15/00 ZNAA (72) Invention Noriaki Kume 68-9 Uminokicho, Ichijo-ji, Sakyo-ku, Kyoto-shi, Kyoto (72) Inventor Nankai 5-1-301, Umeda-cho, Moriyama-shi, Shiga (72) Inventor Daiji Kataoka Ichijoji-daishinkaicho, Sakyo-ku, Kyoto, Kyoto 41-1 −308 (72) Inventor Kazutaka Hayashida Role of entering the west of Nakaritorigawadori, Kamigyo-ku, Kyoto-shi, Hitomachi 260 Palace Royal Nishijin Sawarabi 705 F term (reference) 4B024 AA11 BA63 CA04 DA02 DA06 EA04 GA11 HA12 HA15 4B063 QA01 QA18 QQ20 QR59 QR69 QR77 QR80 QS24 QS28 QX07 4B065 AA26X AA90X AA93Y AB01 AC14 BA02 BB23 CA24 CA46 4C084 AA06 AA07 AA13 BA19 BA35 CA53 CA56 MA24 MA52 MA55 MA66 NA14 ZA451 ZC781 A4A0MAA MAA MAA AA11 AA20 AA30 BA10 CA40 DA50 DA76 DA86 EA50 F A72 FA74

Claims (16)

[Claims] 1. A protein according to any one of the following 1) to 3): 1) amino acids 1 to 254 of SEQ ID NO: 2 in the sequence listing;
A protein consisting of an amino acid sequence represented by any one of amino acids 1 to 246 of SEQ ID NO: 4 in the sequence listing and amino acids 1 to 250 of SEQ ID NO: 6 in the sequence listing; 2) an amino acid number of SEQ ID NO: 2 in the sequence listing 1 to 254,
In the amino acid sequence represented by any one of amino acids 1 to 246 of SEQ ID NO: 4 in the sequence listing and amino acids 1 to 250 of SEQ ID NO: 6 in the sequence listing, one or more amino acids are added or deleted. A protein comprising an inserted and / or substituted amino acid sequence and having scavenger receptor activity; 3) amino acids 1 to 254 of SEQ ID NO: 2 in the sequence listing;
An amino acid sequence having at least 50% homology with any one of amino acid numbers 1 to 246 of SEQ ID NO: 4 in the sequence listing and amino acid numbers 1 to 250 of SEQ ID NO: 6 in the sequence listing A) a protein having a scavenger receptor activity; coli XL1 Blue
pME18S-hSR-PSOX SANK 7130
0 (FERM BP-7260); coliXL1
Blue pME18S-mSR-PSOX SAN
K 71400 (FERM BP-7261) and E.C. coli XL1 Blue pME18S-pS
R-PSOX SANK 71500 (FERM BP
A protein comprising an amino acid sequence encoded by DNA inserted into the multiple cloning site of pME18S in a plasmid carried by any one of the above-mentioned (7262). 2. A protein comprising an amino acid sequence represented by amino acid numbers 1 to 254 of SEQ ID NO: 2 in the sequence listing. 3. A protein comprising an amino acid sequence represented by amino acid numbers 1 to 246 of SEQ ID NO: 4 in the sequence listing. 4. A protein comprising an amino acid sequence represented by amino acid numbers 1 to 250 of SEQ ID NO: 6 in the sequence listing. A DNA encoding the protein according to any one of claims 1 to 4. 6. A DNA according to any one of the following a) to d): a) nucleotide numbers 97 to 8 of SEQ ID NO: 1 in the sequence listing.
58, nucleotides 1 to 7 of SEQ ID NO: 3 in the sequence listing
38 and nucleotide number 13 of SEQ ID NO: 5 in the sequence listing
A) a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 762 to 762; b) nucleotide numbers 97 to 8 of SEQ ID NO: 1 in the sequence listing.
58, nucleotides 1 to 7 of SEQ ID NO: 3 in the sequence listing
38 and nucleotide number 13 of SEQ ID NO: 5 in the sequence listing
To 762, comprising a nucleotide sequence that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 762 to 762; and c) a DNA encoding a protein having scavenger receptor activity; Nucleotides 97 to 8
58, nucleotides 1 to 7 of SEQ ID NO: 3 in the sequence listing
38 and nucleotide number 13 of SEQ ID NO: 5 in the sequence listing
Consisting of a nucleotide sequence having a nucleotide sequence identity of at least 67% with a DNA consisting of the nucleotide sequence represented by any one of SEQ ID NOS: to 762, and encoding a protein having scavenger receptor activity.
A; d) Transformed E. coli. coli XL1 Blue
pME18S-hSR-PSOX SANK 7130
0 (FERM BP-7260); coliXL1
Blue pME18S-mSR-PSOX SAN
K 71400 (FERM BP-7261) and E.C. coli XL1 Blue pME18S-pS
R-PSOX SANK 71500 (FERM BP
DNA inserted into the multiple cloning site of pME18S in the plasmid carried by any one of the above-mentioned DNAs. 7. A recombinant vector comprising the DNA according to claim 5 or 6. 8. The recombinant vector according to claim 7, which is an animal cell expression vector. 9. The transformed E. coli E. coli. coli XL1 B
lue pME18S-hSR-PSOX SANK
71300 (FERM BP-7260); col
i XL1 Blue pME18S-mSR-PSO
X SANK 71400 (FERM BP-726
1) and E. coli XL1 BlueME18
S-pSR-PSOX SANK 71500 (FER
9. The recombinant vector according to claim 7 or 8, wherein the plasmid is a plasmid carried by any one of the recombinant vectors (MBP-7262). A host cell carrying the recombinant vector according to any one of claims 7 to 9. 11. The transformed E. coli E. coli. coli XL1
Blue pME18S-hSR-PSOX SANK
71300 (FERM BP-7260); co
li XL1 Blue pME18S-mSR-PS
OX SANK71400 (FERM BP-726
1) and E. coli XL1 Blue pME1
8S-pSR-PSOX SANK 71500 (FE
RM BP-7262), wherein the host cell is selected from the group consisting of: 12. A host cell transformed with the recombinant vector for animal cell expression according to claim 8 or 9 oxidized LD.
By culturing with the addition of L, oxidized LD
In a system that incorporates or binds L, culture is performed with or without the addition of a test substance when oxidized LDL is added, and then the test substance inhibits the uptake or binding of oxidized LDL to the host cell. A method for testing an agent for preventing or treating arteriosclerosis, characterized by examining whether or not the agent is atherosclerosis. 13. A host cell transformed with the recombinant vector for expressing an animal cell according to claim 8 or 9 cultivated on a plastic plate coated with phosphatidylserine, whereby the host cell adheres to the plate. In the system, the culture is performed with or without the addition of a test substance to the host cell during or before the culture on the plate, and then the adhesion of the test substance to the host cell is measured. A method for testing a preventive or therapeutic agent for arteriosclerosis, which comprises examining whether or not the drug is inhibited. 14. A nucleic acid comprising an antisense sequence of a nucleotide sequence consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. 15. A pharmaceutical composition comprising the nucleic acid according to claim 14 as an active ingredient. 16. An antibody that specifically binds to the protein according to any one of claims 1 to 4.