WO2002012482A1 - Scavenger receptor and use thereof - Google Patents

Abstract

A novel protein having a scavenger receptor activity which is usable as a target of preventives or remedies for arteriosclerosis. More particularly speaking, a protein having the amino acid sequence of the amino acids at the positions 1 to 254 in SEQ ID NO:1 in Sequence Listing; a DNA encoding this protein; a recombinant DNA vector containing this DNA; a host transformed by this recombinant DNA vector; and use of the above protein. By using this method, it is possible to search for a novel compound which has an inhibitory activity specific to the scavenger receptor as described above and is useful as a remedy or a preventive for arteriosclerosis.

Description

 Specification

 Scavenger receptor and its use

[Technical field]

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

[Background technology]

The concept of the macula phage force-venger receptor was proposed by Brown and Goldstein of the University of Texas in 1979 from a study of the pathogenesis of arteriosclerosis. Brown et al. Studied low-density lipoprotein (LDL) receptors, which are responsible for cholesterol uptake in cells. In familial hypercholesterolemia with severe atherosclerosis, LDL receptors are inherited. Was found to be physically defective. It was shown that even if the LDL receptor was deficient, cholesterol derived from LDL was taken up by macrophages on the blood vessel wall via the receptor, and atherosclerosis was generated. The incorporation of acetylated LDL into macrophages is competed by a wide range of negatively charged macromolecules such as nucleic acids such as polye and poly G, sugar chains such as fucoidan, and polymers such as maleated albumin and polybuteyl sulfate. However, this receptor is widely known as a scavenger receptor having a broad ligand recognition function. The scavenger receptor cDNA was first cloned in 1990 [Kodama, T et al. Nature, 343, 531-535, 1990]. The scavenger receptor, a molecular weight of about 2 20,000 saccharide protein consisting of trimer of Sabuyuni' bets a molecular weight of about _70,000. Subsequently, as a scavenger receptor, type I (about 45 amino acids), which has a cysteine-rich domain, and about 100 amino acids more than type I Type II, a short terminal region, has become known. These scavenger receptors penetrate the cell membrane only once and consist of an intracellular domain of about 50 amino acids, and an extracellular domain of about 380 amino acids for type I and about 280 amino acids for type II. You. 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 function as a receptor with a monomer, and need to form a trimer to exhibit activity. Approximately 50 amino acids at the N-terminus of the human force benger receptor are the intracellular region of the receptor. The 7th), and the 4th (49th in the mouse) Ser-Xaa-Lys, Arg-Xaa-Xaa-Thr, Ser_Xaa-Lys (Xaa can be any amino acid) phosphorylatable amino acid sequences Exists (Takeshi Doi et al., Experimental Medicine 10, 19-26, 1992).

The type I scavenger receptor consists of a domain with six cysteines, and a series of membrane proteins are SR-mediated by homology with this domain of the scavenger receptor, resulting in R protein (scavenger receptor cystem rich domain like protein). Recognized and recognized. The SRCR proteins include a number of superreceptors and lymphocytes CD5 and CD6. In addition, the scavenger receptor has a collagen-like domain consisting of 22 amino acids at the C-terminal side and consisting of four lysines forming a cluster, and this domain forms a ligand binding site and can bind to collagen. It is assumed that they play an important role in the treatment of foreign matter and waste products. The scavenger receptor recognizes a variety of nucleoacids, acetic acids, denatured proteins, polymers, etc., and takes them up into the cell and removes them. However, it does not mean to take in anything but to treat substances that bind to biological components, negatively charged foreign substances and wastes very efficiently. For example, normal LDL uptake is hardly observed, but negative charge due to oxidation etc. Increased LDL is very well taken up. The scavenger receptor is a receptor that binds and takes up extremely well with denatured biological components such as oxidized LDL. The accumulation of lipid-deposited foam cells from macrophages and smooth muscle cells (hereafter “SMC”) is one of the characteristic early changes in the atrial intima of ongoing atherosclerotic plaques . It is thought that macrophage force-venger receptors are involved in the process leading to the conversion of macrophages and SMCs to foam cells, and the results of many in vitro and in vivo studies indicate that this We support the model. For example, after incubating the modified LDL in vitro and then adding Chinese hamster ovary cells (hereinafter referred to as “CHO cells”) forcibly expressing the cis-force-venger receptor, the CHO cells are transformed into macrophages in plaques. Can be converted into lipid-depositing cells similar to In addition, mRNA encoding the macrophage scleroderma receptor, its receptor protein and modified LDL are detected in atherosclerotic plaques (scavenger receptor in the symptoms of atherosclerosis). See Krieger et al, J. Biol. Chem. 268 (7): 4569-4572 (1993) and the references cited therein. Scavenger receptors are expressed not only in tissue macrophages but also in a state where macrophages differentiate from monocytes and invade in accordance with various disease states. The most frequently studied expression is in foam cells in the early stage of atherosclerosis (Shin Naito et al., Experimental Medicine, 10, 31-35, 1992). According to the high-lipid diet model of egrets and pigs, which are most commonly observed in the course of atherosclerosis, LDL-like particles adhere to the matrix of the blood vessel wall several days after starting a high-cholesterol diet Then, monocytes are deposited in such a way that a large number of monocytes adhere to endothelial cells, enter the intima and become macrophages to form lesions called fatty striae. These fatty glands are clusters of hundreds or thousands of macrophages, where the scavenger receptor is also most abundantly expressed. As the lesion progresses further and smooth muscle cells enter the intima and become proliferative, macrophages can be located at the edge or deeper of the lesion. And the expression of scavenger receptors is greatly reduced. When macrophages take up foreign substances via scavenger receptors, proteins and nucleic acids are rapidly degraded and released in the case of proteins and nucleic acids. However, when macrophages take up lipoproteins via scavenger receptors, they cannot degrade the cholesterol ring in the macrophages, so they have the ability to transfer to plasma receptors, such as HDL, or the cholesterol called ACAt in the macophage phage. Cholesterol ester lipid droplets accumulate in macrophages, which are transformed into foam cells.

Also recently, one of a new family of novel force receptor receptors, called the Marcos force receptor receptor (MMarco SR), has been cloned from a mouse macrophage cDNA library (Eloma et al., Cell, 80: 603-609). (1995)). The Marcos Force Benger receptor is also involved in binding Gram-positive and negative bacteria. By the way, in addition to these known scavenger receptors, the existence of unknown scavenger receptors has been suggested (Cl inica Chimica Acta 286 (1999) 191-205). And features were not disclosed. That is, an object of the present invention is to provide a novel scavenger receptor protein useful for searching or evaluating a drug for preventing or treating arteriosclerosis, a DNA encoding the protein, and a use of the protein. . Such a new type of drug candidate search can be performed by screening for a substance that inhibits the scavenger receptor activity of the protein of the present invention. [Disclosure of the Invention]

We do not express CD36, a type of scavenger receptor THP-1 cells differentiated by PMA in a macrophage-like manner are known to bind to phosphatidylserine, which is considered to be a physiological ligand of the force-venger receptor. We successfully cloned the cDNA encoding the scavenger receptor, and cloned the scavenger receptor from mouse and pig. Furthermore, the present inventors, when expressing this cDNA in cultured cells, revealed that the transformed cells acquired the ability to take in oxidized LDL, which is a substrate of scavenger receptor. The present inventors have found that it is possible to search for or evaluate a preventive or therapeutic agent for arteriosclerosis using various transformed cells, and completed the present invention.

The present invention firstly relates to a protein described in any one of the following 1) to 4):

 1) It is represented by any one of amino acid numbers 1 to 254 of SEQ ID NO: 2 in the Sequence Listing, 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 A protein consisting of an amino acid sequence;

 2) Any one of amino acid numbers 1 to 254 of SEQ ID No. 2 in the sequence listing, amino acid numbers 1 to 246 of SEQ ID No. 4 in the sequence listing and any one of amino acid numbers 1 to 250 of SEQ ID NO. 6 in the sequence listing A protein comprising an amino acid sequence shown in which one or more amino acids are added, deleted, inserted, and substituted or substituted with one or more amino acids, and which has a force-venger receptor activity;

 3) Any one of amino acid numbers 1 to 254 of SEQ ID NO: 2 in the sequence listing, amino acids 1 to 246 of SEQ ID NO: 4 in the sequence listing, and amino acids 1 to 246 of SEQ ID NO: 6 in the sequence listing A protein consisting of an amino acid sequence having 50% or more homology with the amino acid sequence to be identified and having scavenger receptor activity;

 4) Transformed Escherichia coli E.co1 i XL 1 Blu e pME l 8 S— h S R— P SOX SANK 7 1300 (F ERM B P— 7260), E.coli

XL 1 B lue pME 18 S—mS R—PS OX SANK 7 1 400 (FERM BP-726 1) and E. coli XL 1 B lue pME 18 8 Sp S RP SOX SANK 7 1 500 (FERM BP— 7 26 2 ) A protein that comprises the amino acid sequence encoded by the DNA inserted into the multicloning site of pME18S in the plasmid carried by any one of the above. Second, the present invention relates to a DNA encoding the above protein. Examples of such DNA of the present invention include: a) a DNA described in any one of the following a) to d): a) nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing; SEQ ID NO: 3 in the sequence listing A DNA comprising the nucleotide sequence shown in any one of nucleotide numbers 1 to 738 of the sequence listing and nucleotide numbers 13 to 762 of the sequence listing 5; b) nucleotide number of SEQ ID NO. 1 in the sequence listing 9 7 to 8 58, a DNA comprising the nucleotide sequence shown in any one of nucleotide numbers 1 to 7 38 of SEQ ID NO: 3 in the Sequence Listing and nucleotide numbers 13 to 762 of SEQ ID NO: 5 in the Sequence Listing And a nucleotide sequence that hybridizes under stringent conditions and encodes a protein having scavenger receptor activity;

c) Any one of nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the Sequence Listing, nucleotide numbers 1 to 732 of SEQ ID NO: 3 in the Sequence Listing and nucleotide numbers 13 to 762 of SEQ ID NO: 5 in the Sequence Listing A DNA comprising a nucleotide sequence having a nucleotide sequence identity of 67% or more with the DNA consisting of the nucleotide sequence shown in FIG. 1 and encoding a protein having a scavenger receptor activity;

d) Transformed E. coli E. c 0 1 i XL 1 Blue pME 18 S—h SR— P SOX SANK 7 13 00 (FE RM BP— 7260), E. coli XL 1 Blue pME l 8 S -mS RP SOX SANK 7 1 400 (FERM BP—726 1) or E. coli XL 1 Blue pME 18 S—p SR—P SOX SANK 7 1 500 (FERM BP— 7 2 6 2) DNA that has been inserted into the pME18S multi-cloning site,

Although the present invention is not limited thereto, the present invention includes all DNAs having a nucleotide sequence encoding the above protein. Third, the present invention relates to a recombinant DNA vector containing the above DNA. Such a preferred vector of the present invention is a transformed E. coli E.c01iXL1Blue pME18S-hSR-PS OX SANK 7 1300 (FE RM B P-726 0 ) _N E. coli XL 1 Blu pME 18 S -m SR—PS〇X SANK 7 1400 (FERM BP—726 1) and E.co 1 i XL 1 B luep ME 18 S—p SR—PSOX A recombinant DNA vector which is a plasmid carried by any one of SANK 711 500 (FERM BP-7272), or a recombinant DNA vector for animal cell expression in which the DNA of the present invention is incorporated. However, the present invention is not limited to this. Fourth, the present invention relates to a host cell transformed with the above-described recombinant DNA vector. Such suitable host cells of the present invention include transformed E. coli E.co1iXL1Bluep ME18S—hSR—PSOX SANK71300 (FERMBP-7272), E E. coli XL 1 B lue pM E l 8 S-mS RP SOX SANK 7 1 400 (F ERM BP— 7 26 1) and E. coli XL 1 B lue pME 18 S— p SR— PS〇X SANK 7 1 A host cell selected from the group consisting of 500 (FERM BP-7262), or an animal cell transformed with the above-described recombinant vector for expressing an animal cell, but the present invention is not limited thereto. Fifth, the present invention relates to a method for testing an agent for preventing or treating arteriosclerosis using host cells transformed with the above-described recombinant vector for expressing animal cells. Specifically, the test method is performed by adding oxidized LDL to a host cell transformed with the above-described recombinant vector for animal cell expression and culturing the same, thereby allowing the host cell to incorporate oxidized LDL. In the system to be bound, culture is performed with or without the addition of the test substance at the time of addition of the oxidized LDL, and then whether or not the test substance inhibits the uptake or binding of the oxidized LDL into the host cell A system for attaching the host cells to the plate by examining or culturing host cells transformed with the above-described recombinant vector for animal cell expression on a plastic plate coated with phosphatidylserine. At During or before culturing on the plate, culture is performed with or without the addition of a test substance to the host cells, and then the test substance inhibits attachment of the host cells to the plate. Or not. The present invention also relates to the use of the above DNA for testing a preventive or therapeutic agent for arterial sclerosis as described above. Sixth, the present invention relates to an antisense nucleic acid of the above DNA and its use. Seventh, the present invention relates to an antibody that specifically binds to the above protein.

That is, the present invention provides a novel protein having scavenger receptor activity, a DNA encoding the protein, a recombinant vector containing the DNA, a host cell transformed with the vector, the use of the protein, and It provides an antibody that specifically binds. In the present invention, “scavenger receptor activity” refers to an activity of binding oxidized LDL on a cell membrane and taking it into cells. The DNA encoding the protein of the present invention is a clone having scavenger receptor activity from a cDNA library obtained by performing a reverse transcription reaction using mRNA extracted and purified from animal cells expressing the D.NA as a cycl type. Can be obtained by screening. The animal cells that supply this mRNA are human monocyte-derived cell line THP-1 (A TCC TIB-202) and phorbol 12-myristate 12-acetate (phorbol 12-myristate 12-acetate). : Stimulated with PMA), spleen of BALBc mouse and whole organ tissue of thymus or fetal pig, etc. are preferred, and various mammals (as long as they produce the protein of the present invention) are preferred. Cells or tissues derived from human (including humans) or cultured cell lines can also be used. For the extraction of mRNA, guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate hot phenol method, guanidine hydrochloric acid method, guanidine acid thiocyanate / phenol clonal form method can be used. A commercially available mRNA separation kit can also be used. Many of the mRNAs in the cytoplasm of eukaryotic cells are known to have a poly (A) sequence at the 3 'end, and this feature is used to make a biotinylated oligo (dT) probe. MRNA is adsorbed to the DNA, and the paramagnetic particles on which streptavidin is immobilized capture the mRNA using the binding between biotin and streptavidin, and after washing, purify the mRNA by elution. can do. Further, a method of adsorbing mRNA on an oligo (dT) cellulose column and then eluting and purifying the mRNA may be used. Further, RNA can be further fractionated by sucrose density gradient centrifugation or the like. '■ In addition, after using the mRNA obtained by the above method as type I to synthesize single-stranded cDNA using reverse transcriptase, double-stranded cDNA can be synthesized from this single-stranded cDNA. Wear. Examples of the method include the S1 nuclease method (Efstratiadis, A. et al. (1976) Cell 7, 279-288) and the Land method (Land, H. et al. (1981) Nucleic Acids Res. 9, 2251- 2266), the 0. Joon Yoo method (Yoo, 0. J. et al. (1983) Proc. Natl. Acad. Sci. USA 79, 1049-1053), and the like. The Okayama-Berg method (Okayama, H. and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) is preferred. Next, the obtained cDNA fragment is inserted into a lambda phage vector and allowed to self-replicate, whereby the recombinant phage having the cDNA fragment can be stably retained and amplified. For example, when lambda phage ZAP II (Stratagene) is used, host E. coli XL1-B1ue MRF 'and JM109 strains form plaques, and 5-plaque 4- '5-Bromo-4-chloro-3-indolyl-β-D-galactopyranosid e (X-ga 1)) 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. Alternatively, 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 the desired scavenger receptor activity from the library obtained as described above, for example, any one of the following various methods is employed. it can.

(1) Screening method using a synthetic oligonucleotide probe: When all or part of the amino acid sequence of the target protein has been elucidated (If the sequence is a plurality of consecutive specific sequences, Any portion may be synthesized, and an oligonucleotide encoding the amino acid sequence may be synthesized (for amino acids having degenerate codons, frequently used codons may be used or possible codons may be used). May be combined to synthesize a plurality of nucleotide sequences, and in the latter case, inosine may be included to reduce the number of nucleotide sequences), which is recognized by ³² P, ³⁵ S, biotin, etc. Recombinant phage DNA was denatured and immobilized using as a probe. 2.Hybridized with trocellulose filter or Nymouth filter. Positive clones find and the, this is selected. '

(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, it corresponds to the N-terminal part of the amino acid sequence. Oligonucleotides of the sense strand and an antisense strand corresponding to a part of the C-terminal side are synthesized, and a polymerase chain reaction (hereinafter, referred to as “PCR”) is performed to amplify a DNA fragment encoding a target protein. As the type I DNA used here, cDNA synthesized by reverse transcription from mRNA of cells producing the protein of the present invention or genomic DNA is used. Can be. The DNA fragment prepared in this manner is labeled with ³² P, ³⁵ S, biotin, or the like, and a cDNA library or genomic library obtained by plaque hybridization or colony hybridization using this as a probe. Perform the screening in the above to select the desired clone.

(3) Screening by producing a protein having scavenger receptor activity in another animal cell line: Plasmid (self-replicating) in which cDNA obtained as described above is inserted into an expression vector Transformants can be used to transform animal cell hosts with plasmids that contain a transcriptional promoter region or can be integrated into the chromosome of animal cells. The scavenger receptor activity of the transformed cells is measured, or the presence of the protein of the present invention is determined by using an antibody that specifically binds to the protein of the present invention and a secondary antibody against the antibody. , A strain having cDNA encoding the protein of the present invention is selected. As animal cell hosts, commonly used cell lines such as COS and CHO can be used.However, in order to facilitate detection of the protein of the present invention as a foreign gene product, the host itself must be maintained under certain culture conditions. It is preferable that the cell does not produce the protein of the present invention.

Collection of DNA encoding the protein of the present invention from the target transformant obtained as described above can be performed by a known method (Maniatis, T. et al. (1982): "olecular Cloning A Laboratory Manual Cold Spring Harbor 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. DNA / RNA amplification by PCR can also be suitably used to obtain the gene encoding the protein of the present invention, especially when full-length cDNA cannot be obtained from the library. (See RACE: Experimental Medicine, (1994), 12 (6), 35-38. The ability to use primers based on partial sequences and mRNA derived from mammals, or commercially available kits and cDNA libraries To obtain both ends of the full-length cDNA. Primers used for adopting such 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 DNA of the present invention obtained in this manner can be determined, for example, by the chemical modification method of Maxam and Gilbert (Maxam, AM and Gilbert, W. (1980): "Methods in Enzymology" 65, 499-559). ) Or the dideoxy nucleotide chain termination method using M13 phage (Messing, J. and Vieira, J. (1982) Gene 19, 269-276). In addition, an automatic DNA sequence analyzer using a fluorescent dye instead of the radioisotope (for example, Model 373A manufactured by Parkin Elma's Japan, Inc. Biosystems) can be used. In addition, a transformed E. coli strain E. c 0 1 i XL 1 Blu e ME 18 Sh SR-PSOX SANK 71300, which contains a plasmid into which cDNA encoding the most preferable protein of the present invention has been inserted, E. coli XL 1 B lue pME 18 S—mS R—PS OX SANK 71400 and E. coli XL 1 B luep El 8 Sp SR-PSOX SANK 715 00 are both August 1, 2000 1-1-1, Higashi, Tsukuba-shi, Ibaraki, Japan 1 Internationally deposited with the National Institute of Advanced Industrial Science and Technology (AIST) and the Patent Organism Depositary Center 1 (formerly known as the Institute of Biotechnology and Industrial Technology) Accession numbers FERM BP-7260, FERM BP-7261 and FE RM BP-7262 are attached. 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 vector DNA, other prokaryotic or eukaryotic host cells can be transformed. Furthermore, by introducing an appropriate promoter and a sequence involved in expression into these vectors, the gene can be expressed in each host. Prokaryotic host cells include, for example, Escherichia coli and Bacillus subtilis. To transform a gene of interest in these host cells, the host cell is transformed with a plasmid vector containing regulatory elements and a replicon or origin of replication from a species compatible with the host. 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-based plasmids are generally used as vectors, but are not limited thereto, and various known strains, And vectors can be used. In Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan 'lactose (tac ₎ promoter, lipoprotein ( _lpp) promoter, polypeptide elongation factor Tu

(tufB) promoter and the like. Any promoter can be used for production of the protein of the present invention. As Bacillus subtilis, for example, 207-25 strains are preferable, and as a vector, pTUB228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) is used. However, the present invention is not limited to this. Extracellular secretory expression is also possible by linking a D こ と sequence that encodes the signal peptide sequence of _α -amylase of Bacillus subtilis as a promoter. Eukaryotic host cells include cells such as vertebrates, insects, and yeast. Examples of vertebrate cells include monkey COS cells (Gluzman, Y. (1981) Cell 23 175-182, ATCC CRL-1 650) and Chinese 'hamster ovary cells (CHO cells, ATCC CCL-61) deficient in dihydrofolate reductase Strain (Urlaub, G. and Chasin, A. (1980) Proc. Natl. Acad. Sci. USA 77, 4 126-4220) and the like are often used, but are not limited thereto. As a vertebrate cell expression promoter, a promoter having an upstream sequence of a gene to be normally expressed, an RNA splice site, a polyadenylation site, and a transcription termination sequence can be used. May have a replication origin. An example of the expression vector is p SV 2 dhfr having an early promoter of SV 40 (Subraraani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864). It is not limited to this. For example, when a COS cell is used as the host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcription promoter and a transcription termination. Signals and those with an RNA splice site can be used. The expression vector is obtained by the following method: getylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, 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), etc. Thus, the cells 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 G4 18 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, J, et al. 1989): "Molecular Cloning AL aboratory Manual Cold Spring Harbor Laboratory, NY) and p SV2-neo

(Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341) and the like, and by selecting G4 18 resistant colonies, A transformed cell stably producing a protein can be obtained. When insect cells are used as host cells, cell lines derived from ovarian cells (3-9 or 3 £ —21) of Spodoptera frugi perda (Lepidoptera: Noctuidae) and Trichoplusia ni Egg-derived High Five cells (Wickham, TJ et al, (1992) Biotechnol. Prog, ΐ: 391-396) are often used as host cells, and the baculovirus transfer vector is autographa nucleopolyhedrovirus (Ac). P VL 1392 Z1 393 using the promoter of the polyhedrin protein of Ρ Ρ V) is frequently used (Kidd, IM and VC Emery (1993) The use of baculoviruses as express ion vectors. ied Biochemi stry and Biotechnology 42, 137-159). In addition, vectors using baculovirus P10 or a promoter of the same basic protein can also be used. Furthermore, the recombinant protein can be expressed as a secreted protein by extending the secretory signal sequence of the envelope surface protein GP67 of Ac NPV 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, and among them, yeast of the genus Saccharomyces, for example, Nodule. Saccharorayces cerevisiae and petroleum yeast Pichia pastoris are preferred. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, an alcohol dehydrogenase gene promoter (Bennetzen, J. Shi and Hall, BD (1982) J. Biol. Chem. 257, 3018-3025) And a promoter of the acid phosphatase gene (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5). When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and an endogenous protease of a host cell or a cleavage site of a known protease at the N-terminal side. . For example, in a system in which human tryptic trypsin-type serine protease is expressed in petroleum yeast, the secretory signal sequence of yeast alpha-phater at the N-terminal side and the Κ Ε Χ2 It has been reported that active tributase is secreted into the medium by connecting and expressing the cleavage site (Andrew, Shi Niles, et al. (1998) Biotechnol. Appl. Biochera. 28,

125-131).

— The transformant obtained as described above can be cultured according to a conventional method. The culture produces the protein of the present invention intracellularly or extracellularly. The medium used for the culture can be appropriately selected from various types commonly used depending on the host cells that have been rubbed. For example, in the case of the above-described COS cells, RPMI 164 medium or Dulbecco's modified medium is used. A medium, such as Eagle's medium (hereinafter referred to as “DMEM”), to which serum components such as fetal calf serum are added as necessary, can be used. The protein of the present invention expressed in this manner is subjected to various separation operations utilizing its physicochemical properties, chemical properties, etc. (Biochemical Data Book II, 1st edition, 1175-1259, Tokyo Chemical Published (1980); Biochemistry, vol. 25, No. 25, p8274-8277 (1986); Eur. J. Biochem., 163, p313-321 (1987)). Examples of the method include, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, freeze-thaw method, supersonic wave crushing, ultrafiltration, gel Filtration, adsorption chromatography, ion-exchange chromatography, affinity chromatography, high-performance liquid chromatography

Examples thereof include various liquid chromatography such as (HPLC), dialysis, and combinations thereof. In addition, by connecting a histidine tag consisting of 6 residues to the recombinant protein to be expressed, the protein can be efficiently purified by a nickel affinity column. By combining the above 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 was confirmed using an automatic protein amino acid sequencer (for example, Model 492, manufactured by PerkinElmer Japan ABRI Biosystems). can do. In order for the protein obtained by the genetic engineering technique from the DNA of the present invention to express the scavenger-receptor activity in this manner, the sequence number in the sequence listing is not necessarily required. The amino acid sequence shown in SEQ ID NO: 2 from amino acid No. 1 to 2564, the amino acid sequence shown in SEQ ID NO: 4 from amino acid No. 1 to 2446 or the amino acid sequence shown in SEQ ID NO: 6 It does not need to consist of a sequence that completely matches the amino acid sequence shown; for example, even if it is a partial sequence, it will be composed of such a partial sequence as long as it exhibits the activity of a S. venja monoreceptor. Those are also included in the protein of the present invention. Further, DNA encoding the protein is also included in the present invention. In general, eukaryotic genes are considered to exhibit polymorphism, as is known for the interferon gene (see, for example, Ni shi, T. et al. (1985) J. Biochem. 97, 153-159), but the polymorphism may result in the substitution of one or more amino acids, or the substitution of a nucleotide sequence may not change the amino acids at all. The amino acid sequence shown in SEQ ID NO: 2 from amino acid Nos. 1 to 254, the amino acid sequence shown in SEQ ID NO: 4 from amino acid No.1 to 246 or the amino acid No. in SEQ ID NO: 6 in SEQ ID NO: 4 At one or more sites in the protein amino acid sequence of the present invention consisting of the amino acid sequence represented by 1 to 250, one or more amino acid residues are deleted, added, Even if the protein has been inserted and / or substituted, it often has the activity of a receptor. (A protein having an amino acid sequence in which the natural amino acid sequence is substituted has the same activity as the natural protein.) As an example, for example, a tag obtained by converting a nucleotide sequence corresponding to cysteine of the interleukin 2 (IL-2) gene into a nucleotide sequence corresponding to serine. It is known that protein protein ^s retains IL-2 activity. (Wang, A. et al. (1984) Science 224, 1431-1433)]. All such proteins are included in the present invention as long as they have scavenger receptor activity. Further, the present invention also includes all DNAs having the same nucleotide sequence encoding these proteins. Such various DNAs of the present invention can be prepared, for example, by the phosphite triester method (Hunkapil method) based on the information on the protein having the scavenger receptor activity. et al. (1984) Nature 310, 105-111), and can also be produced by chemical synthesis of nucleic acids. 'The codon corresponding to the desired amino acid may be arbitrarily selected, and can be determined according to an ordinary method, for example, in consideration of the codon usage of the host to be used. (Grantham, R. et al. (1981) Nucleic Acids Res. 9, 143-174). Furthermore, partial modification of these nucleotide sequence codons can be performed by a site-directed mutagenesis method (site specit cit ic acid) using a primer consisting of a synthetic oligonucleotide encoding the desired alteration according to a conventional method. Mutagenes is / Mark, ϋ. F. 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 cut from the end using exonuclease Ba131 (Toshimitsu Kishimoto et al.) "Seismic Chemistry Laboratory Course 1, Gene Research Method II" 335-354), cassette mutation method (Toshimitsu Kishimoto, "New Biochemistry Laboratory Course 2, Nucleic Acid I II Recombinant DNA Technology" 242-251) it can. '' Among the above-mentioned proteins having scavenger receptor activity, a preferred one is a protein having an N-terminal methionine residue of amino acid No. 1 in the amino acid sequence shown in SEQ ID No. 2 in the sequence listing. Protein consisting of 2 amino acids, a protein consisting of 2 4 6 amino acids having the methionine residue of amino acid number 1 in the amino acid sequence shown in SEQ ID NO: 4 as the N-terminus, or shown in SEQ ID NO: 6 A protein consisting of 250 amino acids having the methionine residue at amino acid number 1 in the amino acid sequence to be N-terminal. 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 a sequence of the sequence listing Whether or not to hybridize with the DNA consisting of the nucleotide sequence represented by nucleotide numbers 13 to 762 of No. 5 can be determined by, for example, random primer method (Anal. Biochem., 132: 6013 (1983)) or the like. Nick translation method (Maniatis, T. et al. (198 2) in "Molecular Cloning A Laboratory Mannual" Cold Spring Harbor Labora tory, according NY.) Or the like, can be examined performs hive Lida I internalization using a probe labeled with [α- ^{3 2} P] d CTP like . 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 light. The membrane is then immersed in a prehybridization solution containing, for example, 6 XSSC, 5% Denhardt's solution and 0.1% sodium dodecyl sulfate (hereinafter referred to as "SDS") at 55 ° C for 4 hours. Keep it warm. Thereafter, the labeled probe prepared above is added to the same prehybridization solution so as to have a final specific activity of 1 × 10 ⁶ cpm / m 1, and the mixture is incubated at 60 ° C. overnight. The procedure 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 or not the noise has been hybridized. it can. Using this method, a nucleotide sequence represented by nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing and a nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing can be obtained from cDNA libraries derived from various animal cells. It is possible to isolate cDNA that hybridizes with the DNA consisting of the nucleotide sequence represented by any one of Nos. 1 to 738 or the nucleotide sequence represented by the nucleotide numbers 13 to 762 of SEQ ID NO: 5 in the sequence listing. (Maniatis, T. et al. (1982) in Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory, NY,) ₀ As described above, genetic engineering techniques such as nucleotide sequence modification and nucleic acid hybridization. Suitable as the DNA of the present invention obtained by utilizing the nucleotide sequence shown in nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing, and the nucleotide sequence represented by nucleotide number 1 in SEQ ID NO: 3 in the sequence listing A nucleotide sequence having nucleotide sequence identity to the nucleotide sequence represented by 738 or the nucleotide sequence represented by nucleotide numbers 13 to 762 of SEQ ID NO: 5 in the sequence listing and having a nucleotide sequence identity of at least 67 <½, Examples include DNA encoding a protein having scavenger receptor activity. More preferably, the nucleotide sequence shown nucleotide number ₉ 7 of SEQ ID No. 1 to 8 5 8, of SEQ ID NO: 3 j Examples include a DNA consisting of the nucleotide sequence represented by nucleotide numbers 1 to 738 or the nucleotide sequence represented by nucleotide numbers 13 to 762 of SEQ ID NO: 5 in the sequence listing, and most preferably the sequence in the sequence listing. A DNA consisting of the nucleotide sequence represented by nucleotide numbers 97 to 858 of No. 1 can be exemplified. The fact that the protein of the present invention has a scavenger receptor activity is, for example, that oxidized LDL (radioisotope, etc.) The cells can be confirmed by measuring the amount of oxidized LDL incorporated into cells after a certain period of time.

However, the method for detecting the activity of the protein of the present invention is not limited to these methods, and other methods well known in the technical field of the present invention can also be used. In the above-described method for detecting scavenger receptor activity, a test sample such as a synthesized compound or an extract from a culture of a microorganism may be allowed to coexist when oxidized LDL is added, and the test sample may be a scavenger receptor for the protein of the present invention. By examining whether or not the body activity is inhibited, scavenger receptor inhibitors can be evaluated or screened. In particular, a substance having a strong effect of specifically inhibiting the scavenger receptor activity of the protein of the present invention can be expected as a scavenger receptor-specific inhibitor to have a drug effect in preventing or treating arteriosclerosis.

Examples of the antibody that specifically binds to the protein of the present invention include a monoclonal antibody that specifically binds to the protein of the present invention. The method for obtaining the monoclonal antibody is as described below. is there. In the production of monoclonal antibodies, the following working steps are generally required. That is, (a) purification of a biopolymer used as an antigen,

 (b) immunizing the animal by injecting the antigen with the antigen, collecting blood, assaying the antibody titer, determining the timing of splenectomy, and preparing antibody-producing cells;

(c) preparation of myeloma cells (hereinafter referred to as "myeloma");

 (d) cell fusion between antibody-producing cells and myeloma,

 (e) Selection of hybridomas producing the desired antibody,

 (f) Split into single cell clones (cloning),

 (g) In some cases, culture of hybrids to produce monoclonal antibodies in large quantities, or breeding animals transplanted with hybridomas,

 (h) Examination of the biological activity of the monoclonal antibody thus produced and its recognition specificity, or assay of its properties as a labeling reagent,

And so on. 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 also 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 step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or Rimiyodiban, and the animal is immunized as an immunogen. 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. Immunization can be performed once or several times at appropriate intervals (preferably at intervals of 1 to 5 weeks). Thereafter, the antibody titer to 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 has increased by + as a source of antibody-producing cells. Generally, it is preferable to use antibody-producing cells derived from an animal 3 to 5 days after the final immunization for subsequent cell fusion. The methods for measuring antibody titer used herein include radioisotope immunoassay (hereinafter referred to as "RIA"), enzyme-linked immunosorbent assay (hereinafter referred to as "ELISA"), fluorescent antibody assay, and passive assay. There are various known techniques such as a hemagglutination reaction method, and the RIA method or the ELISA method is more preferable from the viewpoints of detection sensitivity, quickness, accuracy, and possibility of automation of the operation. The measurement of the antibody titer in the present invention can be performed, for example, by the following procedure according to the ELISA method. 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. After washing, the sample is brought into contact with a serially diluted sample (eg, mouse serum) as the first antibody, and the monoclonal antibody in the sample is allowed to bind to the antigen. Further, by adding an antibody against the mouse antibody that has been enzyme-labeled as the second antibody and binding to the mouse antibody, 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 the antibody titer.

(c) Myeloma preparation process

As myeloma, a cell line generally obtained from a mouse, for example, 8-azaguayun-resistant mouse (derived from BALB / c) myeloma strain P3X63Ag8U.1 (P3-Ul) [Yelton, DE et al. Current Topics in Microbiology and Immunology, 81, 1-7 (1978)], P3 / NSI / 1-Ag4-1 (NS-1) [Kohler, G. et al. European J. Immunology, 6, 511-519 (1976) ], Sp2 / 0-Agl4 (SP-2) [Shulman, M. et al. Nature, 276, 269-270 (1978)], P3X63Ag 8.653 (653) [Kearney, JF et al. J. Immunology, 123, 1548-1550 (1979)], and P3X63Ag8 (X63) [Horibata, K. and Harris, AW Nature, 256, 495-497 (1975)]. These cell lines are grown in a suitable medium, such as 8-azaguan medium [RPM I-

164 ◦ Medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as “FCS”) plus 8-azaguanine], Iscove's modified Dulbecco's medium (Iscove ' s Modified Dulbecco's Medium; hereafter referred to as “IMDM”) or subcultured in DMEM, but 3 to 4 days before cell fusion in normal medium [eg 10% FCS containing ASF 1 04 medium (Ajinomoto Co., Ltd.) at subcultured, set aside 2 X 1 0 ⁷ or more cell number day of fusion.

'' (d) Cell fusion

Antibody-producing cells are plasma cells and their precursor lymphocytes, which may be obtained from any part of an individual, and are generally spleen, lymph nodes, peripheral blood, or a combination thereof as appropriate. Etc., but 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 to use polyethylene dalicol, which has relatively low cytotoxicity and easy fusion. This method includes, for example, the following procedure. The spleen cells and myeloma are thoroughly washed with a serum-free medium (for example, RPMI 164) or phosphate buffered saline (PBS), and the ratio of the number of spleen cells to the number of myeloma cells is determined. Mix so that the ratio is 5: 1 to 10: 1, and centrifuge. After removing the supernatant and thoroughly loosening the precipitated cell group, 1 ml of 50% (w ZV) serum-free medium containing polyethylene glycol (molecular weight: 1,000 to 4,000) is added dropwise with stirring. Then, slowly add 10 ml of serum-free medium, and centrifuge. Discard the supernatant again and transfer the precipitated cells to an appropriate amount of hypoxanthine 'aminopterin' Gin (hereinafter referred to as “HAT”) and a medium containing mouse interleukin-12 (“IL-12”) suspended in a normal medium (hereinafter referred to as “HAT medium”) for culture Dispense into each well of a plate (hereinafter referred to as “plate”) and incubate at 37 ° C for about 2 weeks in the presence of 5% carbon dioxide. Supplement the HAT medium as needed.

(e) Selection of Hypri-Dorma group

 When the myeloma cell is an 8-azaguanine-resistant strain, that is, a hypoxanthine 'guanine' phosphoribosyltransferase (HGPRT) deficient strain, the unfused myeloma cell and a fusion cell of myeloma cells are Cannot survive in 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 hybridomas of the antibody-producing cells and myeoma cells survive, and as a result, the hybridomas can be selected. For the hybridomas that have grown in a colo condition, replace the HAT medium with a medium without aminobuterin (hereinafter referred to as “HT medium”). Thereafter, a part of the culture supernatant is collected, and the antibody titer is measured by, for example, ELISA. The method using an 8-azaguanine-resistant cell line has been described above, but other cell lines can also be used according to the selection method of the hybridoma, and in that case, the composition of the medium used also changes.

(f) Cloning

By measuring the antibody titer in the same manner as described in step (b), the hybridoma that has been found to produce a specific antibody is transferred to another plate and cloned. The cloning method includes a limiting dilution method in which a plate is diluted to contain one hybridoma per well, a soft agar method in which the colony is collected by culturing in a soft agar medium, and a micromanipulator. Take one cell at a time There is a method of culturing the cells, and a “Sotak mouth” in which one cell is separated by a cell sorter. The limiting dilution method is simple and often used. For the gel with an antibody titer, for example, cloning by limiting dilution 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 culturing

 The cloned hybridomas are cultured by replacing the medium with HT medium and normal medium. Large-scale cultivation is performed by rotary culture using large culture bottles 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, by growing the hybridoma in the abdominal cavity of a mouse of the same strain (eg, the above BALB / c) or a Nu / Nu mouse, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained. Can be obtained. As a simple method for purification, a commercially available monoclonal antibody purification kit (for example, MabTrapGII kit; manufactured by Pharmacia) can be used. The monoclonal antibody thus obtained has high antigen specificity for the protein of the present invention.

(h) Monoclonal antibody assay

The isotype and subclass of the obtained monoclonal antibody can be determined as follows. First, identification methods include the Ouchterlony method, the ELISA method, and the RIA method. The Octel mouth A method is simple but requires concentration if the concentration of monoclonal antibody is low. On the other hand, when the ELISA method or the RIA method is used, the culture supernatant is reacted with the antigen-adsorbed solid phase as it is, and monoclonal antibodies can be obtained by using antibodies corresponding to various immunoglobulin isotypes and subclasses as secondary antibodies. It is possible to identify antibody isotypes and subclasses. Also, as a simpler method, A commercially available kit for identification (eg, mouse type kit; manufactured by Biorad) can also be used. Furthermore, quantitation of protein can be performed by Folin-Lowry method, and a method of calculating from the Contact Keru absorbance 2 8 0 nm [1. 4 (OD 2 8 0) = I Takeno globulin 1 mg / m 1]. 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. The 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 usually a DNA consisting of 15 to 30 mer which is complementary to a part of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, or a phosphorothioate, methylphosphonate or morpholino derivative thereof. Can be used as stable DNA derivatives, such as stable RNA derivatives such as 2′- '-alkyl RNA. Such antisense molecules can be introduced into cells by methods well known in the art of the present invention, such as by microinjection, ribosome encapsulation, or expression using a vector having an antisense sequence. Such antisense therapy reduces the activity of the protein encoded by the nucleotide sequence shown by nucleotide numbers 97 to 588 of SEQ ID NO: 1 in the sequence listing, so that, for example, a therapeutic or preventive effect on arteriosclerosis can be obtained. Can be expected. A composition useful as a medicament containing the antisense oligonucleotide can be produced by a known method such as mixing a pharmaceutically acceptable carrier. Examples of such carriers and methods of manufacture are described in Remington's Pharmaceutical 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 and the activity of the gene product are recognized as abnormal is recognized. Administered to humans. The effective amount depends on various factors such as the condition, weight, sex, and age of each individual, as well as subcutaneous, topical, oral, and intramuscular. It can vary with different administration methods. For example, in the case of intravenous injection, 0.0 2 to 2 hours at 0. 2m g / k times, In the case of subcutaneous administration may vary as 1 to 20 Om gZm ² Z day.

[Brief description of drawings]

 Figure 1 shows the results of an experiment to examine the ability of human COS-7 cells transfected with the SR-PSOX gene to adhere to the PS coat plate (the number of adherent cells was 100 (% FIG. Figure 2 shows the results of an experiment to examine the binding ability of oxidized LDL in COS-7 cells transfected with the human SR-PSOX gene (the amount bound per mg of oxidized LDL added to the experimental system). is there. FIG. 3 is a graph showing the results of an experiment for examining the resolution of oxidized LDL in COS-7 cells into which the human SR-PSOX gene was introduced (the amount of degradation per lmG of oxidized LDL added to the experimental system).

[Best Mode for Carrying Out the Invention]

 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. In the following examples, unless otherwise specified, each operation relating to gene manipulation is described in “Molecular Cloning J [Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989] Published in the year] ', or when using commercially available reagents and kits, use them according to the instructions of the commercial product.Unless otherwise specified, the ligation was performed using a standard buffer solution. , With the incubation temperature and time, and approximately equimolar amounts of the ligated DNA fragments,

Performed using approximately 10 units of T4 DNA ligase per NA.

[Example 1] Gene closing of SR—PS OX gene The human mononuclear cell line THP-1 stimulated with PMA consists of a plastic plate coated on the surface with phosphatidylserine (PS), which is considered to be a physiological ligand for scavenger receptors. (PS coated plate). Known class A scavenger receptors (hereinafter "SR-A") have been reported to recognize oxidized LDL and acetylated LDL (Kodama T., et al. Nature 1990 Feb 8; 343 (6258) ): 531-5) However, PMA-activated THP-11 cells do not express CD36, which is known as a class A scavenger receptor.Thus, PMA-stimulated THP-1 cells produce oxidized LDL. It was suggested that they might express a novel submerged receptor for PS and PS. Therefore, we attempted to clone this novel scavenger receptor by the following method. First, to isolate the receptor cDNA on THP-1 cells stimulated with PMA, a cDNA library of THP-1 cells was prepared and introduced into the COS-'7 cell line as follows. Subsequently, 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 RPMI 1640 medium containing 10% fetal calf serum (hereinafter referred to as “FBS”) at a final concentration of 160 nM. The cells were cultured for 72 hours after the addition (the same procedure was followed for all THP-1 PMA stimulations). After preparing total RNA from PMA-stimulated THP-1 cells using total RNA extraction and purification reagents (RNAzo1: Biotex Laboratories), use the mRNA isolation kit (Pharmacia). To prepare mRNA. Using this mRNA as type III, cDNA was prepared 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) to create a cDNA library. Method for introduction into expression vector, transformation into Escherichia coli, selection method, method for recovering plasmid

'Method (Mr. Sambrook et al., Molecular Cloning: A Laboratory Manual ^ Part 2; Konore The method described in DeSpring Harbour Laboratory Press, Cold Spring Harbor, New York (1989) was followed. This cDNA library had 1 × 10 ⁶ independent clones.

CO S-7 cells (ATCC CRL-1651) were cultured in semi-confluent Dulbecco 's modified Eagle' s medium (DMEM) containing 10% FBS, and then trypsinized. treated cells were harvested, as a 2 X 1 0 ⁷ cells / ml K- PB S (8. 1 mM dipotassium phosphate, 1. 46 mM phosphate monobasic potassium, 3 0. 8 mM NaCl And 120.7 mM potassium chloride), and the cDNA library obtained above was introduced by electroporation.

Preparation of the PS-coated plate and examination of binding were performed as follows. That is, after dissolving phosphatidylserine (sodium salt, derived from cereal brain: Avanti-Po-Lipids) at a concentration of 8 μg / m1 in cold methanol,

(Ultrasonic: manufactured by Yamato Scientific Co., Ltd.) for 5 minutes at 4 ° C, and this solution is treated with a plastic plate (Φ35mm untreated petri dish: Iwaki Glass)

And dried at room temperature for 5 hours in a clean bench. As a control, a mixture obtained by pouring methanol alone into a plastic plate was used in the same manner. Then, to reduce non-specific binding to the plate, pour DMEM medium containing 10% FBS and 0.5% gelatin into the PS-coated plate, and incubate at 37 ° C for 30 minutes (blocking). Then, the plate was washed twice with a PBS solution containing 5 mM EDTA and 0.5% gelatin (hereinafter referred to as “solution 'A”). The transformed COS cells were seeded on the PS-coated plate thus obtained, left in solution A for 5 minutes, and non-adhered cells were separated and removed from the plate by pitting.

A vector (episome DNA) containing the cDNA introduced into the cells was extracted and isolated with 0.6% SDS and 1 OmMEDTA solution from the CO S-7 cells remaining on the PS-coated plate and isolated. . Electrification of the obtained episode DN A After introducing into a competent Escherichia coli (Elect-Mouth Max DH10B: Gibco BRL) by the transformation method, the transformed Escherichia coli was cultured to purify the amplified plasmid. The purified plasmid was reintroduced into COS-7 cells and selected by binding to PS-coated plates. In this way, after transfection into COS-7 cells and selection of binding to PS-coated plates were repeated 5 times, a single clone showing remarkable binding to PS-coated plates was isolated and retained in the clone. The gene introduced into the plasmid was designated SR-PS. The nucleotide sequence of this gene was determined by the nucleotide sequencing method using the dideoxy termination method [Sanger F., Nicklen S., Coulson AR, Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977)]. It was decided by As a result, the coding region of this cDNA clone contained an open reading frame starting at the start codon (atg) and ending at the stop codon (tga). In addition, the determined nucleotide sequence in the etasspletion sequence tag

A homology search of the database (hereinafter referred to as “EST”) revealed that one EST clone (GenBank accession number AA2900712) was homologous to SR-PS. This ES ST clone was transformed into SR—PSOX

(scavenger receptor for phophatidyi serine and oxidized lipoprotein: SEQ ID NO: 1). However, Ala at amino acid number 181 in the amino acid sequence (SEQ ID NO: 2 in the sequence listing) encoded by the SR-PSOX gene was Val in SR-PS. The SR-PSOX gene is located on human chromosome 17 and encodes a novel type I membrane protein consisting of 254 amino acids. The transformed plasmid E. coli XL 1 Blue pME 18 S—h SR—P SOX SANK 7 1300 carrying this SR—PS〇X gene-containing plasmid pME18S—hSR—PSOX Dated August 1, 2000, 1-1 1-1 Higashi, Tsukuba, Ibaraki, Japan It was internationally deposited at the Nyo Biological Depositary Center (formerly known as the Institute of Biotechnology and Industrial Technology), and was assigned the deposit number F ERM BP-7260.

[Example 2] Cloning of mouse and porcine SR_PSOX homologous gene The mouse and pig homologous gene of human SR-PSOX gene was isolated by the following RT-PCR method. First, total RNA was prepared from the spleen and thymus of a B ALB / c mouse using an RNA extraction reagent (RNAzo1: Biotex Laboratories) as described in the attached manual did. Using this total RNA as a starting material, cDNA was prepared using a cDNA synthesis kit (First'Strand cDNA synthesis kit: manufactured by Amersham Pharmacia) as described in the attached instruction manual. . Similarly, a porcine fetal (derived from all organ tissues) cDNA library was obtained for cloning of porcine homologous genes. Next, the nucleotide sequence shown below:

5'—tcagaattcc caacaagctc cgcagaa —3 '(upstream primer for mouse: SEQ ID NO: 7 from IJ table);

5 '-tcaggatcct gtaggcgcta gggtcttg-3' (downstream primer for mouse: SEQ ID NO: 8 in Sequence Listing); ·

 5'-tcagaattcg aggccgccgg gagaagatg -3 '(upper primer for pig: SEQ ID NO: 9 of IJ system IJ table); and

 5'-tcactcgagc tttcatcctt agctcaggtg -3 '(downstream primer for pigs: SEQ ID NO.

Were synthesized and used as primers, and PCR was performed under the following conditions. Reaction liquid composition:

 c DNA library 10 0 μ 1

 Upstream primer l O pmo l

Downstream primer 1 0 pmo 1 1 0 XP CR buffer 1 Ι Ο μ Ι

 d NT P s (2.5 mM each) 4 μ 1

 Taq polymerase (Promega) 5 units

 The volume was adjusted to 100 μl with sterile water.

Temperature conditions: After holding at 94 ° C for 2 minutes, then 30 seconds at 94 ° C, 52. 1 minute at C, 7

The temperature cycle of 2 minutes at 2 ° C was repeated 30 times, and then the mixture was further incubated at 72 ° C for 10 minutes. The reaction temperature of all the PCRs in the examples was adjusted using a GeneAmp PCR System 9600 (manufactured by PerkinElmer Japan, Inc.).

A part of the PCR reaction solution was electrophoresed on a 1% agarose (FMC Bioproducts) gel. As a result of estimating the chain length of the PCR product band by comparison with the molecular weight marker band electrophoresed on the same gel, the size of the mouse and butter SR-PSOX genes was about 800 bp in both cases. there were. Next, each PCR product was incorporated into a plasmid vector using an original TA cloning kit (Invitrogen). First, add 50 ng of each PCR reaction solution (corresponding to about 10 ng of the target PCR product by electrophoresis assay) and 50 ng of pCRII vector (attached to the kit) to a ligase reaction buffer (6 mM). Tris hydrochloride (pH 7.5), 6 mM magnesium chloride, 5 mM sodium chloride, 7 mM jS-mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1 mM spermidine, 0.1 mg Four ml of T4 DNA ligase (attached to the kit) was added to the plate at room temperature and kept at 14 ° C for 15 hours. Next, Combinant E. coli with 2 μl of 0.5 M jS-mercaptoethanol Τ 0 Ρ 10 F '(attached to the kit) Add 50% of the ligase reaction mixture to 50 μl' The mixture was mixed and 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, ◦.05% sodium chloride, 2.5m potassium chloride, 1mM magnesium chloride, 2OmM glucose) 1 was added and subjected to reciprocal shaking culture (37.C, II O rpm) for 1 hour. The culture was added to 100 μg / m 1 of ampicillin. L-broth agar medium containing 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 0.1% glucose, 0.6% noct agar (manufactured by Difco) The plate was spread on a plate, and incubated at 37 ° C. for static incubation.A single ampicillin-resistant colony that appeared on the plate was selected, and the colony was picked up with a platinum loop to obtain 100 μg / m After culturing overnight at 37 ° C in 5 ml of L-broth medium containing 1 ml of ampicillin, the culture was centrifuged to collect the precipitated cells, and plasmid was collected by the alkaline method. The nucleotide sequence of the cDNA cloning portion in the plasmid thus obtained was determined by nucleotide sequencing using the dideoxy chain termination method [Sanger F., Nicklen S., Coulson AR, Pro Natl. Acad. Sci. USA, 74: 5463-5467 (1977)]. -As a result, the mouse and pig SR-PSOX homologous genes obtained had an open reading frame of 738 bp and 750 bp, respectively (SEQ ID NO: in the sequence listing). 3 and SEQ ID NO: 5) The homology between these nucleotide sequences was analyzed using an analysis software (Macvector: manufactured by Oxford Morequiula Group) The obtained mouse and pig SR-PS OX The amino acid sequences encoded by the homologous genes (SEQ ID NO: 4 and SEQ ID NO: 6 in the sequence listing) correspond to 44% and 51% of the amino acids encoded by the human SR-PSOX gene, respectively. In the nucleotide sequence of the open reading frame, the mouse and porcine SR-PSOX homologous gene was found to be identical to the human SR-PSOX gene. The expression plasmid pME18S-mSR-PSOX containing the mouse SR-PSOX gene thus obtained was 67% and 70% identical, respectively. E. coli XL 1 Blue pME 18 S—mS R—PS〇X SANK 7 1400 and porcine SR—Transformation with plasmid pME 18 S—p SR—PS 〇X containing the OX gene E. coli XL 1 B lue pME 18 S -p S RP SOX SANK 7-1500, dated August 1, 2000, is the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Tsukuba-Higashi, Ibaraki, Japan. International Depositary with the National Institute of Advanced Industrial Science and Technology (AIST), with deposit numbers F ERM BP-7261 and FE RM BP-7262, respectively.

[Example 3] Scavenger receptor activity

 1) Binding to PS coat plate

In order to confirm the binding activity of the SR—PS OX gene product to PS, the binding of COS-7 cells transfected with the SR-PS OX gene to a PS-coated plate was examined. That is, pME18S-hSR-PSOX was introduced into COS-7 cells by the method described in Example 1, and the transformed COS-7 cells were plated on a PS-coated plate and allowed to stand for 30 minutes. The plate was then washed three times with solution A. '' In addition, various additives (PS ribosome, phosphatidylcholine (PC) ribosome, oxidized LDL (preparation method is described in Example 3 below), po1yI, dextran sulfate When adding cells, pre-incubate the cells and the additives for 15 minutes before performing the binding assay, then seed the cells on a PS-coated plate, let stand for 30 minutes, and then use Solution A three times. Plates were washed. The number of cells attached to each plate was measured with a light microscope. All experiments were performed in triplicate, and the number of adherent cells per square well was calculated as the average. As a result, the COS-7 cells into which the SR-PS OX gene was introduced did not bind to the PS-uncoated plate, but specifically bound to the PS-coated plate. COS-7 cells transfected with mouse SR—PS OX similarly bound to the PS-coated plate. The binding was inhibited by 100 μΜ of PS ribosome, but not by 100 μΜ of phosphatidylcholine (PC) liposome. Furthermore, PS-coated plate of COS-7 cells with SR-PS OX introduced Binding to lipoproteins such as oxidized LDL, poly I, and dextran sulfate was also inhibited by chondroitin sulfate, LD, and acetyl LDL. (Figure 1 ) .

2) Incorporation of oxidized LDL into cells ''

Next, the uptake of oxidized LDL into COS-7 cells into which the SR-PSOX gene had been introduced was examined by the method described below. First, potassium bromide was added to healthy human peripheral blood plasma (specific gravity 006) to a specific gravity of 1.019, and then ultracentrifuged at 4 ° C and 58000 rpm for 20 hours (Ultracentrifuge manufactured by Beckman). ) To remove the upper layer. Next, potassium bromide was added to the obtained lower layer solution so that the specific gravity became 1.063, and the mixture was ultracentrifuged again at 4 ° C and 58,000 rpm for 20 hours. The upper layer after centrifugation was collected as human LDL (New Biochemistry Experiment Lecture 4 Lipid I Neutral Lipid Lipoprotein (edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin)) 7. Fractionation of lipoprotein 'Purification 181, 1993 and Havel RJ, et al., J. Clin. Invest. 34: 1345-1353 (1955)). Then, the oxidized LDL was obtained by incubating the obtained human LDL (hereinafter referred to as “native LDL”) in a 7.5 μΜ copper sulfate solution at 37 ° C. for 24 hours. Also, a native method of native LDL and acetic anhydride [Goldstein J., Ho Y. I., Basu S. II., Brown MS, Proc. Natl. Acad. Sci. UAS, 76: 333-337 (1979)] The reaction was carried out according to the procedure described above to obtain an acetylated form of LDL. The formation of oxidized DL and acetylated LDL was confirmed by a change in mobility using agarose gel electrophoresis. According to the method described in the literature [Voyta JC, Via DP, Butterfield CE, Zetter BR, J. Cell Biol., 99: 2034-2040 (1984)], oxidized and DL was converted to 1'1'-dioctadecyl-3,3,3. Labeled with ', 3'-tetramethylindocarbocyanine perchlorate (hereinafter referred to as “DiI”: manufactured by Molecular Probes). Furthermore, iodine monochloride Motoho the human LD L [MacFarlane AS, Nature, 182: 53 (1958)] by was prepared which was radiolabeled with ^{1 25} I. DiI-labeled oxidized LDL (5 μg / m 1) was added to COS cells transfected with the human SR—PS OX or mouse SR—PS OX gene by the method described in Example 2 above. 00 <ug gm1 Unlabeled oxidized LDL, native LDL or acetylated LDL, p01yI at 100 ^ g / m1 or in the presence or absence of 100 μg / m1 dextran sulfate The reaction was carried out at 37 ° C for 2 hours. Next, the cells were washed three times with DMEM, and the uptake of Di1-labeled lipoprotein into the cells was examined under a fluorescence microscope. As a result, remarkable uptake of Di I-oxidized LDL was observed in CO S-7 cells transfected with human SR-PS OX and mouse SR-PS OX genes. This uptake was suppressed by the coexistence of excessive amounts of unlabeled oxidized LDL, poly I, and dextran sulfate, but not by the coexistence of acetylated LDL and native LDL. From the above results, 5? 3〇 was shown to be a specific receptor for oxidized LDL.

3) Oxidized LDL binding capacity and decomposition activity

Next, using COS-7 cells into which the human SR-PSOX gene had been introduced, the binding ability and proteolytic activity of ¹²⁵ I-labeled oxidized LDL were examined as follows.

The binding of ¹²⁵ I-labeled oxidized LD to COS-7 cells transfected with SR-PS OX gene 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 incubated at 4 ° C for 30 minutes in a cold DMEM medium containing 1 OmM Hepes-sodium hydroxide (pH 7.4) and 10% FBS (hereinafter “medium”). . after removal of the pretreated medium with: ( "hereinafter) 1 ml, the cells in the culture medium C 0. 5 m l containing ^{1 25} I-labeled oxidized LDL at various concentrations was added and incubated for 2 hours at 4 ° C. The medium is removed, and the cells are immediately washed with a Tris washing solution (5 OmM Tris-HCl, 15 OmM NaCl, pH 7.4) containing SmgZml of serum albumin (hereinafter referred to as “BSA”). After washing, B The plate was washed twice with SA-free Tris washing solution for 10 minutes each. To the washed cells, add 0.5 ml of 0.2N sodium hydroxide aqueous solution and shake for 3 to 4 hours to lyse the cells, and measure the radioactivity eluted from the cells using a gamma counter. Was. The ^{1 25} I degradation activity of the labeling oxide LD L was measured as follows. Chi words, the ^{1 25} I-labeled oxidized LD L which initially dissolved in the medium C was 4-6 hours incubation at 3 7 ° C in addition to the cells. Next, trichloroacetic acid was added to the medium, the mixture was allowed to stand at 4 ° C for 30 minutes, and then centrifuged at 15,000 rpm for 30 minutes to collect the supernatant. Potassium iodide and hydrogen peroxide were added to the supernatant, mixed, and the mixture was centrifuged after addition of black hole form. 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. 2, COS-7 cells transfected with SR-PS OX gene bound ¹²⁵ I-labeled oxidized LDL, and this binding was caused by excess unlabeled oxidized LDL. Suppressed but not acetylated LDL or native LDL. Further, SR- PS OX gene CO S- 7 cells introduced has been disassembled ^{1 25} 1 labeled oxide L DL, this decomposition is also inhibited by unlabeled oxidized LDL Excess Asechiru of LD L or native It was not inhibited by LDL (Figure 3). Similar experiments were performed using CHO cells (CHO-SR-A) stably expressing the known scavenger receptor hSR-A gene. As a result, acetylated LDL was transferred to CHO-SR-A cells. Inhibition of DiI monoxide LDL was suppressed, confirming that there was no abnormality in acetylated LDL. Based on the above results, SR-PS OX is a specific receptor for oxidized LDL. Cetyled LDL and native LDL were shown not to be high affinity ligands for SR-PSOX. In the experimental system according to any one of the above 1) to 3), by adding a test substance in the same manner as adding various additives, and observing a change in scavenger receptor activity, It is possible to carry out a test for a Surebenja monoreceptor inhibitor useful as an agent for preventing or treating arteriosclerosis.

[Possibility of industrial use]

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

Claims

The scope of the claims  1. A protein according to any one of 1) to 3) below:  1) Any one of amino acid numbers 1 to 254 of SEQ ID NO: 2 in the sequence listing, amino acid numbers 1 to 246 of SEQ ID NO: 4 in the sequence listing and any one of amino acid numbers 1 to 250 of SEQ ID NO: 6 in the sequence listing A protein consisting of the amino acid sequence shown;  2) Any one of amino acid numbers 1 to 254 of SEQ ID No. 2 in the sequence listing, amino acid numbers 1 to 246 of SEQ ID No. 4 in the sequence listing and any one of amino acid numbers 1 to 250 of SEQ ID NO. 6 in the sequence listing A protein having an amino acid sequence in which one or more amino acids are added, deleted, inserted, and / or substituted in the amino acid sequence shown, and which has a force-venger receptor activity;  3) Any one of amino acid numbers 1 to 254 of SEQ ID NO: 2 in the sequence listing, 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 protein consisting of an amino acid sequence having a homology of 50% or more with the amino acid sequence shown in the above, and having a scavenger receptor activity;  4) Transformed Escherichia coli E.co1 i XL 1 Blue pME 18 S-h SR-P SOX SANK 7 1300 (FERBP-7260), E.coli XL 1 B lue pME 18 S-mS RP S OX SANK 7 1 400 (FE RM BP—726 1) and E. coli XL 1 B luep ME 18 S— p SR— P SOX SANK 7 1 500 (FERM BP — A protein consisting of the amino acid sequence encoded by the DNA inserted into the multicloning site of pME18S, in the plasmid carried by any one of the above 72).  2. Amino acid sequence represented by amino acid numbers 1 to 254 of SEQ ID NO: 2 in Sequence Listing 3 An amino acid sequence represented by amino acid numbers 1 to 246 of SEQ ID NO: 4 in the sequence listing. 4 Amino acid sequence represented by amino acid numbers 1 to 250 of SEQ ID NO: 6 in the sequence listing 5. Code the protein according to any one of claims 1 to 4 DNA.  6. DNA according to any one of the following a) to d): a) Any one of nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the Sequence Listing, nucleotide numbers 1 to 738 of SEQ ID NO: 3 in the Sequence Listing and nucleotide numbers 13 to 762 of SEQ ID NO: 5 in the Sequence Listing DNA consisting of a nucleotide sequence shown as one; b) nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the sequence listing, nuclei of nucleotides 1 to 732 of SEQ ID NO: 3 in the sequence listing and SEQ ID NO: 1 in the sequence listing 5.A protein having a scavenger receptor activity consisting of a nucleotide sequence that hybridizes under stringent and stringent conditions to DN. DNA encoding; c) Any one of nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the Sequence Listing, nucleotide numbers 1 to 738 of SEQ ID NO: 3 in the Sequence Listing and nucleotide numbers 13 to 7 62 of SEQ ID NO: 5 in the Sequence Listing A DNA encoding a protein having a scavenger receptor activity, comprising a nucleotide sequence having a nucleotide sequence identity of 67% or more with a DNA consisting of the nucleotide sequence shown in one of them; d) Transformed Escherichia coli E. c 0 1 i XL 1 Blu e pME 18 S— h S R— P SOX SANK 7 1300 (F ERM B P— 7 260), E.coli XL 1 B lue pME 18 S-mS RP SOX SANK 7100 (FE RM BP— 726 1) and E. coli XL 1 B luep ME 18 S- p S RP SOX SANK 7 1500 (FE RM BP — DNA inserted into the multicloning site of pME18S in the plasmid carried by any one of 726 2).  7. A recombinant vector containing DNA according to claim 5 or 6.  8. The recombinant vector according to claim 7, which is an animal cell expression vector. 9. Transformed E. coli XL 1 BLue pME 18 S— h SR-P SOX SANK 7 1300 (FE RM BP— 7 260), E. coli XL 1 Blue ME 18 S— m SR— PS OX S ANK 7 140 0 (FE RM BP— 7 26 1) and E. coli XL 1 B luep M Claims 7 or 8 characterized in that any one of El 8 S- p S RP SOX SANK 7150 00 (FE RM BP— 7262) is a plasmid held by it. The recombinant vector according to any one of the preceding claims.  10. A host cell carrying the recombinant vector according to any one of claims 7 to 9.  1 E. coli. XL 1 B luep ME 18 S-h SR— P SOX SANK 7 1 300 (FE RM BP — 7 260), E.co 1 i XL 1 B luep ME 1 8 S—m SR—PSOX SANK 71.4 00 (FE RM BP-726 1) and E. coli XL 1 Blue ME 18 S—p SR—PS OX SANK 7 1 500 (FE RM BP—72 6 2 The host cell according to claim 1, wherein the host cell is selected from the group consisting of:  1 2. Add oxidized LDL to a host cell transformed with the recombinant vector for expressing an animal cell according to claim 8 or 9, and allow the host cell to incorporate the oxidized LDL. In the system for binding or binding, the culture is performed with or without the addition of the test substance when adding the oxidized LDL, and then the test substance inhibits the uptake or binding of the oxidized LDL into the host cell. A method for testing an agent for preventing or treating arteriosclerosis, characterized by examining the presence or absence of the agent.  1 3. Culturing a host cell transformed with the recombinant vector for expressing an animal cell according to claim 8 or 9 on a phosphatidylserine-coated plastic plate, thereby obtaining the host cell. In the system to be attached to the plate, during or before culturing on the plate, the host cells are cultured with or without the addition of a test substance. A method for testing an agent for preventing or treating arteriosclerosis, which comprises examining whether or not a test substance inhibits adhesion of a substance.  1 4. Use of D Ν 記載 according to claim 5 or 6 for the manufacture of a test kit for an agent for preventing or treating arteriosclerosis. 1 5. 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. 16. A pharmaceutical composition comprising the nucleic acid according to claim 15 as an active ingredient.  17. Use of the nucleic acid according to claim 15 for the manufacture of an agent for preventing or treating arteriosclerosis.  18. An antibody that specifically binds to the protein according to any one of claims 1 to 4.