WO2002012482A1 - Recepteur capteur et son utilisation - Google Patents

Recepteur capteur et son utilisation Download PDF

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Publication number
WO2002012482A1
WO2002012482A1 PCT/JP2001/006750 JP0106750W WO0212482A1 WO 2002012482 A1 WO2002012482 A1 WO 2002012482A1 JP 0106750 W JP0106750 W JP 0106750W WO 0212482 A1 WO0212482 A1 WO 0212482A1
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seq
amino acid
sequence listing
protein
sequence
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PCT/JP2001/006750
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English (en)
Japanese (ja)
Inventor
Shin Yonehara
Takeshi Shimaoka
Toru Kita
Noriaki Kume
Manabu Minami
Hiroharu Kataoka
Kazutaka Hayashida
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Sankyo Company, Limited
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Priority to AU2001276742A priority Critical patent/AU2001276742A1/en
Publication of WO2002012482A1 publication Critical patent/WO2002012482A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • 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.
  • LDL low-density lipoprotein
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 .
  • SMC smooth muscle cells
  • 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.
  • CHO cells Chinese hamster ovary cells
  • scavenger receptor in the symptoms of atherosclerosis.
  • 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).
  • LDL-like particles adhere to the matrix of the blood vessel wall several days after starting a high-cholesterol diet.
  • 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.
  • macrophages can be located at the edge or deeper of the lesion.
  • scavenger receptors 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.
  • MMarco SR Marcos force receptor receptor
  • the Marcos Force Benger receptor is also involved in binding Gram-positive and negative bacteria.
  • the existence of unknown scavenger receptors has been suggested (Cl inica Chimica Acta 286 (1999) 191-205). And features were not disclosed.
  • 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.
  • the present invention firstly relates to a protein described in any one of the following 1) to 4):
  • the present invention relates to a DNA encoding the above protein.
  • DNA of the present invention examples 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.
  • 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;
  • nucleotide numbers 97 to 858 of SEQ ID NO: 1 in the Sequence Listing 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;
  • the present invention includes all DNAs having a nucleotide sequence encoding the above protein.
  • the present invention relates to a recombinant DNA vector containing the above DNA.
  • 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.
  • 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.
  • the present invention is not limited to this.
  • the present invention relates to a host cell transformed with the above-described recombinant DNA vector.
  • suitable host cells of the present invention include transformed E.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention relates to an antisense nucleic acid of the above DNA and its use.
  • the present invention relates to an antibody that specifically binds to the above protein.
  • 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.
  • 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.
  • guanidine thiocyanate / cesium chloride ultracentrifugation method 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.
  • a method of adsorbing mRNA on an oligo (dT) cellulose column and then eluting and purifying the mRNA may be used.
  • RNA can be further fractionated by sucrose density gradient centrifugation or the like. ' ⁇
  • double-stranded cDNA can be synthesized from this single-stranded cDNA.
  • 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.
  • 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.
  • lambda phage ZAP II Stratagene
  • host E. coli XL1-B1ue MRF 'and JM109 strains form plaques
  • Recombinants can be selected based on the presence or absence of color development due to metabolism.
  • a plasmid vector can be used in addition to a lambda phage vector.
  • various commercially available cDNA libraries for example, Clontech
  • 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.
  • 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.
  • 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.
  • 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.
  • prokaryotic host cells 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.
  • 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.
  • the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.
  • K12 strain and the like are often used as Escherichia coli
  • pBR322 and pUC-based plasmids are generally used as vectors, but are not limited thereto, and various known strains, And vectors can be used.
  • tryptophan (trp) promoter lactose (lac) promoter
  • tryptophan 'lactose (tac ) promoter
  • lipoprotein ( lpp) promoter polypeptide elongation factor Tu
  • tufB tufB promoter and the like. Any promoter can be used for production of the protein of the present invention.
  • 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.
  • the present invention is not limited to this.
  • Extracellular secretory expression is also possible by linking a D ⁇ ⁇ sequence that encodes the signal peptide sequence of ⁇ -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.
  • 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.
  • the expression vector 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.
  • the cells can be incorporated into COS cells, and thus desired transformed cells can be obtained.
  • 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
  • a transformed cell stably producing a protein can be obtained.
  • insect cells 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, ⁇ : 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 ⁇ ⁇ V) is frequently used (Kidd, IM and VC Emery (1993)
  • vectors using baculovirus P10 or a promoter of the same basic protein can also be used.
  • 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). .
  • yeast 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.
  • 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).
  • a secretory protein 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.
  • the secretory signal sequence of yeast alpha-phater at the N-terminal side and the ⁇ ⁇ ⁇ 2 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,
  • 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.
  • 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.
  • DMEM Eagle's medium
  • the protein of the present invention expressed in this manner is subjected to various separation operations utilizing its physicochemical properties, chemical properties, etc.
  • 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
  • the protein of the present invention can be easily produced in large quantities with high yield and high purity.
  • 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.
  • an automatic protein amino acid sequencer for example, Model 492, manufactured by PerkinElmer Japan ABRI Biosystems.
  • 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.
  • 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.
  • 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).
  • 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, ⁇ . F. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666).
  • 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 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.
  • 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.
  • 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.
  • 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
  • Examples include DNA encoding a protein having scavenger receptor activity.
  • 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.
  • 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.
  • 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.
  • scavenger receptor inhibitors can be evaluated or screened.
  • 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,
  • myeloma preparation of myeloma cells (hereinafter referred to as "myeloma");
  • 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.
  • 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.
  • an adjuvant such as Freund's complete or incomplete adjuvant or Rimiyodiban
  • 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).
  • 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.
  • 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.
  • the measurement of the antibody titer in the present invention can be performed, for example, by the following procedure according to the ELISA method.
  • 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 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.
  • a serially diluted sample eg, mouse serum
  • 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.
  • FCS fetal calf serum
  • IMDM Iscove's modified Dulbecco's medium
  • DMEM 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.
  • 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.
  • 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.
  • a serum-free medium for example, RPMI 164
  • PBS phosphate buffered saline
  • HAT hypoxanthine 'aminopterin' Gin
  • IL-12 mouse interleukin-12
  • plate normal medium
  • the myeloma cell is an 8-azaguanine-resistant strain, that is, a hypoxanthine 'guanine' phosphoribosyltransferase (HGPRT) deficient strain
  • HGPRT hypoxanthine 'guanine' phosphoribosyltransferase
  • the unfused myeloma cell and a fusion cell of myeloma cells are Cannot survive in HAT containing medium.
  • 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.
  • the hybridomas can be selected.
  • HT medium a medium without aminobuterin
  • 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.
  • 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.
  • 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.
  • 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.
  • a monoclonal antibody that specifically binds to the protein of the present invention can be obtained.
  • 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.
  • a commercially available monoclonal antibody purification kit for example, MabTrapGII kit; manufactured by Pharmacia
  • the monoclonal antibody thus obtained has high antigen specificity for the protein of the present invention.
  • 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.
  • 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.
  • a commercially available kit for identification eg, mouse type kit; manufactured by Biorad
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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).
  • 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).
  • PS coated plate phosphatidylserine
  • SR-A Known class A scavenger receptors
  • 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.
  • cDNA-transfected COS-7 cells that bind to the PS-coated plate were selected, and DNA was isolated from the cells.
  • 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.
  • FBS fetal calf serum
  • 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.
  • 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.
  • DMEM Dulbecco 's modified Eagle' s medium
  • 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 ⁇ g / m1 in cold methanol,
  • 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.
  • 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
  • EST 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
  • SR-PSOX scavenger receptor for phophatidyi serine and oxidized lipoprotein: SEQ ID NO: 1.
  • 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 The transformed plasmid E.
  • RNAzo1 Biotex Laboratories
  • cDNA was prepared using a cDNA synthesis kit (First'Strand cDNA synthesis kit: manufactured by Amersham Pharmacia) as described in the attached instruction manual.
  • a porcine fetal (derived from all organ tissues) cDNA library was obtained for cloning of porcine homologous genes.
  • the volume was adjusted to 100 ⁇ l with sterile water.
  • 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.).
  • each PCR product was incorporated into a plasmid vector using an original TA cloning kit (Invitrogen).
  • 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
  • T4 DNA ligase Attached to the kit
  • Combinant E. coli with 2 ⁇ l of 0.5 M jS-mercaptoethanol ⁇ 0 ⁇ 10 F ' Add 50% of the ligase reaction mixture to 50 ⁇ l' The mixture was mixed and left on ice for 30 minutes, then at 42 ° C.
  • 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.
  • 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.
  • coli XL 1 B lue pME 18 S -p S RP SOX SANK 7-1500 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.
  • 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 ⁇ of PS ribosome, but not by 100 ⁇ of phosphatidylcholine (PC) liposome.
  • 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 ) .
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne une nouvelle protéine possédant une activité de récepteur capteur, qui peut être utilisée comme cible d'agents prophylactiques et de remèdes pour l'artériosclérose. Plus spécifiquement, l'invention concerne une protéine possédant une séquence d'acides aminés dans les positions 1 à 254 de SEQ ID No:1 de l'ordre des séquences, un ADN codant pour cette protéine, un vecteur d'ADN recombiné contenant cet ADN, un hôte transformé par ce vecteur d'ADN recombiné, ainsi que l'utilisation de la protéine susmentionnée. Par ce procédé, il est possible de chercher un nouveau composé qui possède une activité inhibitrice spécifique au récepteur capteur de cette invention, et qui est utile en tant qu'agent prophylactique ou remède pour l'artériosclérose.
PCT/JP2001/006750 2000-08-09 2001-08-06 Recepteur capteur et son utilisation WO2002012482A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061471A2 (fr) * 1998-05-29 1999-12-02 Incyte Pharmaceuticals, Inc. Proteines transmembranaires humaines
WO2000044784A1 (fr) * 1999-01-27 2000-08-03 Compugen Ltd. Sequences d'acide nucleique et d'acide amine
WO2000055375A1 (fr) * 1999-03-17 2000-09-21 Alphagene, Inc. Proteines secretees et polynucleotides les codant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061471A2 (fr) * 1998-05-29 1999-12-02 Incyte Pharmaceuticals, Inc. Proteines transmembranaires humaines
WO2000044784A1 (fr) * 1999-01-27 2000-08-03 Compugen Ltd. Sequences d'acide nucleique et d'acide amine
WO2000055375A1 (fr) * 1999-03-17 2000-09-21 Alphagene, Inc. Proteines secretees et polynucleotides les codant

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BALJINDER S. ET AL.: "Scavenger receptors and oxidized low density lipoproteins", CLINICA CHIMICA ACTA, vol. 286, 1999, pages 191 - 205, XP002946211 *
CARNINCI P. ET AL.: "High-efficiency full-length cDNA cloning", METHODS IN ENZYMOLOGY, vol. 303, 1999, pages 19 - 44, XP002946208 *
MATLOUBIAN M. ET AL.: "A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo", NAT. IMMUNOL., vol. 1, no. 4, October 2000 (2000-10-01), pages 298 - 304, XP002946210 *
SHIMAOKA T. ET AL.: "Molecular cloning of a novel scavenger receptor for oxidized low density lipoprotein, SR-PSOX, on macrophages", J. BIOL. CHEM., vol. 275, no. 52, December 2000 (2000-12-01), pages 40663 - 40666, XP002946209 *

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