WO2004048574A1 - Immunoreceptor proteins - Google Patents

Abstract

It is intended to provide novel immunoreceptor proteins positively or negatively controlling immune responses and genes encoding these proteins. A protein having the amino acid sequence represented by SEQ ID NO:2 in Sequence Listing or a protein having an amino acid sequence derived from the above-described amino acid sequence by deletion, substitution or addition of one to several amino acids and having a function of inhibiting an immune response of cells; a protein having the amino acid sequence represented by SEQ ID NO:4 in Sequence Listing or a protein having an amino acid sequence derived from the above-described amino acid sequence by deletion, substitution or addition of one to several amino acids and having a function of activating an immune response of cells; and genes encoding these proteins.

Description

 Description Immune receptor protein Technical field

 The present invention relates to a novel immunoreceptor protein, a gene encoding the protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, a method for producing the protein, and the like. Background art

 The immune response is controlled by a balance of positive and negative signals from activating and inhibitory cell membrane receptors. The activation of immune cells, such as lymphocytes and myeloid cells, is achieved by the activation of the cell membrane receptor, which recognizes and binds to its ligand, thereby causing the cell membrane receptor itself or an adapter molecule to associate with the receptor. Y_x_x— L- x (6-8)-Y- x_x-L (where x is the immunoreceptor tyrosine-based activation motif: ITAM) present in the intracellular region of It is initiated by phosphorylation of a tyrosine with a characteristic sequence consisting of any amino acid (Lanier, LL, Curr Op in Immunol, 13, 326-331, 2001; Bol land,

S., Ravetch, J.V., et al., Adv. Immunol., 72, 149-177, 1998). On the other hand, the activated immune cells bind to the immunoreceptor tyrosine-based inhibitory motif (Immunoreceptor tyrosine-based inhibitor), which is present in the intracellular region by binding the inhibitory cell membrane receptor molecule to the ligand. Tory motif: ITIM) (I / V / L / S)-Tyrosine with a characteristic sequence consisting of x-Yxx- (L / V) (χ indicates any amino acid) is a phosphatase Has been found to be repressively controlled through

(Lanier, L.L., Curr Op in Immunol, 13, 326-331, 2001; Ravetch, J.V.,

Lanier, L.L., Science, 290, 84-89, 2000).

The activating receptor and the inhibitory receptor are as follows: (1) Both the extracellular region is highly conserved; (2) The activating receptor has an activation motif ITAM in its intracellular region or transmembrane. Meets an ITAM-bearing adapter molecule such as FcsRIr or DAP12 in the region ④ The inhibitory receptor has the inhibitory motif ITIM in the intracellular region, ⑤ The activating receptor is in the cell compared to the inhibitory receptor The feature is that the area is short. Many of the activating receptors are also paired with inhibitory receptors, and both genes have been shown to be located in small clusters on the chromosome (Martin, AM , et al., Trends Immunol, 23, 81-8, 2002). A family of gene families containing both activating and inhibitory receptors is known, for example, KIR, CD94 / NKG2, and Ly49, a family of metastatic receptors that recognize MHC class I ligands. OLanier, LL, Annu Rev Immunol, 16, 359-93, 1998). Also, CD28 and CTLA4 expressed on T cells bind to the same ligand (eg, CD80 and CD86) and deliver opposite positive and negative signals, respectively (Chambers, CA & JP Allison, Curr Opin Cell Biol, 11, 203-210, 1999). Similarly, B cells and / or myeloid cells are derived from human and mouse FCT receptors (Ravetch, JY & RA Clynes, Annu Rev Immunol, 16, 421-32, 1998; Daeron, M., Annur Rev Immunol, 15, 203-34, 1997), mouse pair immunoglobulin (Ig) -like receptor (PIR) (Kubagawa, H., et al., Proc Natl Acad Sci, USA, 94, 5261-5266, 1997; Hayami, K., et al., J Biol Chem, 272, 7320-7327, 1997) and its human homolog Ig-like transcript

(ILT; CD85) (Samaridis, J. & Colon · Colonna, Eur J Immunol, 27, 660-665, 1997; Borges, L., et al., J Immunol, 159, 5192-5196, 1997; Wegtmann, N , et al., Curr Biol, 7, 615-618, 1997) and signal-regulating proteins.

(SIRP) (Kharitonenkov, A., et al., Nature, 386, 181-186, 1997; Fujioka, Y., et al., Mol Cell Biol, 16, 6887-6899, 1996; Ohnishi, H., et. al., J Biol Chem, 271, 25569-25574, 1996).

These family members are highly conserved in their extracellular domain structure and often recognize common or similar ligands, but the resulting immune cell response is positive (activated). And negative (suppressive). The immunological significance of the phenomena of inducing such completely contradictory immune responses is extremely interesting, and elucidates how positive and negative signals are integrated and controlled in vivo. Is thought to be very important for understanding the immune system.

 An object of the present invention is to obtain a novel pair of activation and inhibitory immune receptor genes involved in an immune response including proliferation, activation, and differentiation of myeloid cells and the like, And to clarify the functional properties. Disclosure of the invention

 The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the inhibitory immunoreceptor protein (MAIR-), which has an ITIM in the intracellular region and transmits an inhibitory signal, is derived from the mouse phage. 1) isolating an activated immunoreceptor protein (MAIR-II) having high homology to the immunoglobulin domain in the extracellular region of the inhibitory immunoreceptor protein and transmitting an activation signal; And succeeded in completing the present invention.

 That is, the present invention includes the following inventions.

 (1) An immunoreceptor protein shown in the following (a) or (b):

 (a) a protein having an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing

 (b) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and which has a function of suppressing a cellular immune response;

 (2) An immunoreceptor protein shown in the following (c) or (d).

 (c) a protein having an amino acid sequence represented by SEQ ID NO: 4 in the sequence listing

 (d) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, and which has a function of activating a cellular immune response

 (3) A gene encoding the immunoreceptor protein of the above (1) or (2).

 (4) A gene comprising DNA shown in the following (a) or (b):

 (a) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing

(b) DNA that encodes a protein that hybridizes under stringent conditions to a complementary sequence of the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, and that has a function of suppressing a cell's immune response. (5) A gene comprising the DNA shown in (c) or (d) below.

 (c) DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing

 (d) a DNA that hybridizes with a DNA complementary sequence consisting of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing under stringent conditions and encodes a protein having a function of activating a cellular immune response.

 (6) A recombinant vector containing the gene of (4) or (5).

 (7) A transformant transformed with the gene of (4) or (5).

 (8) The immunoreceptor protein of (1) or (2), wherein the transformant of (7) is cultured in a medium, and an immunoreceptor protein is collected from the obtained culture. Manufacturing method.

 (9) An antibody that recognizes one or both of the immunoreceptor proteins of (1) and (2) above.

 (10) The method for quantifying an immunoreceptor protein according to the above (1) or (2) using the antibody of the above (9).

 (11) The kit for quantifying the immunoreceptor protein of the above (1) or (2), comprising the antibody of the above (9).

 (12) A method for isolating a pair of activated immune receptor genes using a gene encoding an extracellular conserved region of an inhibitory immune receptor having an ITIM motif as a probe.

 (13) A method for isolating a suppressive immune receptor gene to be paired using a gene encoding an extracellular conserved region of an activated immune receptor having an ITAM motif as a probe.

(14) The immunoreceptor protein of the above (1) or (2), or a cell or cell membrane that expresses the protein, is allowed to act on a test substance expected to contain a ligand, and the protein has an affinity for the protein. The method for screening a ligand for an immunoreceptor protein according to the above (1) or (2), which comprises selecting a substance having the following.

 (15) A ligand for the immunoreceptor protein of (1) or (2) obtained by the method of (14).

 (16) (a) a case where the immunoreceptor protein of the above (1) or (2) is caused to act on a ligand for the immunoreceptor protein in the absence of a test substance;

Comparing the immunoreceptor protein of (1) or (2) with a ligand for the immunoreceptor protein in the presence of a test substance, A method for screening an agonist or agonist against said immunoreceptor protein, comprising selecting a substance which affects the binding between said ligand and said ligand.

 (17) A kit for screening an agonist or agonist against the immunoreceptor protein, comprising the immunoreceptor protein of (1) or (2).

 (18) An antagonist or agonist against the immunoreceptor protein of (1) or (2), obtained by using the method of (16) or the kit of (17).

 (19) A drug for controlling an immune function, comprising, as an active ingredient, the immune receptor protein of the above (1) or (2), or a gene encoding the protein.

 (20) A drug for controlling an immune function, which comprises, as an active ingredient, a ligand for the immunoreceptor protein of (1) or (2) or an antagonist or agonist for the immunoreceptor protein of (1) or (2). .

 (21) The reagent for detecting an immunoreceptor protein of the above (1) or (2), comprising the antibody of the above (9). BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1A shows the immunoreceptor proteins of the present invention, MAIR-Ia, MAIR-Ib, MAIR-IIa, and MAIR.

2 shows a comparison of amino acid sequences of -lib.

 FIG. 1B shows a schematic diagram of the MAIR-1 and MAIR-II proteins.

 FIG. 1C shows Southern blot analysis of the genomes MAIR-1 and MAIR-II.

 FIG. 2A shows the results of lysing BW5147 cells expressing Flag-tagged MAIR-1 or MAIR-II, and immunoblotting the protein with anti-Flag mAb under reducing or non-reducing conditions.

 Figure 2B shows BW5147 cells expressing Flag-tagged MAIR-1 or parental BW5147 cells were lysed, proteins were immunoprecipitated with anti-Flag mAb, and the precipitate was treated or treated with N-glycosidase F. 1 shows the results of immunoplotting with anti-Flag mAb under non-reducing conditions. FIGS. 3A and 3B show the results of analyzing the expression of MAIR-1 and MAIR-II transcripts by Northern blotting.

Figure 4A shows the cell membrane surface of MAIR-1 or MAIR-II in the transformed cells (BW5147 cells). The results of analysis of surface expression using anti-MAIR-1 (TX-8 mAb) or anti-MAIR-II (TX-13 mAb) are shown.

 Fig. 4 B, C, and D show the expression of MAIR-1 or MAIR-II on the cell membrane surface in spleen, bone marrow, or peritoneal macrophages from mice using anti-MAIR-1 (TX-8 mAb) or anti-MAIR-II (Π- 13 shows the results of analysis using 13 mAb).

 Figure 4E shows that B cells were cultured in the presence or absence of LPS, and then MAIR-1 or MAIR-II was expressed on the cell membrane surface using anti-MAIR-1 (TX-8 mAb) or anti-MAIR-11. (TX-13 mAb) shows the results of analysis.

 Figure 5A shows the results of MAIR-1 on peritoneal macrophages cross-linked with anti-MAIR-1 mAb (TX-8), incubated at 4 ° C or 37 ° C, and analyzed by flow cytometry. Show.

 FIG. 5B shows the results of MAIR-1 expressing cells cross-linked with anti-MAIR-1 mAb (TX-8) and analyzed under a confocal laser microscope before and after incubation at 37 ° C.

 Figure 6A shows the results of marrow cells derived from bone marrow stimulated with pervanadate, immunoprecipitated with control IgG or anti-MAIR-1, and immunoprecipitates were immunoblotted with anti-phosphotyrosine mAb (anti-pY). Is shown.

 Figure 6 6 shows bone marrow-derived mast cells stimulated with perpanadate and then immunoprecipitated with control IgG, anti-MAIR-1, anti-SHP-I, anti-SHP-II, or anti-SHIP, and immunoprecipitated. The results of immunoblotting of the product with anti-MAIR-1 are shown.

 FIG. 6C shows the results of IgE-mediated serotonin release from bone marrow-derived mast cells after cross-linking of MAIR-1 and anti-MAIR-1 mAb.

 Figure 7A shows that cells (293T) transformed with MAIR-II + DAP12 or MAIR-II + FcεRI were immunoprecipitated with control IgG, anti-DAP12, and anti-FcεRI, and the immunoprecipitate was -The result of immunoblot with MAIR-II (Π-10) is shown.

 FIG. 7B shows MAIR-II transformed cells (RAW) immunoprecipitated with control IgG, anti-DAP12, or anti-FcsRI, and the immunoprecipitate was anti-MAIR-- (TX-10). Shows the result of the simulated knob control.

FIG. 7C shows the results of immunoprecipitation of spleen cells with control IgG, anti-DAP12, or anti-FcsRI, and immunoblot of the immunoprecipitate with anti-MAIR-II (TX-10). FIG. 7D shows that 293T cells were transformed with Flag-tagged DAP12, MAIR-IL or Flag-tagged DAP12 + MAIR-1 and were treated with a biotin-conjugated anti-MAIR-II and a FITC-conjugated anti-Flag mAb. After staining with APC-conjugated streptavidin, the results of analyzing the cell surface expression of MAIR-II and DAP12 by flow cytometry are shown.

 FIG. 8 (left figure) shows TNF-α production after transfection of the Flag-tagged MAIR-II gene-transformed transformant with an anti-Flag antibody. FIG. 8 (right panel) shows the production of TNF-α after cross-linking the MAIR-II molecule on peritoneal macrophages with an anti-MAIR-II antibody. Hereinafter, the present invention will be described in detail. This application claims the priority of Japanese Patent Application No. 2002-341195, filed on November 25, 2002, and includes the contents as set forth in the specification and Ζ or drawings of the patent application. The immunoreceptor proteins of the present invention are referred to as myeloid-associated immunoglobulin-like receptors MAIR-1 and MAIR-II, whose extracellular regions are highly conserved with each other. It is a novel pair of activating and inhibitory receptor proteins. MAIR-I is expressed on most myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, and has a tyrosine-based localization motif and tyrosine-based immune receptor in the cytoplasmic region. Contains inhibitory motifs, induces internalization of the receptor, and suppresses IgE-mediated degranulation from mast cells. On the other hand, MAIR-II is expressed on macrophages and a subset of B cells, associates with the adapter molecule DAP12, which has a tyrosine-based immunoreceptor activation motif, and enhances inflammatory site force secretion from macrophages. Thus, MAIR-1 and MAIR-II regulate the innate immune response positively or negatively, respectively, and play important regulatory roles in cell signaling and immune responses.

1. Isolation of the gene encoding the immunoreceptor protein of the present invention

The cloning of the gene encoding the immunoreceptor protein of the present invention (hereinafter also referred to as MAIR) has been carried out, for example, in the absence of Ρϋ.1, a transcription factor essential for differentiation of myeloid and B cells. ΡΐΙ. Embryos between mice and wild-type mice using 14-day-old liver It can be performed by PCR subtraction based on the DA (Representative Difference Analysis) method. The RDA method is a method in which an adapter is added to genomic DNA digested with restriction enzymes, and a fragment of genomic DNA A is amplified (representation at ion) by PCR and compared by the subtradition method (subtraction method). It detects mutation deletion 丽 A.

 MAIR-encoding gene Also, cDNA libraries derived from the following immune system cells and tissues should be isolated by screening using DNA probes synthesized based on the DNA fragments prepared by the RDA method described above. Can be. The source of the mRNA for preparing the cDNA library is not particularly limited as long as it is a cell expressing MAIR mRNA, and may be humans or other mammals (eg, mouse, rat, guinea pig, egret, All kinds of immune system cells (eg, macrophages, mast cells, granulocytes (neutrophils, basophils, eosinophils), monocytes, dendritic cells, B cells) NK cells, etc.], or any tissue in which these cells are present (eg, spleen, lymph nodes, bone marrow, liver, etc.).

 Preparation of mRNA can be performed by a method usually used in the art. For example, the above cells or tissues are treated with a guadinin reagent, a phenol reagent, or the like to obtain total RNA, and then an oligo (dT) cellulose column—a poly! Using Sepharose 2B as a carrier; Poly (A) + RNA (mRNA) is obtained by the two-column method or the batch method. Further, poly (A +) RNA may be further fractionated by sucrose density gradient centrifugation or the like.

 Next, using the obtained mRNA as type III, a single-stranded cDNA is synthesized using an oligo dT primer and a reverse transcriptase, and then the DNA is synthesized from the single-stranded cDNA using DNA synthase I, DNA ligase, RnaseH, and the like. Synthesize double-stranded cDNA. The synthesized double-stranded cDNA is blunted with a T4 DNA synthase, ligated with an adapter (for example, EcoRI adapter), phosphorylated, etc., integrated into a vector such as Agtll, and packaged in vivo. Libraries can be created. In addition, a cDNA library can also be prepared using a plasmid other than λ phage.

Screening methods for selecting a strain having the desired 丽 A from the transformants obtained as described above include, for example, purification of MAIR-la (described later) from mouse liver. There is a method of synthesizing a sense primer and an antisense primer corresponding to a noic acid sequence, and performing a polymerase chain reaction (PCR) using the primers.

 The type III DNA used here includes cDNA synthesized from the above-mentioned niRNA by a reverse transcription reaction. Further, primers can be designed based on the above amino acid sequence information, while appropriately considering the expected size of the DNA fragment when amplified or the combination of double codons.

 The amplified DNA fragment obtained in this manner is labeled with 32P, 35S or biotin, etc. to form a probe, which is hybridized with a denatured and fixed nitrocellulose filter of the transformant DNA, and the resulting positive strain is obtained. You can screen by searching.

 The nucleotide sequence of the obtained clone is determined. The nucleotide sequence can be determined by a known method such as a maximum-Gilbert chemical modification method or a dideoxynucleotide chain termination method using M13 phage. Sequence determination is performed using a 373A DNA sequencer manufactured by PERKIN-ELMER, a BcaBEST didoxy sequencing kit manufactured by TAKARA, etc.). The obtained base sequence is analyzed using DNA analysis software such as DNASIS (Hitachi Software Engineering) to find the protein coding portion encoded in the obtained DNA chain.

 Examples of the gene encoding the receptor protein of the present invention obtained by the above method include, for example, a gene encoding a mouse-derived immunosuppressive receptor protein MAIR-la represented by the nucleotide sequence of SEQ ID NO: 1, Gene encoding MMR-lb, a mouse-derived immunosuppressive receptor protein represented by the nucleotide sequence of SEQ ID NO: 5, which is its isotype. Immune activation from the mouse represented by the nucleotide sequence of SEQ ID NO: 3. A gene encoding the receptor protein MAIR-IIa, and a gene encoding a mouse-derived immunostimulatory receptor protein MAIR-lib represented by the nucleotide sequence of SEQ ID NO: 7, which is the isotype thereof, may be mentioned.

The immunoreceptor protein of the present invention comprises (a) a protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, or (b) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. Is deleted, substituted or added to the amino acid sequence And (C) a protein having the amino acid sequence shown in SEQ ID NO: 4 in the sequence list; or (d) a protein having the function of suppressing the immune response of the cell; Alternatively, it is a protein having an amino acid sequence in which several amino acids are deleted, substituted or added, and has a function of activating a cell immune response.

 The range of "1 to several" in the above "amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 (or SEQ ID NO: 4)" is particularly limited. However, it means, for example, about 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably about 1 to 3.

 The deletion, addition and substitution of the amino acids can be performed by modifying the gene encoding the immunoreceptor protein by a method known in the art. Mutations can be introduced into a gene by a known method such as the Kunkel method or the Gapped duplex method or a method analogous thereto. For example, a mutagenesis kit using a site-directed mutagenesis method (eg, Mutant Mutations are introduced using -K (TAKARA) or Mutant-G (TAKARA)) or using the TAKARA LA PCR in vitro Mutagenes is series kit.

 The above “having the function of suppressing the immune response of cells” refers to the activity of suppressing the immune response of cells (for example, suppression of degranulation, reduction of cytokin production, suppression of cytotoxic activity, suppression of antigen presentation function, poor Is substantially equivalent to the activity of the protein having the amino acid sequence of SEQ ID NO: 2.

 In addition, “having the function of activating the immune response of cells” as described above refers to the activity of activating the immune response of cells (for example, promotion of degranulation, enhancement of cytokin production, enhancement of cytotoxic activity, antigen presentation) (Enhancement of function, hyperphagia, etc.) are substantially equivalent to the activity of the protein having the amino acid sequence of SEQ ID NO: 4.

 More specifically, as long as the activities are equivalent (for example, about 0.01 to 100 times, preferably about 0.5 to 20 times, and more preferably about 0.5 to 2 times), Elements may be different from the original protein.

Peptide containing partial amino acid sequence in any of the above immunoreceptor proteins (Also referred to as a partial peptide) is also included in the scope of the present invention. In particular, among the partial peptides of the present invention, the portion that is exposed outside the cell membrane when the immunoreceptor protein is naturally present and has a ligand binding activity is particularly useful in determining the ligand of the immunoreceptor protein. Useful. Examples of such a partial peptide include a portion analyzed as an extracellular region (hydrophilic site) in a hydrophobic plot analysis of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. The number of amino acids constituting the partial peptide of the present invention is at least 10 or more, preferably 30 or more, and more preferably 80 or more.

 The immunoreceptor protein or its partial peptide of the present invention can be provided in the form of a salt, if necessary, preferably in the form of a physiologically acceptable acid addition salt. Such salts include salts of inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartar) Acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

 The immunoreceptor protein of the present invention may be obtained by extracting and separating a human or a mammal expressing the protein from cultured cells or tissues, or by a trait containing DNA encoding the protein as described below. It can also be produced by culturing the transformant. When manufacturing from human or mammalian tissues or cells, the human or mammalian tissues or cells are homogenized, extracted with an acid, etc., and the resulting extract is subjected to hydrophobic mouth chromatography, reverse phase chromatography. Isolation and purification can be performed by combining various types of chromatography such as ion exchange chromatography and the like. The partial peptide can be produced by a known peptide synthesis method or by cleaving the immunoreceptor protein with a suitable peptidase (for example, trypsin, chymotrypsin, arginyl endopeptidase). As a peptide synthesis method, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. The gene encoding the immunoreceptor protein of the present invention may be any gene as long as it encodes the above-described immunoreceptor protein of the present invention.

(a) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, (b) SEQ ID NO in the sequence listing

DNA consisting of the nucleotide sequence complementary to the DNA consisting of the nucleotide sequence shown in 1 and a string (C) a DNA consisting of a DNA encoding a protein that hybridizes under transient conditions and has a function of suppressing a cell's immune response; or (c) a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing; ) Encodes a protein that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing, and that has a function of activating the immune response of cells. And a gene consisting of DNA.

 The above-mentioned “DNA that can hybridize with a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (or SEQ ID NO: 3) under stringent conditions” includes SEQ ID NO: 1 (or SEQ ID NO: DNA comprising a nucleotide sequence having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with the nucleotide sequence represented by 3). Here, the stringent condition means, for example, a condition in which the sodium concentration is 600 to 900 mM and the temperature is 60 to 68 ° C, preferably 65 ° C.

 Once the nucleotide sequence of the gene of the present invention is determined, it is subsequently synthesized by chemical synthesis, by PCR using the cDNA of this gene as type III, or by hybridization using a DNA fragment having the nucleotide sequence as a probe, The gene of the present invention can be obtained.

2. Preparation of recombinant vectors and transformants

 (1) Preparation of recombinant vector

The recombinant vector of the present invention can be obtained by ligating the gene of the present invention to an appropriate vector. The vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA. Plasmid DNA includes E. coli-derived plasmids (eg, pRSET, PBR322, pBR325, pUC118, pUC119, pUC18, pUC19), Bacillus subtilis-derived plasmids (eg, ρϋΒΙΙΟ, ρΤΡ5, etc.), and yeast-derived plasmids (eg, ΥΕρ13 , YEp24, YCp50, etc.) and the phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, AgtlO, AgtlK λΖΑΡ, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, baculovirus And insect virus vectors such as

 To insert the gene of the present invention into a vector, first, the purified DNA is digested with an appropriate restriction enzyme and inserted into an appropriate vector DNA restriction enzyme site or a multiple cloning site. And the like.

 The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, the vector of the present invention contains, in addition to the promoter and the gene of the present invention, a cis element such as an enhancer, a splicing signal, a polyA addition signal, a selection marker, a ribosome binding sequence (SD sequence), if desired. Can be linked. Examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.

 When the host cell is Escherichia coli, examples of such a vector include pET vector (Novagen), pTrxFUS vector (Invitrogen), and pCYB vector (NEW ENGLAMD Bio Labs). When the host cell is yeast, for example, pESP-1 expression vector (manufactured by STRATAGENE), pAIffil23 vector (manufactured by Takara Shuzo), pPIC vector (manufactured by Invitrogen), or when the host cell is an animal cell For example, pMAM-neo expression vector (manufactured by CL0NTECH), pCMA3.1 vector (manufactured by Invitrogen), pBK-CMV vector-1 (manufactured by STRATAGENE), etc. When the host cell is an insect cell, for example, Examples include pBacPAK vector (manufactured by CL0NTECH), pAc31 vector-1 (manufactured by CL0NTECH), pAcP (+) IE1 vector (manufactured by Novagen), and the like.

 (2) Preparation of transformant

 The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express 丽 A of the present invention. For example, the genus Escherichia, such as Escherichia coli, or Bacillus subtilis

A bacterium belonging to the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida, or the genus Rhizobium such as Rhizobium mel iloti; Saccharomyces * cerevisiae

(Saccharomyces cerevis iae), Shizo Saccharomyces. (Sc izosaccharomyces pombe) and other yeast cells; monkey cells COS-7, Vero, Chinese eight muscular ovary cells (CH0 cells), mouse L cells, human GH3, human FL cells and other animal cells; or insect cells such as Si9 and Sf21 Is mentioned.

 When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and comprises a promoter, a ribosome binding sequence, a gene of the present invention, and a transcription termination sequence. Is preferred. Further, a gene controlling a promoter may be included.

 Examples of Escherichia coli include Escherichia coli K12 and DH1, and examples of Bacillus subtilis include Bacillus subtilis. Any promoter can be used as long as it can be expressed in a host such as E. coli. For example, promoters derived from Escherichia coli or phage such as trp promoter, lac promoter, PL promoter, and PR promoter are used. An artificially designed and modified promoter such as a tac promoter may be used. The method for introducing the recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using a potassium ion (Cohen, S.N., et al. (1972) Proc. Natl. Acad. Sci., USA 69, 2110-2114), an elect-portion method and the like can be mentioned.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast.Examples include gall promoter, gallO promoter, heat shock protein promoter, MFal promoter, M05 promoter, PGK promoter, GAP promoter, ADH Promo One night, A0X1 promoter and the like. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, the elect-mouth poration method (Becker, DM et al. (1990) Methods. Enzymol., 194, 182) -187), Spheroplast method (Hinnen, A. et al. (1978) Proc. Natl. Acad. Sci., USA 75, 1929-1933), lithium acetate method (Itoh, H. (1983) J. Bacteriol 153, 163-168) and the like. When animal cells are used as the host, monkey cells COS-7, Vero, Chinese hams evening ovary cells (CH0 cells), mouse L cells, rat GH3, human FL cells, etc. are used. As the promoter, an SRa promoter, an SV40 promoter, an LTR promoter, a CMV promoter, or the like may be used. Alternatively, an early gene promoter of human cytomegalovirus may be used. Methods for introducing the recombinant vector into animal cells include, for example, the electoral poration method, the calcium phosphate method, and the lipofection method.

 When insect cells are used as a host, Sf9 cells, Sf21 cells, and the like are used. As a method for introducing a recombinant vector into an insect cell, for example, a calcium phosphate method, a riboaction method, an electroporation method, or the like is used.

 In addition, the method for introducing a gene into each of the above-described host cells can be performed by a method that does not rely on a recombination vector, such as a particle gun method.

3. Production of the immunoreceptor protein of the present invention

 The immunoreceptor protein of the present invention can be obtained by culturing the transformant and collecting from the culture. The term “culture” means any of cultured cells or cultured cells, or crushed cells or cells, in addition to the culture supernatant.

 The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing the host cell.

The culture medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the microorganisms, so that the cultivation of the transformants is efficient. Either a natural medium or a synthetic medium may be used as long as the medium can be performed in a controlled manner. Any carbon source may be used as long as the organism can assimilate it, and carbohydrates such as dalcose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. Can be Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium or inorganic salts of organic acids or other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor, etc. Used by Can be As the inorganic salts, potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

 Culture is usually performed at 37 ° C for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the culture period, maintain the pH at 7. (! To 7.5. Adjust the pH using an inorganic or organic acid, alkali solution, etc. During culture, use antibiotics such as ampicillin and tetracycline as necessary. Substances may be added to the medium.

 When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the Lac promoter, isopropyl-jS_D-thiogalactopyranoside (IPTG) or the like can be cultured using a microorganism transformed with an expression vector using a trp motor. When culturing is performed, indoleacetic acid (IAA) or the like may be added to the medium.

 As a medium for culturing a transformant obtained by using an animal cell as a host, a commonly used RPMI 1640 medium, DMEM medium, or a medium obtained by adding fetal bovine serum or the like to such a medium is used. Culture is usually performed at 37 ° C for 1 to 30 days in the presence of 5% C0. During culture, antibiotics such as kanamycin and penicillin may be added to the medium as needed.

 After culturing, when the immunoreceptor protein of the present invention is produced in cells or cells, the cells are extracted by disrupting the cells or cells. When the immune system receptor protein of the present invention is produced extracellularly or extracellularly, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, common biochemical methods used for isolating and purifying proteins, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., are used alone or in appropriate combination. The immunoreceptor protein of the present invention can be isolated and purified from the culture.

4. Antibodies against the immunoreceptor protein of the present invention

The antibody of the present invention can be obtained by the following general antibody preparation method. TX-8, which recognizes only MAIR-1; 認識 す る -10, which recognizes both MAIR-1 and MAIR-II; -13, which recognizes only MAIR-II, are described by Shibuya, A., et al., Natl. I Awaken unol., 1, 441-6, 2000. Specifically, as shown in the Examples below, immunization is performed by injecting a fusion protein of Ba / F3 transformant expressing MAIR-I or MAIR-II and the Fc portion of human IgG into the foot. It is prepared by fusing popliteal lymph node cells from adult rat with Sp2 / 0 myeloma cell line.

 (1) Preparation of antigen

 In the present invention, the immunoreceptor protein of the present invention isolated or purified as described above or a partial fragment thereof is used as an antigen.

 (2) Preparation of polyclonal antibody

 Animals are immunized with the antigen prepared as described above. The dose of the antigen per animal is 100 to 500 g in the case of a egret, for example, using an adjuvant. Adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant, and the like.

 Immunization is performed by administering to mammals (for example, rats, mice, and egrets). The site of administration is intravenous, subcutaneous or intraperitoneal. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 3 times at intervals of several days to several weeks, preferably at intervals of 2 to 3 weeks. Then, the antibody titer is measured 6 to 60 days after the last immunization, and blood is collected on the day showing the highest antibody titer to obtain antiserum. The antibody titer can be measured by an enzyme immunoassay (ELISA; enzyme-linked immunosorbent assay), a radioimmunoassay (RIA; rad ioifflmuno assay), or the like.

 When purification of the antibody from the antiserum is required, known methods such as ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and affinity chromatography are appropriately selected, or a combination thereof. It can be purified.

(3) Preparation of monoclonal antibody

 (3-1) Immunization and collection of antibody-producing cells

An animal is immunized with the antigen protein prepared as described above. If necessary, an adjuvant (commercially available Freund's complete adjuvant, Freund's incomplete adjuvant, etc.) may be mixed in the same manner as described above for effective immunization. Immunization is performed by administering to mammals (for example, rats, mice, and egrets). The single dose of antigen is 50 / zg per mouse for mice. The administration site is mainly intravenous, subcutaneous, or intraperitoneal. The interval of immunization is not particularly limited, and the immunization is performed at least 2 to 3 times at intervals of several days to several weeks, preferably at intervals of 2 to 3 weeks. After the final immunization, the antibody-producing cells are collected. Antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, and the like, with spleen cells being preferred.

 (3-2) Cell fusion

 To obtain hybridomas, cell fusion between antibody-producing cells and myeloma cells is performed. As the myeloma cell to be fused with the antibody-producing cell, a cell line derived from an animal such as a mouse and generally available can be used. As a cell line to be used, it has drug selectivity, cannot survive in HAT selection medium (including hypoxanthine, aminopterin and thymidine) in an unfused state, and can survive only in a state fused with antibody-producing cells. Are preferred. For example, specific examples of myeoma cell lines include mouse myeloma cell lines such as P3X63-Ag. 8.U1 (P3U1), P3 / NSI / 1-Ag4-1, and Sp2 / 0-Agl4.

 Next, the myeloma cells are fused with the antibody-producing cells. Cell fusion is performed by mixing antibody-producing cells and myeloma cells at a ratio of 15: 1 to 25: 1 in an animal cell culture medium such as EM or RPMI-1640 medium that does not contain serum. The fusion reaction is performed in the presence of a cell fusion promoter or by electric pulse treatment (eg, electroporation).

 (3-3) Sorting and closing of hybrids

 The desired hybridoma is selected from the cells after the cell fusion treatment. For example, cells that are cultured in a medium containing hypoxanthine, aminopterin and thymidine and grow can be obtained as hybridomas.

Next, it is screened whether the target antibody is present in the culture supernatant of the grown hybridoma. Screening for hybridomas may be performed according to a conventional method, and is not particularly limited. For example, a part of the culture supernatant contained in a well grown as a hybridoma is collected and subjected to enzyme immunoassay (ELISA; enzyme-linked immunosorbent assay), IA (radio immunoassay) and the like. Can be cleaned. Cloning of the fused cells is performed by a limiting dilution method or the like, and finally, a hybridoma, which is a monoclonal antibody-producing cell, is established.

(3-4) collection of monoclonal antibody

As a method for collecting a monoclonal antibody from the established hybridoma, an ordinary cell culture method or the like can be employed. In the cell culture method, in an animal cell culture medium such as Haipurido Ma 10% fetal bovine serum-containing RPMI-1640 medium or MEM medium, under normal culture conditions (e.g. 37 ° C, 5% C0 ₂ concentration) 3-10 After culturing for days, the antibody is obtained from the culture supernatant.

 When antibody purification is required in the above antibody collection method, a known method such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, or gel chromatography is appropriately selected, or Purification can be performed by combining these methods.

5. Method for quantifying immunoreceptor protein using the antibody of the present invention, kit for quantification

 The quantification of an immunoreceptor protein using the antibody of the present invention can be performed by, for example, the immunoblotting method, the enzyme antibody method (EIA), the radioimmunoassay method (RIA), the fluorescent antibody method, immunocellular staining, or the like. Yes, but not limited to them.

 Further, the antibody may be a fragment thereof, and specifically includes a single-chain antibody fragment (scFv) of the antibody.

Specifically, when the ELISA method is used, the procedure is as follows. First, a sample such as diluted blood is adsorbed to a 96-well microphone plate, and then reacted with the antibody of the present invention as a primary antibody. Then, an anti-globulin antibody labeled with a specific enzyme such as POD (peroxidase) required for the color reaction is reacted. After washing, ABTS2,2'-azino-di- (3-ethyl-benzothiazoline-6) is used as a color substrate. -Sulfonic acid) and the like, and the color is developed. The immunoreceptor protein of the present invention in the sample is detected by measurement by a colorimetric method. Alternatively, if the sandwich ELISA method is used, proceed as follows. First, a diluted sample such as blood is added to a 96-well microplate on which the antibody of the present invention has been adsorbed in advance, and incubated for a certain period of time. Thereafter, the plate is washed, a purified antibody labeled with biotin is added to each well, and the plate is incubated for a certain time. After the addition, wash the plate, add enzyme-labeled avidin, and incubate further. After incubation, the plate is washed, and color is developed by adding orthophenylenediamine or the like as a color-developing substrate, and the colorimetric measurement is performed.

 The antibody of the present invention can also be used in a kit for quantifying an immunoreceptor protein. The kit only needs to include at least the antibody of the present invention.When the antibody is immobilized on a solid phase, the kit has an antigen recognition site different from that of the antibody, and includes an antibody used as a secondary antibody. You may go out. The antibody used as the secondary antibody may be labeled with, for example, an enzyme or the like, and may contain various reagents (eg, an enzyme substrate, a buffer, a diluent, etc.) in addition to these two antibodies. .

6. Isolation of paired immunoreceptor genes

 The inhibitory immune receptor having the ITIM motif and the activated immune receptor having the ITAM motif have highly conserved extracellular regions. Therefore, in the present invention, if any of the immunoreceptor genes having the above motif can be obtained, a pair of immunoreceptor genes can be obtained using the gene encoding the extracellular conserved region as a probe. It can be isolated from a DNA library or the like. For example, in the case of MAIR-I, as an extracellular region that can serve as a probe, of the nucleotide sequence shown in SEQ ID NO: 1, the 63rd to 596th nucleotide sequence, preferably the 135th to 488th nucleotide sequence is used. A DNA containing a base sequence is exemplified. .

7. A method for screening a ligand for the receptor protein of the present invention

The method for screening a ligand for a receptor protein of the present invention comprises the steps of: causing an immunoreceptor protein of the present invention or a cell or cell membrane expressing the protein to act on a test substance expected to contain a ligand; It is characterized by selecting substances having affinity for it. As a specific method for selecting a substance having an affinity, a test substance is allowed to act on the immunoreceptor protein of the present invention, or on a cell or cell membrane expressing the receptor protein, and the receptor protein is reacted with the receptor protein. It is performed by measuring the amount of the test substance bound or by measuring the immune response signal of the cells. Here, as the immune response signal, for example, degranulation (Release of chemicals such as histamine and serotonin), synthesis of leukotriene ェ platelet activating factor, release of cytodynamic force, activation of complement, production of antibodies, phosphorylation of intracellular proteins, deprivation, cytotoxicity, etc. Say. A test substance having a large amount of binding in the measurement or a test substance having a strong immune response signal can be selected as a candidate substance for a ligand to the receptor protein of the present invention.

 As the test substance, any substance can be used, and its type is not particularly limited. Specific examples of test substances include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, natural product extracts (plant extracts, animal tissues and animal cell extracts), or compound libraries, phage display libraries or It may be a pinatorial library. Construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.

8. An agonist or antagonist screening system for the receptor protein of the present invention

 The receptor protein of the present invention is used to screen for a substance (antagonist) that inhibits the binding of the receptor protein to a ligand, and a substance (agonist) that binds to the receptor and causes an immune response similar to that of the ligand. (Screening method, screening kit).

 The screening method of agonist or angonisto for the receptor protein of the present invention comprises the steps of (a) reacting an immunoreceptor protein with a ligand for the immunoreceptor protein in the absence of a test substance; b) affecting the binding of the immunoreceptor protein to the ligand by comparing with the case where the immunoreceptor protein acts on the ligand for the immunoreceptor protein in the presence of the test substance It is characterized by selecting a substance. If a test substance whose binding amount between the immunoreceptor protein and the ligand decreases or increases when the test substance is added, the test substance inhibits or antagonizes the binding between the protein of the present invention and the ligand. It becomes a candidate for an angist or agonist to help stake and control the immune response.

Further, the receptor protein of the present invention is an agonist or It can also be used as a screening kit for antagonists. The kit only needs to include at least the receptor protein of the present invention, and may include a labeled ligand, a ligand standard solution, and various reagents (for example, a buffer solution, a washing solution, a diluting solution, and the like).

 As the test substance, any substance can be used, and its type is not particularly limited. Specific examples of test substances include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, natural product extracts (plant extracts, animal tissues and animal cell extracts), or compound libraries, phage display libraries or It may be a pinatorial library. Construction of compound libraries is known to those skilled in the art, and commercially available compound libraries can also be used.

9. Drug for controlling immune response function of the present invention

 The immunoreceptor protein of the present invention has a function of activating or suppressing an immune response. In addition, the ligand, angelonist, and agonist of the present invention can bind to the receptor protein and perform a function of changing an activation signal and an inhibitory signal from an immune receptor. Therefore, any of the protein, gene, ligand, angelonist and agonist of the present invention can be used as a drug for controlling immune response functions. For example, when the medicament of the present invention is used for a disease caused by deficiency of an immune control mechanism, the disease can be treated by controlling the direction of immune activation and immunosuppression. Such diseases include, for example, autoimmune diseases (eg, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, polymyositis, dermatomyositis, Siedalen syndrome, Behcet's disease, ankylosing spondylitis) , Insulin-dependent diabetes mellitus, pernicious anemia, etc., allergic diseases (eg, hay fever, bronchial asthma, atopic dermatitis, allergic rhinitis, anesthesia angioedema, serum disease, anaphylaxis, drug allergy, etc.) ), Immunodeficiency diseases (primary immunodeficiency syndrome, secondary immunodeficiency syndrome, etc.), tumors (lung cancer, breast cancer, colorectal cancer, bladder cancer, myeloid leukemia, neuroblastoma, melanoma, Parkit lymphoma, etc.) And the like, but are not limited to these.

The medicament of the present invention is prepared in various preparation forms and is orally or parenterally administered systemically or topically. W can be given. When the medicament of the present invention is administered orally, it is formulated into tablets, capsules, granules, powders, pills, solutions for internal use, suspensions, emulsions, syrups, etc., or redissolved when used. It may be a dry product. When the medicament of the present invention is administered parenterally, it is formulated into an intravenous injection (including intravenous drip), an intramuscular injection, an intraperitoneal injection, a subcutaneous injection, a suppository, and the like. If provided, they are provided in unit dosage ampules or multidose containers.

 These various preparations include excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, dissolution aids, preservatives, A flavoring agent, a soothing agent, a stabilizing agent, a tonicity agent, and the like can be appropriately selected, and can be produced by an ordinary method.

 The above-mentioned various preparations may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, Pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, etc. Can be The additives to be used are appropriately or in combination selected from the above depending on the dosage form.

 The dose of the medicament of the present invention varies depending on the age of the subject to be administered, the administration route, and the number of administrations, and can be widely varied. For example, an effective amount administered as a combination of an effective amount of the protein of the present invention with a suitable diluent and a pharmacologically acceptable carrier can range from 0.01 mg to 1000 mg per kg body weight per serving. The dose can be selected and it is administered once a day or several times a day.

When the gene of the present invention is used as a gene therapy agent for an immune system disease, a method of directly administering the gene of the present invention by injection and a method of administering a vector into which the gene has been incorporated can be mentioned. Examples of the above vectors include adenovirus vector, adeno-associated virus vector, herpes virus vector, vaccinia virus vector, retrovirus vector, and the like. The administration can be carried out efficiently by using a teratase. Alternatively, a method of introducing the gene of the present invention into phospholipid vesicles such as ribosomes and administering the ribosome may be employed.

 Gene therapy can be administered in a systemic manner, such as intravenous or intra-arterial administration, or by local administration to immune system tissues (bone marrow, lymph nodes, etc.). In addition, dosage forms in combination with power catheter technology, surgery, and the like can be employed. The dosage of the gene therapy agent of the present invention varies depending on age, sex, symptom, administration route, number of administrations, and dosage form.However, usually, the weight of the gene of the present invention is 0.1 to 100 mg / day for an adult. A range of weight is appropriate.

10. Immunoreceptor protein detection reagent

 Since the antibody of the present invention reacts with the immunoreceptor protein of the present invention or a partial fragment thereof, it can be used as a reagent for detecting the receptor protein. The method for detecting the immunoreceptor protein is not particularly limited, and for example, a Western plotting method or the like can be employed. For example, a test subject (cell components or fractions thereof) is fractionated by electrophoresis or the like, and then reacted with a pre-labeled (radiolabeled, fluorescent stained, etc.) antibody of the present invention to obtain a signal. To detect. The antibody used to detect the immunoreceptor protein of the present invention may be an antibody against a protein having the full-length amino acid sequence of the receptor protein, or may be an antibody against a peptide having a partial amino acid sequence of the receptor protein. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

 Among the antibodies used in the examples, control rat and mouse IgG, anti-mouse CD4, CD8, CD45R / B220, CDl lb (Macl), CDl lc, Ly6G (Gr-1) and CD32 / 16 mAbs, and M-5mAb was obtained from PharMingen (San Diego, CA). Anti-phosphotyrosine mAb (4G10) and anti-FcεRIa!) Were purchased from Update Biotechnology Inc. (Lake

Placid, NY) and anti-Flag mAb from Sigma (St. Louis, Missouri). Anti-SHP-1, anti-SHP-2, and anti-SHIP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rat IgE anti-DNP mAb was obtained from Zymed Laboratories, Inc. (South San Francisco, CA). Anti-DAP12 polyclonal antibody was obtained from Ehime University School of Medicine.

[Example 1] (Cloning and molecular properties of MAIR-1 and MAIR-II)

 (1) Cloning of MAIR-1 and MAIR-II

 To identify a novel gene involved in the myeloid cell-mediated immune response, PCR was performed on 14-day-old fetal liver derived from wild-type and mouse strains lacking myeloid cells. For subtractive hybridization (RDA), which is a subtractive hybridization of DNA.

 First, total RNA was isolated using Isogen LS solution (Nippon Gene), and oligo (dT) -prime double-stranded cDNA was synthesized using the cDM synthesis system (GIBCO BRL) according to the manufacturer's instructions. RDA was performed according to the method described in Iwama, A., et al., Nucleic Acids Res, 26, 3034-43, 1998. From the DNA fragment obtained by the subtraction, cDNA (MAIR-la) specific to myeloid cells was identified. The full-length MAIR-Ia cDNA was cloned by screening a macrophage cDNA library using the cDNA fragment generated by RDA as a probe.

 The amino acid sequence of MAIR-la is shown in SEQ ID NO: 2 in the sequence listing. MAIR-Ia is a type I transmembrane glycoprotein consisting of 314 amino acids, a leader sequence of 27 amino acids,

It has an extracellular region of 152 amino acids, a transmembrane region of 23 amino acids, and an intracellular region of 112 amino acids (FIG. 1A). The intracellular region has the amino acid sequence VEYSTL and

Including LHYSSV (Figure 1A), they were consistent with the consensus sequence of ITIMs (I / V / L / SxYxxL / V). The ITIM-like sequences YVNL and YSEI also showed that MAIR_Ia recruits protein tyrosine phosphatases and mediates inhibitory signals. In addition,

The ITIM-like motif YVNL is also a consensus sequence involved in receptor internalization (Marks, S.M., et al., Trends in Cell Biology, 7, 124-128,

1997; Boll, W., et al., Embo J, 15, 5789-95, 1996).

Next, to determine if MAIR-la is a member of the paired receptor, MAIR-la A macrophage cDNA library was screened using a cDNA encoding the extracellular region of la (the 63rd to 596th base sequences in SEQ ID NO: 1) as a probe. As a result, an extracellular domain with one Ig-like domain with 92% homology to the amino acid sequence of MAIR-la, a transmembrane domain with charged amino acids (Lys), and a short (20 amino acid) intracellular till A clone encoding a protein containing was identified and named MAIR-Ila. In addition, we found isoforms containing four additional amino acids in MAIR-la and two additional amino acids in MAIR-IIa, MAIR-lb (amino acid sequence: SEQ ID NO: 6) and MAIR-lib (amino acid sequence: SEQ ID NO: 8), respectively. It was named.

 FIG. 1A shows a comparison of the amino acid sequences of MAIR-Ia, MAIR-lb, MAIR-IIa, and MAIR-lib. In FIG. 1A, the underlined lines indicate the putative leader sequence and transmembrane region, and the circled portions indicate the N-linked glycosyl site in the extracellular region and the charged amino acid residues in the transmembrane region in bold (FIG. (The 55th and 113th amino acids in the amino acid sequence shown in the figure below) represent cysteine residues involved in disulfide bonds in the Ig-like domain. The boxed area represents the ITIM, ITIM-like or local motif of the intracellular region.

MAIR-I, MAIR-Ib, MAIR-IIa, and MAIR-lib cDNA sequence data are available from EMBL / GenBank / DDBJ under accession numbers AB0091765, AB0091766, AB0091767, and AB0091768, respectively.

 FIG. 1B shows a schematic diagram of the MAIR-1 and MA-II proteins. A pair of cysteine residues in the extracellular region are thought to be involved in the interchain disulfide bond for the formation of the Ig-like domain, and MAIR-1 and MAIR-II are members of the Ig superfamily It indicates that.

 (2) Southern blot analysis of MAIR-1 and MAIR-II

 FIG. 1C shows a Southern plot analysis of the genomes MAIR-1 and MAIR-II. Mouse genomic DNA was converted to restriction enzymes (E; Eco-RLB; Bam-HI, H; Hind-III, X; Xba-1, P;

Pst-I) and probed with cDNA encoding the extracellular region of MAIR-1. MAIR-lb and MAIR-lib, as indicated by the genomic DM sequences of MAIR-I and MAIR-11,

Alternative splicing variants of MAIR-1a and MAI R-IIa. The Southern plot analysis described above revealed a single hybridization pattern, indicating that MAIR-1 and MA-II were encoded by a single gene (Fig. 1C).

 (3) Chromosome FISH analysis

 The direct R-band FISH method was used for chromosome assignment of the MAIR-1 gene in mice and rats. Preparation of R-band chromosomes and FISH was performed according to Matsuda, II., Et al., Cytogenet Cell Genet, 61, 282-5, 1992 and Matsuda, Y. Chapman, VM, Electrophores is, 16, 261-72, 1995. Performed as described in. The MAIR-1 and MAIR-II genes are located in the proximal region of the E2 band on mouse chromosome 11, with six each, as determined by fluorescence in situ hybridization (FISH) and genomic DNA sequence databases. Consists of four exons. According to database research, MAIR-1 and MAIR-II were found to be 44% at the amino acid level with human CMRF-35-H9 (Green, BJ, et al., Int Immunol, 10, 891-9, 1998), respectively. It was found to have 49% homology with human CMRF-35 (Green, BJ, et al., Int Immunol, 10, 891-9, 1998). CMRF-35-H9 and CMRF-35 are located on human chromosome 17 corresponding to mouse chromosome 11 (Clark, GJ, et al., Tissue Antigens, 55, 101-9, 2000). These are considered to be human homologs of mouse MAIR-1 and MAIR-II.

 (4) Immunoblotting

 Mouse T-cell leukocyte BW5147 cells were transformed with MAIR-la and MAIR-Ila cDNAs whose N-terminus was tagged with a Flag peptide. BW5147 cells expressing Flag-tagged MAIR-1a or MAIR-Ila or parental BW5147 cells were transformed with 1% NP-40, protease inhibitors (lmM PMSF and 20 Kallikrein inhibitor / ml aprotinin), and phosphatase inhibitors It was dissolved in Tris-buffered saline (50 mM Tris, 15 mM NaCl, ρΗδ. 0) containing (lmM EGTA, 10 mM NaF, lmM Na4P207, 0.1 lmM j3-glycemic phosphate, lmM Na3V04). Proteins were analyzed by immunoblotting under reduced or non-reducing conditions using anti-Flag mAb. The results are shown in FIG. 2A. Flag-tagged MAIR-la and MAIR-Ila immunoprecipitates with anti-Flag-mAb on BW5147 transformants had molecular weights of 5050 kDa and 〜35 kDa, respectively (FIG. 2A). .

 (5) MAIR-la N-Darcosidase treatment

To investigate N-daricosylation of MAIR-la, express Flag-tagged MAIR-la BW5147 transformants were immunoprecipitated with anti-Flag mAb or control IgG and treated with N-glycosidase F (Boehringer Mannheim Corp., Indianapolis, Ind.) Using conditions recommended by the manufacturer.

 The mobility of MAIR-Ia decreased from 5050 kDa to 4545 kDa after treatment with N-darcosidase F (FIG. 2B). This was consistent with the predicted size of the polypeptide from the MAIR-1 cDNA and the presence of an N-linked darcosyl site in the extracellular region.

[Example 2] (Expression of MAIR-1 and MAIR-II)

 (1) Northern blot analysis

 MAIR-I c bandages were labeled with [32P] -dCTP using Ready to Go DNA Labeling Beads (Amersham Bioscience Corp., Piscataway, NJ). A membrane containing about lOA Ig of total RNA in each lane derived from various mouse tissues was added to a [32P] -dCTP-labeled cDNA probe using a modified Churcli's hybridization buffer (0.5M Church '). s phosphate buffer, 7% SDS, 1% BSA) at 68 ° C. The membrane was washed in Church's washing buffer (40 mM Church's phosphate buffer, 1% SDS) for 1 hour at 68 ° C and subjected to autoradiography. FIGS. 3A and 3B show the results of analysis of the expression of MAIR-1 and / or MAIR-II transcripts by Northern blotting using the cDNA encoding the extracellular portion of MAIR-1 as a probe. Predominant expression of MAIR transcripts was detected in hematopoietic tissues such as spleen, bone marrow, peritoneal macrophages, and osteocytic cell lines (416B, WEHI3, RAW, and Ml cells), but not in non-hematopoietic organs Ivy (Figure 3A).

 (2) Reverse transcription-polymerase chain reaction (RT-PCR)

To further analyze the expression of MAIR-1 and MAIR-II in the blood cell lineage, we separated each cell lineage from bone marrow, spleen, and thymus by flow cytometry, and analyzed the MAIR-I-specific primer and MAIR-II- RT-PCR was performed using a specific primer as follows. First, RNA was obtained from lineage differentiated cells purified from bone marrow, spleen, and thymus by flow cytometry. This RM was reverse transcribed, and relative-adjusted cDNA was prepared from purified cells using a cell sorter using an HPRT control. Using these cDNAs as type III, MAIR-1-specific primers (5'-AAGCTGATAGGCATCCAGAGC-3 ': SEQ ID NOS: 9 and 5, -AAGGAGTCACAGGTAAAGGTC-3': SEQ ID NO: 10), MAIR-II-specific primer (5, -TCCGAGCCTTGAGAGTGGTAGAC-3 ': SEQ ID NO: 11 and 5'-AGGAGCTGTGTTTAGGGACAG-3 ': SEQ ID NO: 12) or HPRT-specific primer (5, -GCTGGTGAAAAGGACCTCT-3': SEQ ID NO: 13 and 5'-CACAGGACTAGAACACCTGC-3 ': SEQ ID NO: 14) did. The amount of cDNA was standardized by quantitative PCR using TadMan Rodent GAPDH control reagent (Perkin-Elmer Applied Biosystems, Foster, City, CA) (Sakamoto, N., et al., Eur J Immunol, 31, 1310 -6, 2001). PCR was performed under the following conditions: denaturation (94 ° C) for 20 seconds, annealing (55 ° C) for 20 seconds, and extension (72 ° C) for 30 seconds. Figure 3B shows the results of RT-PCR analysis. Both MAIR-1 and MAIR-II transcripts are multilineage, including lymphocytic hematopoietic progenitor cells, granulocytes, macrophages, erythroid cells, and B cells in bone marrow, and T, B, and NK cells in spleen. It was expressed in progenitor cells but was not detected in T cells in the thymus (Figure 3B).

 (3) Analysis of cell surface expression of MAIR-1 and MAIR-II proteins

 To analyze expression at the protein level, monoclonal antibodies Π-8 and TX-13, which specifically react with the MAIR-1 and MAIR-II proteins, respectively, were prepared, and the expression of molecules on the cell surface was analyzed by flow cytometry. investigated.

 (3-1) Preparation of antibody

 TX-8 (anti-MMR-1), TX-10 (anti-MAIR-1 and anti-MAIR-II) and TX-13 (anti-MAIR-II) mAbs are available from Shibuya, A., et al., As described in Natl. Immunol., 1, 441-6, 2000, the fusion protein of Ba / F3 transformant expressing MAIR-1 or MAIR-II and the Fc portion of human IgG is injected into the foot. Were prepared by fusing popliteal lymph node cells from rats immunized with E. coli with the Sp2 / 0 myeloma cell line.

 (3-2) Flow cytometry

MAIR-I or MAIR-II, whose N-terminus was tagged with Flag cDNA, was subcloned into pMX retrovirus vector (Kitanmra, T., et al., Proc Natl Acad Sci, USA, 92, 9146- 50, 1995). BW5147, Ba / F3 and RAW cells stably expressing Flag-tagged MAIR-1 and MAIR-II were established according to Shibuya, K., et al., I. unity, 11, 615-23, 1999. Figure 4A shows that BW5147 transformants expressing MAIR-1 or MAIR-11 were control IgG or anti-MAIR-1 (TX-8 mAb) or stake-MAIR-II (TX-13 mAb), followed by Shows the results of staining with streptavidin aroficosyanin (APC).

 Figures 4B, C, and D show spleen, bone marrow, or peritoneal macrophages derived from C57 / BL6 mice with anti-MAIR-1 (TX-8 mAb) or anti-MAIR-II (TX-13 mAb). ) And PE-bound mAb, followed by staining with arophycocynin (APC) -bound streptavidin. Analysis of spleen and bone marrow cells by flow cytometry showed that MAIR-1 was expressed on most myeloid cells, including macrophages, dendritic cells, granulocytes, marrow-derived mast cells, and some subsets of B cells. However, they were not expressed in T cells or NK cells (Fig. 4B, C, D). On the other hand, the MAIR-II protein was detected only on the cell surface of B cells, Cesubsat and a subset of peritoneal macrophages, and its expression was observed in dendritic cells, mast cells, etc., unlike the RT-PCR analysis results. None (Figures 4B, C, D). This suggests that the expression of the MAIR-II molecule on the cell membrane may be regulated by some mechanism, and that even if transcript expression is observed, it does not necessarily occur that protein protein cell surface expression occurs. Was.

 (4) Regulation of MAIR-1 and MAIR-II protein expression on cell surface

 To examine the mechanism of control of protein expression on the cell surface, B cells purified from spleen were cultured in the presence or absence of LPS (10 g / ml) for 48 hours. Stained with MAIR-1 (TX-8 mAb) or anti-MAIR-II (TX-13 mAb). After 48 hours of culture, the MAIR-II molecule was prominently detected on the cell membrane and was further enhanced by LPS stimulation (FIG. 4E).

[Example 3] Functional analysis of MAIR-1 and MAIR-II

 (1) Internalize Atsushi

 The subcellular region of MAIR-1 contains a tyrosine-based localization motif (YVNL),

(Figure 1A), it has been reported that this motif is involved in the endosomal and lysosomal targeting of proteins and is involved in agonisto-induced internalization (Marks, S. Μ ·, et al., Trends in Cel l Biology, 7, 124- 128, 1997; Boll, W., et al., Eibo J, 15, 5789-95, 1996). Therefore, to investigate whether MAIR-1 induces internalization after cross-linking, MAIR-1 expressed on peritoneal macula phage was treated with a biotin-labeled anti-MAIR-1 mAb (TX -8), cross-linked with APC-labeled streptavidin, and incubated at 4 ° C or 37 ° C for 1 hour. Cells were treated with 2.5% trypsin (+) or untreated (1) to remove cell surface MAIR-1 that did not receive internalization and analyzed by flow cytometry. The fluorescence intensity of cells incubated at 4 ° C after cross-linking was significantly reduced by trypsin treatment, whereas the fluorescence intensity of cells incubated at 37 ° C after cross-linking was constant regardless of trypsin treatment. (Fig. 5A). This indicates that cross-linking of MAIR-1 induced receptor internalization during culture at 37 ° C.

 Ba / F3 transformants expressing MAIR-1 with N-terminal Flag-tagged were incubated with anti-Flag mAb and subsequently cross-linked with FITC-labeled anti-mouse IgG secondary antibody. The cells were resuspended in RPMI 1640 containing 10% FBS, mounted on a chamber slide (Lab-TekII, Nalge Nurc, Naperville, ID) previously coated with polylysine and incubated at 37 ° C for 30 minutes. The cells were centrifuged at 1500 rpm for 5 minutes at 4 ° C, washed twice with PBS, and fixed with 3% paraformaldehyde The cells were mounted with a VECTASHIELD containing DAPI (Vector, CA) and confocal scanning laser microscopy The green fluorescence was detected only on the cell surface before incubation, but was abundantly observed in the cells after incubation at 37 ° C for 30 minutes (Fig. 5B). Indicates that an internalization was performed after the bridge, whereas a tyrosine residue (Y-F233) in the localization motif was mutated to internalize MMR-I. Is not detected, this is the tyrosine at position 233 Is responsible for the MAIR-1 in-home narration.

 (2) tyrosine phosphorylation of MAIR-1 and association with phosphatase

The presence of a consensus sequence for ITIM in the intracellular region of MMR-1 suggested that MAIR-1 could undergo tyrosine phosphorylation and recruit SH-2-containing tyrosine phosphatases. Therefore, it was examined whether MAIR-1 expressed on cultured marrow cells derived from bone marrow was tyrosine phosphorylated by stimulation with pervanadate. Bone marrow cells derived from the thighs and face bones of adult Balb / c mice were cultured at a concentration of 2XlOVml in RPMI1640 medium containing 10% FBS, mouse rIL-3 (4ng / ml; PliarMingen) and mouse rSCF (lOng / ml). . Cultured bone marrow-derived mast cells were stimulated with pervanadate at 37 ° C for 10 minutes or not, with 1% ΝΡ-40 (Figure 6A) or 1% digitonin.

(Sigma) buffer (0.12¾ Triton-100 (Katayama Chemical), 150 mM NaCL 20 mM triethylamine (Katayama Chemical), or the above-mentioned 1% NP-40 buffer containing protease and phosphatase inhibitor) (Figure 6B) and immunoprecipitated with control IgG, anti-MAIR-1, anti-SHP-I, anti-SHP-II, or anti-SHIP. Immunoprecipitates were immunoblotted with stake-phosphocytosine syn mAb (anti-pY) (FIG. 6 6) or anti-MAIR-1 (FIG. 6B). As shown in FIG. 6A, pervanadate stimulation significantly induced tyrosine phosphorylation of MAIR-1 (FIG. 6A). Upon immunoprecipitation with protein-tyrosine phosphatases SHP-1 and SHP-12, and polyphosphate inositol 5-phosphatase SHIP, after stimulation of pervanadate in mast cells, MAIR-1 bound SHIP, 1 and did not bind to SHP-2 (FIG. 6B). These results indicate that MAIR-1 may mediate inhibitory signals after tyrosine in the intracellular region is phosphorylated.

 (3) Serotonin-releasing technology

 MAIR-1 has ITIM in the cytoplasmic region.To confirm whether this is actually responsible for inhibitory signal transduction, in a mast cell induced by culturing bone marrow cells with IL-3 and SCF, We analyzed whether MAIR-1 suppressed degranulation via IgE receptor (FcRI). Uehara, T., et al., J Clin

Invest, 108, 1041-50, 2001. First, bone marrow cell-derived mast cells were combined with 3H-serotonin (5- [1,2-3H (N)]-hydroxytryptamine creatinine sulfate) (5 Ci / ml; NEN Life Science Producsts In, Boston, MA). The cells were incubated for 5 hours at 37 ° C, and re-incubated for another 1 hour to reduce background radioactivity. 3H-serotonin-loaded mast cells were treated with 101 rat IgE anti-DNP mAb (25 g / ml) and various concentrations of rat anti-MAIR-1 or control IgG (IgE niAb + anti-MAIR-1 or IgE niAt) + control IgG), incubate for 30 minutes at 4 ° C, wash, and resuspend in 25 L medium. Suspended. Mast cells primed with the antibody were challenged with 25 / xl Fg (anti-rat) Ig F antibody (40 g / ml) F (alD ') 2 fragment for 30-60 minutes at 37 ° C. The reaction was stopped by adding cold PBS. The 3 H-serotonin in the supernatant was measured using a liquid scintillation counter. The total uptake of 3 H-serotonin was measured by lysing the mast cells in PBS containing 1% SDS / 1% NP40, and the rate of serotonin release was determined as 100 X (cpm of supernatant / total uptake of supernatant). Cpm). As shown in FIG. 6C, cross-linking of MAIR-1 and anti-MAIR-1 mAb inhibited IgE-mediated serotonin release from bone marrow-derived mast cells in a concentration-dependent manner.

 (4) Association of MAIR-II adapter molecules

 Unlike MAIR-I, MAIR-II has a short intracellular region and has no sequence involved in signal transduction, and the presence of a positively charged lysine residue in the transmembrane region, It was predicted that an adapto molecule, such as FcsRI APDAP12, having a negatively charged amino acid residue would associate. Therefore, in order to examine non-covalent binding of MAIR-II to an adapter transmembrane protein such as FcsRIr or DAP12, 293T cells that do not express FcSRIT or DAP12 were transformed with MAIR-II cDNA and FcεRIa. Or DAP12. The transformant was dissolved in 1% digitonin buffer, the cell lysate was immunoprecipitated with control IgG, anti-DAP12 or anti-FcRIr, and the isolated protein was anti-MAIR-II. The immunoblot was performed. As shown in Figure 7A, co-immunoprecipitation of MAIR-II and DAP12 was confirmed, and it was clear that MAIR-II was associated with MP12, but co-immunoprecipitation with FcsRI Not confirmed. Similarly, when immunoprecipitation was performed using RAW cells of mouse macrophages into which the MAIR-II gene had been introduced, co-immunoprecipitation of MAIR-11 and DAP12 was confirmed, but co-immunoprecipitation of FcS RIT was not. It was not confirmed (Fig. 7B).

To confirm the physiological binding of MAIR-II to DAP12 in the following cells, spleen cells were stimulated with LPS, dissolved in 1% digitonin buffer, and immunoprecipitated with anti-Fc £ RI or anti-DAP12. . When the isolated protein was again immunoplotted with anti-MAIR-II, MAR-Π co-immunoprecipitated with DAP12 but not with FcεRIa (FIG. 7C). These results indicate that DAP12 is a physiological partner for MAIR-II. Also, 293T cells were transformed with Flag-tagged DAP 12, MAIR-IL or Flag-tagged DAP ^ + MAIR-II, followed by biotin mono-conjugated anti-MAIR-II and FITC-conjugated anti-Flag mAb, followed by And stained with APC-conjugated streptavidin. The result of analyzing the cell surface expression of MMR-II and DAP12 by flow cytometry is shown in FIG. 7D. It was observed that DAP12 expression on the cell surface of 293T cells was dependent on MAIR-II expression (FIG. 7D).

 (5) TNF-α; secretion

DAP12 has ITAM and has been shown to transmit an activation signal (Lanier, LL, et al., Immunol. Today. 21, 611-614, 2000; Tomasel lo, E., et al. , J. Biol. Chem., 273, 34115-34119, 1998; Lanier, LL, et al., Nature, 391, 703-707, 1998) _{0 In} fact, the activation signal is transmitted from MAIR-II. Was analyzed. RAW cells expressing peritoneal macrophages derived from Flag-tagged MAIR-II or C57BL / 6 mice were pretreated with anti-CD32 / 16 (FcrR) and plastic-coated control IgG, anti-Flag or anti-FragR. -Stimulated with MAIR-II mAb and cultured for 48 hours. The TNF-α concentration in the cell supernatant was measured using ELISA kit (e-Bioscience, San Diego, CA) according to the manufacturer's instructions. As shown in Fig. 8, in the transformants into which the MAIR-II gene with a N-terminal flag added, the production of the inflammatory cytokine, TNF-α, was significantly enhanced when cross-linked with an anti-Flag antibody. . In addition, TNF-a secretion was significantly increased even when MAIR-II molecule was directly cross-linked with anti-MAIR-II antibody in peritoneal macrophage. However, no significant increase in IL-6 and IL-12 secretion from peritoneal macrophages was observed after cross-linking of the anti-MAIR-II antibody. These results indicate that MAIR-II mediates a distant signaling pathway and leads to ΤΝ-α secretion in macrophages. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial applicability

According to the present invention, there is provided a novel immunoreceptor protein which positively or negatively regulates an immune response. The immunoreceptor protein of the present invention activates and suppresses the immune response. It is useful for elucidating the control mechanism of the innate immune response, searching for immunomodulators, and developing pharmaceuticals such as antiallergic drugs and antiinflammatory drugs. Sequence listing free text

 SEQ ID NO: 9: synthetic thigh A

 SEQ ID NO: 10: synthetic DNA

 SEQ ID NO: 11: synthetic DNA

 SEQ ID NO: 12: synthetic DNA

 SEQ ID NO: 13: Synthetic DNA

 SEQ ID NO: 14: Synthetic DNA

Claims

1. An immunoreceptor protein shown in the following (a) or (b):  (a) a protein having an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing  (b) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having a function of suppressing a cell immune response;  2. An immunological receptor protein shown in (c) or (d) below.  (c) a protein having an amino acid sequence represented by SEQ ID NO: 4 in the sequence listing  (d) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing A protein having an amino acid sequence in which is deleted, substituted or added, and having a function of activating a cellular immune response  Enclosure  3. A gene encoding the immunoreceptor protein according to claim 1 or 2. 4. A gene consisting of DNA shown in the following (a) or (b):  (a) DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing  (b) a protein having a function of hybridizing under stringent conditions to a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing under stringent conditions, and of suppressing a cell's immune response. DNA to encode  5. A gene consisting of DNA shown in the following (c) or (d):  (c) DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing  (d) has the function of hybridizing with DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 of the sequence listing under stringent conditions, and activating the immune response of cells DNA encoding protein  6. A recombinant vector containing the gene according to claim 4 or 5.  7. A transformant transformed by the gene according to claim 4 or 5.  8. The immunoreceptor protein according to claim 1 or 2, wherein the transformant according to claim 7 is cultured in a medium, and immunoreceptor protein is collected from the obtained culture. Production method. 9. Either of the immunoreceptor proteins according to claim 1 or 2, or a combination thereof. An antibody that recognizes both.  10. A method for isolating a pair of activated immunoreceptor genes using a gene encoding an extracellular conserved region of an inhibitory immune receptor having an ITIM motif as a probe. 11. A method for isolating a pair of inhibitory immune receptor genes using a gene encoding an extracellular conserved region of an activated immune receptor having an ITAM motif as a probe. 12. The immunoreceptor protein according to claim 1 or 2, or a cell or cell membrane that expresses the protein is caused to act on a test substance expected to contain a ligand, and A method for screening a ligand for the immunoreceptor protein, which comprises selecting a substance having high affinity. 13. The ligand for the immunoreceptor protein according to claim 1 or 2, which is obtained by the method according to claim 12.  14. (a) when the immunoreceptor protein according to claim 1 or 2 is allowed to act on a ligand for the immunoreceptor protein in the absence of a test substance, (b) comparing the immunoreceptor protein according to claim 1 or 2 with a ligand for the immunoreceptor protein in the presence of a test substance, and comparing the immunoreceptor protein with the ligand. A method for screening an antagonist or agonist against the immunoreceptor protein, which comprises selecting a substance that affects the immune receptor protein.  15. A screening kit for an antagonist or agonist against the immunoreceptor protein, comprising the immunoreceptor protein according to claim 1 or 2.  16. An antagonist or agonist against the immunoreceptor protein according to claim 1 or 2, which is obtained using the method according to claim 14 or the kit according to claim 15.  17. A drug for controlling an immune function, comprising the immunoreceptor protein according to claim 1 or a gene encoding the protein as an active ingredient. 18. A drug for controlling an immune function comprising, as an active ingredient, a ligand for the immunoreceptor protein according to claim 1 or 2, or an antagonist or agonist for the immunoreceptor protein according to claim 1 or 2. 19. The reagent for detecting an immunoreceptor protein according to claim 1 or 2, comprising the antibody according to claim 9.