WO2002077230A1 - Nr10 splicing variants - Google Patents

Abstract

It is intended to provide novel hemopoietin receptor proteins, genes encoding the same and production and use thereof. Splicing variants of a human hemopoietin receptor gene NR10 are successfully isolated by PCR cloning. The protein encoded by the isolated genes occurs in two types, i.e., a transmembrane type and a soluble type. The expression of the gene encoding the transmembrane protein is found out in a tissue containing hematopoietic cells. Further, a mouse NR10 gene is identified and its full-length cDNA is isolated. Proteins encoded by these genes, which are novel hemopoietin receptor genes participating in immunomodulation and hematopoietic cell regulation (in vivo), are useful in searching for novel hematopoietic factors capable of functionally binding to the above receptor and developing remedies for immunological and hematopoietic diseases.

Description

 NR 1 branch surgery

 The present invention relates to a novel hemopoietin receptor protein, a gene encoding the same, methods and uses thereof. Landscape technology

It has been known that there are a number of site-related proteins as sexual factors that control proliferation and differentiation of various cells, or maintain and activate the function of differentiated and mature cells, and even down to the point of [^]. Have been. Identification of genes encoding novel hemopoietins and novel lymphopoietins, which have physiological activity on lymphohematopoietic system function, in particular, is one of the most effective means for searching for drug discovery target molecules. It is considered to be one. However, the primary structural homology at these sites is low, and there is no significant homology at the amino acid level even between cytokine members belonging to the same subfamily (Murakami, M. et al. Natl. Acad. Sci. USA, 1991, 88, 11349-11353) ₀ Therefore, it is extremely difficult to estimate new member genes from primary structure by sequence homology search. Therefore, the present inventors have developed a motif consisting of 5 amino acids of Tir-Ser-Xaa-Trp_Ser (Xaa is any amino acid) conserved on the primary structure of each receptor family corresponding to the above-mentioned target site force-in. And developed a search for a novel cytokin receptor gene with this motif. In fact, this approach allows the use of IL-11 receptor (Robb, L. et al., J. Biol. Chem. 271 (23), 1996, 13754-13761), leptin receptor (Gains ford T. et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (25), 14564-8), IL-13 receptor (Hi ton DJ et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (1), 497-501) and 6 (Alexander WS et al., Current Biology, 1999, 9, 605-608). The present inventors have found a partial genomic sequence of the novel cytokine receptor gene NR10 by searching the human genome database, and have isolated the full-length human NR10 cDNA based on the partial sequence, It was clear. The transcript of human NR10 can encode two types of tightly-coupled receptor protein and soluble secretory receptor protein, depending on the nucleotide sequence caused by the splicing variant (International Publication No. W000 / 75314). ). Disclosure of the invention

 The present invention provides novel hemopoietin receptor proteins, and DNAs encoding them. The present invention also provides a vector into which the DNA has been inserted, a transformant carrying the DNA, and an i ^ method for a recombinant protein using the transformant. The present invention further provides a method for screening a compound that binds to the protein. The present inventors can encode two types of transmembrane receptor protein and soluble secretory receptor protein in the above-mentioned human NR10 transcript, depending on the nucleotide sequence difference resulting from the splicing variant. (International Publication Number 1) 00/75314). However, although its transmembrane receptor, View 0.3, possesses the Boxl motif, a JAK kinase binding site in its intracellular domain, the NR10 molecule itself is activated by ligand stimulation. It did not carry tyrosine residues to be phosphorylated. Based on these facts, 10 is a site-force receptor α chain capable of binding ligand, but another partner chain (site-force receptor 13) is involved in intracellular signal transduction via NR10. Predicted to be necessary.

The present inventors predicted that there would be further splicing variants in the NR10 gene, and performed PCR cloning for the purpose of identifying them. As a result, Mutants were successfully isolated, and the structural analysis and gene expression of these mutants were performed. As a result, it was revealed that one of the 10 gene splicing variants has a tyrosine residue in the intracellular region. The human NR10 gene exhibits diversity due to alternative splicing, particularly in the structure at the C-terminal side of the transmembrane region. The present inventors named the above-mentioned splicing variant which is predicted to have a signaling function as NR10.4. In addition, new splicing mutants that possess the fine J3 reticulum region but are predicted to have no signal transduction ability are named NR10.5 and NR10.6, respectively. Sequences were distinguished. In addition, a new soluble secreted receptor that does not have the transmembrane region was isolated and designated as N10.7 and NR10.8. It is expected that the regulation of functional expression of the N10 gene group will be controlled by the alternative splicing specific to thread II 戠 or specific cell type. As a result of expression of NR4, NR10.5, and Ml0.6 in humans, strong expression was detected in the responsible thread II 血, hematopoietic tissues, and livestock. From these expression characteristics, it is presumed that these NR10 molecules are involved in the regulation of biological immunity or hematopoietic cells, and the NR10 molecule is used to search for new hematopoietic factors that can functionally bind to the receptor Is considered very useful.

One reason that these human N10 gene cluster splicing variants (collectively "NR10") are considered to be functionally equivalent is their ligand binding ability. Although they show diversity at the C-terminal side due to alternative splicing, they share a cytokine binding region in all clones. That is, in the extracellular region, since they are almost the same in all clones, they have the same structure and are considered to recognize the same specific ligand. On the other hand, the reason that each of the biological function activities is considered to fulfill a different biologic responsibility is due to the difference in their intracellular signaling functions. Before the human NR10.4 gene sequence and below, the presence of the plurality of splicing variants identified in the present invention became apparent, in particular, analysis of NR10. Therefore, it was thought that it was difficult to transmit a strong proliferation signal or differentiation signal into cells by itself because the amino acid sequence extending to the C-terminal side was short. It is based on the Boxl motif (the Pro-Xaa-Pro sequence following several bases' one raw amino acid and multiple hydrophobic amino acids) (Xaa represents any amino acid). Possesses: fAK kinase binding is predicted, but in both cases of NR10. 1 and NR10.3, itself is phosphorylated within the sequence at the C-terminal side of the Box 1 motif. Because no tyrosine residue was present. From the above speculations, like most receptors belonging to the same receptor family, by forming a multi-complex such as a heterodimer or heterotrimer in NR10 together with another receptor subunit, It was predicted to establish the intracellular signal transmission function. However, the identification of the signaling receptor NR10.4 in the present invention also suggests that NR10 does not require a third partner chain and may function as a homodimer. On the other hand, by overexpressing the splicing mutant NR10.6, which is predicted to have no signal transduction function, as a dominant deficient mutant (dominant negative chain) on the cell surface, it is extremely effective to use it for evaluation of a ligand search system. In addition, by using the secretory receptors NR10.7 and R10.8 as decoy receptors, it is possible to competitively inhibit the binding of NRIO ligand molecules to cell surface NR10 molecules. it is conceivable that.

Furthermore, the present inventors identified a mouse NR10 homologous gene corresponding to the human NR10.4 gene, isolated full-length cDNA, and analyzed the primary structure. In the present invention, the mouse NR10 gene has been identified, isolated, and its structure has been elucidated, so that the search for mouse NR10 ligand can be developed by the same means as for the human NR10 molecule. The construction of a mouse NR10 ligand retrieval system is considered to be extremely effective because it allows the NR10 ligand to be used without using human-derived test samples. In addition, the details of NR10 molecular function analysis, which cannot be performed with human subjects, can be analyzed using mouse subjects. It can also be implemented. Furthermore, generation of NR10 gene-deficient mice by using a mouse genomic gene fragment encoding mouse NR10 is also considered to be extremely effective in clarifying the NRIO ^^ function.

 That is, the present invention relates to novel hemopoietin receptors and their genes, and further to their use.

 (1) DNA according to any of (a) to (d) below,

 (a) DNA encoding a protein consisting of the amino acid sequence described in any of 4, 6, 8, 10, 12, 14, 16, or 18;

 (b) System IJ number: DNA containing the coding region of the nucleotide sequence described in any of 3, 5, 7, 9, 11, 13, 15, or 17;

 (c) System IJ number: In the amino acid sequence described in any of 4, 6, 8, 10, 12, 14, 16, or 18, one or more amino acids are substituted, deleted, inserted, and Z or added. Having the following amino acid sequence, and encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, and 18. ,

 (d) System IJ number: The DNA consisting of any one of 3, 5, 7, 9, 11, 13, 15, or 17 and a string IJ number under stringent conditions. DNA encoding a protein functionally equivalent to a protein consisting of the amino acid sequence described in any of 4, 6, 8, 10, 12, 14, 16, or 18;

(2) a DNA encoding a partial peptide of a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, or 18;

 (3) A protein or peptide encoded by the DNA according to (1) or (2)

(4) a vector into which the DNA of (1) or (2) has been inserted,

(5) a transformant carrying the DNA of (1) or (2) or the vector of (4), (6) the step of culturing the transformant according to (5), and recovering the expressed protein from the transformant or a culture supernatant thereof; Production method,

 (7) an antibody that binds to the protein according to (3),

 (8) System IJ number: Polynucleotide containing at least 15 nucleotides complementary to DNA consisting of the base sequence described in any of 3, 5, 7, 9, 11, 13, 15, or 17 or a complementary strand thereof Nucleotides,

 (9) A method for screening a compound that binds to the protein according to (3),

(a) contacting a test sample with the protein or a partial peptide thereof,

 (b) detecting the binding activity between the protein or its partial peptide and a test sample,

 (c) selecting a compound having an activity of binding to the protein or a partial peptide thereof. The present invention provides a novel molecule of the hemopoietin receptor NR10. GenBank database-Based on the results of horny loaf and-PCR, the present inventors found that a new human-derived mopoietin receptor gene NR10.4, NR10.5, NR10.6, NR10.7, and NR10.8 was successfully identified and isolated. Furthermore, the present inventors succeeded in isolating the mouse R10 (R10C and NR10B) genes for the first time. N 10.4, NR10.5, and NIU0.6 encoded a transmembrane receptor, and NR10.7 and NR10.8 encoded a soluble receptor. In addition, both mouse NR10C and M10B encoded a sensitive penetrating receptor.

The nucleotide sequences of the NR10.4, NR10.5, NR10.6, NR10.7, and N10.8 cDNAs are encoded by these cDNAs as SEQ ID NOs: 3, 5, 7, 9, and 11, respectively. The amino acid sequences of the proteins are shown in SEQ ID NOs: 4, 6, 8, 10, and 12, respectively. In addition, the nucleotide sequences of mouse NR10C and NR10B cDNA are shown in SEQ ID NO: The amino acid sequences of the proteins encoded by these cDNAs are shown in SEQ ID NOs: 14 and 16, respectively.

 In the extracellular region, NR10.4, NR10.5, NR10.6, NR10.7, and N10.8 are almost all the same, so that they have the same three-dimensional structure and furthermore have the same specificity. It is believed to recognize the ligand.

 As a result of gene expression analysis in each human organ using the RT-PCR method, N10.4, NR10.5, and 10.6 genes were found to be in charge of evacuation, hematopoiesis, and in living tissues. Strong expression was detected. Specifically, strong expression was detected in the thymus, lymph nodes, peripheral leukocytes, lung, bone marrow, and other medullary fibers, and hematopoietic cells, and in addition, testis, prostate, placenta, uterus, etc. Strong expression was also detected in fiber and endocrine fibers. In addition, although weak expression was detected in kidney, knee, and small intestine, no expression was observed in spleen, tonsils, heart, brain, liver, skeletal muscle, and colon. On the other hand, strong expression was detected in CD14 + monocytes (macrophages), CD4 + T cells, and quiescent CD19 + B cells among blood ffll 戠 cells, but CD8 + T cells and activated CD19 + B cells Showed no expression at all. When these expression distributions are combined, the gene of the present invention shows that the gene of the present invention has a novel hematopoietic factor receptor It is presumed to code the body. In addition, the expression distribution was observed in thread 1 戠 other than the above, suggesting that the gene of the present invention may be able to regulate not only the ^ S system and hematopoietic system but also a wide variety of physiological functions in vivo. are doing.

The protein of the present invention can be applied to medical treatment. The expression of the residue of the present invention in thymus and peripheral leukocytes suggests that it may be an unknown hematopoietic factor receptor. Therefore, the protein of the present invention is considered to provide a useful material for obtaining this unknown hematopoietic factor. It is also conceivable to search for an peptide capable of functionally binding to the protein of the present invention or an gonist for a peptide library or a synthetic chemical material to isolate and identify it. Further, the protein of the present invention Clinical applications such as control of living body immunity and control of hematopoietic cells by searching for novel antibodies capable of functionally binding to white matter and specific antibodies that can limit the function of the protein of the present invention are expected.

 In addition, it is assumed that the expression of the residue of the present invention may be specifically expressed in a limited group of these hematopoietic cells. Antibodies are useful. The cells isolated in this way can be applied to cell transplantation therapy. Further, the antibody is expected to be applied to diagnosis or treatment of disease types such as leukemia.

 On the other hand, soluble proteins containing the extracellular domain of the protein of the present invention, or soluble proteins such as 10.7 and NR10.8 are expected to be used as decoy receptors as inhibitors of NR10 ligand, and NR10 is involved. It can be expected to be applied to the treatment of leukemia and other diseases.

The present invention includes a protein functionally equivalent to the protein consisting of the amino acid sequence described in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18. Such proteins include, for example, human R10.4, ..5, 麵 .6, fraction.7, or NR10.8 protein (SEQ ID NOs: 4, 6, 8, 10, 12, or 14, respectively), Alternatively, a homologous protein and a variant of another organism corresponding to the mouse N10 protein (NR10C or NR10B) (SEQ ID NO: 16 or 18, respectively) are included. In the present invention, “functionally equivalent” means that the target protein is the human NR10.4, NR10.5, NR10.6, R10.7, or R10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14) or mouse NR10 protein (NR10C or NR10B; SEQ ID NO: 16 or 18, respectively). The biological activity includes, for example, a method well known to those skilled in the art for preparing a protein that is functionally equivalent to a protein that is a membrane-bound or soluble hematopoietic factor receptor protein. It is known to introduce a mutation into a protein. For example, in the field If available, site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275; Zoller, MJ and Smith, M. (1983) Methods Enzymol. 100, 468-500, Kramer , W. et al. (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz, HJ (1987) Methods Enzymol. 154, 350-367; Kunkel, TA (1985) Proc. Natl. Acad Sci. US A. 82, 488-492; Kunkel TA (1988) Methods Enzymol. 85, 2763-2766), and human 1、10.4 ^ 0.5, 1110.6, 110.7, or NR10.8 protein (respectively). SEQ ID NO: 4, 6, 8, 10, 12, or 14) or mouse R10 protein (10C or NR10B) (SEQ ID NO: 16 or 18) by introducing appropriate mutations into the amino acids of these proteins and their functions. It is possible to prepare a protein which is substantially equivalent. Amino acid mutations can also occur in nature. Thus, the human NR10.4, NR10.5, NR10.6, NR10.7, or NR10.8 protein (SEQ ID NOs: 4, 6, 8, 10, 12, or 14, respectively), or mouse N 10 protein (NR10C or 10B) (SEQ ID NO: 16 or 18, respectively) having an amino acid sequence in which one or more amino acids are mutated, and the corresponding original protein (SEQ ID NO: 4, 6, respectively) A protein functionally equivalent to 8, 10, 12, 14, 16, or 18) is also included in the protein of the present invention.

Specific examples of the protein functionally equivalent to the protein of the present invention include one or two or more in the amino acid sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18. Preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less amino acids are deleted, as shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18. An amino acid sequence having one or more amino acids, preferably two or more and no more than 30 amino acids, more preferably two or more and no more than 10 amino acids; SEQ ID NOs: 4, 6, 8, 10, 12, 14, One or two or more, preferably two or more and thirty or less, more preferably two or more and ten or less amino acids in the amino acid sequence represented by 16, or 18 are substituted with another amino acid. No. Preferably, the mutated amino acid residue is mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, the properties of amino acid side chains are sparse

7K amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, E, Q, G, H, K, S, T), fatty females Amino acids with side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, Τ, Υ), amino acids with sulfur atom-containing side chains (C, Μ), Amino acids with carboxylic acid and amide containing side chains (D, N, E, Q), Amino elimination with base containing side chains (R, K, Η), Amino acids with aromatic containing side chains (H, F, Y,) can be mentioned (all the letters in parentheses represent the one-letter code of amino acids). Thus, proteins in which amino acids have been conservatively substituted are included in the proteins of the present invention.

 It is already known that a protein having an amino acid sequence modified by deletion or addition of one or more amino acid residues to a certain amino acid sequence and substitution by Z or another amino acid maintains its biological activity. Known (Mark, DF et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666; Zoller, MJ and Smith, M. (1982) Nucleic. Acids Res. 10, 6487-6500; Wang, A. et al. (1984) Science 224, 1431-1433; Dalbadie-McFarland, G. et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6409-6413).

Human NR10.4, NR10.5, NR10.6, NR10.7, or NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14 respectively) or mouse NR10 protein (NR10C Or NR10B) (SEQ ID NO: 16 or 18), respectively, with one or more amino acid residues added to the amino acid sequence, for example, human NR10.4, N10.5, NR10.6 , NR10.7, or NR10.8 protein (SEQ ID NOs: 4, 6, 8, 10, 12, or 14 respectively) or mouse NR10 protein (NR10C or 10B) (SEQ ID NO: 16 respectively) Or a fusion protein comprising 18). The fusion protein is a fusion of these NR10 proteins with other peptides or proteins, and such fusion proteins are also included in the present invention. The method for producing the fusion protein is as follows: The DNA encoding the NR10 protein and the DNA encoding the other peptide or protein may be ligated in such a manner that the frames bind to each other, introduced into an expression vector, and expressed in a host. A known method can be used. The other peptide or protein to be fused with the protein of the present invention is not particularly limited.

 Other peptides to be fused to the protein of the present invention include, for example, FLAG (Hopp, TP et al. (1988) BioTechnology 6, 1204-1210) and six His (histidine) residues. 6 XHis, 10XHis, influenza agglutinin (HA), human c-myc fragment, VSV-GP. Fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, Known peptides such as a-tubulin fragment, B-tag, and Protein C fragment can be used. Other proteins to be fused with the protein of the present invention include, for example, GST (daltathione S-transferase), HA (influenza agglutinin), immunoglobulin constant region, j3-galactosidase And MBP (maltose binding protein). A fusion protein can be prepared by fusing a commercially available DNA encoding the peptide or protein with a DNA encoding the protein of the present invention, and expressing the fusion DNA prepared thereby. Wear.

In addition, the protein of the present invention particularly includes, in the amino acid sequence described in human NR10.4 (SEQ ID NO: 4), one or more amino acids substituted, deleted, inserted, and Z- or added amino acids. A protein having a sequence and comprising at least 7 contiguous amino acids, preferably 8 amino acids or more, more preferably 9 amino acids or more in the amino acid sequence of SEQ ID NO: 21; And functionally equivalent proteins. Further, the protein of the present invention has an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and Z- or added in the amino acid sequence described in human NR10.5 (SEQ ID NO: 6). And at least 7 contiguous amino acids, preferably at least 8 amino acids, more preferably at least 9 amino acids of the amino acid sequence of SEQ ID NO: 24, and the amino acid of SEQ ID NO: 6 Includes proteins functionally equivalent to proteins consisting of acid sequences.

 Proteins functionally equivalent to human NR10.4 (SEQ ID NO: 4) and mouse NR10 protein (NR10C or 〖复 10B) (SEQ ID NO: 16 or 18 respectively) include human NR10.4 or mouse One or more amino acids in the amino acid sequence of the NR10 protein (NR10C or 置換 NR10B) have an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, or Z-added, and B0X1 (Pro-Xaa-Pro) and B0X1 And a protein having a tyrosine residue on the C-terminal side of the protein.

Other methods well known to those skilled in the art for preparing proteins that are functionally equivalent to a protein include hybridization techniques (Sambrook, J. et al. (1989) Molecular Cloning 2nd ed., 9.7-9.58, Cold Spring Harbor Lab. Press). That is, those skilled in the art will recognize that human NR10.4, 10.5, N10.6, N10.7, or NR10.8 protein (SEQ ID NOs: 4, 6, 8, 10, 12, or 14, respectively) or mouse NR10 protein ( NR10C or N10B) (SEQ ID NO: 16 or 18, respectively) (based on the sequence II number: 3, 5, 7, 9, 11, 11, 13, 15, or 17) or a portion thereof Usually, it is also possible to isolate a DNA highly homologous thereto and to isolate a protein functionally equivalent to the above protein from the DNA. Thus, human NR10.4, NR10.5, NR10.6, NR10.7, or N10.8 protein (SEQ ID NOs: 4, 6, 8, 10, 12, 12, or 14, respectively), or mouse NR10 protein (NR10C or «NR10B) (SEQ ID NOs: 16 and 18 respectively) are proteins encoded by DNAs that hybridize with DNAs consisting of DNAs consisting of a part thereof or humans NR10.4, N10.5, and NR10, respectively. .6, NR10.7, or NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14 respectively) or mouse R10 protein (N10C or NR10B) (SEQ ID NO: 16 or 18 respectively) ) Are also included in the protein of the present invention. Such proteins include, for example, homologues of mammals other than human and mouse (for example, proteins encoded by monkey, rat, puppies, pelvis genes). You. Human NR10.4, NR10.5, NR10.6, NR10.7, or NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14 respectively) or mouse N10 protein (NR10C or N 10B) (SEQ ID NO: 16 or 18, respectively) When isolating a cDNA highly homologous to the DNA from an animal, the hematopoietic cell line tissue such as spleen, thymus, lymph node, bone marrow, peripheral leukocytes, etc. , And immunocompetent cell line tissues may be preferred, but are not limited to B.

 Human NR10.4, NR10.5, NR10.6, NR10.7, or NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14, respectively) or mouse NR10 protein (NR10C or R10B) As a condition for hybridization for isolating a DNA encoding a protein functionally equivalent to (SEQ ID NO: 16 or 18, respectively), those skilled in the art can select. The conditions for hybridization include, for example, stringent conditions. The stringent condition is, for example, using HpressHyb Hybridization Solution (manufactured by Clontech) as a hybridization buffer, 42 ° (: preferably, after 50 hours at 50 ° C for one hour, and then after hybridization. Washing conditions include 42 ° C, 2XSS 0.1% SDS, preferably 50 ° C, 2XSSC, 0.1% SDS, and more preferably, more stringent conditions. The conditions are, for example, after hybridization in a hybridization buffer at 65 ° C for 1 hour in the above solution, and then washing conditions after hybridization include 65 ° C, 2XSSC and 0.1% SDS. Under these conditions, not only DNAs with high homology as the temperature is lowered, but also DNAs with low homology are linguistic.

:Obtainable. Conversely, it can be expected that as the temperature is increased, only DNA having high homology can be obtained. However, the hybridization story

There may be a plurality of factors, such as salt concentration, other than temperature, which affect the water content. A person skilled in the art can realize a similar stringency by appropriately selecting these factors. In addition, instead of the hybridization, human NR10.4, NR10.5, NR10.6, NR10.7, or NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14, respectively), Alternatively, the sequence information of the DNA (SEQ ID NO: 3, 5, 7, 9, 11, 13, 13, 15 or 17) encoding the mouse NR10 protein (N10C or NR10B) (SEQ ID NO: 16 or 18 respectively) is obtained. It is also possible to isolate a target DNA by performing a gene amplification method using a primer synthesized based on the base, for example, a polymerase chain reaction (PCR) method.

 Human NR10.4, 10.5, 10.6, 10.7, or 10.8 protein encoded by DNA isolated by these hybridization or gene amplification techniques (SEQ ID NO: 4, 6, 8, 10, 12, or 14) or a protein functionally equivalent to the mouse NR10 protein (B10C or NR10B) (SEQ ID NO: 16 or 18, respectively) is usually human NR10.4, NR10.5, NR10.6, NR10.7, Or the NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14 respectively) or the mouse N10 protein (NR10C or NR10B) (SEQ ID NO: 16 or 18 respectively) and the amino acid sequence Have high homology. The protein of the present invention includes human NR10.4, NR10.5, NR10.6, NR10.7, or NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14, respectively) or mouse Functionally equivalent to the NR10 protein (NR10C or N10B) (SEQ ID NO: 16 or 18, respectively) and the amino acid shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 16 or 18 Proteins with high homology to the sequence are also included. High homology usually refers to 70% or more homology, preferably 80% or more homology, more preferably 90% or more, more preferably 95% or more identity. To determine protein homology, the algorithm described in the literature (Wilbur, WJ and Lipman, DJ (1983) Proc. Natl. Acad. Sci. USA 80, 726-730) may be used.

As the protein of the present invention, in particular, a DNA (cDNA or the like) which can be hybridized with a DNA consisting of the nucleotide sequence of SEQ ID NO: 19 or a part thereof under stringent conditions is used. A human NR10.4, NR10.5, NR10.6, N10.7, or NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14 respectively) )) Are preferred. Above all, it is a protein encoded by a DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 20 or a part thereof, and is a transitive human NR10.4 (SEQ ID NO: 4) A protein equivalent to is preferred as the protein of the present invention. Furthermore, a human NR10.5 protein (SEQ ID NO: 6), which is a protein encoded by DNA (such as cDNA) that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 22 or a part thereof. ) Are particularly preferred as proteins of the present invention. Among them, a protein encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 23 or a part thereof, and is functional with human N10.5 protein (SEQ ID NO: 6). A protein equivalent to the above is particularly preferable as the protein of the present invention.

 The protein of the present invention may vary in amino acid sequence, amount, isoelectric point, presence / absence and form of a short chain, etc., depending on the cell, host, or purification method that produces the protein as described below. However, the protein obtained was the human protein .4, 麵 .5, cell .6, cell .7, or NR10.8 protein (SEQ ID NO: 4, 6, 8, 10, 12, or 14) or as long as it has a function equivalent to that of mouse NR10C or NR10B (SEQ ID NO: 16 or 18, respectively). For example, when the protein of the present invention is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the N-terminal of the amino acid sequence of the original protein. When expressed in eukaryotic cells, for example, mammalian cells, the N-terminal signal sequence is removed. The proteins of the present invention also include such proteins.

The primary structures of the amino acid sequences of human NR10.4, δ.5, δ.6, γ.7, and NM0.8 protein (1; 4, 6, 8, 10, 12, and 14) to a protein structure analysis program (European Molecular Bio ht tp: // 丽. emb heidelberg. de / predictprotein /) of “The PredictProtein server” from logy Laboratory, Heidelberg, Germany. As a result, the signal sequence was 1st Met to 51st

It was estimated to Ala. Accordingly, the present invention includes a protein consisting of Ala at position 52 to Val at position 764 in the amino acid sequence of SEQ ID NO: 4. Similarly, in the amino acid sequence of SEQ ID NO: 6, it includes a protein consisting of Ala at position 52 to Val at position 764. Furthermore, in the amino acid sequence of SEQ ID NO: 8, it includes a protein consisting of Ala at position 52 to Pro at position 627. Furthermore, it includes a protein consisting of Ala at position 52 to Ser at position 581 in the amino acid sequence of SEQ ID NO: 10. Furthermore, the amino acid sequence of SEQ ID NO: 12 includes a protein consisting of Ala at position 52 to Asn at position 549. In addition, SEQ ID NO:

The amino acid sequence according to 14 includes a protein consisting of Ala at position 52 to Arg at position 548.

 The protein of the present invention can be prepared as a recombinant protein or a natural protein by methods known to those skilled in the art. In the case of a recombinant protein, a DNA encoding the protein of the present invention (eg, a DNA having the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 13 or 15) is used. After integration into an appropriate expression vector, introducing it into an appropriate host cell, recovering the transformant, obtaining an extract, and performing chromatography such as ion exchange, reverse phase, gel filtration, etc. Alternatively, the protein can be purified and prepared by subjecting the antibody to the protein of the present invention to affinity chromatography immobilized on a column, or by further combining a plurality of these columns.

When the protein of the present invention is expressed in host cells (eg, animal cells, Escherichia coli, etc.) as a fusion protein with the dalhithione S-transferase protein or as a recombinant protein to which a plurality of histidines are added. The purified recombinant protein is purified using a glutathione column or a nickel column. be able to. After purification of the fusion protein, if necessary, a region other than the target protein in the fusion protein can be cleaved with thrombin or Factor Xa and removed.

 If the protein is a natural protein, a method known to those skilled in the art, for example, an affinity in which an antibody that binds to the protein of the present invention described below is bound to a tissue or cell extract expressing the protein of the present invention, as described below. Isolation can be performed by purifying the reaction with one column. The antibody may be a polyclonal antibody or a monoclonal antibody.

 The present invention also includes partial peptides of the protein of the present invention. The partial peptide having an amino acid sequence specific to the protein of the present invention has an amino acid sequence of at least 7 amino acids, preferably at least 8 amino acids, more preferably at least 9 amino acids. The partial peptide can be used, for example, for preparing an antibody against the protein of the present invention, screening for a compound that binds to the protein of the present invention, and screening for promoters and inhibitors of the protein of the present invention. In addition, it can be an agonist for the ligand of the protein of the present invention. The partial peptide of the protein of the present invention includes, for example, a protein comprising the amino acid sequence J! Shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, or 18. A partial peptide consisting of an active center is exemplified. Further, a partial peptide containing one or more of a hydrophobic region and a hydrophilic region estimated from a hydrophobic plot analysis may be mentioned. These partial peptides may include part or all of one hydrophobic region, or may include part or all of one hydrophilic region. Further, for example, soluble proteins of the protein of the present invention and proteins consisting of extracellular regions are also included in the present invention.

The partial peptide of the protein of the present invention particularly includes a protein containing SEQ ID NO: 21 (an amino acid sequence encoded by exon CP10.4 of NR10.4) or a part thereof. Also included are proteins containing SEQ ID NO: 24 (amino acid sequence encoded by exon CP10.5 of NR10.5) or a part thereof. The partial peptide of the present invention can be produced by genetic engineering techniques, known peptide synthesis methods, or by cleaving the protein of the present invention with an appropriate peptidase. As the peptide synthesis method, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.

 The protein containing a partial peptide of the present invention includes a chimeric protein of the protein of the present invention and another hemopoietin receptor protein. For example, as described in Examples, a chimeric receptor in which the fine domain of the protein of the present invention is linked to an intracellular domain containing the transmembrane domain of another hemopoietin receptor protein is the present invention. It is useful for transmitting a signal of the other hemopoietin receptor protein in response to a ligand of the protein. In addition, a chimeric receptor in which the extracellular domain of the hemopoietin receptor other than the protein of the present invention is linked to the intracellular domain of the protein of the present invention including the fine W communication domain comprises the other hemopoietin receptor protein. It is useful for transmitting the signal of the protein of the present invention in response to the above ligand. The signal transduction effect of the protein of the present invention is examined by preparing a chimeric receptor with the protein of the present invention using the extracellular domain (ligand binding region) of the known hemopoietin receptor known as a ligand. It is possible. As described above, the present invention provides a chimeric receptor in which the intracellular domain of the protein of the present invention is linked to the intracellular domain containing the transmembrane domain of another hemopoietin receptor protein, and a hemopoietin other than the protein of the present invention. It includes a chimeric receptor in which the extracellular domain of the receptor is linked to the intracellular domain of the protein of the present invention including the transmembrane domain.

The present invention also provides a DNA encoding the protein of the present invention. The DNA of the present invention is used for production of the protein of the present invention in vivo or in vitro as described above, and for example, is a gene for a disease caused by an abnormality in the gene encoding the protein of the present invention. Application to treatment etc. is also conceivable. The DNA of the present invention may be in any form as long as it can encode the protein of the present invention. That is, it does not matter whether it is cDNA synthesized from mRNA, genomic DNA, or chemically synthesized DNA. In addition, the present invention DNAs having an arbitrary base sequence based on the degeneracy of the genetic code are included as long as they can encode the above protein.

 The DNA of the present invention can be prepared by a method known to those skilled in the art. For example, a cDNA library is prepared from cells expressing the protein of the present invention, and the sequence of the DNA of the present invention (for example, SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17) It can be prepared by performing hybridization using a part or a part thereof as a probe. The cDNA library may be prepared, for example, by the method described in «(Sambrook, J. et al., Molecular Clonings Cold Spring Harbor Laboratory Press (1989)), or a commercially available DNA library may be used. . In addition, after preparing a thigh from cells expressing the protein of the present invention and synthesizing cDNA with a reverse transcriptase, the sequence of the DNA of the present invention (for example, SEQ ID NO: 3, 5, 7, 9, 1) It can also be prepared by synthesizing oligo DNA based on (1), (13), (15), or (17)) and performing a PCR reaction using this as a primer to amplify the cDNA encoding the protein of the present invention. It is.

 Further, by determining the nucleotide sequence of the obtained cDNA, the translation region encoded by the cDNA can be determined, and the amino acid sequence of the protein of the present invention can be obtained. Genomic DNA can be isolated by screening the genomic DNA library using the obtained cDNA as a probe.

Specifically, the following may be performed. First, cells or tissues expressing the protein of the present invention, β (for example, thymus, lymph node, peripheral leukocyte, or hematopoietic cell line fiber such as bone marrow, and cell line cell fiber for nail), mRNA, etc. Is isolated. mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al. (1979) Biochemis try 18, 5294-5299), AGPC method (Chomczynski, P. and Sacci, N. (1987) ) The total RNA can be prepared by Anal. Biochem. 162, 156-159) or the like, and mRNA can be purified from the total RNA using mRNA Purification Cat Kit (Pharmacia). Also, by using QuickPrep mRNA Purification Cat (Pharmacia), mRNA can also be prepared directly.

 CDNA is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis can also be performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Shii-Dagaku Kogyo) or the like. In addition, using the DNA sequence and the like described in the present specification, 5′-MCE method using 5′-Ampli FINDER RACE Kit (manufactured by Clontech) and polymerase chain reaction (PCR) 3, -RACE method (Frohman, Μ · A. Et al. (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002; Belyavsky, A. et al. (1989) Nucleic Acids Res. 17 , 2919-2932) to synthesize and amplify cDNA.

 A target DNA fragment is prepared from the obtained PCR product and ligated to a vector DNA. Furthermore, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. The nucleotide sequence of the target DNA can be confirmed by a known method, for example, the dideoxynucleotide chain-one-minute method.

In the DNA of the present invention, a nucleotide sequence having higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al. (1981) Nucleic Acids Res. 9, r43-74) _Q The DNA of the present invention can be modified by a commercially available kit or a known method. Modifications include, for example, digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of a linker, insertion of an initiation codon (ATG) and Z or a stop codon (TM, TGA, or TAG). No.

The DNA of the present invention includes a DNA consisting of a protein coding sequence (CDS) in the nucleotide sequence of the system (1, 3, 5, 7, 9, 11, 13, 15, or 17). Specifically, the DNA of the present invention is specifically a DNA consisting of base A at position 7 to base C at position 2298 in the base sequence of SEQ ID NO: 3, and position 7 in the base sequence of SEQ ID NO: 5. A DNA consisting of base C at position 2298 from base A of SEQ ID NO: 7; DNA consisting of base C at position 7, DNA consisting of base C at position 1749 from base A at position 7 in the base sequence of SEQ ID NO: 9, nucleotide 1613 at base 7 of position 7 in base sequence of SEQ ID NO: 11 DNA consisting of T, DNA consisting of base A at position 7 to base A at position 1650 in the base sequence of SEQ ID NO: 13, consisting of base A at position 331 to base C of position 2478 in base sequence of SEQ ID NO: 15 DNA, including DNA consisting of base A at position 421 to base C at position 2568 in the base sequence of SEQ ID NO: 17.

 The DNA of the present invention is also a DNA that is eight-hybridized under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17, and DNAs encoding proteins functionally equivalent to the proteins of the invention are included.

 Stringent conditions can be appropriately selected by those skilled in the art, and include, for example, the above-described stringent conditions. That is, for example, using ExpressHyb Hybridization Solution (manufactured by Clontech) as a hybridization buffer, the mixture was subjected to hybridization for 1 hour at 42 ° C., preferably 50 ° C., and then washed at 42 ° C. for 42 hours. C, 2XSS 0.1% SDS, and preferably 50 ° C, 2XSSC, 0.1% SDS. More preferably, more stringent conditions can be mentioned. More stringent conditions include, for example, using xpressHyb Hybridization Solution (manufactured by Clontech) as a hybridization buffer, hybridizing at 42 ° C., preferably 50 ° C. for 1 hour, and then after hybridization. Wash conditions include 65 ° C, 2XSSC and 0.1% SDS. Under these conditions, DNA with higher homology can be obtained as the temperature is increased. The above hybridizing DNA may preferably be a naturally occurring DNA, for example, a cDNA or a chromosomal DNA.

Particularly, the DNA of the present invention is a DNA (such as a cDNA) that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ ID NO: 19 or a part thereof, and includes human NR10.4, NR10.5, and R10. .6, NR10.7, or NR10.8 protein SEQ ID NO: 4, 6, 8, 10, 12, or 14) DNA that encodes a functionally equivalent protein is preferred. DNA that hybridizes under stringent conditions with the DNA consisting of the NR10.4 part and that encodes a protein functionally equivalent to human NR10.4 (SEQ ID NO: 4) is preferred. Furthermore, it is a DNA (such as a cDNA) that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 22 or a part thereof, and is functionally similar to the human NR10.5 protein (SEQ ID NO: 6). DNAs encoding equivalent proteins are particularly preferred. Above all, DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of SEQ ID NO: 23 or a part thereof, and is functionally equivalent to human NR10.5 protein (SEQ ID NO: 6) Is particularly preferred.

 The present invention also provides a vector into which the DNA of the present invention has been inserted. The vector of the present invention is useful for retaining the DNA of the present invention in a host cell or expressing the protein of the present invention.

For example, when Escherichia coli is used as a host, the vector is amplified in Escherichia coli to amplify the vector in large amounts in Escherichia coli (e.g., JM109, DH5o !, HB10K XLl-Blue), etc. Ori, and a transformed gene of Escherichia coli (for example, a drug jffl live gene that can be identified by any drug (ampicillin-tetracycline, kanamycin, chloramphenicol, etc.)) There is no particular limitation. Examples of vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, and the like. In addition, for the purpose of subcloning and excision of cDNA, pGEM-T, pDIRECT, pT7 and the like can be mentioned in addition to the above vectors. When a vector is used for the purpose of producing the protein of the present invention, an expression vector is particularly useful. As an expression vector, for example, in the case of expression in E. coli, in addition to having the above-mentioned characteristics such that the vector is amplified in E. coli, the host may be used in the form of 109, DH5α, HB101, XLl-Blue, etc. In the case of E. coli, a promoter that can be efficiently expressed in E. coli LacZ promoter (Ward, ES et al. (1989) Nature 341, 544-546; Ward, ES (1992) FASEB J. 6, 2422-2427), araB promoter (Bet ter, M. et. al. (1988) Science 240, 1041-1043), or having a T7 promoter. Such vectors include PGEX-5X-1 (Pharmacia), QIAexpress systemj (Qiagen), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase in addition to the above vectors). BL21 is preferred).

 The vector may also include a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, the pelB signal sequence (Lei, SP et al (1987) J. Bacteriol. 169, 4379) may be used for production in E. coli periplasm. The introduction of the vector into the host cell can be performed using, for example, a calcium chloride method or an electroporation method.

 Other than Escherichia coli, for example, as a vector for producing the protein of the present invention, a mammalian expression vector (for example, pcDNA3 (InvitrogenviS), pEF-BOS (Mizushiraa, S. and Nagata , S. (1990) Nucleic Acids Res. 18, 5322), pEF, pCDM8), insect vector-derived expression vector (for example, `` BAC-TO-BAC Baculovinis Expression Systems J (GIBC0 BRL), pBacPAK8), Plant-derived expression vectors (eg, ρΜΗ1, ρΜΗ2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, ZIPneo), yeast-derived expression vectors (eg, rpichia Express ion KitJ (Invitrogen), pNVll, SP-Q01) and Bacillus subtilis-derived expression vectors (eg, pPL608, pKTH50).

When the expression is intended for expression in animal cells such as CH0 cells, COS cells, and NIH3T3 cells, promoters required for expression in cells, such as the SV40 promoter (Mulligan, RC et al. (1979) Nature 277, 108-114), MMLV-LTR promoter, EFla promoter (Mizushima, S. and Nagata, S. (1990) Nucleic Acids Res. 18, 5322), it is essential to have the CMV promoter all over the world, and it is essential to have a gene for selection of cell transformation (for example, drug resistance that can be determined by drug (neomycin, G418, etc.)) It is more preferable to have a gene). Vectors having such characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, and the like.

 Furthermore, in order to amplify the copy number of the gene in the cell and stably express the gene, a vector (for example, pCHOI, etc.) having the DHFR gene complementing it in CH0 cells lacking the nucleic acid synthesis pathway is used. And then amplifying it with methotrexate (MTX). For the purpose of transient gene expression, use COS cells that have a gene that expresses the SV40 T antigen on the chromosome. Transformation with a vector (such as pcD) having the SV40 replication origin can be mentioned. As the origin of replication, those derived from poliovirus, adenovirus, sipapipioma virus (BPV) and the like can also be used. Furthermore, the expression vector is used as a selectable marker for aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt) gene because of the gene copy number increase in the host cell system. Or a dihydrofolate reductase (dhfr) gene or the like.

On the other hand, as a method for expressing the DNA of the present invention in an animal body, the DNA of the present invention is incorporated into an appropriate vector and, for example, a retrovirus method, a ribosome method, a cationic liposome method, an adenovirus method, or the like is used. For example. This makes it possible to perform gene therapy for a disease caused by mutation of the gene of the present invention. Examples of the vector used include, but are not limited to, an adenovirus vector (eg, pAdexlcw) and a retrovirus vector (eg, pZIPneo). General manipulations such as introduction of the DNA of the present invention into a vector can be performed according to a conventional method (Sambrook, J. et al. (1989) Molecular Cloning 2nd ed., 5.61-). 5.63, Cold Spring Harbor Lab. press). Administration into a living body may be an ex vivo method or an in vivo method. The present invention also provides a transformant into which the DNA or the vector of the present invention has been introduced. The host cell into which the vector of the present invention is introduced is not particularly limited, and for example, Escherichia coli and various animal cells can be used. The host cell of the present invention can be used, for example, as a production system for producing or expressing the protein of the present invention. Production systems for protein production include in vitro and in vivo production systems. Examples of the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells.

 When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts. Animal cells include mammalian cells, for example, CH0, COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells, for example, African Xenopus oocytes (Val le, et al. 1981) Nature 291, 358-340) or insect cells such as Sf9 and Sf2 Tn5 are known. CH0 cells are, in particular, CH0 cells deficient in the DHFR gene (Mr-CHO (Urlaub, G. and Chas in, LA (1980) Proc. Natl. Acad. Sci. USA 77, 4216-4220) and Natl. Acad. Sci. USA 60, 1275-1281) CHO K-1 (Kao, FT and Puck, TT (1968) Proc. Natl. Acad. Sci. USA 60, 1275-1281) For large-scale expression in animal cells In this case, the CH0 cell is particularly preferable.The introduction of the vector into the host cell is performed by, for example, a calcium phosphate method, a DEAE dextran method, a method using Cationic ribosome D0TAP (Boehringer Mannheim), It can be done by election port method, lipofection, etc.

As a plant cell, for example, a cell derived from Nicotiana tabacum is known as a protein production system, which may be callus cultured. Fungal cells include yeast, for example, Saccharomyces genus, for example, Saccharomyces cerevisiae, filamentous fungi, for example, genus Aspergillus, for example, Aspergillus-Nigaichi lus niger) is known.

 When prokaryotic cells are used, there are production systems using bacterial cells. Examples of bacterial cells include Escherichia coli (E. coli), for example, JM109, DH5o !, HB101, and the like, and Bacillus subtilis.

 These cells are transformed with the target DNA, and the transformed cells are cultured in vitro to obtain a protein. The culturing can be performed according to a known method. For example, as a culture solution of animal cells, for example, DMEM, MEM, RPMI 1640 and IMDM can be used. At that time, a serum replacement solution such as fetal calf serum (FCS) can be used together, or serum-free culture may be performed. The pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.

 On the other hand, examples of a system for producing a protein in vivo include a production system using animals and a production system using plants. The target DNA is introduced into these animals or plants, and proteins are produced and recovered in the animals or plants. The “host” in the present invention includes these animals and plants.

 When using animals, there are production systems using mammals or insects. As mammals, goats, pigs, sheep, mice, mice, etc. can be used (Vicki Glaser, SPECTRUM Biotechnology Applicat ions, 1993). When a mammal is used, a transgenic animal can be used.

For example, the target DNA is prepared as a fusion gene with a gene such as goat / 3 casein, which encodes a protein uniquely produced in milk. Next, the DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. The target protein can be obtained from milk produced by the transgenic goat born from the goat that has received the embryo or its progeny. Hormones may be used as appropriate in transgenics and squirrels to increase the amount of milk containing proteins produced by transgenic goats (Ebert, KM et al. (1994) Bio / Technology 12, 699-702).

In addition, silkworms can be used as insects, for example. When a silkworm is used, the target protein can be obtained from the body fluid of the silkworm by infecting the silkworm with a baculovirus into which the DNA encoding the target protein has been introduced (S Kasumi, M. et al. (1985) Nature 315, 592-594) ₀

 Furthermore, when using a plant, for example, tobacco can be used. When tobacco is used, DNA encoding the protein of interest is introduced into a plant expression vector, for example, pMON530, and this vector is introduced into bacteria such as Agrobacteriuin tumefaciens. This bacterium is infected to tobacco, for example, Nicotiana tabacum, and the desired polypeptide can be obtained from the leaves of this tobacco (Ma, JK et al. (1994) Eur. J. Immunol. 24 , 131-138).

 The protein of the present invention thus obtained can be isolated from inside or outside the host cell (such as a medium) and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be carried out by using the separation and purification methods used in ordinary protein purification, and is not limited at all. For example, chromatographic columns, filters, gel filtration, salting out, solvent precipitation, elution, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. By properly selecting and combining, proteins can be separated and purified.

Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization). ion: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. The present invention relates to these purification methods. It also includes proteins highly purified using the method.

 The protein can be arbitrarily modified or partially removed by the action of an appropriate protein-modifying enzyme before or after protein purification. As the protein-modifying enzyme, for example, trypsin, chymotrypsin, lysylendopeptidase, protein kinase, dalcosidase and the like are used.

 The present invention also provides an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a monoclonal antibody as well as a polyclonal antibody. Also included are antisera obtained by immunizing immunized animals such as rabbits with the protein of the present invention, polyclonal antibodies and monoclonal antibodies of all classes, as well as human antibodies and humanized antibodies obtained by genetic recombination. .

 The protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal, for example, a human, a mouse or a rat, and particularly preferably a protein derived from a human. Human-derived proteins can be obtained using the gene sequences or amino acid sequences disclosed herein.

 In the present invention, the protein used as the sensitizing antigen may be a full-length protein or a partial peptide of the protein. The partial peptide of the protein includes, for example, an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein. As used herein, “antibody” refers to an antibody that reacts with the full length or fragment of a protein. In addition, for example, an antibody that binds to an amino acid sequence of SEQ ID NO: 21 or 24 or a peptide consisting of a part thereof is particularly preferable as the antibody of the present invention.

A gene encoding the protein of the present invention or a fragment thereof is inserted into a known expression vector system, and the host cell described in this specification is transformed with the vector. These can be obtained by a known method and used as a sensitizing antigen. Alternatively, a cell expressing the protein, a lysate thereof, or a protein of the present invention synthesized in a ligatory manner may be used as the sensitizing antigen. Short bubble wrap It is preferable that the antigen be appropriately bound to a carrier protein such as keyhole limpet mosquitoin, human serum albumin, and ovalbumin to obtain an antigen.

 The mammal to be sensitized is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Eyes, egrets, and primates are used.

 As rodent animals, for example, mice, rats, hamsters and the like are used.動物 As an heronoid animal, for example, a heron is used. For example, monkeys are used as primates. As the monkeys, monkeys of the lower nose (old world monkeys), for example, cynomolgus monkeys, macaques, baboons, and chimpanzees are used.

 To sensitize an animal to a sensitizing antigen, a known method is used. In the HIS method, a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, a normal adjuvant, for example, Freund's complete adjuvant, is mixed with an appropriate amount of the sensitizing antigen diluted and suspended with PBS (Phosphate-Buffed Saline) or physiological saline, etc., if desired. After emulsification, it is administered to mammals. Thereafter, it is preferable to administer the sensitizing antigen mixed with an appropriate amount of Freund's incomplete adjuvant several times every 4 to 21 days. In addition, a suitable carrier can be used at the time of immunization with the sensitizing antigen. Immunization is performed in this manner, and it is confirmed by a conventional method that the level of the desired antibody in the serum is increased.

Here, in order to obtain a polyclonal antibody against the protein of the present invention, the blood of a mammal sensitized with the antigen is removed after confirming that the desired antibody level in serum has increased. The serum is separated from the blood by a known method. As the polyclonal antibody, a serum containing the polyclonal antibody may be used, or if necessary, a fraction containing the polyclonal antibody may be further isolated from this serum and used. . For example, using an affinity column to which the protein of the present invention is coupled, a fraction that recognizes only the protein of the present invention is obtained. Alternatively, immunoglobulin G or immunoglobulin G can be prepared by purification using protein G protein.

 In order to obtain a monoclonal antibody, after confirming that the level of the desired antibody is increased in the serum of a mammal sensitized with the antigen, dead cells may be removed from the mammal and subjected to cell fusion. In this case, splenocytes are particularly preferred as preferred ^ $ cells used for cell fusion. The other parent cell to be fused with the immunocyte is preferably a mammalian myeloma cell, more preferably a myeloma cell that has acquired characteristics for selecting a fused cell by a drug.

 The cell fusion between the cells and myeloma cells is basically a known method, for example, the method of Milstein et al. (Gal re, G. and Milstein, C. (1981) Methods Enzymol. 73, 3-46) It can be performed according to.

 The eight hybridomas obtained by cell fusion are selected by culturing them in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminobuterin and thymidine). Culturing in the HAT culture solution is continued for a time sufficient for the death of cells other than the desired hybridoma (non-fused cells), usually for several days to several weeks. Next, a conventional limiting dilution method is performed to screen and clone a hybridoma producing the desired antibody.

 In addition to immunizing non-human animals with an antigen to obtain the above-mentioned antibodies, human lymphocytes, for example, human lymphocytes infected with ΪΒ virus, can be treated in vitro with proteins, protein-expressing cells or lysates thereof. After sensitization, the sensitized lymphocytes can be fused with human-derived myeloma cells capable of permanent division, for example, U266, to obtain a hybridoma that produces the desired human antibody having protein binding activity. Published Japanese Patent Application No. 63-17688.

Next, the obtained hybridoma was transplanted into the abdominal cavity of a mouse, ascites was recovered from the mouse, and the obtained monoclonal antibody was subjected to, for example, ammonium sulfate®, protein A, protein G column, DEAE ion exchange chromatography, Power the invention protein It can be prepared by purification using a coupled affinity column or the like. The antibody of the present invention is used for purification and detection of the protein of the present invention, and is also a candidate for an agonist or antagonist of the protein of the present invention. It is also conceivable to apply this antibody to antibody therapy for diseases involving the protein of the present invention. When the obtained antibody is used for the purpose of administration to the human body (antibody therapy), a human antibody or a human antibody is preferable in order to reduce the nucleogenicity.

 For example, a transgenic animal having a repertoire of human antibody genes is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain antibody-producing cells, and a hybridoma obtained by fusing this with myeloma cells is obtained. Can be used to obtain a human antibody against the protein (see International Publication Nos. W092-03918, W093-2227, W094-02602, W094-25585, W096-33735 and 34096).

 In addition to producing antibodies using hybridomas, cells in which immune cells such as sensitized lymphocytes that produce antibodies are immortalized with oncogenes may be used. The monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a genetic recombination technique (for example, Borrebaeck, CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United States). Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Recombinant antibodies are produced by cloning DNA encoding the same from ^ $ cells, such as hybridomas or sensitized lymphocytes, which produce the antibody, incorporating the DNA into an appropriate vector, and introducing it into a host to produce it. The present invention includes this recombinant antibody.

Further, the antibody of the present invention may be a modified antibody fragment thereof as long as it binds to the protein of the present invention. For example, antibody fragments include Fab, F (ab ') 2, Fv, or a single chain Fv (scFv) obtained by linking an Fv of an H chain and an L chain with an appropriate linker (Huston, JS et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Specifically, the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed, and Is introduced into an expression vector and then expressed in an appropriate host cell (eg, Co, MS et al. (1994) J. Immunol. 152, 2968-2976; Better, M. and Horwitz, AH (1989) Methods Enzymol. 178, 476-496; Pluckthun, A. and Skerra, A. (1989) Methods Enzymol. 178, 497-515; Lamoyi, E. (1986) Methods Enzymol. 121, 652-663; Rousseaux, J. et al. (1986) Methods Enzymol. 121, 663-669; Bird, RE and Walker, BW (1991) Trends Biotechnol. 9, 132-137).

 As the modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) can be used. The “antibody” of the present invention also includes these modified antibodies. Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.

 In addition, the antibody of the present invention can be prepared by using a chimeric antibody composed of a variable region derived from a non-human antibody and a constant region derived from a human antibody or a CDR (complementarity determining region) derived from a non-human antibody and a human antibody using known techniques. It can be obtained as a humanized antibody consisting of a FR (framework region) and a constant region derived therefrom.

The antibody obtained as described above can be purified to homogeneity. The separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins. For example, if chromatographic columns such as affinity chromatography, filters, gel filtration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. are selected and combined, antibodies can be separated. It can be purified (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but is not limited thereto. The concentration measurement of the antibody obtained above is carried out by measuring the absorbance. Alternatively, it can be performed by an enzyme-linked ^ SP binding assay (Enzyme-1 inked immunosorbent assay; ELISA) or the like. Examples of the force column used for affinity chromatography include a protein A column and a protein G column. For example, using a protein A column Columns include Hyper D, POROS, Sepharose FF (Pharmacia), etc. Examples of chromatography other than affinity chromatography include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, Examples include adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography such as HPLC and FPLC.

 Examples of the method for measuring the antigen-binding activity of the antibody of the present invention include, for example, measurement of the degree, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme-linked immunosorbent assay), and RIA (enzyme-linked immunosorbent assay). (Radioimmunoassay) or a fluorescent antibody method can be used. When using ELISA, the protein of the present invention is added to a plate on which the antibody of the present invention has been immobilized, and then a sample containing the target antibody, for example, antibody-producing cells,

 P

Add culture supernatant and purified antibody. A secondary antibody that recognizes an enzyme, for example, an antibody labeled with alkaline phosphatase, is added, the plate is incubated, washed, and then the enzyme substrate such as P-nitrophenyl phosphate is added to measure the degree. Antigen binding activity can be evaluated. As the protein, a protein fragment, for example, a C-terminal fragment or an N-terminal fragment thereof may be used. BIAcore (Pharmacia) can be used to evaluate the activity of the antibody of the present invention. By using these techniques, the antibody of the present invention is brought into contact with a sample expected to contain the protein of the present invention, and a complex of the antibody and the protein is detected or measured. In addition, the method for detecting or measuring the protein of the present invention can be carried out.

Since the protein detection or measurement method of the present invention can specifically detect or measure a protein, it is useful for various experiments and the like using proteins. In addition, the present invention Of the present invention is strongly expressed in 爐, hematopoietic crane お よ び, and scar fi ^, and is thought to be involved in immune and / or hematopoietic signal transduction as a hemopoietin receptor. Through the detection or measurement of the protein of the present invention, it is also possible to examine and diagnose a structural abnormality or abnormal expression of the protein of the present invention. The present invention also relates to the human NR10 or the human NR10 protein (SEQ ID NO: 4, 6, 8, 10, 12, 12, or 14). DNA (SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17) encoding a protein (NR10C or NR10B) (SEQ ID NO: 16 or 18, respectively) or complementary to its complementary strand Provide a polynucleotide comprising at least 15 nucleotides. A polynucleotide may be, for example, DNA or fragile. The polynucleotide of the present invention is useful, for example, for detecting, amplifying, detecting the expression of, or controlling the expression of the DNA encoding the protein of the present invention. Detection of DNA includes detection of mutations in DNA.

 As used herein, the term “complementary strand” refers to one strand of a double-stranded polynucleotide consisting of A: T (but RNA: Α in the case of RNA) and G: C base pairs and the other strand. The term "complementary" is not limited to a sequence completely complementary to at least 15 contiguous nucleotide regions, but is at least 70, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is that they have homology on the base sequence of at least%. The algorithm described in the present specification may be used as an algorithm for determining homology.

Such polynucleotides include human NR10.4, NR10.5, NR10.6, 10.7, or R8 protein (SEQ ID NOs: 4, 6, 8, 10, 12, or 14, respectively), or mouse NR10 protein. (R10C or ^ NRIOB) (SEQ ID NO: 16 or 18 respectively) DNA (SEQ ID NO: 3, 5, 7, 9, 11, 13, 15 or 17) or its complementary strand Polynucleotides containing at least 15 nucleotides are included. Preferably, the polynucleotide is a human N 10.4, NR10.5, NR10.6, NR10.7, or N10.8 protein (SEQ ID NO: No .: 4, 6, 8, 10, 12, or 14) or DNA encoding mouse NR10 protein (10C or N10B) (SEQ ID NO: 16 or 18 respectively) (SEQ ID NO: 3) , 5, 7, 9, 11, 13, 15 or 17) or its complementary strand. The term "specifically hybridizes" means that the DNA does not significantly hybridize with DNA encoding another protein under ordinary hybridization conditions, preferably under the above-mentioned stringent hybridization conditions.

 In particular, a polynucleotide comprising at least 15 nucleotides complementary to the nucleotide sequence of SEQ ID NO: 19 or a complementary strand thereof is a human NR10.4, NR10.5, NR10.6, NR10.7, or NR10.8 It is useful for detecting or isolating a gene containing an exon that is specific to E. coli. In addition, a polynucleotide comprising at least 15 nucleotides complementary to the nucleotide sequence of SEQ ID NO: 22 or its complementary strand is useful for detecting or isolating a gene containing exon specific to human NR10.5 It is.

 Such polynucleotides include probes and primers used for detection and amplification of DNA encoding the protein of the present invention, probe primers for detecting expression of the DNA, and proteins of the present invention. A nucleotide or a nucleotide derivative for controlling expression (for example, an antisense oligonucleotide or a lipozyme, or a DNA encoding the same) is included. Such a polynucleotide can also be used for producing a DNA chip or a microarray. The polynucleotide of the present invention includes DNA and RNA. It also includes sense nucleotides and antisense nucleotides.

Antisense oligonucleotides include, for example, antisense oligonucleotides that hybridize at any position in the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17 Nucleotides. This antisense oligonucleotide preferably has a nucleotide sequence of at least 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, or 17. It is an antisense oligonucleotide. Even more preferably, it is an antisense oligonucleotide in which at least 15 or more consecutive nucleotides contain a translation initiation codon.

 When used as a primer, the 3 ′ region can be complementary, and a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 ′ region.

 As the antisense oligonucleotide, derivatives and modifications thereof can be used. Examples of the modified product include a modified lower alkylene phosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothioate modified product or a phosphoroamidate modified product.

 Antisense oligonucleotides include not only those in which all nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are complementary sequences, but also those in which DNA or mRNA and oligonucleotides have their own sequence numbers: 3, 5, 7 , 9, 11, 13, 15 or 17 may have a mismatch of one or more nucleotides as long as the nucleotide sequence can be specifically hybridized to the nucleotide sequence.

 The antisense oligonucleotide derivative of the present invention acts on a cell producing the protein of the present invention and binds to DNA or mRNA encoding the protein, thereby inhibiting its transcription or translation or inhibiting the mRNA. By promoting ^ and suppressing the expression of the protein of the present invention, it has the effect of suppressing the action of the protein of the present invention.

 The antisense oligonucleotide derivative of the present invention can be mixed with a suitable base material that is inactive against the derivative to form an external preparation such as a coating agent or a puff.

 If necessary, excipients, isotonic agents, solubilizing agents, stabilizers, preservatives, soothing agents, etc. may be added to tablets, splinters, granules, capsules, ribosome capsules, It can be a lyophilized agent such as a propellant, a liquid, a nasal drop and the like. These can be prepared using conventional techniques.

The antisense oligonucleotide derivative of the present invention is applied directly to the affected area of a patient. Or applied to the patient so that it can eventually reach the affected area, such as by intravenous administration. Furthermore, an antisense-encapsulated material that enhances durability and membrane permeability can be used. For example, ribosome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives thereof can be mentioned.

 The dosage of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.

 The antisense oligonucleotide of the present invention inhibits the expression of the protein of the present invention and is therefore useful in suppressing the biological activity of the protein of the present invention. Further, the expression inhibitor containing the antisense oligonucleotide of the present invention is useful in that it can suppress the biological activity of the protein of the present invention.

 The protein of the present invention is useful for screening for a compound that binds to the protein. That is, the protein of the present invention is brought into contact with a test sample expected to contain a compound that binds to the protein, the binding activity between the protein of the present invention and the compound contained in the test sample is detected, and It is used in a method for screening a compound that binds to the protein of the present invention, which comprises selecting a compound having an activity of binding to the protein of the present invention.

The protein of the present invention used for screening may be a recombinant protein or a naturally-derived protein. It may also be a partial peptide of the protein of the present invention. It may also be in a form expressed on the cell surface or as a membrane fraction. The test sample is not particularly limited, and for example, any sample containing a test compound can be used. Specifically, for example, cell extracts, cell culture supernatants, fermented microbial products, marine organism extracts, plant extracts, purified or crudely purified proteins, peptides, non-peptide compounds, synthetic low liver compounds , And Tenshodarigo. The protein of the present invention to be brought into contact with a test sample may be, for example, a purified protein, a soluble protein, a form bound to a carrier, a fusion protein with another protein, As a form expressed above, it can be applied to a test sample as a membrane fraction.

 As a method for screening a protein (ligand or the like) binding to the protein using the protein of the present invention, for example, many methods known to those skilled in the art can be used. Such screening can be performed, for example, by a sedimentation method. Specifically, it can be performed as follows. By inserting the gene encoding the protein of the present invention into a vector for expressing a foreign gene such as pSV2neo, pcDNAI, or pCD8, the gene is expressed in animal cells or the like. The promoters used for expression include the SV40 early promoter (Rigby In Williamson ½d.), Genetic Engineering, Vol. 3.Academic Press, London, p. 83-141 (1982)) and the EF-1 promoter ( Kim, DM et al. (1990) Gene 91, 217-223), CAG promoter (Niwa, H. et al. (1991) Gene 108, 193-200), RSV LTR promoter (Cul en, BR (1987)) Methods in Enzymology 152, 684-704), SR a promoter (Takebe, Y. et al. (1988) Mol. Cel l. Biol. 8, 466-472), CMV immediate early promoter (Seed, B. and Aruffo, A. (1987) Proc. Natl. Acad. Sci. USA 84, 3365-3369), SV40 late promoter (Gheysen, D. and Fiers,. (1982) J. Mol. Appl. Genet. 1, 385-394. ), Adenovirus late promoter (Kaufman, RJ et al. (1989) Mol. Cell. Biol. 9, 946-958), HSV TK promoter, and other commonly used promoters. Is also good.

In order to express a foreign gene by introducing the gene into animal cells, the electroporation method (Chu, G. et al. (1987) Nucleic Acid Res. 15, 1311-1326) and the calcium phosphate method (Chen, C and Okayama, H. (1987) MoL Cel l. Biol. 7, 2745-2752), DEAE dextran method (Lopata, MA et al. (1984) Nucleic Acids Res. 12, 5707-5717; Sussman, DJ and Milman, G. (1985) Mol. Cel l. Biol. 4, 1642-1643), lipofectin method (Derijard, B. (1994) Cel l 7, 1025-1037; Lamb, BT et al. (1993) Nature Genetics 5, 22-30; Rabindran, SK et al. (1993) Science 259, 230-234), but any of these methods may be used. By introducing a recognition site (epitof) of a monoclonal antibody whose specificity is known to the N-terminus or C-terminus of the protein of the present invention, a fusion protein having a recognition site of a monoclonal antibody of the present invention can be obtained. The protein can be expressed. As the epitope-antibody system to be used, commercially available ones can be used (Experimental Medicine, 85-90 (1995)). Vectors capable of expressing a fusion protein with 8-galactosidase, maltose-binding protein, daltuthione S-transferase, green fluorescent protein (GFP), and the like via a multicloninda site are commercially available.

 In order to minimize the properties of the protein of the present invention by changing it into a fusion protein, a small peptide consisting of several to a dozen or so amino acids! Methods for preparing fusion proteins have also been reported. For example, polyhistidine (His-tag), influenza agglutinin HA, human c-myc, FLAG, vesicular s tomat it is viral glycoprotein (VSV-GP), T7 genelO protein (T7-tag), human simple herb Epitopes such as virus virus glycoproteins (HSV-tags) and E-tags (epitopes on a monoclonal phage) and a monoclonal antibody recognizing them can be used for screening proteins that bind to the protein of the present invention. It can be used as an antibody system (Experimental Medicine 185-90 (1995)).

In immunoprecipitation, these antibodies are added to a cell lysate prepared with appropriate surfactants to form a ^ $ complex. This; ^ complex comprises the protein of the present invention, a protein capable of binding thereto, and an antibody. In addition to using antibodies against the above-mentioned epitopes, immunoprecipitation can also be performed using antibodies against the protein of the present invention. Antibodies against the protein of the present invention include, for example, a gene encoding the protein of the present invention introduced into an appropriate E. coli expression vector, expressed in E. coli, and the expressed protein is purified. It can be prepared by immunizing goats and chickens. In addition, the synthesized present invention It can also be prepared by immunizing the above animal with a partial peptide of the above protein.

 For example, if the antibody is a mouse IgG antibody, the escape complex can be precipitated using Protein A Sepharose or Protein G Sepharose. When the protein of the present invention is prepared, for example, as a fusion protein with an epitope such as GST, the protein of the present invention can be prepared using a substance that specifically binds to these epitopes such as daltathione-Sepharose 4B. An immune complex can be formed as in the case of using the above antibody.

 The specific method of evacuation is described, for example, in accordance with the method described in the literature (Harlow, E. and Lane, D .: Ant ibodies, pp. 511-552, Cold Spring Harbor Laboratory publicat ions, New York (1988)). , Or according to it.

SDS-PAGE is generally used to analyze precipitated proteins, and the bound proteins can be analyzed based on the molecular weight of the proteins by using an appropriate concentration gel. In this case, the protein bound to the protein of the present invention is generally a radioisotope, since it is difficult to detect the protein by ordinary staining methods such as Kumashi staining and silver staining. ³⁵ S- cells were cultured in Mechionin or culture medium containing ³⁵ S- cysteine and labeled proteins in said cell, it is possible to improve the detection sensitivity by detecting this. Once the amount of protein is known, the target protein can be purified directly from SDS-polyacrylamide gel and sequenced.

 Isolation of a protein binding thereto using the protein of the present invention is carried out, for example, using the ウ エ ス タ ン western blotting method (Skolnik, EY et al. (1991) Cell 65, 83-90). be able to. That is, cells that are expected to express a binding protein that binds to the protein of the present invention,

(Agtll, ZAP, etc.) to prepare a cDNA library, express it on LB-agarose, fix the expressed protein on a filter, purify and label the protein of the present invention and the above filter. And reacting the protein with the protein of the present invention. The expressed plaque may be detected by a label. Examples of the method for labeling the protein of the present invention include a method using the binding property of biotin and avidin, and a method of specifically binding to the protein of the present invention or a peptide or polypeptide (eg, GST or the like) fused to the protein of the present invention. Examples include a method using an antibody, a method using a radioisotope, and a method using fluorescence.

In addition, as another embodiment of the screening method of the present invention, a 2--8 hybrid system using cells (Fields, S., and Sternglanz,. (1994) Trends Genet. 10, 286-292; Dalton S, and Treisman R (1992) Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element, Cel l 68, 597-612, `` Kasumi CHMAKER Two-Hybrid System '', `` Mammalian MATCHMAKER Two-Hybrid Assay Kit ”and“ MATCHMAKER One-Hybrid SystemJ (all manufactured by Clontech); rHybriZAP Two-Hybrid Vector SystemJ (Stratagene). ”2-Hybrid In the system, the protein of the present invention or a partial peptide thereof is fused with an SRF DNA binding region, a GAL4 fragment A binding region, or the like, and expressed in yeast cells to express a protein that binds to the protein of the present invention. VP16 or GAL4 translocation region A cDNA library that can be expressed in a form fused with E. coli, etc., is prepared, introduced into the above yeast cells, and the cDNA derived from the library is isolated from the detected positive clonal cells. When a protein that binds to the protein of the present invention is expressed, the binding of the two activates the reporter gene, and a positive clone can be confirmed.) By introducing the isolated cDNA into Escherichia coli and expressing it, the cDNA can be expressed. Thus, a protein that binds to the protein of the present invention or a gene thereof can be prepared by using the HIS3 gene in addition to the HIS3 gene. , Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen act ivator inhibitor typel) gene, etc. Not limited to these. Screening by the two-hybrid method is not only for yeast It can also be performed using mammalian cells and the like.

 Screening for a protein that binds to the protein of the present invention can also be performed using affinity chromatography. For example, the protein of the present invention is immobilized on an affinity carrier, and a test sample which is expected to express a protein that binds to the protein of the present invention is applied thereto. The test sample in this case includes, for example, a cell extract, a cell lysate, and the like. After applying the test sample, the column is washed to prepare a protein bound to the protein of the present invention.

 The obtained protein is analyzed for its amino acid sequence, and based on it, an ori: iDNA is synthesized, and the DNA encoding the protein is obtained by screening a cDNA library using the DNA as a probe. Can be.

 In the present invention, a biosensor utilizing a surface plasmon resonance phenomenon can be used as a means for detecting or measuring the bound protein. The biosensor using the surface plasmon resonance phenomenon as an IJ is a real-time surface plasmon resonance signal without capping the interaction between the protein of the present invention and the test protein using a trace amount of protein. (For example, BIAcore, manufactured by Pharmacia). Therefore, it is possible to enhance the binding between the protein of the present invention and the test compound by using a biosensor such as BIAcore.

Methods for isolating not only proteins but also compounds (including agonists and antagonists) that bind to the protein of the present invention include, for example, immobilized proteins of the present invention, synthetic compounds, and natural compounds. Method of screening ^ that bind to the protein of the present invention by using a product bank or a random phage peptide display library, and a screening method using high throughput by combinatorial chemistry technology (Wright on, NC et al. al. (1996) Smal l pept ides as potent mimetics of the protein hormone erythropoietin, Science 273, 458-64; Verdine, GL (1996) The combinatorial chemistry of nature, Nature 384, 11-13; Hogan, JC Jr. (1996) Directed combinatorial chemistry, Nature 384, 17-19) are known to those skilled in the art.

 Screening for a ligand that binds to the protein of the present invention comprises linking the extracellular domain of the protein of the present invention with the intracellular domain of the hemopoietin receptor protein having known signal transduction ability, including the transmembrane domain. Expressing the chimera receptor prepared above on the cell surface of a suitable cell line, preferably a cell line that can survive and proliferate only in the presence of a suitable growth factor (growth factor-dependent cell line). It can be performed by culturing the cell line with addition of a material expected to contain various growth factors, cytodynamic factors, hematopoietic factors, and the like. This method takes advantage of the fact that the above-mentioned growth factor-dependent cell line can survive and proliferate only when the test material contains a ligand that specifically binds to the extracellular domain of the protein of the present invention. are doing. Examples of known hemopoietin receptors include, for example, thrompopoietin receptor, erythropoietin receptor, G-CSF receptor, gpl30, and the like.Partners of the chimeric receptor used in the screening system of the present invention include those known It is not limited to the hemopoietin receptor, and any cytoplasmic domain having a structure necessary for signal transduction activity may be used. As a growth factor-dependent cell line, for example, an IL3-dependent cell line such as BaF3 or FDC-P1 can be used.

The ligand that specifically binds to the protein of the present invention, though rare, may be a finely-coupled protein instead of a soluble protein. In such a case, it is rather preferable to express the ligand after labeling a protein containing only the extracellular domain of the protein of the present invention or a fusion protein obtained by adding a partial sequence of another soluble protein to the extracellular domain. It is possible to screen by measuring the expected binding to cells. The protein containing only the extracellular domain of the protein of the present invention includes, for example, a soluble receptor protein artificially prepared by inserting a stop codon at the N-terminal side of the fine transmembrane domain, or NR10.7 or可 Soluble proteins such as NR10.8 are available. On the other hand, in the fine domain of the protein of the present invention, As a fusion protein to which a partial sequence of the soluble protein is added, for example, a protein prepared by adding an Fc site of 赚 globulin or a FLAG peptide to the C-terminus of the extracellular domain can be used. These soluble labeled proteins can also be used for detection in the above-mentioned West Western method.

 For example, a chimeric protein comprising the extracellular region of the protein of the present invention and the Fc region of an antibody (eg, a human IgG antibody) can be purified using a protein A column or the like. Since such an antibody-like chimeric protein has ligand-binding activity, it can be used for screening ligands after labeling with radioisotopes and the like as appropriate (Suda, T. et al.). al., Cell, 175, 1169-1178 (1993)). In addition, since certain cytokines such as TNF family molecules are also membrane-bound, i.e., they react with various cells and anti-a chimeric protein to show the binding activity. It may be possible to isolate the ligand. In addition, a ligand can be isolated in the same manner using cells into which the cDNA library has been introduced. Furthermore, an antibody-like chimeric protein can be used as an antagonist.

 Compounds that can be isolated by screening become drug candidates for promoting or inhibiting the activity of the protein of the present invention, and are applicable to the treatment of diseases caused by abnormal expression or function of the protein of the present invention. Can be considered. The substance obtained by using the screening method of the present invention and having a part of the structure of the compound having the activity of binding to the protein of the present invention, which is converted by addition, deletion, Z or substitution, is also a substance of the present invention. It is included in the compounds obtained by using the screening method.

Compounds obtained by the screening method of the present invention and proteins of the present invention (deco type (soluble type)) can be used in human animals, such as mice, rats, guinea pigs, 'herons', chickens, cats, dogs, and higgies. When used as a medicinal product, as a drug, it is not only that the isolated compound itself is administered directly to the patient, but also that it is administered in a well-known pharmaceutical manner. It is also possible to do. For example, sugar-coated as needed, capsules, elixir 1 micro force It can be used orally as a capsule or parenterally in the form of a sterile solution or suspension in water or other pharmaceutically acceptable liquid. For example, pharmacologically acceptable carriers or vehicles, specifically, sterile water or physiological water, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles It can be formulated by mixing with preservatives, binders, etc., in admixture in unit dosage form required for generally accepted pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate capacity within the specified range can be obtained.

 Additives that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, corn starch, gelatinous alginic acid, and the like. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose are used. When the unit dosage form is a capsule, the above materials may further contain a night carrier such as an oil or fat. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

Aqueous injection solutions include, for example, physiologically sterile water, isotonic solutions containing glucose and other adjuvants, such as W-sorbitol, D-mannose, D-mannitol, sodium chloride. Oils, such as sesame oil, which may be used in combination with alcohols, for example, ethanol, polyalcohols, for example, propylene glycol, polyethylene glycol, nonionic surfactants, for example, polysorbate 80 (TM), HC0-50. And soybean oil, which may be used in combination with benzoic acid or benzyl alcohol as a solubilizing agent. It may also be combined with a buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant. . Prepared Injectables are usually filled into appropriate inflation.

 Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by methods known to those skilled in the art. Can be done. The dose varies depending on the weight and age of the patient, the administration method, and the like, and those skilled in the art can select an appropriate dose. In addition, if the compound can be encoded by DNA, the DNA may be incorporated into a vector for gene therapy to perform gene therapy. The dose and administration method vary depending on the patient's weight, age, symptoms, and the like, and those skilled in the art can select an appropriate dose. For example, the dose of the protein of the present invention (decoy type (soluble type)) varies depending on the administration subject, subject fl, symptom, and administration method. (As 60 kg) would be considered to be about 100 g to 20 mg per day, preferably about 100 / g to 10 mg per day.

 For example, the dose of the compound that binds to the protein of the present invention or the dose of the compound that inhibits the activity of the protein of the present invention varies depending on the symptoms. , The amount is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.

 In the case of parenteral administration, the single dose varies depending on the subject, subject, symptoms, and administration method. For example, in the case of an injection, it is usually 1 day for an adult (with a body weight of 60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per iv. In the case of other animals, the dose can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the nucleotide sequence of NI 0.3 detected from the human genome. Amino acid coding column ¾ The column is shown in uppercase, and the predicted untranslated nucleotide sequence is shown in lowercase. Indicated by

 FIG. 2 is a continuation of FIG.

 FIG. 3 is a view showing an amino acid sequence which can be encoded by N 10.3 detected in the human genome.

 FIG. 4 is a diagram showing the nucleotide sequence of NR10.4 detected by human genome analysis. The predicted nucleotide sequence of the amino acid coding region is shown in capital letters. The predicted nucleotide sequence of the untranslated region is shown in lower case.

 FIG. 5 is a continuation of FIG.

 FIG. 6 is a diagram showing an amino acid sequence that can be encoded by NR10.4 and detected from the human genome.

 FIG. 7 is a diagram showing the nucleotide sequence of NR10.4 isolated from a human peripheral leukocyte cDNA library and the amino acid sequence encoded thereby. In addition, the amino acid sequence predicted to be the transmembrane region is underlined.

 FIG. 8 is a continuation of FIG.

 FIG. 9 is a continuation of FIG.

 FIG. 10 shows the nucleotide sequence of NR10.5 isolated from a human thymus cDNA library and the amino acid sequence encoded by it. In addition, the amino acid sequence predicted to be a penetrating region is underlined.

 FIG. 11 is a continuation of FIG. 10.

 FIG. 12 is a continuation of FIG. 11.

 FIG. 13 shows the nucleotide sequence of NR10.6 isolated from a human thymus cDNA library and the amino acid sequence encoded by it. In addition, the amino acid sequence predicted as a fine ^ S penetrating region is underlined.

 Fig. 14 shows the yarn sales of Fig. 13.

 FIG. 15 is a continuation of FIG.

Figure 16 shows the nucleotide sequence of NR10.7 isolated from the human thymus cDM library, and It is a figure showing the amino acid sequence encoded by it.

 FIG. 17 is a continuation of FIG.

 FIG. 18 is a continuation of FIG.

 FIG. 19 shows the nucleotide sequence of R10.8 isolated from a human thymus cDNA library and the amino acid sequence encoded by it.

 FIG. 20 is a continuation of FIG.

 FIG. 21 is a continuation of FIG.

 FIG. 22 is a photograph showing the results of RT-PCR of NR10 gene expression in various human organs. The size of the specific PCR amplification product of NR10 is indicated by an arrow.

 FIG. 23 is a photograph showing the results of analysis of the expression state of the NR10 gene in human glue β and human peripheral blood cells by RT-PCR.

 FIG. 24 is a photograph showing the results of quantitative analysis of N10 gene expression in various human organs by Southern blotting. The specific signal of NR10 detected is indicated by an arrow.

 FIG. 25 is a photograph showing the results of Southern blotting on the expression of NR10 in various human organs and various blood cells of human peripheral blood. Arrows indicate the specific signals of NR10 detected.

 FIG. 26 is a photograph showing the result of analyzing the expression of 10 genes in various human cell lines. The results of analysis of 10 gene expression patterns in various human cell lines by RT-PCR were shown (upper panel). In addition, the results of quantitative spinning by Southern blotting were shown (middle panel). In addition, it was confirmed using a set of G3PDH primers that the mRNA used as type III in the above RT-PCR analysis was standardized among the samples (lower panel).

FIG. 27 is a diagram showing a structural schematic diagram of an expressible protein encoded by NR10 constructed in a plasmid vector that can be expressed in mammalian cells. FIG. 28 shows the nucleotide sequence of niNRlO detected from a mouse genome database search. The predicted nucleotide sequence of the exon region is shown in bold. The amino acid sequences that can be encoded are also shown.

 Figure 29 shows the amino acids that were predicted to be exonable in the Roh10 base sequence that showed a positive response to the human NR10.'4 amino acid sequence used in the query in the mouse genome database search. It is the figure which compared the sequence.

 FIG. 30 is a diagram showing the nucleotide sequence of mouse N 1 OB isolated from one Balb / c testis cDNA library and the amino acid sequence encoded thereby. Also, the amino acid sequence predicted as a fine β-penetrating region is underlined.

 FIG. 31 is a continuation of FIG. 30.

 FIG. 32 is a continuation of FIG. 31.

 FIG. 33 shows the nucleotide sequence of mouse NR10C isolated from a C57BL / 6 day8.5 embryo cDNA library and the amino acid sequence encoded thereby. The amino acid sequence predicted as a transmembrane region is underlined.

 FIG. 34 is a continuation of FIG. 33.

 FIG. 35 is a continuation of FIG.

FIG. 36 shows a comparison between the full-length amino acid sequence of human NR10.4 (hidden 10) and the full-length amino acid sequence of mouse N10B derived from Balb / c testis (MNR10). Common amino acids are indicated by an asterisk. The sequence predicted to be the penetrating region is underlined. FIG. 37 is a diagram schematically showing the genomic structure of the C-terminal region from FN-III, which shows diversity in various splicing variants of human N10. The exon site is indicated by a square in the genome structure. In addition, in the exon site, a sequence portion capable of encoding a continuous amino acid translation frame by splicing was colored. BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. All documents cited in this specification are incorporated as a part of this specification.

 [Example 1] Identification of human N10 genomic sequence by TblastN search

 As a result of vigorous decipherment of the human genome gene sequence by various human genome horns, the draft sequence and physical map have been almost completely edited. In fact, even in the GenBank database, about 90% or more of all human genome sequences are already used as draft sequences. However, there is no regularity in the arrangement of these sequences, and future transcript (transcriptome) keratotomy, functional protein (proteome) analysis, and SNP (base mutation polymorphism) analysis will be important issues. (Nature, 1999, 402, p489-495; Nature, 2000, 405, p311-319). The present inventors performed a Blast search to detect a full-length genomic gene capable of encoding the human N10 gene. Using the full-length amino acid sequence of N 10.3 (International Publication No.TO00 / 75314) as a query, htgs (sorted in the GenBank database, one of the public databases, was searched) High Throughput Genome Sequence; human genome draft database) was selected. Expect value = 50, Descriptions value = 100, and Alignments value = 100 were used as parameters for search conditions. The setting of the filter was set to the default value. As a result of the above search using the Tblas tN (Advanced Tblas tN 2.0.13) program, multiple human BAC clones capable of encoding the human NR10.3 sequence were detected. Among these positive clones, the clone having the least determined nucleotide sequence was selected. As a result, two human BAC clones, GenBank accession number: AC022265 (accession number: AC022265) and accession number: AC008857 (accession number: AC008857), could be selected. The human BAC clone sequence selected here was provided as an NR10 human genome positive clone to the NR10 human genome construct described below.

[Example 2] Structure of NR10 human genome gene! For the purpose of structural analysis of a full-length genomic sequence capable of encoding the human NR10 gene, identification of the exon sequence of the human NR10 gene encoded by the BAC clone found above was attempted. The identification of the exon site was solved by conducting a BlastN (Advanced BlastN 2.0.13) search using the nucleotide sequence of human NR10.3 as a query in the aforementioned BAC clone AC022265. This search used programs in the NCBI server (http://www.ncbi.nlm.nih.gov/), and all search conditions were set to the default values. As a result, it was detected that the entire nucleotide sequence of the 5 ′ untranslated region of the human NR10.3 gene and the 3 ′ untranslated region including the caro signal with polyA can be encoded in the forward direction. Similarly, a BlastN search was performed on the BAC clone AC008857 to re-verify the search results. As a result, it was detected that the human N10.3 sequence could be encoded in the opposite direction as in AC022265. However, it was predicted that in the BAC clone AC008857, not only the human NR10.3 sequence but also a new human clothing lOniRNA sequence could be encoded due to the sequence difference near the C-terminus of NR10. The novel sequence detected and predicted here was named NR10.4. Genome analysis results In NR10.4, the intron during splicing to the final exon of NR10.3 is not skipped, and the amino acid translation frame continues as it is. Therefore, compared to NR10.3, which is predicted to have a short intracellular region and no signal transduction function, a long amino acid translation frame can be encoded at the C-terminus. Furthermore, the presence of three tyrosine residues in the intracellular region of NR10.4 was predicted to possess intracellular signal transduction. Incidentally, the BAC clone AC022265 cannot encode the nucleotide sequence of NR10.4 that could be encoded by BAC clone AC008857. This is considered to be misregistration of the registrant. Structure of human genome sequence Fig.1 shows the base sequence of N 10.3 (SEQ ID NO: 1) detected from lucidity! 2 and the amino acid sequence of NR10.3 (SEQ ID NO: 2) encoded by it is shown in FIG. On the other hand, is it expected to be detected from the human genome? The nucleotide sequence of NR10.4 (SEQ ID NO: 3) is shown in Figs. Was. N 10.4 could be expected to be present as a transcript due to alternative splicing of NR10. Thus, a downstream orientation: iDNA primer was designed near the predicted amino acid translation initiation codon of NR10.4, while an upstream oligo DNA primer was designed in the 3 'untranslated region of NR10.4. By performing RT-PCR using these primer sets, it was considered possible to prove that the mRNA encoding NR10.4 was correct; The oligonucleotide primer designed as described above was synthesized and purified under the following conditions.

 [Example 3] Synthesis and purification of oligonucleotide primer

 As described above, the exon site is predicted in the BAC clone sequence, and based on the predicted sequence, the following oligonucleotides specific to the human NR10.4 gene are shown.

— Designed. As primers, NIU0.4-MET was synthesized on the sense side (downstream direction) and NR10.4-UTR was synthesized on the sense side (upstream direction). Primers were synthesized using ABI 394 DNA / RA Synthesizer under the conditions of adding a 5'-terminal trityl group. Thereafter, the full-length synthetic product was purified using an OPC column (ABI # 400771) and provided to the RT-PCR method described later.

Picture. 4-MET (SN) (SEQ ID NO: 25)

5 'One CTG GGA ATG TGC ATC AGG CM CTC AAG—3'

NR10.4- UTR (AS) (SEQ ID NO: 26)

5'One TTT TTT AGT AGT CM TGC AGT CTA AAT TGG GG -3 '

 [Example 4] Identification of human NR10.4 gene by RT-PCR cloning method

To isolate the full-length coding sequence (CDS) of human N 10.4, the NR10.4-MET primer of Example 3 was used as a sense primer, and the NR10.4-UTR primer was used as an antisense primer. The RT-PCR cloning method used was tried. Human PBL (human peripheral leukocyte) 1st strand cDNA (Clontech # K142-1) was used as type Advantage, and Advantage cDNA Polymerase Mix (Clontech # 8417-l) was used for PCR experiments. Using a PerMn Elmer Gene Amp PCR System 2400 thermal cycler As a result of performing the PCR under the following PCR conditions, an amplification product having the expected size was obtained94. 4 minutes at C

 [20 seconds at 94 ° C, 10 seconds at 62 ° C, 90 seconds at 70 ° C] 10 cycles

 30 cycles of [20 seconds at 94 ° C, 10 seconds at 60 ° C, 90 seconds at 68 ° C]

 3 minutes at 72 ° C

 Terminates at 4 ° C

The obtained PCR product was subcloned into pGEM-T Easy vector (Promega # A1360) and the nucleotide sequence was determined. For the recombination of the PCR product into the pGEM-T Easy vector, a reaction was performed at 16 ° C / 2 hours using T4 DNA Ligase (Promega # A1360). PCR products and recombinants of the pGEM-T Easy vector were obtained by transforming E. coli strain DH5 o! (Toyobo # DNA-903). In addition, Insert Check Ready Blue (Toyobo # PIK-201) was used for selecting the recombinants. Furthermore, the determination of the salt Motohai歹¹ j uses the BigDye Terminator Cycle Sequencing Ready React ion Ki t (ABI / Perkin Elmer # 4303154), was subjected to angular晰Te ABI PRISM 377 DNA Sequencer Niyotsu. The nucleotide sequence of all the insert fragments was determined for 12 independent clones of the recombinant, and it was confirmed that the NR10.4 gene sequence derived from the specific PCR amplification reaction was present. Furthermore, the nucleotide sequence of a cDNA clone capable of encoding the full-length CDS of NR10.4 including the transmembrane region was determined. The determined base sequence of NI 0.4 (SEQ ID NO: 5) and the amino acid sequence encoded by it (SEQ ID NO: 6) are shown in FIGS.

[Example 5] Search for human NR10 gene splicing variants by RT-PCR method RT-PCR cloning was attempted for the purpose of identifying further splicing variants capable of encoding the human NR10 gene. Human Thymus Marathon-Ready cDNA Library (Clontec # 7415-1) was used as type 铸. All other KR conditions were as in Example 4 above. That is, NR10.4-MET primer and NR10.4-UTR primer And the same amplification reaction was carried out using Advantage cDNA Polymerase Mix. As a result, an amplification product showing multiple sizes near the predicted size was obtained. Thus, according to the method of Example 4, the obtained PCR product was subcloned into a pGEM-T Easy vector, and the nucleotide sequence was determined. The nucleotide sequence of all insert fragments was determined for 24 independent clones, and it was confirmed that the NR10.4 gene sequence derived from the specific PCR amplification reaction was present. However, among the 24 clones of the transgenic plants that had been subjected to the cultivation, several clones having an insert sequence different from that of NR10.4 were simultaneously obtained. As a result of clarity based on those nucleotide sequences, the clones identified were named NR10.5, NR10.6, N10.7, and R10.8, respectively. The determined nucleotide sequence of NR10.5 (SEQ ID NO: 7) and the amino acid sequence encoded by it (SEQ ID NO: 8) are shown in FIGS. The determined nucleotide sequence of N 10.6 (SEQ ID NO: 9) and the amino acid sequence encoded by it (SEQ ID NO: 10) are shown in FIGS. The determined nucleotide sequence of NI 0.7 (SEQ ID NO: 11) and the amino acid sequence (SEQ ID NO: 12) encoding it are shown in FIGS. The determined nucleotide sequence of NR10.8 (SEQ ID NO: 13) and the amino acid sequence encoded by it

(SEQ ID NO: 14) is shown in FIGS. The primary structures of these clones showing different insert sequences were compared with the results of the structural analysis of the NR10 human genomic gene according to Example 2 described above, and as a result, all clones showed NR10 splicing variants derived from alternative splicing. It turned out to be. Here, although NR10.5 and R10.6 can encode a transmembrane receptor protein having a transmembrane domain, they do not have tyrosine residues in the intracellular domain, and therefore have an intracellular signaling function. Was not expected to be held. In addition, since NR10.7 and NR10.8 did not possess the transmembrane region, it was predicted that they could encode a soluble secretory receptor protein.

Table 1 summarizes the structural features of the splicing variants of the NR10 gene. The genomic structure of these NR10 gene splice variants is shown in the schematic diagram of Figure 37. In particular, the structure at the C-terminal side of the Hosoto penetration region shows diversity due to selective splicing. It is expected that the regulation of functional expression of the 0 gene group will be strictly controlled by alternative splicing that is specific to 戠 or specific cell types. Table 1 Structural features of the NR10 splice variant Clone CBM FN—III ™ Boxl CP-Tyr

NRIO. 4 + + + +

 NRIO. 5 + + + +

 NRIO. 6 + +

 NRIO. 7 + +

 NRIO. 8 + +

CBM; Cytokine Binding Module FN-I I; FibronecUn type I I I domain

 TM: Transmembrane domain

 Boxl ； Jak kinase binding domain

(Pro-Xaa-Pro mot i f)

 CP-Tyr; Cytoplasmic Tyr-residues

[Example 6] Search for NRIO gene-expressing tissues by RT-PCR method and keratocarp expression expression distribution of NR10.4, NRIO.5 and NRIO.6 genes in each human fl¾ For the purpose of analyzing E. coli, mRNA was detected by the RT-PCR method. For RT-PCR analysis An oligonucleotide primer having the following sequence was newly synthesized. The NR10.4-TM primer was used as the sense (downstream) primer, and the R10.4-STP primer was used as the antisense (upstream) primer. The synthesis and purification of the primers were performed in accordance with Example 3 described above. Here, the gall 0.4-TM primer is designed in the narrow penetration region of NR10. On the other hand, the NR104-STP primer is designed near the amino acid translation stop codon of N10.4. Therefore, it was considered that the sequences of the three splicing mutants of NR10.4, NR10.5, and NR10.6 could be amplified and edible by these primer sets. However, the NR10.3 gene, which has a small J! »Penetrating region but has a different 3 'untranslated region sequence in the final exon, and NR10.7, which has no further fine penetrating region, and 8 is not amplified.

Marauder 0.4. 4-TM (SEQ ID NO: 27)

5 '-GAG ATT ATC CTC ATA ACT TCT CTG ATT GGT GG -3'

hN 10.4-STP (SEQ ID NO: 28)

 5 '-GCT ATG GTC GCA TTT AGA CTT CTC CCT TGG TGT G -3'

 Types 錶 include Human Multiple Tissuue cDNA (MTC) Panel I (Clontech # K1420-1), Human MTC Panel II (Clontech # K1421-l), Human Immune System MTC Panel (Clontech # K1426-l), and: Human Blood Fract ions MTC Panel (Clontech # K1428-l) was used. PCR was performed using an Advantage cDNA Polymerase Mix (Clontech # 8417-l) and a Perkin Elmer Gene Amp PCR System 2400 thermal cycler. The PCR reaction was performed under the following cycle conditions to try to amplify the target gene.

 4 minutes at 94 ° C

 5 cycles of [20 seconds at 94 ° C, 1 minute at 72 ° C]

 5 cycles of [20 seconds at 94 ° C, 1 minute at 70 ° C]

 25 cycles of [20 hectares at 94 ° C, 1 minute at 68 ° C]

3 minutes at 72 ° C Finish at 4 ° C

 As a result, as shown in FIG. 22 and FIG. 23, when the expression distribution of R10.4, NR10.5, and U0.6 genes were comprehensively evaluated, it was found that immunity 担当 | 戠, hematopoietic ffi 戠, and live 戠Strong expression was detected. In addition, by detecting the expression of the housekeeping gene G3PDH under the above PCR conditions using a human G3PDH primer for all the 铸 types used for It has been confirmed that it has been standardized. In the following, when the above-mentioned BNR10 gene expression was detected, β was listed. Strong expression was detected in thymus, lymph nodes, peripheral leukocytes, lungs, bone marrow, etc., and in hematopoietic fibers. In addition, strong expression was detected in testis, ^ m, placenta, uterus, etc., and in endocrine II. In addition, although weak expression was detected in kidney, kidney, and small intestine, no expression was observed in spleen, tonsil, heart, brain, liver, skeletal muscle, and colon. On the other hand, among hemocyte tissue cells, strong expression was detected in CD14 + monocytes (macrophages), CD4 + T cells in J3 package, and quiescent CD19 + B cells, but CD8 + T cells and activated CD19 + In + B cells, no expression was observed. Here, the size of the detected RT-PCR amplification product matches the predicted size of NR10. Therefore, these were considered to be products of specific PCR amplification reactions. By further confirming this by the Southehrn Blotting method described in the next section, the possibility that they were products of non-specific PCR amplification was denied.

 [Example 7] Confirmation of specificity of RT-PCR product by Southern blotting method

The target gene product amplified by RT-PCR in Example 6 above was subjected to Southern Blotting using a specific cDNA fragment common to each of the NR10.4, NR10.5, and NR10.6 genes as a probe. As a result, they were confirmed to be specific amplification products. At the same time, by quantitatively detecting the RT-PCR product, an attempt was made to perform a comparative measurement evaluation of the gene expression state among human organs. The agarose gel electrophoresis of the RT-PCR product described in the preceding paragraph was followed by blotting on a charged nylon with Hybond N (+) (Amersham, cat # RPN303B), followed by hybridization. As a probe The NR10.4 cDNA fragment obtained in Example 4 was used. Preparation of probe, Mega Prime Ki t (Amersham, cat # RPN1607) using ^{[α - 32 P] dCTP (} Amersham, cat # M0005) were radio § iso taupe labeled with. Use Perfect Hyb (Toyobo # HYB-101) for pre-hybridization at 68 ° C / 30 min, add heat-denatured labeled probe, and add 68 ° C / 120 min. No. 8 I carried out an education. After washing under the following conditions, the plate was exposed to an imaging plate (FUJI # BAS-III), and an NR10-specific signal was detected with an image analyzer (FUJIX, BAS-2000 II).

 Washing conditions (1) lx SSC I 0.1% SDS, room temperature for 10 minutes

 (2) lx SSC I 0.1% SDS, 60. 45 minutes at C

 (3) 0.lx SSC I 0.1% SDS, 60 ° C for 45 minutes

 As a result, as shown in Fig. 24 and Fig. 25, all the PCR products amplified by RT-PCR in the previous section may be amplification products specific to N10.4, NR10.5 and NR10.6. confirmed. In addition, the comparative quantification of the expression level in each case supported the evaluation described in the previous section. On the other hand, the method for detecting target gene expression, which combines RT-PCR and Southern blotting, is an extremely sensitive detection method even when compared to other expression analysis methods. No expression was observed in spleen, tonsils, heart, brain, liver, skeletal muscle, and colon. Furthermore, no expression was detected in CD8 + T cells and activated CD19 + B cells.

 [Example 8] Identification of NR10 gene-expressing cell line by RT-PCR method

In order to identify a human cell line that overexpresses the NR10.4 gene, mRNA was detected by RT-PCR. The human cell lines shown in Table 2 were selected for the search. Table 2 Expression of the NR10.4 gene Jarkat human T cell line (Human T-cell ll ine)

 HL-60 Promyelocytic leukemia

 Raj i's lymphoma (Burkitt's lymphoma)

K-562 Chronic myelogenous leukemia

YTN-17 Human NK cell line (Human NK cel l line)

The above-mentioned human cell line was cultured according to a conventional method, and after harvesting about 10 ⁶ cells, niRNA was prepared. Preparation of mRNA A high-purity mRNA was purified by performing polyA positive selection using mMACS mRNA Isolate ion Kit (Miltenyi Biotec # 130-075-201) according to the manufacturer's manual. Each purified mRNA was used in lOOng to obtain RT-PCR type II. RiverTraDash (Toyobo # PCR-401) was used for the synthesis of the primary strand cDNA, and the random priming method was used. In addition, KOD Dash (Toyobo # LDP-101) was used for the RT-PCR reaction conditions according to the manual specified. The primer set used for the RT-PCR amplification reaction is the same as in Example 6 described above. Furthermore, the Southern Blotting method was carried out according to Example 7 in the previous section to confirm that they were specific amplification products. As a result, as shown in FIG. 26, strong expression gene expression was detected in HL-60 and -562.

Example 9 Construction of NR10 Ligand Retrieval System Using Growth Factor-Dependent Cell Line Screening of a ligand protein that specifically binds to the protein of the present invention is performed using the extracellular domain and the Hosoki trans domain of the protein of the present invention. A chimeric receptor linked to the intracellular domain of the hemopoietin receptor protein capable of signaling »can survive and proliferate only in the presence of a suitable cell line, preferably a suitable growth factor. After being expressed on the cell surface of a cell line (growth factor-dependent cell line), the cell line is cultured with the addition of materials that are expected to contain various growth factors, cytodynamic factors, hematopoietic factors, etc. This can be implemented. Since the above-mentioned growth factor-dependent cell line rapidly dies in the absence of growth factors, survival and survival only occur when a test substance contains a ligand that specifically binds to the fine domain of the protein of the present invention. Establish a screening system by utilizing the possibility of multiplication. Examples of difficult hemopoietin receptors include, for example, thrompopoietin receptor, erythropoietin receptor, G-CSF receptor, and gpl30.The chimeric receptor used in this screening system is the known hemopoietin receptor. The present invention is not limited to this, and any material may be used as long as it has a structure necessary for signal transduction activity in the cytoplasmic domain. In addition, as growth factor dependent cell lines, it is possible to use IL-3 dependent cell lines such as Ba / F3 and FDC-P2 for IJ. Therefore, first, the cDNA sequence encoding the extracellular domain and transmembrane domain of NR10.4 (amino acid sequence; Ala at position 52 to Lys at position 576) is amplified by PCR, and this DNA fragment is A fusion sequence encoding a chimeric receptor was produced by ligating the intracellular region of the mopoetin receptor to a DNA fragment encoding the same in the same translational frame. Here, as described above, several candidates were listed as known partner hemopoietin receptors, and the human TP0 receptor (Human MPL-P) was selected from them. In addition, the chimeric receptor sequence prepared above was ligated downstream of the mouse IL-3 signal sequence and the FLAG peptide sequence with the same amino acid translation frame, and inserted into a plasmid vector pCOS that can be expressed in mammalian cells. . FIG. 27 shows a schematic structural diagram of the chimeric receptor (pCOS I NR10MPL) constructed here. This chimeric receptor expression vector is introduced into a growth factor-dependent cell line Ba / F3 and strongly expressed, and stable transfected cells are selected. Here, the selection of gene-introduced cells is based on the fact that the above-mentioned expression vector has a drug (neomycin) resistance gene, and selects only the gene-introduced cells that have acquired drug resistance in the same medium containing the drug. It is possible to proliferate. As described above, the cell line expressing the chimeric receptor obtained above is switched to a culture system in the absence of a growth factor (here, IL-3), and is expected to contain the target ligand. With the ingredients A novel cytokine that functions as a ligand for NR10 by developing a screening system that utilizes the ability to survive only when a ligand that specifically binds to NR10 functionally binds to E Screening can be performed.

 [Example 10] Construction of NR10.4 and 6 gene expression vectors

From the NR10 cDNAs identified and isolated as described above, we attempted to construct an expression vector that can be expressed in mammalian cells using NR10.4 and NR10.6. The respective cDNA clones were ligated, and the sequence from the sequence excluding the secretion signal to the coding region up to the amino acid translation stop codon was amplified by conventional PCR in a conventional manner. ^ Advantage cDNA Polymerase Mi was used for the amplification reaction. Each amplification product was ligated to the mouse IL-3 signal sequence and the FLAG peptide sequence downstream of the same amino acid translation frame to construct a plasmid vector pCOS that can be expressed in mammalian cells. Sequencing of the constructed nucleotide sequence of the expression vector confirmed that there was no point mutation involving amino acid sequence substitution, and that it was inserted in the forward direction. . Figure 27 shows a schematic diagram of these constructed expression vectors. The pCOS / NR10.4 NPL vector constructed here contains from Ala at position 52 of NR10.4 to Val at position 764, and the pCOS / NR10.6 NFL vector is the Ala at position 52 of NR10.6. From position 581 to Ser 581 are included as insert sequences capable of expressing functions. The pCOS / NR10.4 NFL vector can be introduced into a growth factor-dependent cell line Ba / F3 and strongly expressed in the same manner as in Example 9 to select stable gene-transfected cells. The NR10.4 expressing cell line obtained in this way was switched to a culture system in the absence of growth factors (here, IL-3), and alternatively, a material expected to contain the target ligand was added. By culturing, it is possible to identify new cytokines by developing a screening system utilizing the fact that they can survive / proliferate only when there is a ligand that specifically binds to NR10. In addition, overexpression of the pCOS / NR6 NFL vector in the cell line responding to the cytodynamic force (NR10 ligand) thus identified. Therefore, it is expected that NR10.6, which has no intracellular signal transduction function, can function as a dominant deficient (dominant negative chain. Therefore, the C0S / NR10.6 NFL vector was In addition, the pCOS / NR10.6 NFL vector expression system can be used to evaluate the cell responsiveness to NR10 ligand, that is, the details of the biological activity possessed by NR10 ligand. It is anticipated that it is possible to make horns.

Example 11 Construction of Expression System for Cellular Secretory, Soluble Recombinant N10 Protein The ligand protein that specifically binds to the protein of the present invention may be a rare but soluble protein, but not a soluble protein. Is also assumed. In such a case, it is rather expected that the ligand is expressed after labeling a protein containing only the extracellular domain of the protein of the present invention or a fusion protein in which a partial sequence of another soluble protein is added to the extracellular domain. Screening is possible by measuring the binding to the cells that are performed. In the former case, for example, NR10.2, NR10.7 encoding a soluble receptor protein artificially created by inserting a stop codon at the N-terminal side of the narrow transduction domain, or a soluble form of NR10 , And the sequences of R10.8 are available. In the latter case, it can be prepared, for example, by adding a labeled peptide sequence such as an Fc site of immunoglobulin or a FLAG peptide to the C-terminus of the extracellular domain. These soluble labeled proteins can also be used for detection by the Westwes Even method. Therefore, the present inventors amplified the cDNA sequence encoding the extracellular region of NR10.4 (amino acid sequence; Ala at position 52 to Glu at position 256) by PCR, and amplified this DNA fragment with the mouse IL-3 signal. The sequence was further linked downstream of the FLAG peptide sequence with the same amino acid translation frame to construct a plasmid vector PCH0 that can be expressed in mammalian cells. Thus, the sequence encoding the soluble marker protein was completed. Here, a stop codon due to point mutation is inserted at the position of Glu at position 257 and below Glu at position 256 in NR10.4. Figure 27 shows a schematic diagram of the constructed NR10 soluble receptor signature protein (pCHO / N 10.2 NFL). This expression vector was introduced into the mammalian cell line CH0 cells and strongly expressed. Transgenic cells were selected. The above expression cells are cultured in a large halo, and the recombinant protein secreted in the culture supernatant is immunoprecipitated with an anti-FLAG peptide antibody, and further subjected to Western blotting to obtain the soluble receptor protein. Expression was confirmed. In addition, large-scale purification was carried out with an affinity ram loaded with an anti-FLAG peptide antibody. The recombinant protein obtained as described above can be used not only for the above-mentioned assays and the like, but also for the specific binding activity in the presence of a material predicted to contain the target ligand, for example, in the BIA-C0RE system (Pharmacia). ), And is considered to be extremely useful for searching for new hemopoietin capable of functionally binding to NR10.

 [Example 1 2] Construction of NR10.8 gene expression vector

Among the NR10 cDNAs identified and isolated as described above, NR10.8 was used to construct an expression vector that can be expressed in mammalian cells. Using a cDNA clone of NR10.8 as type II, PCR amplification was carried out from the sequence excluding the secretory signal to the coding region up to the amino acid translation stop codon according to a conventional method. Advantage cDNA Polymerase Mix was used for the amplification reaction. The obtained amplification product was ligated with the same amino acid translation frame downstream of the mouse IL-3 signal sequence and the FLAG peptide sequence to construct a plasmid vector pCHO that can be expressed in mammalian cells. By performing sequencing on the insert base sequence of the constructed expression vector, it was confirmed that there was no point mutation involving amino acid sequence substitution, and that it was inserted in the forward direction. Figure 27 shows a schematic diagram of the constructed expression vector. The thus constructed CHO / NRlO.8 NFL vector contains from Ala at position 52 to Arg at position 548 in 10.8 as an expressible insert sequence. The pCHO / NR10.8 NFL vector can be introduced into a mammalian cell line CH0 cell and strongly expressed as in Example 11 described above, and a stable gene-transfected cell can be selected. After confirming the expression of the soluble protein, the expression cells are cultured in a large amount, and the recombinant protein secreted in the culture supernatant can be immunoprecipitated with an anti-FLAG peptide antibody. In addition, affinity loaded with anti-FLAG peptide antibody Large-scale purification with a single column is possible. The above 1 obtained above

 By applying the protein to the Atsushi BIA-CORE system of Example 11 described above, it can also be used for measurement of specific binding activity in the presence of a material predicted to contain a target ligand.

 [Example 13] Identification of mouse NR10 homologous gene

 Identification of human NTR10 cDNA genes, analysis of human NR10 genomic gene structure, construction of human NR10 gene expression system, construction of recombinant human NR10 protein expression system in mammalian cells, and human R10 ligand search system The construction of was described. Further, the present inventors have attempted to identify a mouse homologous gene.

 To detect the mouse NR10 gene, we performed a Bias phagocytosis using the human MU0 gene sequence as a query. To prepare the query, the cDNA sequence of human NR10.4 was translated into an amino acid sequence, and the full-length amino acid sequence was used. A mouse gss (Genomic Survey Sequence; a genomic sequence obtained by randomly decoding the terminal sequence of a BAC clone by shotgun analysis) selected in the GenBank database, one of the public databases, is selected as a search target. did. The parameters used as search conditions were Expect 50, Descriptions value = 100, and Alignments value = 100. Filter setting ^ Default value. As a result of the above search using the Tblas tN (Advanced Tbl as tN 2.0.13) program, the mouse N10 genomic gene showing homology to the human NR10 amino acid sequence (GenBank accession number: AZ618234 / accession number) : AZ618234) could be detected in the reverse direction. The AZ618234 mouse gss base sequence selected here (SEQ ID NO: 29) and the predicted amino acid sequence of the exon site (SEQ ID NO: 30) are shown in FIG. In addition, FIG. 29 shows a sequence comparison between the two sites at a site showing a positive result for the human Νΐθ.4 amino acid sequence. Oligonucleotide primers were designed on the mouse NR10 genomic gene sequence detected as described above, and synthesis and purification were performed under the following conditions.

[Example 14] Design of an oligonucleotide primer specific for mouse NR10 An exon site was predicted in the AZ618234 mouse gss base sequence identified as described above, and based on the predicted sequence, oligonucleotide primers specific to the mouse NR10 gene shown below were designed. As primers, mNRlO-S1 and mNRlO-S2 were synthesized on the sense side (downstream direction), and πΜΙΟ-A1 and ιιΙΟ-Α2 were synthesized on the antisense side (upstream direction). The primer was synthesized according to Example 3 described above, using a 394 DNA / RNA Synthesizer manufactured by ABI under the condition of adding a 5'-terminal trityl group. Thereafter, the full-length synthetic product was purified using an OPC column, and provided to the 5′-RACE method and the 3′-RACE method described below.

Consideration 10-S1 (SEQ ID NO: 31)

5 '-TAT ATC AGT GTA TCC AGT GTT GGG ACA CCG -3'

mN 10-S2 (SEQ ID NO: 32)

5 '-ACA CCG AGT TGG AGA GCC GTA TTC AAT C -3'

Consideration 10-A1 (SEQ ID NO: 33)

5 '-TCT TTG GCA TAA GCT TGG ATT GAA TAC -3'

mN 10-A2 (SEQ ID NO: 34)

5 '-GGA TTG AAT ACG GCT CTC CM CTC GGT GTG -3'

 [Example 15] Isolation of mouse R10 gene by RACE method

In order to isolate the N-terminal sequence of the cDNA clone corresponding to full-length mouse N10, the mNRlO-A1 primer described in Example 14 above was used for the primary PCR, and the mNRlO-A2 primer was used; ^^! Use for ^-! ^^? I tried ^^. On the other hand, to isolate the C-terminal sequence of a cDNA clone corresponding to full-length mouse NR10, the mNRlO-S1 primer of Example 14 was used for primary PCR, and the 10-32 primer was used for primary PCR. Next, we tried 3'-1 ^ ∑ PCR. Using Mouse Test is (Balb / c testis) Marathon-Ready cDNA Library (Clontech # 7455-1) as a 铸 type, ^ Advantage cDNA Polymerase Mix was used for PCR experiments. Using the Perkin Elmer Gene Amp PCR System 2400 thermal cycler under the following PCR conditions, a PCR product of the desired size was obtained. One; ^ CR conditions:

 94. 4 minutes at C

 5 cycles of [20 seconds at 94 ° C, 90 seconds at 72 ° C]

 5 cycles of [20 seconds at 94 ° C, 90 seconds at 70 ° C]

 [20 seconds at 94 ° C, 90 seconds at 68 ° C] 28 cycles

 3 minutes at 72 ° C

 Terminates at 4 ° C

Two conditions:

 94. 4 minutes at C

 5 cycles of [20 seconds at 94 ° C, 90 seconds at 70 ° C]

 25 cycles of [20 seconds at 94 ° C, 90 seconds at 68 ° C]

 3 minutes at 72 ° C

 Terminates at 4 ° C

 The obtained PCR product was subcloned into pGEM-T Easy vector according to Example 4 described above, and the nucleotide sequence was determined. As a result of determining the nucleotide sequence of all the insert fragments from multiple independent recombinant strains, it was confirmed that the nucleotide sequence was the full-length nucleotide sequence of mouse NR10 gene derived from the specific PCR amplification reaction. The determined sequence was designated as mouse N10B, and its nucleotide sequence (SEQ ID NO: 15) and the amino acid sequence encoded by it (SEQ ID NO: 16) are shown in FIGS. A similar gene was cloned using Mouse day 8.5 embryo (C57BL / 6 embryo 8.5 day mouse embryo) cDNA Library (GIBCO BRL) as a type III, and the target gene was isolated. The sequence determined in the same manner as above was named mouse NR10C, its base sequence (SEQ ID NO: 17) and the amino acid sequence encoded by it

(SEQ ID NO: 18) is shown in FIGS. There are slight sequence differences between the mouse N10B and mouse NR10C with mutations in the amino acid ffi sequence. This is presumed to be sequence differences from different mouse strains, : It is not the difference of the origin transcript. FIG. 36 shows a comparison between the full-length amino acid sequence of mouse NR10B determined as described above and the full-length amino acid sequence of human NR10.4 determined in Example 4 described above. Mouse NR10B retains 59.426% homology to human NR10.4. Based on this high homology and structural features, mouse N10 molecule is presumed to have an activity that is functionally equivalent to the biological activity of human NR10 liver. Industrial applicability

 According to the present invention, a novel hemopoietin receptor protein and a DNA encoding the same are provided. Also provided are a vector into which the DNA has been inserted, a transformant carrying the DNA, and a method for producing a recombinant protein using the transformant. Further, a method for screening for a natural ligand or compound that binds to the protein was provided. Since the protein of the present invention is considered to be directly involved in the regulation of biological immunity or the regulation of hematopoietic cells, the understanding of the ^^ response in the living body or the fundamental properties of the hematopoietic mechanism and the furnace-related diseases based on it are And application to diagnosis and treatment of hematopoietic diseases.

In particular, it is important to isolate an unknown hematopoietic factor capable of functionally binding to the protein of the present invention, and it is extremely useful to use the gene of the present invention in screening for the purpose. It is considered to be. Further, it is also effective to conduct a search for an agonist or an angonist capable of functionally binding to the protein of the present invention with respect to a peptide library or a synthetic chemical material to isolate and identify it. As described above, the gene of the present invention is considered to provide a useful material for obtaining unknown hematopoietic factors and agonists capable of functionally binding to the receptor: protein encoded by the gene. In vivo administration of such a function-binding substance or a specific antibody capable of activating the ^^ function of the receptor protein can enhance the cellular ^ S and the hematopoietic function of the living body. J is done. In other words, the proliferation of immunocompetent cells or hematopoietic cells It is considered that clinical application as an enhancer, a differentiation inducer, or an activator of immune cell function is possible. In addition, it is thought that it is possible to enhance the cytotoxicity of certain types of cancer thread II そ れ ら through them. Furthermore, the expression of the gene in the present invention is

However, it is supposed that the antibody is specifically expressed in a limited group of 13 cells in these hematopoietic fibers, and an antibody that specifically binds to the protein of the present invention is useful as a means for separating the group. It is. It can be applied to cell transplantation therapy for small bandages isolated in this way.

 On the other hand, R10.8 having no transmembrane domain, soluble deletion protein, and the like are expected to be used as decoy-type receptors as inhibitors of NR10 ligand. In addition, it is possible to suppress the cellular immunity of the living body and suppress the growth of hematopoietic cells by in vivo administration of an agonist, which can functionally bind to the NR10 molecule, or other inhibitors, or a specific antibody that can inhibit the function of the NR10 molecule. Is expected. Such an inhibitor is considered to be clinically applicable as an immunosuppressive cell or hematopoietic cell proliferation inhibitor, a differentiation inhibitor, or an immunosuppressant—an anti-inflammatory agent. Specifically, it can be applied to the suppression of the onset of autologous furnace disease caused by autologous thread II 戠 injury, and the suppression of rejection of the thread by the living body, which is the biggest problem in the field of transplant immunity. There is also. Furthermore, it is considered to be extremely effective against a disease area caused by abnormally high response, and is also effective against various antigen-specific allergies to metals and pollen using the above-mentioned inhibitor. Is considered to be effective.

Claims

The scope of the claims

1. DNA described in any one of (a) to (d) below.

 (a) DNA encoding a protein consisting of the amino acid sequence of any one of 4, 6, 8, 10, 12, 14, 16, and 18.

 (b) a DNA comprising the coding region of the nucleotide sequence of any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, and 17;

 (c) System IJ number: Substitution, deletion, insertion, and Z or addition of one or more amino acids in the amino acid sequence described in any of 4, 6, 8, 10, 12, 14, 16, or 18 SEQ ID NOs: 4, 6, 8, 10, 1

DNA encoding a protein functionally equivalent to a protein consisting of the amino acid sequence of any of 2, 14, 16, or 18.

 (d) SEQ ID NOs: 4, 5, 6, 7, 8, 9, 13, 13, 15, or 17 are hybridized under stringent conditions with a DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 4, 5, and 6, DNA encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of any one of 8, 10, 12, 14, 16, and 18.

 2. System ij number: DNA that encodes a partial peptide of a protein consisting of the amino acid sequence described in any of 4, 6, 8, 10, 12, 14, 16, and 18.

 3. A protein or peptide encoded by the DNA according to claim 1 or 2.

4. A vector into which the DNA according to claim 1 or 2 has been inserted.

 5. Retains the DNA of claim 1 or 2 or the vector of claim 4

6. Production of the protein or peptide according to claim 3, comprising a step of culturing the transformant according to claim 5, and recovering the expressed protein from the transformant or a culture supernatant thereof. Method.

7. An antibody that binds to the protein of claim 3.

8. System IJ number: At least 15 nucleotides complementary to the DNA consisting of the base sequence described in any of 3, 5, 7, 9, 11, 11, 13, 15 or 17 or its complementary strand A polynucleotide containing

 9. A method for screening a compound that binds to a protein according to claim 3,

(a) contacting a test sample with the protein or a partial peptide thereof,

 (b) a step of detecting a binding activity between the protein or a partial peptide thereof and a test sample

(c) selecting a compound having an activity of binding to the protein or a partial peptide thereof.