JP2008099690A - Novel hemopoietin receptor protein, nr12 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel hemopoietin receptor gene (NR12) isolated by extracting motifs conserved among the amino acid sequences of known hemopoietin receptors and by using the predicted sequence. <P>SOLUTION: The expression of the NR12 gene is detected in tissues containing hematopoietic cells, and the NR12 gene has two forms, a transmembrane type and a soluble type. NR12 is a novel hemopoietin receptor molecule involved in the regulation of immune system and hematopoietic cells in vivo. NR12 has a transmembrane type and a soluble type. NR12 is useful in the search for novel hematopoietic factors functionally binding to the NR12 receptor and in the development of therapeutic drugs for diseases associated with immunity or hemopoiesis. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel hemopoietin receptor protein, a gene encoding the same, a production method thereof, and use.

  The existence of many cytokines has been known so far as a humoral factor that controls the growth and differentiation of various cells, or maintains and activates the functions of differentiated and mature cells, and further leads to cell death. Each of these cytokines has specific receptors, and these receptors are classified into several families based on structural similarity (Hilton DJ, in "Guidebook to Cytokines and Their Receptors" edited by Nicola NA (A Sambrook & Tooze Publication at Oxford University Press), 1994, p8-16 (Non-Patent Document 1)).

  On the other hand, compared with the similarity between receptors, the homology on the primary structure between cytokines is low, and no remarkable homology at the amino acid level is recognized even among cytokine members belonging to the same receptor family. This explains the functional specificity of individual cytokines as well as the similarity of cellular responses induced by individual cytokines.

  Representative of the above cytokine receptor families are the tyrosine kinase receptor, hemopoietin receptor, tumor necrosis factor (TNF) receptor, and transforming growth factor (TGF) receptor. Involvement of different signal transduction systems has been reported. Among these receptor families, most of the hemopoietin receptor family is expressed in blood cells or immunocompetent cells, and the cytokine serving as its ligand is often referred to as hematopoietic factor or interleukin. Some of these hematopoietic factors or interleukins are present in living blood and are considered to be involved in systemic hematopoiesis or humoral regulation of immune function.

  This is in contrast to the fact that cytokines corresponding to other receptor families are often thought to be involved only in local regulation, and some of these hemopoietins are regarded as hormone-like factors. Is possible. Conversely, receptors for growth hormone, prolactin, or leptin, which are typical peptide hormones, also belong to the hemopoietin receptor family. From the above-mentioned hormone-like systemic regulation mode, application to the treatment of various diseases by administering these hemopoietins is expected. In fact, among many cytokines, erythropoietin, G-CSF, GM-CSF, and IL-2 have been clinically applied, and IL- 11, In addition to LIF and IL-12, the peptide hormones growth hormone and prolactin are considered together, and by searching for a new cytokine that binds to the hemopoietin receptor family from the various cytokine receptor superfamily described above. It is considered possible to find cytokines that can be clinically applied with higher probability.

As stated above, cytokine receptors have structural similarities between family members. Many attempts have been made to discover new receptors using this similarity, and in particular, with respect to the tyrosine kinase receptor, many receptors have already been used by utilizing a highly conserved sequence at the catalytic site. It has been cloned (Matthews W. et al., Cell (UNITED STATES), 1991, 65 (7) p1143-52 (Non-patent Document 2)). In contrast, the hemopoietin receptor does not have an enzyme active domain like tyrosine kinase in its cytoplasmic region, and its signal transduction is associated with another tyrosine kinase protein that exists in the cytoplasm in a free state. It is known to be done. Although the binding sites on the receptors of these cytoplasmic tyrosine kinases, called JAK kinases, are conserved among family members, their homology is not very high (Murakami M. et al., Proc Natl. Acad. Sci. USA, 1991, 88, p11349-11353 (Non-patent Document 3)). On the other hand, the sequence that best characterizes these hemopoietin receptors exists rather in the extracellular region, and in particular, the motif consisting of 5 amino acids of Trp-Ser-Xaa-Trp-Ser (Xaa is an arbitrary amino acid) has almost all hemopoietin receptors. Stored in the body. Therefore, it is expected to isolate a novel hemopoietin receptor gene by searching for a new family member using this motif sequence. In fact, the IL-11 receptor (Robb, L. et al., J. Biol. Chem. 271 (23), 1996, 13754-13761 (non-patent document 4)), the leptin receptor (Gainsford T. et al. al., Proc. Natl. Acad. Sci. USA, 1996, 93 (25) p14564-8 (non-patent document 5)) and IL-13 receptor (Hilton DJ et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (1) p497-501 (Non-Patent Document 6)) has been identified by this approach.
Hilton DJ, in "Guidebook to Cytokines and Their Receptors" edited by Nicola NA (A Sambrook & Tooze Publication at Oxford University Press), 1994, p8-16 Matthews W. et al., Cell (UNITED STATES), 1991, 65 (7) p1143-52 Murakami M. et al., Proc. Natl. Acad. Sci. USA, 1991, 88, p11349-11353 Robb, L. et al., J. Biol. Chem. 271 (23), 1996, 13754-13761 Gainsford T. et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (25) p14564-8 Hilton DJ et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (1) p497-501

  The present invention provides novel hemopoietin receptor proteins and DNA encoding them. The present invention also provides a vector into which the DNA has been inserted, a transformant retaining the DNA, and a method for producing 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 have heretofore accepted a novel receptor by a plaque hybridization method using an oligonucleotide encoding a Trp-Ser-Xaa-Trp-Ser motif (WS motif) as a probe, or a method such as RT-PCR. I have tried to search for the body. However, since the oligonucleotide tggag (t / c) nnntggag (t / c) (n is an arbitrary base) encoding this motif is as short as 15 base pairs, an annealing temperature lower than usual is required. On the contrary, since the above sequence has a high g / c content rate, it is difficult to select only those in which 15 bases are completely hybridized unless the annealing temperature is higher than usual. For these reasons, it has been extremely difficult to develop screening under normal hybridization experimental conditions.

  In order to solve these problems, motifs conserved in the hemopoietin receptor family at sites other than the WS motif were examined. As a result, a tyrosine residue or a histidine residue located 13 to 27 amino acids upstream from the WS motif is conserved in the extracellular region of the family with a high probability, and further, from the Tyr / His residue to the C-terminal direction. As a result of examining a consensus sequence frequently occurring in the 6 amino acids of (A), (Tyr / His) -Xaa- (Hydrophobic / Ala)-(Gln / Arg) -Hydrophobic-Arg amino acid sequence (hereinafter referred to as YR motif) It was possible to find out. However, this YR motif cannot always be determined as a complete consensus sequence, and combinations of base sequences encoding this motif are complex. Therefore, it is practically impossible to synthesize and provide oligonucleotides that can encode all of this amino acid sequence as probes for hybridization as a practical means of screening or primers for RT-PCR. Nearly possible.

  Therefore, as a result of investigating means for searching for specific novel hemopoietin receptor family members using the above two types of motifs as probes, partial amino acid sequences obtained by fragmenting known hemopoietin receptors so as to include both motif sequences. A database search on a computer using a query formula as a query was deemed valid. As a result of actually repeating TBlastN search against the GenBank htgs database using partial amino acid sequences of a plurality of known hemopoietin receptors as a query, in each case, a large number of known hemopoietin receptors are included. A positive clone was obtained. Next, for the clone obtained by the above search, the base sequence around the positive sequence with high probability is converted into an amino acid sequence and compared with the amino acid sequence of a known hemopoietin receptor. We selected genes thought to encode members. By the above two-step Blast search approach, two clones of known hemopoietin receptor and one clone of a new hemopoietin receptor gene were finally identified.

  Next, a specific oligonucleotide primer was designed based on the exon site sequence that could be predicted from the obtained base sequence. 5'-RACE method and 3'-RACE method using these primers are used for human fetal liver, adult thymus and adult testis cDNA library as templates, and correspond to N-terminal region and C-terminal region of NR12 Each clone was obtained. The base sequences of these clones were determined, and the entire base sequence of the full-length cDNA was clarified by connecting the two at the overlapping central site.

  As a result of structural analysis, differences in at least three kinds of transcripts derived from splicing mutants were observed. Among them, a cDNA clone consisting of 337 amino acids out of splicing variants and capable of encoding a cell-secreted soluble receptor protein was named NR12.1, while NR12.2 and NR12.3 were 428 amino acids and 629 amino acids, respectively. It was possible to encode a transmembrane receptor protein consisting of In common with the primary structure of all of these isolated NR12 cDNA clones, the repeat structure of cysteine residues conserved among other family members in the extracellular region, the YR motif, the WS motif, etc. It was well conserved and thought to encode a typical hemopoietin receptor.

  Thereafter, RT-PCR analysis using a primer set specific to NR12.1, NR12.2 and NR12.3 is further performed on mRNA derived from each human organ, and the expression tissue of the gene is searched. At the same time, the expression distribution and expression mode of the same gene in each human organ were analyzed. The target gene amplified by the RT-PCR method was subjected to Southern blotting using a cDNA fragment specific for these clones as a probe, denying the possibility of non-specific amplification, Quantitative evaluation of RT-PCR products was performed. As a result, it was confirmed that the main production tissues of these clones were a hematopoietic cell line tissue and an immunocompetent cell line tissue.

  Furthermore, the present inventors performed PCR cloning on a human thymus cDNA library, so that NR12.2, NR12.3, two clones (NR12. 4, NR12.5) (5 isolated clones are collectively referred to as “NR12”).

  From the above characteristics of NR12, NR12 is presumed to be a novel hemopoietin receptor molecule involved in biological immune regulation or hematopoietic cell regulation. It is considered extremely useful to use.

  Furthermore, the present inventors isolated a mouse homologous genomic gene fragment of the receptor by performing cross-species crossover hybridization cloning using a human NR12 cDNA as a probe on a mouse genomic DNA library. succeeded in. By producing NR12 gene-deficient mice using the mouse gene fragment, further elucidation of the biological function of the receptor protein is expected.

That is, the present invention relates to novel hemopoietin receptors and their genes, and uses thereof, more specifically,
(1) The DNA according to any one of (a) to (d) below,
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10;
(B) DNA containing the coding region of the base sequence according to any one of SEQ ID NOs: 1, 3, 5, 7, or 9,
(C) an amino acid sequence according to SEQ ID NO: 2, 4, 6, 8, or 10 having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added, DNA encoding a protein that is functionally equivalent to a protein consisting of the amino acid sequence of any of Nos. 2, 4, 6, 8, or 10;
(D) hybridizes under stringent conditions with DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9, and SEQ ID NOs: 2, 4, 6, 8, or A DNA encoding a protein functionally equivalent to the protein comprising the amino acid sequence according to any one of 10;
(2) DNA encoding a partial peptide of a protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10;
(3) a protein or peptide encoded by the DNA according to (1) or (2),
(4) A vector into which the DNA according to (1) or (2) is inserted,
(5) A transformant carrying the DNA according to (1) or (2) or the vector according to (4),
(6) The method for producing a protein or peptide according to (3), comprising a step of culturing the transformant according to (5) and recovering a protein expressed from the transformant or a culture supernatant thereof,
(7) an antibody that binds to the protein according to (3),
(8) a polynucleotide comprising at least 15 nucleotides complementary to DNA consisting of the base sequence of SEQ ID NO: 1, 3, 5, 7, or 9 or a complementary strand thereof,
(9) A method for screening a compound that binds to the protein according to (3),
(A) contacting the test sample with the protein or a partial peptide thereof,
(B) detecting the binding activity between the protein or a partial peptide thereof and a test sample;
(C) A method comprising a step of selecting a compound having an activity of binding to the protein or a partial peptide thereof.

  The present invention provides a novel hemopoietin receptor “NR12”. The present inventors succeeded in identifying and isolating a novel hemopoietin receptor gene NR12 from the results of GenBank database analysis and analysis by 5′-RACE and 3′-RACE. The presence of at least three splice variants was confirmed in the transcript of NR12. Among these, a cDNA clone encoding a soluble receptor-like protein was designated as NR12.1. On the other hand, among the cDNA clones that are thought to encode a transmembrane receptor protein, NR12.2 is a clone encoding a protein expected to have a short intracellular region of 51 amino acids, and a long intracellular region of 252 amino acids. A clone encoding a protein expected to have NR12.3 was named.

  In addition, we performed PCR cloning on a human thymus cDNA library and re-isolated consecutive full-length coding sequences (CDS). Among these, a clone having a full-length ORF almost identical to NR12.2 was named NR12.4, and a clone having a full-length ORF almost identical to NR12.3 was designated NR12.5.

  The nucleotide sequence of NR12.1 cDNA is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 2. The base sequence of NR12.2 cDNA is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 4. The base sequence of NR12.3 cDNA is shown in SEQ ID NO: 5, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 6. Further, the base sequence of NR12.4 cDNA is shown in SEQ ID NO: 7, the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 8, the base sequence of NR12.5 cDNA is shown in SEQ ID NO: 9, and the cDNA The amino acid sequence of the protein encoded by is shown in SEQ ID NO: 10.

  In the extracellular region, NR12.1, NR12.2, NR12.3, NR12.4, and NR12.5 are almost the same, so they have the same conformation and recognize the same specific ligand. I think that.

  As a result of gene expression analysis in each human organ using RT-PCR method, strong NR12 gene in adult hematopoietic cell line tissues such as adult spleen, thymus, lymph node, bone marrow and peripheral leukocytes, and immunocompetent cell line tissues Expression was observed, and the expression was also detected in the testis, liver, lung, kidney, pancreas, small intestine, and colonic tract. Moreover, the gene expression was recognized also in mRNA derived from all human fetal organs analyzed. When these gene expression distributions of NR12 are combined, NR12 encodes a novel hematopoietic factor receptor, as strong expression localization was detected mainly in immunocompetent cell line tissues and tissues thought to contain hematopoietic cells. Estimated. In addition, the expression distribution was also observed in tissues other than the above, suggesting that NR12 may regulate not only the immune system and hematopoietic system, but also various physiological functions in vivo.

  The NR12 protein can be applied to medicine. The expression of NR12.1 in the thymus, peripheral leukocytes and spleen suggests the possibility of being an unknown hematopoietic factor receptor. Therefore, NR12 protein is considered to provide a useful material for obtaining this unknown hematopoietic factor. It is also possible to search for an agonist or an antagonist capable of functionally binding to the NR12 molecule against a peptide library or a synthetic chemical material, and to identify and identify it. Furthermore, clinical applications such as bioimmune response control and hematopoietic cell control are expected by searching for new molecules that functionally bind to NR12 molecules and specific antibodies that can limit NR12 molecule functions.

  Further, it is assumed that the expression of NR12 is specifically expressed in a limited cell population in these hematopoietic tissues, and anti-NR12 antibody is useful as a means for separating this cell population. The cell population separated in this manner can be applied to cell transplantation therapy. Furthermore, anti-NR12 antibodies are expected to be applied to diagnosis and treatment of diseases such as leukemia.

  On the other hand, a soluble protein containing the extracellular domain of NR12 protein, or NR121, a splice variant of NR12, is expected to be used as an inhibitor of NR12 ligand as a decoy-type receptor, including leukemia involving NR12. It can be expected to be applied to the treatment of diseases.

  The present invention includes proteins functionally equivalent to the NR12 protein. Such proteins include, for example, homologous proteins of other organisms corresponding to human NR12 protein and mutants of human NR12 protein. In the present invention, “functionally equivalent” means that the target protein has biological activity equivalent to that of the NR12 protein. The biological activity is, for example, membrane-bound or soluble hematopoietic factor receptor protein activity.

  As a method well known to those skilled in the art for preparing a protein functionally equivalent to a certain protein, a method of introducing a mutation into the protein is known. For example, those skilled in the art will recognize 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 (1988) Methods Enzymol. 85, 2763-2766), etc. A protein equivalent to can be prepared. Amino acid mutations can also occur in nature. Thus, a protein having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of human NR12 protein and functionally equivalent to human NR12 protein is also included in the protein of the present invention.

  As a protein functionally equivalent to the NR12 protein of the present invention, specifically, 1 or 2 or more, preferably 2 in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10 is preferable. 1 to 2 or more in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10, preferably from 2 to 10, more preferably from 2 to 10 amino acids deleted 2 or more and 30 or less, more preferably 2 or more and 10 or less amino acids added, 1 or 2 or more in the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10, Preferably, 2 or more and 30 or less, more preferably 2 or more and 10 or less amino acids are substituted with other amino acids.

  The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids with amino acids (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino groups with base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all parentheses indicate single letter amino acids).

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

  Examples of the protein in which one or more amino acid residues are added to the amino acid sequence of human NR12 protein (SEQ ID NO: 2, 4, 6, 8, or 10) include fusion proteins containing human NR12 protein. . The fusion protein is a fusion of human NR12 protein and another peptide or protein, and is included in the present invention. A method for producing a fusion protein includes the step of ligating DNA encoding the human NR12 protein of the present invention and DNA encoding another peptide or protein so that the frames coincide with each other, introducing this into an expression vector, and expressing it in a host. Any method known to those skilled in the art can be used. Other peptides or proteins that are subjected to fusion with the protein of the present invention are not particularly limited.

  Other peptides to be subjected to fusion with the protein of the present invention include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210) and 6 His (histidine) residues. 6 × His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck Known peptides such as tag, α-tubulin fragment, B-tag, protein C fragment and the like can be used. Examples of other proteins attached to the fusion of the protein of the present invention include, for example, GST (glutathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP (maltose binding). Protein) and the like.

  A fusion protein can be prepared by fusing commercially available DNA encoding these peptides or proteins with the DNA encoding the protein of the present invention and expressing the prepared fusion DNA.

  Another method well known to those skilled in the art for preparing a protein functionally equivalent to a certain protein is a hybridization technique (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring). A method using Harbor Lab. Press, 1989). That is, those skilled in the art can isolate DNA having high homology with a DNA sequence (SEQ ID NO: 1, 3, 5, 7, or 9) encoding human NR12 protein or a part thereof. A protein functionally equivalent to the human NR12 protein can usually be isolated from the DNA. Thus, a protein encoded by a DNA that hybridizes with a DNA encoding a human NR12 protein or a DNA comprising a part thereof, and a protein functionally equivalent to the human NR12 protein is also included in the protein of the present invention. . Examples of such proteins include homologues of mammals other than humans (for example, proteins encoded by monkey, mouse, rat, rabbit, and bovine genes). When isolating a cDNA highly homologous to the DNA encoding the human NR12 protein from an animal, use a hematopoietic cell line tissue such as spleen, thymus, lymph node, bone marrow, and peripheral leukocyte, and an immunocompetent cell line tissue. Although it is considered preferable, it is not limited to those organs.

  Those skilled in the art can appropriately select hybridization conditions for isolating DNA encoding a protein functionally equivalent to the human NR12 protein. Examples of hybridization conditions include low stringency conditions. Low stringent conditions include, for example, 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, highly stringent conditions are included. Examples of highly stringent conditions include 65 ° C., 2 × SSC, and 0.1% SDS. Under these conditions, not only DNA having high homology as the temperature is lowered but also DNA having low homology can be obtained comprehensively. On the contrary, it can be expected that only DNA having high homology can be obtained as the temperature is increased. However, multiple factors other than temperature, such as salt concentration, can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by selecting these factors as appropriate. It is.

  Further, in place of hybridization, a gene amplification method using a primer synthesized based on sequence information of DNA (SEQ ID NO: 1, 3, 5, 7, or 9) encoding human NR12 protein, for example, polymerase chain reaction It is also possible to isolate the target DNA using the (PCR) method.

  A protein functionally equivalent to the human NR12 protein encoded by the DNA isolated by these hybridization techniques or gene amplification techniques usually has high homology in amino acid sequence with the human NR12 protein. The protein of the present invention includes a protein that is functionally equivalent to the human NR12 protein and has high homology with the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10. High homology generally refers to a homology of 70% or higher, preferably 80% or higher, more preferably 90% or higher, more preferably 95% or higher. To determine protein homology, the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730) may be used.

  The protein of the present invention may vary in amino acid sequence, molecular weight, isoelectric point, presence / absence of sugar chain, form, etc., depending on the cell, host, or purification method that produces the protein described later. However, as long as the obtained protein has a function equivalent to the human NR12 protein of the present invention (SEQ ID NO: 2, 4, 6, 8, or 10), it is included in the present invention. For example, when the protein of the present invention is expressed in prokaryotic cells such as E. coli, a methionine residue is added to the N-terminus of the original protein amino acid sequence. In addition, when expressed in a eukaryotic cell such as a mammalian cell, the N-terminal signal sequence is removed. The protein of the present invention includes such a protein.

  For example, as a result of analyzing the protein of the present invention based on the method described in the literature (Von Heijne, G. Nucleic Acids Research (1986) 14, 4683-4690), the signal sequence is SEQ ID NO: 2, 4, 6, In the amino acid sequences of 8 and 10, it was estimated from the 1st Met to the 23rd Gly. Therefore, the present invention includes a protein consisting of Gly at position 24 to Cys at position 337 in the amino acid sequence shown in SEQ ID NO: 2. Similarly, the amino acid sequence of SEQ ID NO: 4 includes a protein consisting of Gly at position 24 to Ser at position 428. Similarly, in the amino acid sequence set forth in SEQ ID NO: 6, a protein comprising Gly at position 24 to Lys at position 629 is included. Similarly, in the amino acid sequence set forth in SEQ ID NO: 8, a protein comprising Gly at position 24 to Ser at position 428 is included. Similarly, in the amino acid sequence set forth in SEQ ID NO: 10, a protein comprising Gly at position 24 to Lys at position 629 is included.

  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. If it is a recombinant protein, a DNA encoding the protein of the present invention (for example, a DNA having the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9) is incorporated into an appropriate expression vector, and this is appropriately used. After recovering the transformant obtained by introduction into a suitable host cell and obtaining an extract, chromatography such as ion exchange, reverse phase, gel filtration, or affinity chromatography in which an antibody against the protein of the present invention is immobilized on a column It can be purified and prepared by graphy or by further combining a plurality of these columns.

  In addition, when the protein of the present invention was expressed in a host cell (eg, animal cell or E. coli) as a fusion protein with glutathione S-transferase protein or as a recombinant protein to which a plurality of histidines were added, it was expressed. The recombinant protein can be purified using a glutathione column or a nickel column.

  After purification of the fusion protein, the region other than the target protein in the fusion protein can be cleaved and removed with thrombin or factor Xa as necessary.

  In the case of a natural protein, a method well known to those skilled in the art, for example, an affinity column in which an antibody that binds to the NR12 protein described later is allowed to act on a tissue or cell extract expressing the protein of the present invention. Can be isolated by purification. 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 consisting of an amino acid sequence specific for the protein of the present invention consists of an amino acid sequence of at least 7 amino acids, preferably 8 amino acids or more, more preferably 9 amino acids or more. 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 a promoter or inhibitor of the protein of the present invention. It can also be an antagonist to the ligand of the protein of the present invention. Examples of the partial peptide of the protein of the present invention include a partial peptide consisting of an active center of a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, or 10. Moreover, the partial peptide containing one or several area | regions of the hydrophobic region estimated from the hydrophobic plot analysis and a hydrophilic region is mentioned. These partial peptides may contain part or all of one hydrophobic region, or part or all of one hydrophilic region. Further, for example, a soluble protein of the protein of the present invention and a protein comprising an extracellular region are also included in the present invention.

  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 a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used.

  The present invention also provides DNA encoding the protein of the present invention. The DNA of the present invention is used for in vivo and in vitro production of the protein of the present invention as described above, for example, for gene therapy for diseases caused by abnormality of the gene encoding the protein of the present invention. Applications are also possible. 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. Moreover, as long as the protein of the present invention can be encoded, DNA having any base sequence based on the degeneracy of the genetic code is included.

  The DNA of the present invention can be prepared by methods known to those skilled in the art. For example, a cDNA library is prepared from cells expressing the protein of the present invention, and a part of the DNA sequence of the present invention (eg, SEQ ID NO: 1, 3, 5, 7, or 9) is used as a probe. It can be prepared by performing hybridization. The cDNA library may be prepared by the method described in, for example, Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989), or a commercially available DNA library may be used. In addition, RNA is prepared from a cell expressing the protein of the present invention, and an oligo DNA is synthesized based on the sequence of the DNA of the present invention (for example, SEQ ID NO: 1, 3, 5, 7, or 9). It can also be prepared by performing PCR reaction using this as a primer and amplifying cDNA encoding the protein of the present invention.

  Further, by determining the base 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. In addition, genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.

  Specifically, it may be performed as follows. First, mRNA is isolated from cells, tissues, and organs that express the protein of the present invention (eg, hematopoietic cell line tissues such as spleen, thymus, lymph node, bone marrow, peripheral leukocytes, and immune cell line tissues). . Isolation of mRNA is performed by known methods such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC (Chomczynski, P. and Sacchi, N., Anal. Total RNA is prepared by Biochem. (1987) 162, 156-159) etc., and mRNA is purified from the total RNA using mRNA Purification Kit (Pharmacia) etc. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).

  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 (Seikagaku Corporation). In addition, using the primers described in this specification, 5′-Ampli FINDER RACE Kit (manufactured by Clontech) and 5′-RACE method using polymerase chain reaction (PCR) (Frohman, MA et al.) al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) It can be performed.

  A target DNA fragment is prepared from the obtained PCR product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector. The base sequence of the target DNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.

  In addition, in the DNA of the present invention, a base sequence with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucelic Acids Research (1981). ) 9, r43-74). Moreover, the DNA of the present invention can be modified by a commercially available kit or a known method. Examples of modifications include digestion with restriction enzymes, insertion of synthetic oligonucleotides and appropriate DNA fragments, addition of linkers, insertion of start codon (ATG) and / or stop codon (TAA, TGA, or TAG). .

  Specifically, the DNA of the present invention is a DNA consisting of the base A at position 98 to the base C at position 1108 in the base sequence of SEQ ID NO: 1, and the base position A from position 98 to the position 1138 in the base sequence of SEQ ID NO: 3. DNA consisting of the base C of SEQ ID NO: 5, DNA consisting of the base A at position 98 to base G of the position 1984, and the base sequence of SEQ ID NO: 7 from base A at the position 1 to base C at the position 1284 And DNA consisting of base A at position 1 to base G at position 1887 in the base sequence of SEQ ID NO: 9.

  The DNA of the present invention is also a DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9, and is functional with the protein of the present invention. Contains DNA encoding the equivalent protein.

  Stringent conditions can be appropriately selected by those skilled in the art, and examples thereof include low stringent conditions. Low stringent conditions include, for example, 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, highly stringent conditions are included. Examples of highly stringent conditions include 65 ° C., 2 × SSC, and 0.1% SDS. Under these conditions, DNA having high homology can be obtained as the temperature is increased. Said hybridizing DNA may preferably be naturally derived DNA, such as cDNA or chromosomal DNA.

  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 for expressing the protein of the present invention.

  As the vector, for example, when Escherichia coli is used as a host, the vector is amplified in Escherichia coli (for example, JM109, DH5α, HB101, XL1Blue), etc. As well as a selection gene of transformed E. coli (for example, a drug resistance gene that can be discriminated by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like. In addition, for the purpose of subcloning and excision of cDNA, in addition to the above vector, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned. An expression vector is particularly useful when a vector is used for the purpose of producing the protein of the present invention. As an expression vector, for example, for the purpose of expression in E. coli, in addition to the above characteristics that the vector is amplified in E. coli, the host is E. coli such as JM109, DH5α, HB101, XL1-Blue, etc. In some cases, promoters that can be efficiently expressed in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al. , Science (1988) 240, 1041-1043), or the T7 promoter or the like. In addition to the above vectors, such vectors include pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase). BL21 is preferred).

  The vector may contain a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the protein is produced in the periplasm of E. coli. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

  In addition to E. coli, for example, vectors used to produce the protein of the present invention include mammalian-derived expression vectors (for example, pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8], insect cell-derived expression vectors (for example, “Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8), plant-derived expression vectors (for example, pMH1, pMH2], animal virus-derived expression vectors [eg, pHSV, pMV, pAdexLcw], retrovirus-derived expression vectors [eg, pZIpneo], yeast-derived expression vectors [eg, “Pichia Expression Kit” (manufactured by In vitrogen) PNV11, SP-Q01], expression vectors derived from Bacillus subtilis [eg, pPL608, pKTH50], and the like.

  When intended for expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and genes for selecting transformation into cells (for example, More preferably, it has a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

  Furthermore, when the gene is stably expressed and the purpose is to amplify the copy number of the gene in the cell, a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway (for example, , PCHOI, etc.) and amplifying with methotrexate (MTX), and for the purpose of transient expression of the gene, COS having a gene expressing SV40 T antigen on the chromosome An example is a method of transforming with a vector (such as pcD) having an SV40 replication origin using cells. As the replication origin, those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used. Furthermore, for gene copy number amplification in host cell systems, the expression vectors are selectable markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene and 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 introduced into the living body by a retrovirus method, a liposome method, a cationic liposome method, an adenovirus method, or the like. The method of doing is mentioned. Thereby, it is possible to perform gene therapy for a disease caused by the mutation of the NR12 gene of the present invention. Examples of the vector to be used include, but are not limited to, an adenovirus vector (for example, pAdexlcw) and a retrovirus vector (for example, pZIPneo). General genetic manipulations such as insertion of the DNA of the present invention into a vector can be performed according to conventional methods (Molecular Cloning, 5.61-5.63). Administration to 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 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, E. 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 production and expression of the protein of the present invention. Production systems for protein production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems that use eukaryotic cells and production systems that use prokaryotic cells.

  When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as the host. Animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells such as Xenopus eggs Mother cells (Valle, et al., Nature (1981) 291, 358-340) or insect cells such as Sf9, Sf21, Tn5 are known. Examples of CHO cells include dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Proc. Natl. Acad.), Which are CHO cells deficient in the DHFR gene. Sci. USA (1968) 60, 1275) can be preferably used. In animal cells, CHO cells are particularly preferred for the purpose of mass expression. Introduction of a vector into a host cell can be performed by, for example, a calcium phosphate method, a DEAE dextran method, a method using a cationic ribosome DOTAP (Boehringer Mannheim), an electroporation method, a lipofection method, or the like.

  As plant cells, for example, cells derived from Nicotiana tabacum are known as protein production systems, and these may be cultured in callus. As fungal cells, yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, filamentous fungi such as the genus Aspergillus, such as Aspergillus niger, are known. .

  When prokaryotic cells are used, there are production systems using bacterial cells. Examples of bacterial cells include E. coli, for example, JM109, DH5α, HB101, and others, and Bacillus subtilis is known.

  These cells are transformed with the target DNA, and the proteins are obtained by culturing the transformed cells in vitro. The culture can be performed according to a known method. For example, DMEM, MEM, RPMI1640, and IMDM can be used as the culture medium for animal cells. At that time, a serum supplement such as fetal calf serum (FCS) can be used in combination, or serum-free culture may be performed. The pH during culture is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C. for about 15 to 200 hours, and medium exchange, aeration, and agitation are added as necessary.

  On the other hand, examples of the 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 collected in the animals or plants. The “host” in the present invention includes these animals and plants.

  When animals are used, there are production systems using mammals and insects. As mammals, goats, pigs, sheep, mice and cows can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Moreover, when using a mammal, a transgenic animal can be used.

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

  Moreover, as an insect, a silkworm can be used, 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 inserted with a DNA encoding the target protein (Susumu, M. et al., Nature (1985) 315, 592-594).

  Furthermore, when using plants, for example, tobacco can be used. When tobacco is used, a DNA encoding a target protein is inserted into a plant expression vector such as pMON 530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. This bacterium can be infected with tobacco, for example, Nicotiana tabacum, and the desired polypeptide can be obtained from the leaves of this tobacco (Julian K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

  The protein of the present invention thus obtained can be isolated from the inside of the host cell or outside the cell (such as a medium) and purified as a substantially pure and uniform protein. Separation and purification of proteins may be carried out using separation and purification methods used in usual protein purification, and are not limited in any way. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected, When combined, proteins can be separated and purified.

  Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and 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 chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. The present invention also includes highly purified proteins using these purification methods.

  In addition, the protein can be arbitrarily modified or the peptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after the protein is purified. Examples of protein modifying enzymes include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase, and the like.

  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 monoclonal antibodies as well as polyclonal antibodies. Also included are antisera obtained by immunizing animals such as rabbits with the protein of the present invention, all classes of polyclonal and monoclonal antibodies, human antibodies and humanized antibodies 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 such as a human, mouse or 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 in this specification.

  In the present invention, the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of the protein partial peptide include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein. The “antibody” described in the present specification means an antibody that reacts with the full length or fragment of a protein.

  A gene encoding the protein of the present invention or a fragment thereof is inserted into a known expression vector system, the host cell described herein is transformed with the vector, and the target protein or fragment thereof is known from inside or outside the host cell. These may be used as a sensitizing antigen. Alternatively, cells expressing the protein, lysates thereof, or chemically synthesized proteins of the present invention may be used as the sensitizing antigen.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of the compatibility with the parent cell used for cell fusion. Rabbit eyes, primate animals are used.

  Examples of rodent animals include mice, rats, and hamsters. For example, a rabbit is used as the animal of the order of rabbit eyes. For example, monkeys are used as the primate animals. As monkeys, monkeys (old world monkeys) with narrow nose, such as cynomolgus monkeys, rhesus monkeys, baboons, chimpanzees and the like are used.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. As a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, sensitizing antigen is diluted with PBS (Phosphate-Buffered Saline) or physiological saline to an appropriate amount and suspended, and if necessary, a normal adjuvant, for example, Freund's complete adjuvant, is mixed in an appropriate amount. After emulsification, it is administered to mammals. Furthermore, it is preferable to administer a sensitizing antigen mixed with an incomplete Freund's adjuvant several times every 4 to 21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen. Immunization is carried out in this manner, and it is confirmed by a conventional method that the desired antibody level is increased in the serum.

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

  In order to obtain a monoclonal antibody, after confirming that a desired antibody level rises in the serum of a mammal sensitized with the antigen, immune cells are taken out from the mammal and subjected to cell fusion. In this case, spleen cells are particularly preferable as immune 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 fused cells by a drug.

  The cell fusion between the immunocytes and myeloma cells is basically performed by a known method such as the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). It can be done according to this.

  The hybridoma obtained by cell fusion is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT culture solution is carried out for a time sufficient for the cells other than the target hybridoma (non-fusion cells) to die, usually several days or weeks. Subsequently, a normal limiting dilution method is performed to screen and clone hybridomas that produce the target antibody.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized in vitro with proteins, protein-expressing cells, or lysates thereof. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain a hybridoma that produces a desired human antibody having binding activity to the protein (Japanese Patent Laid-Open No. 63-17688). .

  Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purification using an affinity column coupled with a protein. The antibody of the present invention can be used for the purification and detection of the protein of the present invention and can also be a candidate for the 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 administering it to the human body (antibody treatment), a human antibody or a human-type antibody is preferable in order to reduce immunogenicity.

  For example, a transgenic animal having a repertoire of human antibody genes is immunized with an antigen protein, a protein-expressing cell or a lysate thereof to obtain an antibody-producing cell, and a hybridoma obtained by fusing this with a myeloma cell is used for the protein. Human antibodies can be obtained (see International Publication Nos. WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735 and WO96-34096).

  In addition to producing antibodies using hybridomas, cells obtained by immortalizing immune cells such as sensitized lymphocytes that produce antibodies with an oncogene may be used.

  The monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using genetic recombination technology (for example, Borrebaeck, CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Recombinant antibodies are produced by cloning DNA encoding them from immune cells such as hybridomas or sensitized lymphocytes producing antibodies, incorporating the DNA into an appropriate vector, and introducing it into a host. The present invention encompasses this recombinant antibody.

  Furthermore, the antibody of the present invention may be an antibody fragment or a modified antibody thereof as long as it binds to the protein of the present invention. For example, antibody fragments include Fab, F (ab ′) 2, Fv, or single chain Fv (scFv) in which Hv and L chain Fv are linked by an appropriate linker (Huston, JS et al., Proc. Natl). Acad. Sci. USA (1988) 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 introduced into an expression vector, and then an appropriate host cell. (Eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

  Antibodies modified with various molecules such as polyethylene glycol (PEG) can also be used as modified antibodies. The “antibody” of the present invention includes these modified antibodies. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.

  In addition, the antibody of the present invention is derived from a chimeric antibody comprising 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 the FR (framework region) and constant region.

  The antibody obtained as described above can be purified to homogeneity. Separation and purification of antibodies used in the present invention may be carried out using separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but is not limited thereto. The antibody concentration obtained above can be measured by measuring absorbance, enzyme-linked immunosorbent assay (ELISA), or the like.

  Examples of columns used for affinity chromatography include a protein A column and a protein G column. For example, Hyper D, POROS, Sepharose F. F. (Pharmacia) etc. are mentioned as a column using a protein A column.

  Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and 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 chromatography 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 absorbance measurement, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme immunoassay), and RIA (radioimmunoassay). Measurement method) or 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 is immobilized, and then a sample containing the target antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added. A secondary antibody that recognizes an antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated, washed, and then an enzyme substrate such as p-nitrophenyl phosphate is added to measure the absorbance. Binding activity can be evaluated. A protein fragment such as a fragment consisting of the C terminus or a fragment consisting of the N terminus may be used as the protein. BIAcore (manufactured by Pharmacia) can be used for evaluating 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 contained in the sample, and an immune complex between the antibody and the protein is detected or measured. The method for detecting or measuring the protein of the present invention can be implemented.

  Since the protein detection or measurement method of the present invention can specifically detect or measure the protein, it is useful for various experiments using the protein.

  The present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to DNA encoding human NR12 protein (SEQ ID NO: 1, 3, 5, 7, or 9) or its complementary strand.

  Here, “complementary strand” refers to the other strand of one strand of a double-stranded polynucleotide comprising A: T, G: C base pairs. Further, the term “complementary” is not limited to a case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is just to have the homology on the above base sequences. The algorithm described in the present specification may be used as an algorithm for determining homology.

  Such polynucleotides include probes and primers used for detection and amplification of DNA encoding the protein of the present invention, nucleotides or nucleotide derivatives for suppressing the expression of the protein of the present invention (for example, antisense oligonucleotides and ribozymes). Etc.). Moreover, such a polynucleotide can also be used for production of a DNA chip.

  Antisense oligonucleotides include, for example, antisense oligonucleotides that hybridize to any position in the base sequence of SEQ ID NO: 1, 3, 5, 7, or 9. This antisense oligonucleotide is preferably an antisense oligonucleotide against at least 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, or 9. More preferably, it is an antisense oligonucleotide in which at least 15 or more consecutive nucleotides contain a translation initiation codon.

  As antisense oligonucleotides, derivatives or modifications thereof can be used. Examples of the modified body include a lower alkyl phosphonate modified body such as a methylphosphonate type or an ethyl phosphonate type, a phosphorothioate modified body, or a phosphoramidate modified body.

  In the antisense oligonucleotide, not only nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are all complementary sequences, but also DNA or mRNA and oligonucleotide are represented by SEQ ID NOs: 1, 3, 5, 7 As long as it can specifically hybridize to the base sequence shown in 9 or 9, there may be one or more nucleotide mismatches.

  The antisense oligonucleotide derivative of the present invention acts on the cells producing the protein of the present invention and binds to DNA or mRNA encoding the protein, thereby inhibiting transcription or translation, or promoting mRNA degradation. Thus, by suppressing the expression of the protein of the present invention, the effect of suppressing the action of the protein of the present invention is obtained.

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

  If necessary, excipients, isotonic agents, solubilizers, stabilizers, preservatives, soothing agents, etc., and tablets, powders, granules, capsules, liposome capsules, injections Furthermore, it can be made into a freeze-drying agent, such as a liquid preparation and a nasal drop. These can be prepared according to conventional methods.

  The antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or applied to the patient so that it can reach the affected area as a result, for example, by intravascular administration. Furthermore, an antisense encapsulating material that enhances durability and membrane permeability can also be used. For example, liposome, 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 patient's condition, 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. Moreover, 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 compounds that bind 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 test sample is detected, and the protein binds to the protein of the present invention. And a method for screening a compound that binds 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. Moreover, the form expressed on the cell surface or the form as a membrane fraction may be sufficient. The test sample is not particularly limited. For example, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, purified or crude protein, peptide, non-peptidic compound, synthetic low Examples include molecular compounds and natural compounds. The protein of the present invention to be contacted with the test sample is, for example, a purified protein, a soluble protein, a form bound to a carrier, a fusion protein with other proteins, a form expressed on a cell membrane Moreover, it can be made to contact a test sample as a membrane fraction.

  For example, many methods known to those skilled in the art can be used as a method of screening for proteins (ligands, etc.) that bind to the protein of the present invention. Such screening can be performed, for example, by immunoprecipitation. Specifically, it can be performed as follows. A gene encoding the protein of the present invention is inserted into a vector for expressing a foreign gene such as pSV2neo, pcDNA I, or pCD8 to express the gene in animal cells or the like. The promoter used for expression is SV40 early promoter (Rigby In Williamson (ed.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)), EF-1 α promoter (Kim et al. Gene 91 , p.217-223 (1990)), CAG promoter (Niwa et al. Gene 108, p.193-200 (1991)), RSV LTR promoter (Cullen Methods in Enzymology 152, p.684-704 (1987), SR α promoter (Takebe et al. Mol. Cell. Biol. 8, p.466 (1988)), CMV immediate early promoter (Seed and Aruffo Proc. Natl. Acad. Sci. USA 84, p.3365-3369 (1987 )), SV40 late promoter (Gheysen and Fiers J. Mol. Appl. Genet. 1, p. 385-394 (1982)), Adenovirus late promoter (Kaufman et al. Mol. Cell. Biol. 9, p. 946 ( 1989)), HSV TK promoter, and any other promoter that can be generally used may be used.

  In order to express foreign genes by introducing genes into animal cells, electroporation (Chu, G. et al. Nucl. Acid Res. 15, 1311-1326 (1987)), calcium phosphate method (Chen, C and Okayama, H. Mol. Cell. Biol. 7, 2745-2752 (1987)), DEAE dextran method (Lopata, MA et al. Nucl. Acids Res. 12, 5707-5717 (1984); Sussman, DJ and Milman, G. Mol.Cell. Biol. 4, 1642-1643 (1985)), Lipofectin method (Derijard, B. Cell 7, 1025-1037 (1994); Lamb, BT et al. Nature Genetics 5, 22-30 (1993); Rabindran, SK et al. Science 259, 230-234 (1993)), etc., but any method may be used. By introducing a recognition site (epitope) of a monoclonal antibody whose specificity is known to the N-terminus or C-terminus of the protein of the present invention, the protein of the present invention is expressed as a fusion protein having the recognition site of the monoclonal antibody. be able to. A commercially available epitope-antibody system can be used (Experimental Medicine 13, 85-90 (1995)). A vector capable of expressing a fusion protein with β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP) and the like via a multicloning site is commercially available.

  In order to avoid changing the properties of the protein of the present invention as much as possible by using a fusion protein, a method for preparing a fusion protein by introducing only a small epitope consisting of several to a dozen amino acids has also been reported. Yes. For example, polyhistidine (His-tag), influenza agglutinin HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV) -tag), E-tag (epitope on monoclonal phage) and other monoclonal antibodies that recognize them can be used as an epitope-antibody system for screening proteins that bind to the protein of the present invention (Experimental Medicine 13). , 85-90 (1995)).

  In immunoprecipitation, an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant. This immune complex consists of the protein of the present invention, a protein capable of binding to the protein, and an antibody. In addition to using an antibody against the above epitope, immunoprecipitation can also be performed using an antibody against the protein of the present invention. The antibody against the protein of the present invention can be expressed, for example, by introducing a gene encoding the protein of the present invention into an appropriate E. coli expression vector and expressing it in E. coli, and purifying the expressed protein. It can be prepared by immunizing goats, chickens and the like. It can also be prepared by immunizing the above animal with the synthesized partial peptide of the protein of the present invention.

  For example, when the antibody is a mouse IgG antibody, the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose. In addition, when the protein of the present invention is prepared, for example, as a fusion protein with an epitope such as GST, a substance that specifically binds to these epitopes such as glutathione-Sepharose 4B is used to produce the protein of the present invention. Similar to the case of using an antibody, an immune complex can be formed.

  For general methods of immunoprecipitation, for example, according to the method described in the literature (Harlow, E. and Lane, D .: Antibodies, pp.511-552, Cold Spring Harbor Laboratory publications, New York (1988)), or It may be done according to this.

SDS-PAGE is generally used for the analysis of immunoprecipitated proteins, and by using a gel with an appropriate concentration, it is possible to analyze the proteins bound by the molecular weight of the protein. At this time, in general, the protein bound to the protein of the present invention is difficult to detect by ordinary protein staining methods such as Coomassie staining and silver staining. Therefore, ³⁵ S-methionine, which is a radioisotope, is used. Detection sensitivity can be improved by culturing cells in a culture solution containing ³⁵ S-cysteine, labeling the protein in the cells, and detecting the protein. Once the molecular weight of the protein is known, the protein of interest can be purified directly from an SDS-polyacrylamide gel and its sequence determined.

  In addition, isolation of a protein that binds to the protein of the present invention can be performed using, for example, the West Western blotting method (Skolnik, EY et al., Cell (1991) 65, 83-90). it can. That is, a cDNA library using a phage vector (λgt11, ZAP, etc.) was prepared from cells, tissues, and organs that are expected to express a binding protein that binds to the protein of the present invention, and this was prepared as LB-agarose. The protein expressed above and immobilized on the filter is immobilized, purified and labeled with the protein of the present invention, and the filter is reacted, and the plaque expressing the protein bound to the protein of the present invention is detected with the label. . Examples of the method for labeling the protein of the present invention include a method using the binding property of biotin and avidin, an antibody that specifically binds to the protein of the present invention or a peptide or polypeptide (eg, GST) fused to the protein of the present invention. , A method using a radioisotope, a method using fluorescence, and the like.

  As another embodiment of the screening method of the present invention, a two-hybrid system using cells (Fields, S., and Sternglanz, R., Trends. Genet. (1994) 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.Cell 68, 597-612, MATCHMAKER Two-Hybrid System, Mammalian MATCHMAKER Two-Hybrid Assay Kit ”,“ MATCHMAKER One-Hybrid System ”(all manufactured by Clontech) and“ HybriZAP Two-Hybrid Vector System ”(manufactured by Stratagene)). In the 2-hybrid system, it is expected that the protein of the present invention is fused with the SRF DNA binding region or the GAL4 DNA binding region and expressed in yeast cells, and the protein that binds to the protein of the present invention is expressed. A cDNA library that can be expressed in a form that fuses with the VP16 or GAL4 transcriptional activation region, introduces it into the yeast cell, and isolates the cDNA from the detected positive clone. (When a protein that binds to the protein of the present invention is expressed in yeast cells, 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, a protein encoded by the cDNA can be obtained. This makes it possible to prepare a protein that binds to the protein of the present invention or a gene thereof. Examples of the reporter gene used in the 2-hybrid system include, but are not limited to, the HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor type 1) gene, and the like. Not.

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

  By analyzing the amino acid sequence of the obtained protein, synthesizing oligo DNA based on the amino acid sequence, and screening the cDNA library using the DNA as a probe, DNA encoding the protein can be obtained.

  In the present invention, a biosensor using the surface plasmon resonance phenomenon can also be used as a means for detecting or measuring a bound compound. The biosensor using the surface plasmon resonance phenomenon can observe the interaction between the protein of the present invention and the test compound in real time as a surface plasmon resonance signal without using a trace amount of protein and labeling. There are (eg BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the protein of the present invention and the test compound by using a biosensor such as BIAcore.

  Further, 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, synthetic compounds, natural product banks, or random A method of screening a molecule that binds to the protein of the present invention by allowing a phage peptide display library to act, or a screening method using high throughput by combinatorial chemistry technology (Wrighton NC; Farrell FX; Chang R; Kashyap AK; Barbone FP; Mulcahy LS; Johnson DL; Barrett RW; Jolliffe LK; Dower WJ., Small peptides as potent mimetics of the protein hormone erythropoietin, Science (UNITED STATES) Jul 26 1996, 273 p458-64, Verdine GL., The combinatorial chemistry of nature Nature (ENGLAND) Nov 7 1996, 384 p11-13, Hogan JC Jr., Directed combinatorial chemistry. Nature (ENGLAND) Nov 7 1996, 384 p17-9) It is known to those skilled in the art.

  In addition, screening for a ligand that binds to the protein of the present invention was carried out by linking the extracellular domain of the protein of the present invention and the intracellular domain containing the transmembrane domain of the hemopoietin receptor protein having a known signal transduction ability. After the body is expressed on the cell surface of a suitable cell line, preferably a cell line that can only survive and grow in the presence of a suitable growth factor (growth factor-dependent cell line). It can be carried out by adding and culturing a material expected to contain factors, cytokines, hematopoietic factors and the like. This method utilizes the fact that the growth factor-dependent cell line can survive and grow only when a ligand that specifically binds to the extracellular domain of the protein of the present invention is present in the test material. Yes. Known hemopoietin receptors include, for example, thrombopoietin receptor, erythropoietin receptor, G-CSF receptor, gp130, etc. The partner of the chimeric receptor used in the screening system of the present invention is the known hemopoietin receptor. It is not limited to the body, and anything may be used as long as it has a structure necessary for signal transduction activity in the cytoplasmic domain. As the growth factor-dependent cell line, for example, IL3-dependent cell lines such as BaF3 and FDC-P1 can be used.

  The ligand that specifically binds to the protein of the present invention may be a rare but soluble protein and a cell membrane-bound protein. In such a case, it is expected that a 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. It is possible to screen by measuring the binding to the cells to be screened. Examples of the protein containing only the extracellular domain of the protein of the present invention include a soluble receptor protein artificially prepared by inserting a stop codon at the N-terminal side of the transmembrane domain, or a soluble receptor protein such as NR12-1 Type proteins are available. On the other hand, as a fusion protein in which a partial sequence of another soluble protein is added to the extracellular domain of the protein of the present invention, for example, an Fc site of immunoglobulin or a FLAG peptide is added to the C-terminus of the extracellular domain. Protein is available. These soluble labeled proteins can also be used for detection in the above-described 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, human IgG antibody) can be purified using a protein A column or the like. Since such antibody-like chimeric protein has ligand binding activity, it can be appropriately labeled with a radioisotope or the like and then used for screening for a ligand (Suda, T. et al., Cell, 175, 1169-1178 (1993)). In addition, since some cytokines such as TNF family molecules exist in membrane-bound form, ligands are isolated from cells that show binding activity by reacting various cells with antibody-like chimeric proteins. There is also a possibility of being able to do. Similarly, a ligand can be isolated using a cell into which a cDNA library has been introduced. Furthermore, antibody-like chimeric proteins can be used as antagonists.

  Compounds that can be isolated by screening become candidates for drugs that promote or inhibit the activity of the protein of the present invention, and can be applied to the treatment of diseases caused by abnormal expression or abnormal function of the protein of the present invention. . A substance obtained by using the screening method of the present invention, wherein a part of the structure of the compound having the activity of binding to the protein of the present invention is converted by addition, deletion and / or substitution is also used for the screening method of the present invention. It is contained in the compound obtained by using.

  The compound obtained by using the screening method of the present invention and the protein of the present invention (decoy type (soluble type)) are used in humans and animals such as mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cows, When used as a medicine for monkeys, baboons, and chimpanzees, the isolated compound itself can be directly administered to a patient, and can also be prepared by a known pharmaceutical method. For example, tablets, capsules, elixirs, microcapsules as required, or aseptic solutions or suspensions with water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to prepare a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

  Additives that can be mixed into tablets and capsules include, for example, binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry are used. When the dispensing unit form is a capsule, the above material can further contain a liquid carrier such as fats and oils. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

  Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.

  Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  For example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, percutaneous, or oral administration to patients are performed by methods known to those skilled in the art. sell. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. In addition, if the compound can be encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

  For example, the dose of the protein of the present invention (decoy type (soluble type)) varies depending on the administration subject, target organ, symptom, and administration method. (As 60 kg) is considered to be about 100 μg to 10-20 mg per day.

  For example, the dose of the compound that binds to the protein of the present invention or the compound that inhibits the activity of the protein of the present invention varies depending on the symptom, but in the case of oral administration, generally in adults (weight 60 kg), About 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

  When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, and administration method. For example, in the form of an injection, it is usually about an adult (with a body weight of 60 kg) per day. 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 by intravenous injection. In the case of other animals, an amount converted per 60 kg body weight or an amount converted per body surface area can be administered.

EXAMPLES Next, although an Example demonstrates this invention further more concretely, this invention is not limited to the following Example.
[Example 1] Isolation of NR12 gene (1) Primary screening by TblastN search Even though the human genome analysis project of each institution has energetically promoted the decoding of the human genome gene sequence, the analysis has already been completed. The current situation is that even less than 10% of all human genome sequences are present. However, when searching for a target gene, determining a base sequence, or mapping a gene, use of the information provided so far by the above project is one of effective means. The information base of the above sequence is the future complete database while forming an enormous amount of information by aligning bacterial artificial chromosome (BAC) clones and yeast artificial chromosome (YAC) clones. Aiming to be made. The present inventors identified a human genome gene encoding a part of a novel hemopoietin receptor protein in the BAC clone sequence in GenBank's “High Throughput Genomic Sequence (htgs)” database, which is one of public databases. did.

As described above, the present inventors used (Tyr / His) -Xaa- (Hydrophobic / Ala)-(Gln / Arg) -Hydrophobic-Arg as a motif sequence conserved in the hemopoietin receptor family. We found both the motif (YR motif) and the Trp-Ser-Xaa-Trp-Ser motif (WS motif) located near the C-terminal. However, it has been extremely difficult to design an oligonucleotide probe sequence that comprehensively contains both of these motif sequences. Therefore, a database search on a computer was performed using a partial amino acid sequence obtained by fragmenting a known hemopoietin receptor so as to include both sequences as a query. Each human receptor shown in Table 1 was used as a known hemopoietin receptor sequence, and fragmentation of a partial amino acid sequence that could be used as a query for each was examined. Here, on the genome structure of the known hemopoietin receptor, these YR motifs and exons encoding WS motifs (WS exons) are approximately 50 to 70 amino acids, and are further adjacent to the N-terminal side of this exon. Focusing on the fact that exons (PP exons) are also about 50 to 70 amino acids in the same way, about 120 amino acids were cut out so as to include both exons for convenience and used as a query sequence. Therefore, the length of the partial amino acid sequence used as the query sequence varies depending on each known hemopoietin receptor, but in all query sequences, one or more Pro residues located near the starting point of PP exon. Thus, after the WS motif on the WS exon is terminated, the structural features are conserved by cutting out about 10 amino acids to the C-terminal side.
The table lists known hemopoietin receptors used as query formulas for database searches. Amino acid residues conserved in the motif sequence are shown in bold underlined.

Using the above sequence as a query formula, a search using the TblastN (Advanced TblastN 2.0.9) program was performed on the htgs database of GenBank. The search parameters were Expect value = 100, Descriptions value = 250, Alignments value = 250, and the filters were default values. As a result of the search, a large number of false positive clones were hit. Here, those in which the YR motif and WS motif were not encoded in the same reading frame, or those having a stop codon between both motif sequences were excluded. In addition, clones that had the YR motif but did not preserve the WS motif were also excluded from the search. This is due to the support of the conservation of the WS motif over the YR motif which is not a fully established consensus sequence as described above. As a result of the above selection, the primary search positive clones shown in Table 2 were narrowed down from about 1000 false positive clones that were positive by the above TblastN search.
In the table, of the positive clones obtained from the results of the primary search against the htgs database, positive clones possessing the target motif sequence with high probability were selected and listed. In the motif sequence, conserved amino acid residues are indicated by bold lines with underlining.

(2) Secondary screening by BlastX search As a means of subsequent secondary search, first, each of the 28 TblastN primary search positive clones listed in Table 2 was positive for the primary search query sequence. The base sequence around the position was cut out. Using this extracted sequence as a query formula for further secondary search, a BlastX (Advanced BlastX 2.0.9) search was performed on the GenBank nr database. The interrogation sequence was prepared here for the convenience of a base sequence of 240 bp in total including about 200 bp upstream from the sequence capable of encoding the WS motif. As described above, since the exon encoding this WS motif is relatively small, about 50 to 70 amino acids, in the genomic structure of the known hemopoietin receptor, the 240 bp interrogation sequence prepared here is sufficient. This is due to the prediction that the exon part can be covered. BlastX search parameters were Expect value = 100, Descriptions value = 100, Alignments value = 100, and the filters were default values. By this search, at least a plurality of different known hemopoietin receptors among positive search positive clones and positive clones that can show homology are selected as secondary search positive clones that can encode the receptor family members. I expected.

  Through the above two-step Blast search, among the human genome clones shown in Table 2, AC008048, AC007174, and AL109843 clones were successfully identified as secondary search positive clones. However, AC008048 and AC007174 were found to be genomic sequences encoding human IL-2 receptor beta chain and human IL-5 receptor, respectively. On the other hand, since it was speculated that the remaining AL109843 could only encode the targeted novel hemopoietin receptor, this clone was named NR12 and decided to aim at isolation of the full-length cDNA.

  AL109843 is a genomic draft sequence derived from human chromosome 1 which was just registered in the htgs database on August 16, 1999, and has a total length of 149104 bp. However, at present, the base sequence of about 8000 bp is undecided in the middle of 10 places. In the AL109843 sequence, the peripheral sequence that was positive by the TblastN primary search was able to predict the presence of the WS exon, as shown in FIG. In this sequence, the [YVFQVR] sequence was recognized as the YR motif, and the [WQPWS] sequence was recognized as the WS motif. Furthermore, FIG. 2 shows an amino acid sequence comparison with a known hemopoietin receptor whose homology was detected in a BlastX secondary search. Based on the above results, specific oligonucleotide primers were designed on the exon sequence that could be predicted within the AL109843 sequence, and these primers were provided to the 5′-RASE method and 3′-RACE method described below. .

(3) Design of oligonucleotide primer As described above, an exon site was predicted in the AL109843 sequence, and an NR12-specific oligonucleotide primer shown in the following sequence was designed based on the predicted sequence. There are three primers, NR12-S1, NR12-S2, and NR12-S3 on the sense side (downstream direction), and NR12-A1, NR12-A2, and NR12-A3 on the antisense side (upstream direction). Each book was synthesized. Primer synthesis was performed using ABI's 394 DNA / RNA Synthesizer under conditions for adding a 5'-terminal trityl group. Thereafter, the full-length synthesized product was purified by OPC column (ABI # 400771).

NR12-S1; 5'- GCA ACA GTC AGA ATT CTA CTT GGA GCC -3 '(SEQ ID NO: 11)
NR12-S2; 5'-CAT TAA GTA CGT ATT TCA AGT GAG ATG TC -3 '(SEQ ID NO: 12)
NR12-S3; 5'- GGT ACT GGC AGC CTT GGA GTT CAC TG -3 '(SEQ ID NO: 13)
NR12-A1; 5′-CAG TGA ACT CCA AGG CTG CCA GTA CC -3 ′ (SEQ ID NO: 14)
NR12-A2; 5'-GAC ATC TCA CTT GAA ATA CGT ACT TAA TG -3 '(SEQ ID NO: 15)
NR12-A3; 5'- GGC TCC AAG TAG AAT TCT GAC TGT TGC -3 '(SEQ ID NO: 16)

  In designing the oligonucleotide primer sequences shown above, NR12-S1 and NR12-A3, NR12-S2 and NR12-A2, and NR12-S3 and NR12-A1 each have a completely complementary sequence. is doing.

(4) Cloning of N-terminal cDNA by 5'-RACE Method In order to isolate the N-terminal sequence of a cDNA clone corresponding to full-length NR12, the NR12-A1 primer of (3) above is used for primary PCR, 5'-RACE PCR was attempted using NR12-A2 primer for secondary PCR. Human Fetal Liver Marathon-Ready cDNA Library (Clontech # 7403-1) was used as a template, and Advantage cDNA Polymerase Mix (Clontech # 8417-1) was used for PCR experiments. As a result of using the Perkin Elmer Gene Amp PCR System 2400 thermal cycler under the following PCR conditions, PCR products having two sizes shown in FIG. 3 were obtained.
The conditions for primary PCR are 94 ° C for 4 minutes, 5 cycles of "94 ° C for 20 seconds, 72 ° C for 90 seconds", 5 cycles of "94 ° C for 20 seconds, 70 ° C for 90 seconds", "at 94 ° C “20 seconds, 68 ° C. for 90 seconds” ends at 28 cycles, 72 ° C. for 3 minutes, and 4 ° C.
The conditions for secondary PCR are 94 ° C for 4 minutes, 5 cycles of "94 ° C for 20 seconds, 70 ° C for 90 seconds", "94 ° C for 20 seconds, 68 ° C for 90 seconds", 25 cycles, at 72 ° C. Terminate at 3 minutes and 4 ° C.

  Both of the obtained two kinds of PCR products were subcloned into pGEM-T Easy vector (Promega # A1360), and the nucleotide sequences were determined. Recombination of the PCR product into pGEM-T Easy vector was carried out with T4 DNA Ligase (Promega # A1360) at 4 ° C / 12 hours. A gene recombinant of the PCR product and pGEM-T Easy vector was obtained by transforming E. coli strain DH5α (Toyobo # DNA-903). In addition, Insert Check Ready Blue (Toyobo # PIK-201) was used for selection of gene recombinants. Furthermore, the base sequence was determined using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI / Perkin Elmer # 4303154) and analyzed by ABI PRISM 377 DNA Sequencer. As a result of determining the base sequences of all insert fragments for 10 independent clones of the recombinants, the same sequence group of 4 clones showing an insert size of 1.3 kb due to the length of base pairs and the difference in sequence, It was possible to distinguish between 6 clones of identical sequence group showing an insert size of 1.0 kb. However, the former 1.3 kb 5′-RACE PCR product was found to be a non-specific PCR amplification product. This sequence is derived from the minor band of the 5′-RACE PCR product of FIG. On the other hand, the latter 1.0 kb 5′-RACE PCR product was confirmed to be a partial base sequence of NR12 derived from the correct PCR amplification reaction.

(5) Cloning of C-terminal cDNA by 3'-RACE method In order to isolate the C-terminal sequence of a cDNA clone corresponding to full-length NR12, the NR12-S1 primer of (3) above was used for primary PCR, 3'-RACE PCR was attempted using NR12-S2 primer for secondary PCR. Except for using Human Thymus Marathon-Ready cDNA Library (Clontech # 7415-1) as a template, PCR was carried out under exactly the same PCR conditions as the 5′-RACE method in the previous section. That is, the Perkin Elmer Gene Amp PCR System 2400 thermal cycler was used for PCR experiments using Advantage cDNA Polymerase Mix. As a result of carrying out under the same PCR conditions as in (4) above, a 750 bp 3′-RACE amplification product having a single size was obtained as shown in FIG. The obtained PCR product was subcloned into the pGEM-T Easy vector as described above, and the nucleotide sequence was determined. Recombination of the PCR product into pGEM-T Easy vector was carried out with T4 DNA Ligase for 4 ° C / 12 hours. PCR products and vector genetic recombinants were obtained by transforming E. coli strain DH5α. Further, Insert Check Ready Blue was used for selection of the gene recombinants as described above. In the determination of the base sequence, the BigDye Terminator Cycle Sequencing Ready Reaction Kit was used and the analysis was carried out by ABI PRISM 377 DNA Sequencer. As a result of determining the base sequences of all insert fragments of the two independent clones of the gene recombinants, it was confirmed that the C-terminal sequence of the full-length NR12 cDNA clone having the PolyA sequence was included.

  By combining the nucleotide sequence determined as a result of this 3′RACE-PCR with the nucleotide sequence of the 5′RACE-PCR product determined in the above (4), the cell-soluble soluble receptor protein is finally obtained. The entire nucleotide sequence of a cDNA clone capable of encoding the DNA was determined, and this cDNA clone was named NR12.1. FIG. 4 shows the determined nucleotide sequence of NR12.1 cDNA (SEQ ID NO: 1) and the amino acid sequence encoded by it (SEQ ID NO: 2).

(6) Cloning of C-terminal splicing mutant by 3'-RACE method The NR12.1 clone, which has been isolated and structurally analyzed, satisfies the characteristics of the known hemopoietin receptor but possesses a transmembrane region. Not done. Therefore, it was presumed to encode a soluble receptor protein as described above. Therefore, the present inventors predicted the presence of a splicing variant possessing a transmembrane region, particularly in the C-terminal region of the transcript of the gene, and continued to use the 3′-RACE method to further isolate a single NR12 cDNA clone. I tried to leave.

  Therefore, 3'-RACE PCR was attempted using the NR12-S2 primer of (3) above for primary PCR and the NR12-S3 primer for secondary PCR. Except for using the Human Testis Marathon-Ready cDNA Library (Clontech # 7414-1) as a template, the PCR was carried out under the same PCR conditions as the 5′-RACE method in (4) above. As a result, 3'-RACE amplification products having different sizes were obtained. All of the obtained PCR products were subcloned into the pGEM-T Easy vector as described above, and the nucleotide sequence was determined. As a result of determining the base sequence of all insert fragments of 6 independent clones, one of them must be the same clone as NR12.1, whose base sequence was determined by the previous section. There was found. The remaining 5 clones could all encode cell membrane proteins possessing the targeted transmembrane region. Therefore, as expected, the present inventors confirmed the presence of a splicing variant of NR12. The five cDNA clones further show differences due to alternative splicing in the intracellular region on the C-terminal side. That is, 2 clones possessed only a short intracellular region, which was named NR12.2, while 3 clones encoded a long intracellular region. This long ORF-encoding cDNA clone was identified as NR12.3 to distinguish the sequences.

  As a result of this 3′RACE-PCR, the base sequence that has been determined and the base sequence of the 5′RACE-PCR product determined in (4) above are finally combined to encode the transmembrane receptor protein. The complete nucleotide sequence of possible cDNA clones was determined. The determined nucleotide sequence of NR12.2 cDNA (SEQ ID NO: 3) and the amino acid sequence encoded by it (SEQ ID NO: 4) are shown in FIG. 5, and further the nucleotide sequence of NR12.3 cDNA (SEQ ID NO: 5), The amino acid sequence encoded by it (SEQ ID NO: 6) is shown in FIG. 6 and FIG.

  In addition, the exon site sequence prediction in (2) above does not use a program such as genomic gene analysis software, but rather a splicing consensus sequence for RNA transcription (Hames, BD and Glover, DM, Transcription and Splicing (Oxford, IRL Press ), 1988, p131-206). As a result of determining the entire nucleotide sequence of the isolated cDNA clone, the exon site predicted in the partial sequence of AL109843 shown in FIG. 1 completely matched the exon site used for actual NR12 gene transcription. However, only the NR12.1 cDNA clone was found to be a transcript that reads through the same sequence as the genome structure and enters the 3 ′ untranslated region without splicing after the completion of WS exon.

(7) Structural features and functional prediction of NR12
As a result of determining the entire nucleotide sequences of NR12.1, NR12.2, and NR12.3, it was revealed by selective splicing that these were transcripts showing structural diversity on the C-terminal side. NR12.1 can encode a soluble secreted hemopoietin receptor protein consisting of 337 amino acids, while NR12.2 and NR12.3 have a transmembrane type consisting of 428 amino acids and 629 amino acids, respectively. It could encode hemopoietin receptor protein. The following structures are recognized as features of these NR12.

  First, in the extracellular region common to these clones, a typical secretory signal sequence is predicted from Met at amino acid number 1 to Gly at position 23. Here, since there is an in-frame stop codon at position minus 32 from Met at position 1, this Met residue is presumed to be the translation initiation site. Next, an Ig-like region is constructed from Gly at 24th position to Pro at 124th position. It is presumed that the Cys residue at position 133 and the Cys residue at position 144 form one of the loop structures, which is the ligand binding site. Furthermore, from Tyr at position 290 to the Arg residue at position 295 is a highly conserved YR motif, and a typical WS motif is observed from Trp at position 304 to the Ser residue at position 308. .

  Here, NR12.1 encodes 29 amino acids after the WS motif sequence, and the translation frame is terminated by the next stop codon. This encodes a soluble hemopoietin receptor protein that does not retain the transmembrane domain. On the other hand, in NR12.2 and NR12.3, following each of the above conserved motifs, a typical transmembrane domain is observed in 26 amino acids from Gly at position 352 to Asn residue at position 377. In addition, up to the Gln residue at position 413 in the intracellular region, NR12.2 and NR12.3 encode exactly the same amino acid sequence, but they are different exons due to alternative splicing in the subsequent C-terminal region. Due to the connection, the difference in structure occurs. That is, NR12.2 encodes 428 amino acids and has a short intracellular region of 51 amino acids because the translation frame is terminated by the next stop codon. On the other hand, NR12.3 encoded 629 amino acids and possessed an intracellular region of 252 amino acids. From the above structural features, it was confirmed that the NR12 gene satisfactorily satisfied the features of the novel hemopoietin receptor protein.

[Example 2] Search of NR12 gene expression tissue by RT-PCR method and analysis of expression mode In order to analyze the expression distribution and gene expression mode of NR12.1 gene in each human organ, mRNA by RT-PCR method Was detected. An NR12-PPD primer having the following sequence was newly synthesized as a sense-side (downstream) primer for use in RT-PCR analysis. As the antisense side (upstream direction) primer, the NR12-A1 primer synthesized in Example 1 (3) was used. The synthesis and purification of NR12-PPD primer were in accordance with Example 1 (3). The primer set of [NR12-PPD vs. NR12-A1] is expected to amplify and detect an N-terminal region common to all splicing variants of NR12.1, NR12.2, and NR12.3.
hNR12-PPD; 5'- CCG CCA GAT ATT CCT GAT GAA GTA ACC -3 '(SEQ ID NO: 17)

  Templates include Human Multiple Tissue cDNA (MTC) Panel I (Clontech # K1420-1), Human MTC Panel II (Clontech # K1421-1), Human Immune System MTC Panel (Clontech # K1426-1), and Human Fetal MTC Panel (Clontech # K1425-1) was used. For PCR, Advantage cDNA Polymerase Mix (Clontech # 8417-1) was used, and a Perkin Elmer Gene Amp PCR System 2400 thermal cycler was used. PCR reaction is 4 minutes at 94 ° C, 5 cycles of "94 ° C for 20 seconds, 72 ° C for 1 minute", 5 cycles of "94 ° C for 20 seconds, 70 ° C for 1 minute", "94 ° C for 20 seconds , 1 minute at 68 ° C. ”was performed under the cycle conditions of 25 cycles, termination at 72 ° C. for 3 minutes, and 4 ° C. to attempt amplification of the target gene.

  As a result, as shown in FIG. 8, strong gene expression of NR12 was recognized in hematopoietic cell line tissues such as adult spleen, thymus, lymph node, bone marrow, peripheral leukocytes, and immune cell line tissues, and further, testis, liver, lung Expression was also detected in the digestive tract of the kidney, pancreas, small intestine and colon. Moreover, the gene expression was recognized also in mRNA derived from all human fetal organs analyzed. Here, for all the templates used in the analysis, the copy number of the template mRNA was normalized between samples in advance by detecting the expression of the housekeeping gene G3PDH under the above PCR conditions using human G3PDH primers. Make sure that it is.

  The size of the RT-PCR amplification product detected here is 561 bp, which matches the size calculated from the determined nucleotide sequence of NR12 cDNA. Therefore, these were considered to be products of a specific PCR amplification reaction. By further confirming this by the Southern blotting method in the next section, the possibility of non-specific PCR amplification was denied.

  In the analysis results of NR12 gene expression distribution and gene expression mode by RT-PCR method, there are limitations on the organs and tissues where the expression was detected, and a large deviation was also observed in the expression level between each organ It was. When these gene expression distributions of NR12 are combined, NR12 can function as a novel hemopoietin receptor, since strong expression localization was detected mainly in immunocompetent cell line tissues and tissues thought to contain hematopoietic cells. The possibility was suggested more strongly. In addition, the expression distribution was also observed in tissues other than the above, suggesting that NR12 may regulate not only the immune system and hematopoietic system, but also various physiological functions in vivo.

  In addition, the presence of splicing mutants was confirmed. In these specific cell groups, transcriptional regulation to determine functional specificity, transcriptional induction by exogenous stimulating factors, and selective splicing regulation are performed, and NR12 gene expression is strictly regulated. It strongly suggests the possibility of receiving transcriptional control.

[Example 3] Confirmation of specificity of RT-PCR product by Southern blotting method The target gene product amplified by RT-PCR in Example 2 is subjected to Southern blotting method using NR12-specific cDNA fragment as a probe. This confirmed that it was a specific amplification. At the same time, we attempted comparative comparative evaluation of gene expression between human organs by quantitatively detecting RT-PCR products based on the label signal intensity. The RT-PCR product in the previous section was subjected to agarose gel electrophoresis, blotted on a charged nylon membrane with Hybond N (+) (Amersham, cat # RPN303B), and subjected to hybridization. As a probe specific for NR12, the cDNA fragment of the 5′RACE PCR product corresponding to the N-terminus of NR12 obtained in Example 1 (4) was used. The probe was prepared by radioisotope labeling with [α- ³² P] dCTP (Amersham, cat # AA0005) using Mega Prime Kit (Amersham, cat # RPN1607). For hybridization, Express Hyb-ridization Solution (Clontech # 8015-2) is used. After pre-hybridization at 68 ° C / 30 minutes, a heat-denatured labeled probe is added and hybridization is performed at 68 ° C / 120 minutes. did. (1) 1x SSC / 0.1% SDS, 5 minutes at room temperature, (2) 1x SSC / 0.1% SDS, 30 minutes at 50 ° C, (3) 0.1x SSC / 0.1% SDS, 30 minutes at 50 ° C After washing, the plate was exposed to an Imaging Plate (FUJI # BAS-III), and an NR12-specific signal was detected by Image Analyzer (FUJIX, BAS-2000 II).

  As shown in FIG. 9, the PCR product amplified by RT-PCR in the previous section was confirmed to be an amplification product specific for NR12. In addition, the comparative quantification of the expression level in each organ also supported the evaluation in the previous section. On the other hand, the target gene expression detection method here, which combines the RT-PCR method and the Southern blotting method, is an extremely sensitive detection means compared to other expression analysis methods. No NR12 gene expression was detected in skeletal muscle, adult brain, prostate, ovary, or placenta.

[Example 4] NR12 gene expression analysis by Northern blotting method NR12 gene expression analysis by Northern blotting method for the purpose of analysis of gene expression mode of NR12 in each human organ and human cancer cell line and identification of NR12 transcription size Tried. Blots include Human Multiple Tissue Northern (MTN) Blot (Clontech # 7760-1), Human MTN Blot II (Clontech # 7759-1), Human MTN Blot III (Clontech # 7767-1), and Human Cancer Cell Line MTN Blot (Clontech # 7757-1) was used.

The 5′-RACE product cDNA fragment obtained in Example 1 (4) was used as the probe. The probe was prepared by radioisotope labeling with [α- ³² P] dCTP using Mega Prime Kit as in Example 3. For hybridization, Express Hyb-ridization Solution was used. After prehybridization at 65 ° C./30 minutes, a heat-denatured labeled probe was added and hybridization was carried out at 65 ° C./16 hours. (1) 1x SSC / 0.1% SDS at room temperature for 5 minutes, (2) 1x SSC / 0.1% SDS at 48 ° C for 30 minutes, (3) 0.5x SSC / 0.1% SDS at 48 ° C for 30 minutes After washing, the imaging plate was exposed to light as in the previous section, and an attempt was made to detect a signal specific to NR12 by using Image Analyzer.

  However, as a result, no signal was detected in any human organ. As a cause, in the Northern analysis method, it is considered that mRNA having a low expression level could not be detected because the detection sensitivity was considerably lower than the RT-PCR level.

[Example 5] Construction of an NR12 ligand search system using a growth factor-dependent cell line Screening for a ligand protein that specifically binds to the protein of the present invention is performed using hemopoietin having a known signal transduction ability in the extracellular domain of the protein of the present invention. A chimeric receptor linked to an intracellular domain including the transmembrane domain of the receptor protein can survive and grow in an appropriate cell line, preferably only in the presence of an appropriate growth factor (growth factor-dependent After expression on the cell surface of the cell line), the cell line can be cultured by adding materials expected to contain various growth factors, cytokines, hematopoietic factors and the like. Since the above growth factor-dependent cell lines die rapidly in the absence of growth factors, they can survive only when a ligand that specifically binds to the extracellular domain of the protein of the present invention is present in the test material. A screening system is established by utilizing the fact that proliferation is possible. Examples of known hemopoietin receptors include thrombopoietin receptor, erythropoietin receptor, G-CSF receptor, gp130, etc., but the partner of the chimeric receptor used in this screening system is limited to the above known hemopoietin receptor Instead, any cytoplasmic domain may be used as long as it has a structure necessary for signal transduction activity. In addition, as a growth factor-dependent cell line, IL-3 dependent cell lines such as Ba / F3 and FDC-P1 can be used.

  Therefore, first, a cDNA sequence encoding the extracellular region of NR12 (amino acid sequence; from Met at position 1 to Gly at position 319) was amplified by PCR, and this DNA fragment was then transmembrane region of a known hemopoietin receptor, And a fusion sequence encoding a chimeric receptor was made by binding in the same translation frame with a DNA fragment encoding the intracellular region. Here, as a known hemopoietin receptor as a partner, several candidates were mentioned as described above, and a human TPO receptor (Human MPL-P) was selected from them. Further, the chimeric receptor sequence prepared as described above was inserted into a plasmid vector pME18S / neo that can be expressed in mammalian cells. FIG. 10 shows a structural schematic diagram of the constructed chimeric receptor (pME18S / NR12-TPOR). These chimeric receptor expression vectors are introduced into the growth factor-dependent cell line Ba / F3 and strongly expressed to select stable gene-transferred cells. Here, the selection of the gene-transferred cells is based on the fact that the expression vector has a drug (neomycin) resistance gene, and selectively propagates only the gene-transferred cells that have acquired drug resistance performance in the same drug-added medium. It is possible to make it. As described above, the chimeric receptor-expressing cell line obtained above is switched to a culture system in the absence of growth factor (IL-3), and materials that are expected to contain the target ligand are added instead. By culturing the cells, new hemopoietin can be screened by developing a screening system utilizing the fact that the cells can survive / proliferate only in the presence of a ligand that specifically binds to NR12.

[Example 6] Construction of an expression system for cell secreted and soluble recombinant NR12 protein It is assumed that the ligand protein that specifically binds to the protein of the present invention is rare but not a soluble protein but a cell membrane-bound protein. The In such a case, it is 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.

  In the former case, for example, a soluble receptor protein artificially prepared by inserting a stop codon at the N-terminal side of the transmembrane domain, or a sequence of NR12.1 encoding a soluble protein of NR12 can be used. is there. In the latter case, for example, it can be prepared 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 in the West Western method.

  Accordingly, the present inventors amplified the cDNA sequence encoding the extracellular region of NR12 (amino acid sequence; from Met at position 1 to Gly at position 319) by PCR, and in the same translation frame at the C-terminus of this DNA fragment. The addition of a FLAG peptide sequence was chosen to create a sequence encoding the soluble labeled protein. The sequence prepared here was inserted into a plasmid vector pCHO that can be expressed in mammalian cells. FIG. 10 shows a structural schematic diagram of the constructed NR12 soluble receptor-labeled protein (pCHO / NR12-FLAG). This expression vector is introduced into a mammalian cell line CHO cell and strongly expressed to select a stable gene-transferred cell. After confirming the expression of the soluble protein, the expressed cells are cultured in large quantities, and the recombinant protein secreted into the culture supernatant can be immunoprecipitated with an anti-FLAG peptide antibody. Furthermore, purification using an affinity column or the like is possible.

  The recombinant protein obtained as described above can be used for the above-described assay and the like.For example, specific binding activity in the coexistence with a material expected to contain a target ligand can be expressed in the BIA-CORE system (Pharmacia). Therefore, it is considered to be extremely useful for searching for novel hemopoietin capable of functionally binding to NR12.

[Example 7] Re-isolation of full-length human NR12CDS (1) Design of oligonucleotide primers The present inventors have succeeded in isolating full-length cDNA of NR12 gene so far. Since the 5′-RACE method and the 3′-RACE method were used, the target gene was an isolated product of the N-terminal sequence and the C-terminal sequence, respectively. Therefore, re-isolation of NR12.2 and NR12.3 genes containing continuous full-length coding sequences was attempted.

First, a sense primer (NR12.1-MET) having the following sequence including a Met sequence that is a translation initiation codon, which is a base sequence common to each cDNA clone of NR12, was designed. On the other hand, primers (NR12.2-STP and NR12.3-STP) containing translation stop codons that are specific nucleotide sequences for NR12.2 and NR12.3 were designed as antisense primers. Primer synthesis was in accordance with Example 1 (3). That is, ABI 394 DNA / RNA Synthesizer was used under the conditions of 5'-terminal trityl group addition, and then the full-length synthesized product was purified by OPC column (ABI # 400771).
NR12.1-MET; 5'- ATG AAT CAG GTC ACT ATT CAA TGG -3 '(SEQ ID NO: 18)
NR12.2-STP; 5'- GCA GTC CTC CTA CTT CAG CTT CCC -3 '(SEQ ID NO: 19)
NR12.3-STP; 5'- TTG ATT TTG ACC ACA CAG CTC TAC -3 '(SEQ ID NO: 20)

(2) PCR cloning
In order to isolate the full-length CDS of NR12, the NR12.1-MET primer of (1) above was used as the sense primer, and the NR12.2-STP and NR12.3-STP primers were used as the antisense primer. PCR cloning was attempted for each. Human Thymus Marathon-Ready cDNA Library (Clontech # 7415-1) was used as a template, and Advantage cDNA Polymerase Mix (Clontech # 8417-1) was used for PCR experiments. The Perkin Elmer Gene Amp PCR System 2400 thermal cycler was used under the following PCR conditions.As a result, a 1301 bp amplification product was obtained with the primer set `` NR12.1-MET vs. NR12.2-STP ''. The sequence was NR12.4. On the other hand, an amplification product of 1910 bp was obtained with the primer set of “NR12.1-MET vs. NR12.3-STP”, and this sequence was distinguished as NR12.5.

  PCR conditions were 94 ° C for 4 minutes, 5 cycles of "94 ° C for 20 seconds, 72 ° C for 90 seconds", "94 ° C for 20 seconds, 70 ° C for 90 seconds", and "94 ° C for 20 seconds. Sec, 90 seconds at 68 ° C ", 28 cycles, 3 minutes at 72 ° C, and 4 ° C.

  The obtained PCR product was subcloned into pGEM-T Easy vector (Promega # A1360) according to Example 1 (4), and the nucleotide sequence was determined. The PCR product was recombined into pGEM-T Easy vector using T4 DNA Ligase (Promega # A1360) for 4 ° C / 12 hours. A gene recombinant of the PCR product and pGEM-T Easy vector was obtained by transforming E. coli strain DH5α (Toyobo # DNA-903). In addition, Insert Check Ready Blue (Toyobo # PIK-201) was used for selection of gene recombinants. Furthermore, the base sequence was determined using the BigDye Terminator Cycle Sequencing SF Ready Reaction Kit (ABI / Perkin Elmer # 4303150) and analyzed with the ABI PRISM 377 DNA Sequencer. The nucleotide sequence of the insert fragment was analyzed for the independent gene recombinants of NR12.4 and NR12.5, and the sequence of a cDNA clone capable of encoding full-length CDS was determined.

  As a result, NR12.4 has a full-length ORF of NR12.2 due to the design of the primers used in the PCR experiments, but it does not have a 5 'untranslated region, and the 3' untranslated region is also a primer-derived sequence. It was not coded except. Similarly, NR12.5 has the full-length ORF of NR12.3, but does not have a 5 ′ untranslated region, and the 3 ′ untranslated region does not encode any sequence other than the primer-derived sequence. The determined nucleotide sequence of NR12.4 and the amino acid sequence encoded by it are shown in FIGS. 11 and 12, while the nucleotide sequence of NR12.5 and the amino acid sequence encoded by it are shown in FIGS.

The Escherichia coli strain DH5α transformed with the pGEM-T Easy vector (pGEM / NR12.5CDS) containing the NR12.5 cDNA of the present invention was deposited internationally on July 31, 2000 as follows.
Name and address of depositary institution Name: Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry Address: 1-3 1-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan (Postal code 305-8566)
Deposit date July 31, 2000 Deposit number Life Research Institute Article 7259 (FERM BP-7259)

[Example 8] Isolation of mouse NR12 homologous genomic gene (1) Preparation of human NR12 probe fragment With the aim of analyzing the genomic structure of the mouse NR12 gene, plaque hybridization to a mouse genomic DNA library was attempted. In order to perform cross-species hybridization hybridization on a mouse genomic DNA library, a probe fragment of human NR12 cDNA was prepared. Using the 5'-RACE product of human NR12 obtained in Example 1 (4), the insert fragment was excised with Not I, and the purified product was used as the probe fragment. The QIAquick Gel Extraction Kit (QIAGEN # 28704) was used for excising the insert fragment from the agarose gel and purification. In the same manner as in Example 3, the probe was prepared by radioisotope labeling with [α- ³² P] dCTP using the Mega Prime Kit and subjected to plaque hybridization.

(2) Plaque hybridization The mouse 129SVJ strain Genomic DNA (Stratagene # 946313) constructed in Lambda FIX II was used as a library. About 320,000 plaques of Genomic library were developed on NZY agar medium and blotted onto a charged nylon membrane with Hybond N (+) (Amersham # RPN303B), and then subjected to primary screening. For hybridization, Perfect-Hyb Solution (Toyobo # HYB-101) was used. After prehybridization at 60 ° C / 30 min, a heat-denatured labeled probe was added and hybridization was performed at 60 ° C / 16 hr. . 1x SSC / 0.1% SDS at room temperature for 5 minutes, 1x SSC / 0.1% SDS at 50 ° C for 30 minutes, 0.5x SSC / 0.1% SDS at 50 ° C for 30 minutes, then X-ray Film (Hyperfilm MP: Amersham, # RPN8H) was exposed to detect NR12 positive plaques.

  As a result, 6 independent clones showing positive or false positive were obtained. As a result of conducting a secondary screening in the same manner for these 6 clones obtained by the primary screening, plaques of 2 independent clones showing NR12 positivity were successfully isolated. The isolated plaque Lambda DNA was prepared in large quantity by plate lysis method. When the insert fragments were cut out with the restriction enzyme Sal I and their sizes were confirmed, they were estimated to be about 18.5 kb and about 16.0 kb, respectively.

  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 retaining the DNA, and a method for producing a recombinant protein using the transformant. Furthermore, a screening method for a natural ligand or compound that binds to the protein is provided. Since the protein of the present invention is considered to be directly involved in biological immune regulation or hematopoietic cell regulation, an understanding of the fundamental characteristics of the immune response or hematopoietic mechanism in the living body, and immune-related diseases and hematopoiesis-related Application to diagnosis and treatment of diseases is expected.

  In particular, it is important to isolate the unknown hematopoietic factor capable of functionally binding to the NR12 molecule of the present invention, and it is extremely useful to use the gene of the present invention for screening for that purpose. It is thought that. Furthermore, it is also effective to perform isolation and identification of peptide libraries or synthetic chemical materials by searching for agonists or antagonists capable of functionally binding to the NR12 molecule.

  As described above, the NR12 gene is considered to provide a useful material for obtaining an unknown hematopoietic factor or agonist that can be functionally linked to the receptor protein encoded by the NR12 gene. By in vivo administration of such a functional binding substance or a specific antibody capable of activating the NR12 molecule function, it is predicted that cellular immunity and hematopoietic function of the living body can be enhanced. That is, it is considered that clinical application is possible as an immunocompetent cell or hematopoietic cell proliferation promoter, differentiation inducer, or immune cell function activator. In addition, it is also possible to enhance cytotoxic immunity against certain types of cancer tissue via them. Furthermore, it is assumed that the expression of NR12 is specifically expressed in a limited cell population in these hematopoietic tissues, and anti-NR12 antibody is useful as a means for separating this cell population. The cell population separated in this manner can be applied to cell transplantation therapy.

  On the other hand, NR12.1, which is a splice variant of NR12, is expected to be used as an inhibitor for NR12 ligand as a decoy type receptor. In addition, it is possible to suppress the cellular immunity of the living body and the growth of hematopoietic cells by in vivo administration of an antagonist that can functionally bind to the NR12 molecule, other inhibitors, or a specific antibody that can inhibit the function of the NR12 molecule. is expected. It is considered that such an inhibitory substance can be clinically applied as an inhibitor of proliferation of immunocompetent cells or hematopoietic cells, a differentiation inhibitor, or an immunosuppressant or anti-inflammatory agent. Specifically, there is a possibility of application to suppression of autoimmune disease onset due to self-tissue damage and suppression of tissue rejection response by living immunity, which is the biggest problem in the field of transplantation immunity. Furthermore, it is considered to be extremely effective for the disease region caused by abnormally enhanced immune response, and immunosuppression using the above inhibitors against various antigen-specific allergies to metals, pollen, etc. It is considered that the solution by is effective.

FIG. 1 is a diagram showing a partial base sequence of AL109843 identified in the htgs database. The base sequence of the exon sequence that could be predicted was written in capital letters, and the amino acid sequence was also written. The amino acid sequences of the target YR motif sequence and WS motif sequence are shown in boxes. FIG. 2 shows the partial amino acid sequences of known hemopoietin receptors that show homology with the partial amino acid sequence of NR12 found in the AL109843 sequence. Matching amino acid sequences are shown with a frame, and amino acid sequences showing similar properties are shaded. In addition, the gap space was supplemented by a bar. In order from the top, human gp130, human NR9, human Prolactin receptor, human IL-7 receptor, and human LIF receptor are described. FIG. 3 is a photograph showing PCR products amplified by 5′-RACE method and 3′-RACE method using oligonucleotide primers designed on the WS exon predicted within the AL109843 sequence. Amplification products from specific PCR reactions are indicated by arrows. FIG. 4 is a diagram showing the base sequence of the full-length cDNA of NR12.1 in which the products obtained by 5′-RACE and 3′-RACE are combined. The amino acid sequence encoded by NR12.1 is also shown. In addition, the secretory signal sequence and the predicted amino acid sequence are underlined. Furthermore, the conserved cysteine residues and the amino acid sequences of the YR motif and WS motif are shown with a frame. FIG. 5 is a diagram showing the base sequence of the full-length cDNA of NR12.2 in which the products obtained by 5′-RACE and 3′-RACE are combined. The amino acid sequence encoded by NR12.2 is also shown. In addition, the secretory signal sequence and the predicted amino acid sequence are underlined. Furthermore, the amino acid sequence predicted to be a transmembrane region is shaded. The conserved cysteine residues in the extracellular region and the amino acid sequences of the YR motif and WS motif are shown with a frame. FIG. 6 shows the nucleotide sequence of the full-length cDNA of NR12.3 in which the products obtained by 5′-RACE and 3′-RACE are combined. The amino acid sequence encoded by NR12.3 is also shown. In addition, the secretory signal sequence and the predicted amino acid sequence are underlined. Furthermore, the amino acid sequence predicted to be a transmembrane region is shaded. The conserved cysteine residues in the extracellular region and the amino acid sequences of the YR motif and WS motif are shown with a frame. FIG. 7 is a continuation of FIG. FIG. 8 is a photograph showing the results of analyzing the gene expression distribution of NR12 in each human organ by RT-PCR. The size of the specific PCR amplification product of NR12 is indicated by an arrow. FIG. 9 is a photograph showing the results of quantitative analysis of the gene expression distribution of NR12 in each human organ by Southern blotting. The size of the specific signal of NR12 detected is indicated by an arrow. FIG. 10 is a structural schematic diagram of an NR12 fusion protein that can be expressed in mammalian cells constructed in an expression vector. FIG. 11 shows the base sequence of the full-length cDNA of NR12.4. The amino acid sequence encoded by NR12.4 is also shown. In addition, the secretory signal sequence and the predicted amino acid sequence are underlined. In addition, the amino acid sequence predicted to be a transmembrane region was shaded. The conserved cysteine residues in the extracellular region and the amino acid sequences of the YR motif and WS motif are shown in bold. FIG. 12 is a continuation of FIG. FIG. 13 shows the nucleotide sequence of the full-length cDNA of NR12.5. The amino acid sequence encoded by NR12.5 is also shown. In addition, the secretory signal sequence and the predicted amino acid sequence are underlined. In addition, the amino acid sequence predicted to be a transmembrane region was shaded. The conserved cysteine residues in the extracellular region and the amino acid sequences of the YR motif and WS motif are shown in bold. FIG. 14 is a continuation of FIG.

Claims (7)

DNA described in any of (a) to (c) below
(A) The amino acid sequence from the 24th Gly to the 337th Cys in the amino acid sequence of SEQ ID NO: 2, the amino acid sequence from the 24th Gly to the 428th Ser in the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: In the amino acid sequence from the 24th Gly to the 629th Lys in the amino acid sequence of 6, the amino acid sequence from the 24th Gly to the 428th Ser in the amino acid sequence of SEQ ID NO: 8, or in the amino acid sequence of SEQ ID NO: 10 DNA encoding a protein having any amino acid sequence from the 24th Gly to the 629th Lys
(B) In the amino acid sequence of SEQ ID NO: 2, the amino acid sequence from the 24th Gly to the 337th Cys, in the amino acid sequence of SEQ ID NO: 4, the amino acid sequence from the 24th Gly to the 428th Ser, SEQ ID NO: In the amino acid sequence from the 24th Gly to the 629th Lys in the amino acid sequence of 6, the amino acid sequence from the 24th Gly to the 428th Ser in the amino acid sequence of SEQ ID NO: 8, or in the amino acid sequence of SEQ ID NO: 10 A protein having an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in any one of the amino acid sequences from the 24th Gly to the 629th Lys, and having hematopoietic factor receptor protein activity DNA encoding gene (c) and hybrida under conditions of 65 ° C., 2 × SSC, 0.1% SDS A gene that encodes a protein having the activity of a hematopoietic factor receptor protein.   A protein encoded by the DNA of claim 1.   A vector into which the DNA according to claim 1 is inserted.   A transformant carrying the vector according to claim 3.   A method for producing a protein, comprising a step of culturing the transformant according to claim 4 and recovering the protein expressed from the transformant or a culture supernatant thereof.   An antibody that binds to the protein of claim 2. A method for screening a compound that binds to the protein according to claim 2, comprising:
(A) contacting the test sample with the protein or a partial peptide thereof,
(B) detecting the 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,
Including methods.

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