WO2001066720A1

WO2001066720A1 - Mouse adipocyte-origin genes

Info

Publication number: WO2001066720A1
Application number: PCT/JP2001/001863
Authority: WO
Inventors: Toshio Kitamura; Hiromichi Tsuruga
Original assignee: Toshio Kitamura; Hiromichi Tsuruga
Priority date: 2000-03-10
Filing date: 2001-03-09
Publication date: 2001-09-13
Also published as: AU2001241080A1

Abstract

By screening molecules having signal peptides with the use of an originally developed efficient signal sequence trapping method, 8 novel genes likely encoding membranes or soluble proteins are successfully identified. These genes show expression in association with the differentiation of adipocytes.

Description

Mouse adipocyte-derived gene

The present invention relates to a novel protein derived from an adipocyte and having a signal sequence, a gene thereof, and their production and use. Background art

Adipose tissue is simply fat-free until it is known that the mutation causes hereditary obesity in mice (Zhang, Y. et al. (1994) Nature 372, 425-432). It was thought to be just a storage. At present, adipose tissue is recognized as the largest secretory organ.

Other secreted proteins produced by adipocytes include TNF-chicken plasminogen activator inhibitor (PAI-1). TNF-H affects the metabolism of lipids and darcos and is overexpressed in adipose tissue of genetically obese rodents (Hotamisligil, GS et al. (1993) Science 259, 87-91 ), Leads to insulin resistance (Hotamisligil ₃ GS et al. (1995) J. Clin. Invest. 95, 240 9-2415) o PAI-1 is highly expressed in visceral adipocytes and It has been implicated in the onset of the disease (Shimomura J. et al. (1996) Nature medicine 2, 800-803).

To date, very few cytokines have been identified in fat cells, but it is thought that adipose tissue secretes other soluble factors that regulate lipid and glucose metabolism. Disclosure of the invention The present invention provides novel proteins derived from adipocytes and having a signal sequence, their genes, molecules functionally equivalent thereto, and their production and use.

In order to solve the above-mentioned problems, the present inventors have developed a 3T3-L1 cell line (Meuth ₅ M. and Green, H. (1974) Cell 3, 367-A) which is a well-characterized model for adipocyte differentiation. _{_{374; Green 5 H. and Kehinde 3}} 0. (1975) Cell 5, a cDNA library prepared from 19-27), retrovirus-mediated expression cloning system (. Kojima, T and K itamura 3 T. (1999) Nature Biotechnol.17, 487-490), and has a signal peptide (von Heijne, (1985) J. Mol. Biol. 184, 99-105) using an efficient signal sequence trapping method. Molecular screening was performed. In the SST-REX method, a cDNA encoding a protein capable of expressing MPL on the cell surface is searched for by screening a library that expresses a fusion protein with the constitutively active cytokine receptor MPL. In this method, the target clone can be easily selected because the expression of the autonomic proliferation ability to an IL-3-dependent cell line by expression of MPL on the cell surface is used as an index.

As a result of screening 9 × 10 ⁵ clones, the present inventors succeeded in identifying eight novel genes that may encode a membrane or a soluble protein. Seven of them were named "# 101" to "# 107", respectively.

These genes showed expression associated with adipocyte differentiation. In addition, although the expressed tissues were different, they showed tissue-specific expression. Therefore, although these genes have different functional sites, they are thought to be molecules that play a role related to adipocyte differentiation in vivo.

The present invention relates to a novel protein derived from an adipocyte and having a signal sequence, a gene thereof, a molecule functionally equivalent thereto, and production and use thereof, and more specifically,

(1) a DNA according to any of the following (a) to (f), (a) a DNA encoding a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 8 to 11;

(b) DNA comprising the coding region of the nucleotide sequence of any one of SEQ ID NOs: 1 to 7.

(c) one or more amino acids in the amino acid sequence of any of SEQ ID NOs: 8 to 11 having substitution, deletion, insertion, and / or addition of an amino acid sequence, and SEQ ID NOs: 8 to 11 A DNA encoding a protein functionally equivalent to the protein comprising the amino acid sequence of any one of the above.

(d) one or more amino acids in the amino acid sequence of the protein encoded by the full-length DNA corresponding to MA consisting of the nucleotide sequence of any of SEQ ID NOs: 1, 2, and 7; substitution, deletion, insertion, And / or has an added amino acid sequence and encodes a protein functionally equivalent to the protein encoded by the full-length DNA.

(e) hybridizes with a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 3 to 6 under stringent conditions, and functions as a protein comprising the amino acid sequence of SEQ ID NOs: 8 to 11; DNA that encodes a protein equivalent to

(f) a protein that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 1, 2, and 7, and functions as a protein encoded by the full-length MA corresponding to these MAs DNA that encodes proteins that are equivalent to each other.

(2) a DNA encoding a partial peptide of a protein consisting of the amino acid sequence of any of SEQ ID NOs: 8 to 11,

(3) a protein or peptide encoded by the DNA of (1) or (2),

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

(5) a host cell carrying the DNA of (1) or (2) or the vector of (4), (6) The method for producing the protein or peptide according to (3), comprising culturing the host cell according to (5) and recovering the expressed protein from the host cell 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 a DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 7 or a complementary strand thereof, and

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

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

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

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

The present invention provides a gene encoding a novel protein derived from an adipocyte and having a signal sequence. The present inventors searched for fat cell-derived cDNAs encoding secretory and membrane proteins by the recently established novel signal sequence trapping method, SST-REX method, and found 63 known cDNAs and 8 novel cDNAs. Got a piece. Among the novel cDNAs isolated by the present inventors using the SST-REX method, seven cDNAs (these clones were named “# 101” to “# 107”, respectively. Are collectively referred to as "# 10X.") The nucleotide sequences of The amino acid sequence of the protein encoded by the # 103 to # 106 cDNA is shown in SEQ ID NOs: 8-11.

All 63 cDNAs isolated by the SST-REX method were found to contain 28 cMA 5 'sequences of 28 known proteins, all of which were membrane or secreted proteins. Of 63 clones (79%), 50 were secreted proteins. This is a fat cell It shows that it has a population as cells that make up a urinary organ. In particular, 28 out of 63 clones (44%) were extracellular matrices such as collagen or related proteins. It has been shown that the ability of fibroblasts to secrete extracellular matrix proteins is enhanced upon differentiation into adipocytes. Collagen sph-1 (IV) and sph-2 (IV) synthesis and secretion are negligible in fibroblasts, but markedly increased in adipocytes. Enhancement of endocin secretion was also observed (Aratani, Y. and Kitagawa ₃ Y. (1988) J. Biol. Chem. 263, 16163-16169). The results shown by the present inventors are consistent with these results, and suggest that the synthesis of extracellular matrix proteins is important for adipose tissue morphogenesis.

Also, 10 clones were 30 kDa (Acrp30) adipocyte complement-related proteins with collagen 'repeats (Scherer, PE et al. (1995) J. Biol. Chem. 270, 26746-26749). . Acrp30 mRNA is induced more than 100-fold during adipocyte differentiation. Therefore, 10 out of 50 (20%) secreted proteins were Acrp30.

There were also two lipoprotein lipase (LPL) clones. LPL is an adipocyte-specific gene involved in fat accumulation, and its expression is enhanced in adipocytes by PPARa. It is appropriate that the LPL gene was obtained by screening the adipocyte cDNA library by the SST-REX method. On the other hand, proteins such as syndecan-1 and lysosomal membrane glycoprotein type A may be housekeeping genes.

By Northern blot analysis, most SST clones obtained from adipocyte cDNA libraries showed an adipocyte-specific expression pattern. Differentiation of preadipocytes into adipocytes may dramatically alter gene expression patterns. Clone # 103 was hardly expressed in 3T3-L1 preadipocytes, but was found in confluent and adipocytes. One possibility may be that it is one of the early markers of adipocyte differentiation such as lipoprotein lipase, collagen and FAAR (fatty acid activated receptor) (MacDougald, 0.A. and Lane, MD (l 995) Annu. Rev. Biochem. 64, 345-375). Expression of the early marker gene is essential and important for subsequent preadipocyte differentiation. In particular, the level of clone # 106 mRNA increased during differentiation. Thus, this may be related to adipocyte differentiation. The expression of clone # 105 is interesting. Two transcripts were detected by Northern blot analysis. The two transcripts can be from different genes.

Furthermore, Northern blot analysis showed that expression of # 103 was almost temporarily lost in the early stage after induction of differentiation. It is theorized that when 3T3-L1 is induced to differentiate into adipocytes, some cells will first divide and proliferate and become adipocytes. In addition, the expression level is low in actively growing logarithmic 3T3-L1 cells, and the expression level is increased in confluent cells in which cell growth has stopped or in differentiated adipocytes. It may not only play an important role in cell differentiation, but may also be involved in cell growth and arrest in general, or may be a marker gene. In # 105, high-molecular-weight bands are observed until the second day after differentiation induction, but only low-molecular-weight bands are seen from the third day, indicating that there is an important meaning in considering adipocyte differentiation. There is.

In addition, since the addition of insulin suppressed the expression of the # 105 gene, this gene may be involved in insulin-mediated signal transduction, which has important implications for the treatment and prevention of diabetes and other diseases. There may be.

In addition, the nucleotide sequence of the human homolog of the # 104 gene has been registered in the gene bank, and according to the description, the expression is enhanced by canceration in the liver system. Expression of # 104 was observed in various cancer cell lines, and their expression patterns were different. Depending on the type of cancer, the expression of the # 104 gene may or may not be seen, and there may be some causal relationship between the # 104 gene and cancer.

Of the # 10X clones isolated by the inventors, # 101, key, and # 107 are not full-length sequences. However, those skilled in the art will Techniques for isolating full-length genes have become common technical knowledge. For example, those skilled in the art can isolate the full-length gene by performing colony hybridization using these fragments as probes. Therefore, the present invention includes full-length genes corresponding to these fragments.

The present invention also provides a functionally equivalent to # 10X protein (a protein consisting of the amino acid sequence of SEQ ID NOs: 8 to 11; a protein encoded by full-length MA corresponding to SEQ ID NOs: 1, 2, and 7). Various proteins. Such proteins include, for example, mutants of these proteins, homologs of organisms other than mice, and the like. Here, “functionally equivalent” means that the target protein has a function as a protein having a signal sequence, like the # 10X protein. As such a function, for example, a function described in International Publication No. WO 00/00610 or a function as a secretory protein can be considered. Whether or not the target protein is a secretory protein can be determined by the following method.

First, a fusion gene in which a gene encoding a commercially available peptide (eg, His-tag or FLAG) is ligated to the 3rd end of the # 10X gene is prepared. Next, the fusion gene is introduced into animal cells (eg, COS cells, etc.) using an expression vector for animal cells (eg, pcDNA3 or pCOS-1), and the # 10X protein is converted as a fusion protein with the peptide. To be expressed. Whether or not this fusion protein is secreted into the culture supernatant is evaluated by ELISA, Western blotting or immunoprecipitation using an antibody against the peptide.

Secretory protein S has various industrial advantages. For example, when it is desired to obtain a certain recombinant protein, if the protein is expressed in a cell as a fusion protein with a secretory protein or a partial peptide capable of secreting the same, the fusion protein is secreted out of the cell. However, there is an advantage that the purification of the recombinant protein is facilitated. In addition, since many secreted proteins are useful drugs, they can be applied as drugs themselves. The protein of the present invention was derived from adipocytes and showed expression associated with adipocyte differentiation. Accordingly, the gene or protein of the present invention, or a compound that regulates the expression or activity of the protein, is not limited thereto. For example, for the treatment or prevention of diseases such as obesity, hyperlipidemia, diabetes, and arteriosclerosis. The application of is considered.

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 a protein is known. For example, those skilled in the art can use a site-directed mutagenesis method (Hashimoto-Gotoh ₅ 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, 944 9456, Kramer W, and Fritz HJ (1987) Methods.Enzymol. 154, 350-367, Kunkel, TA (1985) ) Proc Natl Acad Sci USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 276 3-2766) and the like, using the # 10X protein (from the amino acid sequence described in SEQ ID NOs: 8 to 11). A protein functionally equivalent to the protein can be prepared by appropriately mutating amino acids of the protein (a protein encoded by full-length DNA corresponding to SEQ ID NOs: 1, 2, and 7). Amino acid mutations can also occur in nature. As described above, a protein having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the # 10X protein and functionally equivalent to the protein is also included in the protein of the present invention. The number of amino acids to be mutated in such a mutant is usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (eg, within 5 amino acids).

It is desirable that the amino acid residue to be mutated is mutated to another amino acid that preserves the properties of the amino acid side chain. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, QG, H, K, S, T), an amino acid having an aliphatic side chain (A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, Τ, Υ), an amino acid having a sulfur atom-containing side chain Acids (Μ), carboxylic acids and amino acids with amide-containing side chains (D, N, E, Q ), Amino acids having base-containing side chains (R, K, Η), and amino acids having aromatic-containing side chains (H, F, Y,). Character mark).

It is already known that a protein having an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acid residues relative to an amino acid sequence maintains its biological activity. Nat. Acad. Sci. USA (1984) 81, 5662-5666, Zoller ₃ 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).

# 1 For proteins with multiple amino acid residues added to the amino acid sequence of OX protein

And fusion proteins containing these proteins. The fusion protein is a fusion of these proteins with another peptide or protein, and is included in the present invention. The fusion protein is prepared by encoding the # 10X protein (a protein consisting of the amino acid sequence of SEQ ID NOs: 8 to 11; a protein encoded by the full-length DNA corresponding to SEQ ID NOs: 1, 2, and 7). DNA and DNA encoding another peptide or protein may be ligated in frame so that they are introduced into an expression vector and expressed in a host, and a method known to those skilled in the art can be used. Other peptides or proteins to be fused with the protein of the present invention are not particularly limited.

Other peptides to be fused to the protein of the present invention include, for example, FLAG (Høρρ, Τ.Ρ. et al., BioTechnology (1988) 6, 1204-1210) and 6 His (histidine). ) Residues consisting of 6 xHis,! O xHiS influenza agglutinin (HA), human cmyc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen Known peptides such as fragments, lck tags, a-tubulin fragments, B-tags, and Protein C fragments can be used. Other proteins to be fused with the protein of the present invention include, for example, GST (Gluythione-S-transferase), H A (influenza agglutinin), immunoglobulin constant region, 領域 -galactosidase, MBP (maltose binding protein) and the like. A fusion protein can be prepared by fusing commercially available MA encoding the peptide or the protein with DNA encoding the protein of the present invention, and expressing the fusion DNA prepared thereby. .

In addition, proteins in which a plurality of amino acid residues are deleted from the amino acid sequence of the # 10X protein include proteins in which signal sequences have been removed from these proteins.

Other methods well known to those skilled in the art for preparing proteins functionally equivalent to a protein include the hybridization technique (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). That is, those skilled in the art can isolate a DNA having high homology with the DNA sequence (SEQ ID NOS: 1 to 7) encoding the # 10X protein or a part thereof, and extract the # 10X protein from the DNA. Isolation of a protein that is functionally equivalent to this can also usually be performed.

The present invention includes proteins encoded by DNA that hybridizes to MA encoding the protein and functionally equivalent to the # 10X protein. Such proteins include, for example, homologs of mouse and other mammals (eg, proteins encoded by humans, rodents, rabbits, magpies, etc.).

Hybridization conditions for isolating MA encoding a protein functionally equivalent to the # 10X protein can be appropriately selected by those skilled in the art. The conditions for the hybridization include, for example, low stringent conditions. The low stringent conditions are, for example, conditions of 42 ° C and 0.1% SDS of O.l SSC in washing after hybridization, preferably 50 ° C, 0.1 x SSC and 0.1% SDS. Is the condition. More preferable hybridization conditions include high stringent conditions. Highly stringent conditions are, for example, conditions of 65 ° C., 5 × SSC and 0.1% SDS. In these conditions Thus, it can be expected that DNA with higher homology can be obtained more efficiently as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of the hybridization, and those skilled in the art can realize similar stringency by appropriately selecting these factors. It is possible to

In addition, a gene amplification method using primers synthesized based on the sequence information of DNA (SEQ ID NOS: 1 to 7) encoding the # 10X protein instead of the hybridization method, for example, polymerase chain reaction (PCR) method It can also be used for isolation.

Proteins functionally equivalent to the # 10X protein encoded by DNA isolated by these hybridization techniques or gene amplification techniques are usually the # 10X protein (the amino acid sequence described in SEQ ID NOs: 8 to 11). , A protein encoded by the full-length DNA corresponding to SEQ ID NOs: 1, 2, and 7) and has a high homology in the amino acid sequence. The proteins of the present invention also include proteins that are functionally equivalent to the # 10X protein and that have high homology to the amino acid sequence of the protein. High homology usually means at least 50% identity, preferably 75% identity, more preferably 85% identity, and even more preferably 95% identity at the amino acid level. . The identity of amino acid sequences and nucleotide sequences can be determined by the algorithm BLAST (Proc. Natl. Acad. Sei. USA 90: 5873-5877, 1993) by Karl in and Altschul. Based on this algorithm, programs called BLASTN and BL ASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When analyzing the nucleotide sequence by BLASTN, the parameters are, for example, score = 100 and wordlength = 12. When analyzing amino acid sequences by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known (https://www.ncbi.nlm.nih.gov.) ₀ The protein of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chains, etc., depending on the cell, host, or purification method for producing the protein described below. However, as long as the obtained protein has a function equivalent to that of the # 10X protein, it is included in the present invention. For example, when the protein of the present invention is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the N-terminal of the amino acid sequence of the original protein. The proteins of the present invention also include such proteins.

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 nucleotide sequence of SEQ ID NOS: 1 to 7) is inserted into an appropriate expression vector and introduced into an appropriate host cell. The resulting transformant is collected and an extract is obtained, which is then subjected to 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. The protein of the present invention can be used as a fusion protein with glutathione S-transferase protein or histidine. When expressed in host cells (eg, animal cells, E. coli, etc.) as multiple added recombinant proteins , The set seen recombinant protein expressed can and child purified using Guru evening Chionkaramu or nickel column. After purification of the fusion protein, if necessary, regions of the fusion protein other than the target protein can be cleaved with thrombin or Factor Xa and removed.

If the protein is a natural protein, a method known to those skilled in the art, for example, an affinity in which an antibody that binds to the protein of the present invention described below is bound to a tissue or cell extract expressing the protein of the present invention, as described below. Isolation can be achieved by purifying using one column. it can. 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 of the present invention has an amino acid sequence of at least 7 amino acids or more, preferably 8 amino acids or more, and 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, for screening a compound binding to the protein of the present invention, and for screening for a promoter or inhibitor of the protein of the present invention. Further, it can be an antagonist of the protein of the present invention or a competitive inhibitor. The partial peptide of the present invention can be produced by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase. The peptide may be synthesized by, for example, either a solid phase synthesis method or a liquid phase synthesis method.

The DNA encoding the protein of the present invention is used for in vivo and in vitro production of the protein of the present invention as described above, and also includes, for example, diseases caused by abnormalities in the gene encoding the protein of the present invention. And gene therapy for diseases treatable by the protein of the present invention. 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. Also, as long as it can encode the protein of the present invention, MA having an arbitrary nucleotide sequence based on the degeneracy of the genetic code is included.

The DNA of the present invention can be prepared by a method known to those skilled in the art. For example, it is prepared by preparing a cDNA library from cells expressing the protein of the present invention and performing hybridization using a part of the sequence of the DNA of the present invention (for example, SEQ ID NOS: 1 to 7) as a probe. it can. The cDNA library may be prepared, for example, by the method described in the literature (Sambrook, J. et al., Molecular Clonings Cold Spring Harbor Laboratory Press (1989)), or using a commercially available DNA library. Good. In addition, cells expressing the protein of the present invention After synthesizing cDNA using reverse transcriptase, oligo DNA is synthesized based on the sequence of the DNA of the present invention (for example, SEQ ID NOS: 1 to 7), and the PCR reaction is performed using this as a primer. It can also be prepared by amplifying a cDNA encoding the protein of the present invention.

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

Specifically, the following may be performed. First, mRNA is isolated from a cell, tissue, or organ that expresses the protein of the present invention (eg, an adipocyte or a tissue in which expression has been observed by Northern blotting in this example). mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), and AGPC method (Chomczynski, P. and Sacchi, N. ₃ Anal. Total RNA is prepared by Biochem. (1987) 162, 156-159) and the like, and mRNA is purified from the total RNA using the mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (Pharmacia).

CDNA is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis, AMV

Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) can also be used. In addition, using the primers and the like described in the present specification, the 5, -RACE method (Frohman, MA) using the 5, -Ampli FINDER RACE Kit (manufactured by Clontech) and the polymerase chain reaction (polymerase chain reaction; PCR). Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic.

According to Acids Res. (1989) 17, 2919-2932), cDNA can be synthesized and amplified.

A target DNA fragment is prepared from the obtained PCR product, and ligated to a vector DNA. Furthermore, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected. To prepare the 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 the DNA of the present invention, a nucleotide sequence with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al. ₃ Nucelic Acids Research ( 1981) 9, r43-74). The MA of the present invention can be modified by a commercially available kit or a known method. Modifications include, for example, digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of a linker, insertion of an initiation codon (ATG) and / or stop codon (TAA, TGA, or TAG). And the like.

Specifically, the DNA of the present invention is a DNA consisting of a base sequence from base c at position 1 to base g at position 165 in the base sequence of SEQ ID NO: 1, or a DNA containing the base sequence, A DNA consisting of a base sequence from the 1st base to the 438th base t in the base sequence or a DNA containing the base sequence, and a base sequence from the 83rd base a to the 1408th base a in the base sequence of SEQ ID NO: 3 DNA consisting of a base sequence or DNA containing said base sequence, DNA consisting of a base sequence from base a at position 214 to base g at position 1662 in base sequence of SEQ ID NO: 4, or DNA containing said base sequence, SEQ ID NO: A DNA consisting of the base sequence from the base a at position 289 to the base c at position 1407 in the base sequence of 5, or a DNA containing the base sequence, and the base a from position 86 to position 3244 of base 86 at position 86 in the base sequence of SEQ ID NO: 6. DNA consisting of a base sequence up to base a or Nucleotide sequence including MA, and SEQ ID NO: encompasses DNA containing the DNA or nucleotide sequence consisting of the nucleotide sequence of the 7 from nucleotide position 1 of the nucleotide c to 522-positional base c.

The DNA of the present invention also includes a DNA that hybridizes with the MA having the nucleotide sequence shown in SEQ ID NOs: 1 to 7, and that encodes a protein functionally equivalent to the protein of the present invention. The conditions for the hybridization can be appropriately selected by those skilled in the art. Specifically, the conditions described above can be used. You. Under these conditions, DNA with higher homology can be obtained as the temperature is increased. The hybridizing DNA is preferably a naturally occurring DNA, for example, cDNA or chromosomal DNA.

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

For example, when E. coli is used as a host, the vector

(E.g., DH9, DH5, Ver.1, XLlBlue) for large-scale amplification and preparation of large amounts of E. coli for `` ori '' amplification and selection of transformed E. coli There is no particular limitation as long as it has a gene (for example, a drug resistance gene that can be identified by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). Examples of vectors include M13-based vectors, pUC-based vectors, pBR322. PBluescript, pCR-Script, and the like. In addition, for the purpose of subcloning and excision of cDNA, in addition to the above vectors, examples include pGEM-T, pDIRECT, pT7, and the like. When a vector is used for the purpose of producing the protein of the present invention, an expression vector is particularly useful. For example, when the expression vector is intended for expression in E. coli, the expression vector may have the above-mentioned characteristics such that the vector is amplified in E. coli, and may be used in a host such as JM109, DH5a, HB101, or XU-Blue. In the case of Escherichia coli, a promoter that can be efficiently expressed in Escherichia coli, for example, lacZ promoter, Yuichi (Ward et al., Nature (1989) 341,

544-546; it is essential to have FASEB J. (1992) 6, 242Z-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or T7 promoter. Such vectors include pGEX-5X-1 (Pharmacia), QIAexpress systemj (Qiagen), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase in addition to the above vectors). BL21 is preferred). The vector may also include a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, the pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) may be used for production in the periplasm of Escherichia coli. The introduction of the vector into the host cell can be performed using, for example, a calcium chloride method or an electroporation method.

In addition to Escherichia coli, for example, as a vector for producing the protein of the present invention, a mammalian expression vector (for example, pcDNA3 (manufactured by Invitrogen) or pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8), expression vector derived from insect cells (eg, “Bac-to-BAC baculovairus expression systemj (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vector ( For example, ρΜΗ1, pMH2), an expression vector derived from an animal virus (eg, pHSV, pMV, pAdexLcw), an expression vector derived from a retrovirus (eg, pZIPneo), an expression vector derived from a yeast (eg, rpichia Expression Kitj (Invitrogen) ), PNVU, SP-Q01) and Bacillus subtilis-derived expression vectors (eg, pPL608, pKTH50).

In the case of expression in animal cells such as CH0 cells, COS cells, and NIH3T3 cells, promoters necessary for expression in cells, such as the SV40 promoter (Muligan et al., Nature (1979) 277, 108) ), MMLV-LTR promoter, EF1 promoter Yuichi (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter — It is essential to have a promoter and transform cells. It is more preferable to have a gene (for example, a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.)) for selecting Cp. Examples of vectors with such characteristics include, p negation, pDR2, pBK-RSV, can be mentioned up such pBK-CMVヽp0PRSV _s p0P13.

Furthermore, when the gene is to be stably expressed and the number of copies of the gene is to be increased in a cell, a vector having a DHFR gene complementing the nucleic acid synthesis pathway-deficient CH0 cell is used. (For example, pCHOI) and methotrexe (MTX). In the case of transient expression of a gene, replication of SV40 is performed using COS cells having a gene expressing the SV40 T antigen on the chromosome. Transformation can be performed using a single vector (eg, pcD) having an origin. As the replication origin, those derived from poliovirus, adenovirus, sipapirovirus (BPV) and the like can also be used. In addition, the expression vector is used as a selection marker for amplification of gene copy number in the host cell system, such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, and Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogp t). 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, for example, by a retrovirus method, a ribosome method, a cationic ribosome method, an adenovirus method, or the like. There is a method for introduction into a living body. This makes it possible to carry out gene therapy for a disease caused by a mutation in the # 10X gene of the present invention. Examples of the vector used include, but are not limited to, an adenovirus vector (eg, pAdexlcw) and a retrovirus vector (eg, pZIPneo). General genetic manipulations such as insertion of the DNA of the present invention into a vector can be performed according to a conventional method (Molecular Cloning, 5.61-5.63). Administration into a living body may be an ex vivo method or an in vivo method.

The present invention also provides a host cell into which the vector of the present invention has been introduced. The host cell into which the vector of the present invention is introduced is not particularly limited, and for example, Escherichia coli and various animal cells can be used. The host cell of the present invention can be used, for example, as a production system for producing or expressing the protein of the present invention. Production systems for protein production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells. When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts. As animal cells, mammalian cells, for example, CHO (J. Exp. Med.

(1995) 108, 945), COS-3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells such as African Megafrog oocytes (Valle, et al.,

Nature (1981) 291, 358-340) or insect cells such as Sf9, Sf21, and Tn5 are known. As the CH0 cells, in particular, dhfr-CHO (Proc. Natl. Acad. Scad. USA (1980) 77, 4216-4220) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) can be suitably used. In animal cells, when large-scale expression is intended, CH0 cells are particularly preferred. Vector vectors can be introduced into host cells by, for example, the calcium phosphate method, the DEAE dextran method, the method using Cationic Ribosome D0TAP (manufactured by Behringer Mannheim), the electoral poration method, or the Lipofexion method. Is possible.

As plant cells, for example, cells derived from Nicotiana tabacum (Nicotiana tabacum) are known as protein production systems, which may be callus cultured. Fungal cells include yeast, for example, the genus Saccharomyces, for example, Saccharomyces cerevisiae, filamentous fungi, for example, the genus Aspergillus, for example, Aspergillus niger s. Are known.

When prokaryotic cells are used, there are production systems using bacterial cells. Examples of bacterial cells include Escherichia coli (E. coli) such as JM109, DH5 and HB101, and Bacillus subtilis.

The protein is obtained by transforming these cells with the target DNA and culturing the transformed cells in vitro. The culturing can be performed according to a known method. For example, as a culture solution of animal cells, for example, DMEM, MEM, RPMI1640, and IMDM can be used. At this time, serum replacement fluid such as fetal calf serum (FCS) They may be used in combination, or may be serum-free culture. The pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.

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

When using animals, there are production systems using mammals and insects. As mammals, goats, bushes, ovines, mice, and mice can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.

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

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

Furthermore, when using a plant, for example, tobacco can be used. When tobacco is used, the DNA encoding the target protein is inserted into a plant expression vector, for example, pMON530, and this vector is inserted into Agrobacterium 'mmfemaciens. (Agrobacterium tumefaciens). The bacterium is 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 inside or outside the host cell (such as a medium) and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be carried out by using the separation and purification methods used in ordinary protein purification, and is not limited at all. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. When combined, proteins can be separated and purified.

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

The protein can be arbitrarily modified or partially removed by reacting the protein with an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, lysylendopidase, protein kinase, glucosidase and the like are used.

The present invention also provides an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a monoclonal antibody as well as a polyclonal antibody. Further, antiserum obtained by immunizing an immunized animal such as a heron with the protein of the present invention; This includes all classes of polyclonal and monoclonal antibodies, as well as human and recombinant humanized antibodies.

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

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 partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein. As used herein, “antibody” refers to an antibody that reacts with the full length or fragment of a protein.

A gene encoding the protein of the present invention or a fragment thereof is inserted into a known expression vector system, and the vector is used to transform the host cell described in the present specification. Fragments may be obtained by a known method, and these may be used as a sensitizing antigen. Further, a cell expressing the protein, a lysate thereof, or a chemically synthesized protein of the present invention may be used as the sensitizing antigen. It is preferable that the short peptide is appropriately bound to a carrier protein such as keyhole lysine hepatocyanin, pepsin albumin, ovalbumin, etc. to form an antigen.

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

As rodent animals, for example, mice, rats, hamsters and the like are used.動物 As an heronoid animal, for example, a heron is used. For example, monkeys are used as primates. As the monkeys, monkeys of the lower nose (old world monkeys), for example, cynomolgus monkeys, macaques, baboons, and chimpanzees are used. Immunization of an animal with a sensitizing antigen is performed according to a known method. As a general method, a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and then mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if desired. After emulsification, it is administered to mammals. Thereafter, it is preferable to administer the sensitizing antigen mixed with an appropriate amount of incomplete Freund's adjuvant several times every 4 to 21 days. In addition, a suitable carrier can be used at the time of immunization with the sensitizing antigen. Immunization is performed in this manner, and an increase in the desired antibody level in the serum is confirmed by a conventional method.

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

To obtain a monoclonal antibody, after confirming that the desired antibody level is increased in the serum of the mammal sensitized with the antigen, the immune cells may be removed from the mammal and subjected to cell fusion. At this time, preferred immune cells used for cell fusion include splenocytes in particular. The other parent cell to be fused with the immunocyte is preferably a mammalian myeloma cell, more preferably a myeloma cell that has acquired the properties for fusion cell selection by a drug. Cell fusion between the immune cells and myeloma cells is basically performed according to a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C, Methods Enzymol. (1981) 73, 3-46). It can be carried out.

The hybridoma obtained by cell fusion is selected by culturing it in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT culture solution is continued for a period of time sufficient to kill cells other than the target hybridoma (non-fused cells), usually for several days to several weeks. Next, screening and cloning of hybridomas producing the desired antibody are performed by the usual limiting dilution method.

In addition, in addition to obtaining the above-mentioned hybridoma by immunizing non-human animals with an antigen, human lymphocytes, for example, human lymphocytes infected with EB virus are sensitized in vitro with proteins, protein-expressing cells or lysates thereof, It is also possible to fuse a sensitized lymphocyte with a human-derived myeloma cell having permanent division ability, for example, U266, to obtain a hybridoma that produces a desired human antibody having a protein binding activity (Japanese Patent Application Laid-Open No. -17 688 publication).

Next, the obtained hybridoma is transplanted into the peritoneal cavity of a mouse, ascites is collected from the mouse, and the obtained monoclonal antibody is subjected to, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, and the present invention. It can be prepared by purifying the above protein using a coupled affinity column or the like. The antibody of the present invention is used for purification and detection of the protein of the present invention, and is also a candidate for an agonist and an angoniist 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 administration to the human body (antibody therapy), a human antibody or a humanized antibody is preferable in order to reduce immunogenicity.

For example, a transgenic animal having a human antibody gene repertoire is immunized with a protein serving as an antigen, protein-expressing cells or a lysate thereof to obtain antibody-producing cells. A human antibody against the protein can be obtained using a hybridoma fused with myeloma cells (International Publication Nos. W092--03918, W093-2227, W094-02602, W094-25585, W096-33735 and W096). -See 34096).

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

Further, the antibody of the present invention may be a modified antibody fragment thereof as long as it binds to the protein of the present invention. For example, as an antibody fragment, Fab, F (ab ') 2, Fv, or a single chain Fv (scFv) in which an Fv of an H chain and an L chain 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, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragments is constructed and introduced into an expression vector. Expressed in cells (for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better ₅ M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun ₅ A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E. ₅ 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.) As the modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) can be used. The “antibody” of the present invention also includes these modified antibodies. Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.

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

The antibody obtained as described above can be purified to homogeneity. The separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins. For example, chromatography columns such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. can be appropriately selected and combined. Antibodies can be separated and purified (Antibodies: A Laboratory Manual; Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but are not limited thereto. The concentration of the antibody obtained above can be measured by measuring the absorbance or by using an enzyme-linked immunosorbent assay (ELISA) or the like. Columns used for affinity chromatography include a protein A column and a protein G column. For example, columns using a protein A column include Hyper D, POROS, Sepharose F.F. (Pharmacia), and the like. Examples of chromatography other than affinity chromatography include, for example, 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 chromatographys can be performed using liquid phase chromatography such as HPI and FPLC.

Methods for measuring the antigen-binding activity of the antibody of the present invention include, for example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme-linked immunosorbent assay), Immunoassay) or a fluorescent antibody method can be used. When ELISA is used, 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. Add a secondary antibody that recognizes an enzyme, for example, an antibody labeled with alkaline phosphatase, etc., incubate the plate, wash the plate, and then add an enzyme substrate such as P-nitrophenylphosphoric acid and measure the absorbance. Antigen binding activity can be evaluated. As the protein, a protein fragment, for example, a fragment comprising the C-terminus thereof may be used. For evaluation of the activity of the antibody of the present invention, BI Acore (Pharmacia) can be used.

By using these techniques, the antibody of the present invention is brought into contact with the sample contained in the sample which is expected to contain the protein of the present invention, and an immune complex of the antibody and the protein is detected or measured. Thus, the method for detecting or measuring a protein of the present invention can be carried out. INDUSTRIAL APPLICABILITY The method for detecting or measuring a protein of the present invention can specifically detect or measure a protein, and thus is useful for various experiments and the like using proteins.

The present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to the DNA encoding the # 10X protein (SEQ ID NOS: 1 to 7) or its complementary strand.

Here, the “complementary strand” refers to one strand of a double-stranded nucleic acid consisting of A: T (U in the case of RNA) and G: C base pairs with respect to the other strand. The term “complementary” is not limited to a case where the sequence is completely complementary to at least 15 contiguous nucleotide regions, but is at least 70 °, preferably at least 80%, more preferably 90%, and more preferably It is preferable that they have a homology of 95% or more on the base sequence. The algorithm for determining homology may use the algorithm described in this specification.

Such nucleic acids include probes and primers used for detection and amplification of DNA encoding the protein of the present invention, probe primers for detecting the expression of the DNA, and expression of the protein of the present invention. Nucleotides or nucleotide derivatives (eg, antisense oligonucleotides, ribozymes, or DNA encoding them) for control are included. Such a nucleic acid can also be used for producing a DNA chip.

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

Antisense oligonucleotides include, for example, antisense oligonucleotides that hybridize at any position in the base sequence of SEQ ID NOs: 1 to 7. This antisense oligonucleotide is preferably an antisense oligonucleotide for at least 15 or more consecutive nucleotides in the nucleotide sequence of SEQ ID NOs: 1 to 7. More preferably, it is an antisense oligonucleotide in which at least 15 or more consecutive nucleotides contain a translation initiation codon.

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

Antisense oligonucleotides include not only those whose nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are all complementary sequences, but also DNAs or mRNAs and oligo nucleotides shown in SEQ ID NOs: 1 to 7. As long as it can specifically hybridize to the base sequence to be used, one including one or more nucleotide mismatches is also included. The antisense oligonucleotide derivative of the present invention acts on cells producing the protein of the present invention to bind to DNA or mRNA encoding the protein, thereby inhibiting the transcription or translation of the protein or producing mA. By suppressing the expression of the protein of the present invention, for example, by promoting the degradation, 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 material which is inactive against the derivative to prepare an external preparation such as a liniment or a poultice.

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

The antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is applied to the patient so as to be able to reach the affected area as a result of intravenous administration or the like. Furthermore, an antisense-encapsulated material that enhances durability and membrane permeability can be used. Examples include liposomes, poly-L-lysine, lipids, cholesterol, ribofectin or derivatives thereof.

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

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

The protein of the present invention is useful for screening for a compound that binds to the protein. That is, the protein of the present invention is brought into contact with a test sample expected to contain a compound binding to the protein, and a compound having an activity of binding to the protein of the present invention is selected. And a method of screening for a compound that binds to the protein of the present invention.

The protein of the present invention used for the screening may be a recombinant protein or a protein of natural origin. It may be a partial peptide. It may also be a form expressed on the cell surface or a form as a membrane fraction. The test sample is not particularly limited and includes, for example, a cell extract, a cell culture supernatant, a fermentation microorganism product, a marine organism extract, a plant extract, a purified or crude protein, a peptide, a non-peptidic compound, and a synthesis. Low molecular weight compounds and natural compounds. The protein of the present invention, which is brought into contact with a test sample, may be, for example, a purified protein, a soluble protein, a form bound to a carrier, a fusion protein with another protein, or a form expressed on a cell membrane. The sample can be brought into contact with a test sample as a membrane fraction.

As a method for screening a protein that binds to the protein using the protein of the present invention, for example, many methods known to those skilled in the art can be used. Such screening can be performed, for example, by immunoprecipitation. Specifically, it can be performed as follows. The gene encoding the protein of the present invention is introduced into a vector for expression of a foreign gene such as pSV2neo, pcDNA I, or pCD8, so that the gene is expressed in animal cells or the like. Promoters used for expression include SV40 early promoter (Rigoy In Williamson ea.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)), EF-1 promoter (Kim et al. , P.217-223 (1990)), CAG promoter (Niwa et al. Gene 108, p.193-200 (1991)), RSV LTR promoter ^ Cull en Methods in Enzymology 152, p.684-704 (1987) , SR a promoter (Takebe et al. Mol. Cell. Biol. 8, p. 66 (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. A ppl. Genet. II, p. 385-394 (1982)), Adenovirus late promoter (Kaufman et a 1. Mol. Cell. Biol. 9, p. 946 (1989)), HSV TK promoter, and any other commonly used promoters can be used.

In order to express a foreign gene by introducing a gene into an animal cell, the electroporation method (Chu, G. et al. Nuc 1. Acid Res. 15, 1311-1326 (1987)) and the calcium phosphate method (Chen , C and Okayama, H. Mol. Cell. Biol. 7, 2745-2752 (1 987)), DEAE dextran method (Lopata, MA et al. Nuc 1. Acids Res. 12, 5707-5717 (1984); Sussman, DJ and Milman ₃ G. Mol. Cell. Biol., 1642-1643 (1 985)), 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)) and the like.

The protein of the present invention is expressed as a fusion protein having a monoclonal antibody recognition site by introducing a recognition site (epitope) of the monoclonal antibody of which specificity is known into the N-terminal or C-terminal of the protein of the present invention. be able to . A commercially available epitope-antibody system can be used (Experimental Medicine 1 ^, 85-90 (1995)). Vectors that can express a fusion protein with β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP), and the like via a multicloning site are commercially available.

In order to minimize the property of the protein of the present invention by changing it into a fusion protein, a method for preparing a fusion protein by introducing only a small epitope portion consisting of several to several tens of amino acids has been reported. I have. For example, polyhistidine (His-tag), influenza agglutinin HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 genel0 protein (T7-tag), human pure virus We developed epitopes such as viral glycoproteins (HSV-tags) and E-tags (epitopes on monoclonal phages) and monoclonal antibodies that recognize them. It can be used as an Epitoboo antibody system for screening proteins that bind to the Myoprotein (Experimental Medicine, 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 comprises the protein of the present invention, a protein capable of binding thereto, and an antibody. In addition to using antibodies against the above-mentioned epitopes, immunoprecipitation can also be performed using antibodies against the protein of the present invention. Antibodies against the protein of the present invention include, for example, a gene encoding the protein of the present invention introduced into an appropriate E. coli expression vector, expressed in E. coli, and the expressed protein is purified. It can be prepared by immunizing goats and chickens. Alternatively, it can be prepared by immunizing the above animal with the synthesized partial peptide of the protein of the present invention.

If the antibody is a mouse IgG antibody, for example, the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose. Further, when the protein of the present invention is prepared as a fusion protein with an epitope such as GST, for example, a substance specifically binding to these epitopes such as glutathione-Sepharose 4B is used to prepare the protein of the present invention. An immune complex can be formed in the same manner as in the case where an antibody of the above protein is used.

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 performed according to.

SDS-PAGE is generally used for analysis of immunoprecipitated proteins. By using an appropriate concentration of gel, the bound proteins can be analyzed by the molecular weight of the protein. In addition, at this time, it is generally difficult to detect the protein bound to the protein of the present invention by ordinary staining methods such as Coomassie staining and silver staining, so that ³⁵ S- Cells in culture containing methionine and ³⁵ S-cysteine Is cultured, the protein in the cell is labeled, and the detection is performed, whereby the detection sensitivity can be improved. Once the molecular weight of the protein is known, the protein of interest can be purified directly from the SDS-polyacrylamide gel and its sequence determined.

Examples of the method for isolating a protein that binds to the protein using the protein of the present invention include, for example, the West Western blotting method (Skolnik, EY et al., Cell (1991) 65, 83-90). ). That is, cells, tissues, and organs that are expected to express the protein that binds to the protein of the present invention (for example, fat cells and tissues in which expression has been observed by Northern blotting in the Examples) are phage-derived. A cDNA library using a vector (human gtll, ZAP, etc.) was prepared, and expressed on LB-agarose. The protein expressed on a filter was fixed, purified and labeled with the protein of the present invention. The plaque expressing the protein bound to the protein of the present invention may be detected by labeling by reacting the above-mentioned filter with the protein. Examples of the method for labeling the protein of the present invention include a method utilizing the binding property of biotin and avidin, a method of specifically binding to the protein of the present invention or a peptide or polypeptide fused to the protein of the present invention (for example, GST). Examples of the method include a method using an antibody, a method using a radioisotope, and a method using fluorescence.

Further, as another embodiment of the screening method of the present invention, a 2-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, `` MATCHMARKER Two-Hybrid Systemj, '' Mamm alian MATCHMAKER Two-Hybrid Assay Kit ”,“ MATCHMAKEE One-Hybrid System ”(all manufactured by Clontech), and“ HybriZAP Two-Hybrid Vector Systemj ”(Strata Gene) are used. In the system, the protein of the present invention or a partial peptide thereof is fused with an SRF DNA binding region or GAL4 DNA binding region and expressed in yeast cells, and the protein of the present invention and From the cells that are expected to express the protein to be bound, a cDNA library was prepared that could be expressed in a form fused with the VP16 or GAL4 transcription activation region, and introduced into the yeast cells described above for detection. The library-derived cDNA is isolated from the obtained positive clones (when the 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). The protein encoded by the cDNA can be obtained by introducing the isolated cDNA into Escherichia coli for expression. As a result, it is possible to prepare a protein that binds to the protein of the present invention or a gene thereof. The repo overnight gene used in the 2-hybrid system includes, for example, the HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor typel) gene, etc. But not limited to these. Screening by the two-hybrid method can be performed using mammalian cells in addition to yeast cells.

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

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

In the present invention, a biosensor utilizing the surface plasmon resonance phenomenon can be used as a means for detecting or measuring the bound compound. A biosensor utilizing the surface plasmon resonance phenomenon can be used to detect the interaction between the protein of the present invention and a test compound using a trace amount of protein and without labeling the surface plasmon resonance signal. It can be observed in real time as a null (for example, BIAcore, manufactured by Pharmacia). Therefore, it is possible to evaluate the binding between the protein of the present invention and a test compound by using a biosensor such as BIAcore.

Methods for isolating not only proteins but also compounds that bind to the protein of the present invention (including agonist and angelic gonist) include, for example, immobilized protein of the present invention, synthetic compounds, and natural compounds. A method for screening molecules that bind to the protein of the present invention using a product bank or a random phage peptide display library, and 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. ₅ Small peptides as potent mimetics of the protein hormone erythropoietin, Science (ring ITED STATES) Jul 26 1996, 273 p458-64, Verdine GL., The combinatorial chemistry of nature.Nature (ENGLAND) Nov 7 1996, 384 pi 1-13, Hogan JC Jr., Directed combinatorial chemistry.Nature (ENG LAND) Nov 7 1996, 384 pl7-9) are known to those skilled in the art.

The compound that can be isolated by the screening of the present invention is a candidate for the agent for regulating the activity of the protein of the present invention, and is a disease caused by abnormal expression or dysfunction of the protein of the present invention or the activity of the protein of the present invention. It can be applied to the treatment of diseases that can be treated by controlling the disease. Substances that can be converted by addition, deletion, and / or substitution of a part of the structure of a compound that can be isolated using the screening method of the present invention are also included in the compounds that bind to the protein of the present invention.

The protein of the present invention, or a compound that can be isolated by the screening of the present invention, is used in a human mammal, such as a mouse, a rat, a guinea pig, a heron, a chicken, a cat, a dog, a sheep, a bush, a monkey, a monkey, a baboon, When used as a chimpanzee medicament, the protein or isolated compound itself can be administered directly to a patient, or can be formulated and administered by a known pharmaceutical method. For example, need Injection of tablets, capsules, elixirs, microcapsules orally, or sterile solutions or suspensions with water or other pharmaceutically acceptable liquids, as appropriate for sugar coating It can be used parenterally in dosage form. For example, pharmacologically acceptable carriers or vehicles, specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, It is possible to formulate the drug product by combining it with a preservative, binder and the like as appropriate and mixing it in the unit dosage form required for accepted pharmaceutical practice. The effective components of these preparations are intended to provide a suitable volume in the specified range.

Additives that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Suitable leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used. When the unit dosage form is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat. A sterile composition for injection can be formulated using a potable vehicle such as distilled water for injection according to normal pharmaceutical practice.

Aqueous injection solutions include, for example, physiological saline, isotonic solutions containing pudose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. It may be used in combination with an agent, for example, an alcohol, specifically ethanol, a polyalcohol, for example, propylene glycol, polyethylene glycol, a nonionic surfactant, for example, polysorbate 80 (TM) or HC0-50.

The oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. Also, buffers such as phosphate buffers, sodium acetate buffers, soothing agents such as proforce hydrochloride, stabilizers, such as It may be blended with benzyl alcohol, phenol and antioxidants. The prepared injection solution is usually filled into an appropriate ampoule.

Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by methods known to those skilled in the art. It can do better. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. Further, if the compound can be encoded by DNA, the DNA may be incorporated into a gene therapy vector to perform gene therapy. The dose and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can appropriately select the dose of the protein of the present invention. Depending on the subject, target organ, symptoms and method of administration, for example, in the form of an injection, it is generally considered to be about 100 to 20 mg per day for an adult (with a body weight of 60 kg).

The dose of the compound that binds to the protein of the present invention or the compound that modulates the activity of the protein of the present invention varies depending on the symptoms. However, in the case of oral administration, in general, in adults (with a body weight of 60 kg), 1 It is believed to be about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.

In the case of parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, and administration method. For example, in the case of injection, it is usually 1 dose for adults (with a body weight of 60 kg). It may be convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is an electrophoretic photograph showing the result of detecting the expression of the gene of the present invention in 3T3-LI cells by Northern blot analysis. Lane 1 shows proliferating 3T3-L1 cells, lane 2 shows confluent 3T3-L1 cells, and lane 3 shows 3T3-L1 cells induced to differentiate into adipocytes.

FIG. 2 is an electrophoretic photograph showing the results of detecting the expression of the gene of the present invention in various tissues by Northern blot analysis. The lanes are as follows: 1: heart, 2: brain, 3: spleen, 4: lung, 5: liver, 6: skeletal muscle, ：: kidney, 8: testis. Figure 3 shows the expression of # 103 to # 106 and GAPDH in 3T3-L1 in log phase, 3T3-Ll in confluent state, and 3T3-L1 1 to 9 days after induction of differentiation into fat cells. It is an electrophoresis photograph which shows the result detected by Northern blot analysis. FIG. 4 is an electrophoretic photograph showing the results of Northern blot analysis of the expression of # 105 and GAPDH in adipocytes to which OnM (control: MOCK), 25 nM, and 250 nM insulin was added.

FIG. 5 is an electrophoretic photograph showing the results of detecting the expression of # 104 in various cancer cell lines, adipocytes and 3T3-L1 by Northern blot analysis. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Construction and screening of cDNA library

A cDNA library was constructed and expressed using the retroviral vector pMX-SST (Kojima. And Kitamura ₅ T. (1999) Nature Biotechnol. 17, 487-490).

Differentiated ST3-L1 cells were extracted using Fast Track 2.0 mRNA isolation kit (Invitrogen, Lisbad, Calif.) According to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from poly (A) + RNA using random hexamers using the Superscript 'Thiois' system (Gibco-BRL, Rockville, Maryland, USA). The BstXI adapter was used to insert into the BstXI site of the pMX-SST vector (Invitrogen, Lisbad, CA). To construct the SST-REX library, the ligated DNA was amplified into DH10B cells (Electromax, Gibco-BRL) and the Qiagen Plasmid kit (Qiagen-Inc, Valencia, CA) was used. To prepare library DNA.

A cell line containing a high-potency retrovirus presenting the SST-REX library was packaged (Pear, W. S. et al. (1993) Proc. Natl. Acad. Sci. U. S.A. 90, 839

2-8396) and infected Ba / F3 cells as described. Washing the cells three times after infection day, and clones were selected in the absence of IL- 3 using 96 well multi-evening Ita first plate (10 ³ / Ueru).

Twelve days later, genomic DNA was extracted from factor-independent Ba / F3 clones, subjected to genomic PCR, and the integrated cDNA was recovered using vector primers (GGGG GTGGACCATCCTCTA / SEQ ID NO: 12, and CGCGCAGCTGTAAACGGTAG / sequence). Number: 1 3). Using the GeneAmp PCR System 2400 (Perkin Elmer, Norwalk, Conn.) And LA Taq Polymerase (Yukara, Kyoto, Japan), perform 30 cycles of PCR (denaturation at 98 ° C for 20 seconds, 68 (Ringing and elongation at 2 ° C for 2 minutes). The obtained PCR fragments were sequenced using the Taq Dye Mineral One-Day Cycle Sequencing Kit (Applied Biosystems, Inc., Foss Yuichi City, Calif-Ornier) to obtain an automated sequencer (310 Analyzed by Gene Analyzer, Applied'Biosystems, Inc.). We screened 9 × 10 ⁵ clones. Cells growing in a total of 71 cells out of 1152 cells generated a single PCR band, which was subjected to further analysis. The differentiated 3T3-LI cells used in the experiment were prepared as follows. The 3T 3-L1 cells were cultured in 10% FCS, 50 units / ml penicillin, and 50〃G / ml Sutorebutoma containing leucine DMEM (DMEM-FCS) 5% C0 2 at 37 ° C in. Cells were grown to confluence and maintained for 2 days. To induce differentiation, the medium was changed to DMEM-FCS containing 0.5 IDM trimethyl-3-isobutylxanthine, 0.25 / M dexamethasone, and 10 μg / ml insulin. Two days later, the medium was changed to DMEM-; FCS to differentiate the cells. Ba / F3, a mouse IL-3 dependent pre-B cell line, was cultured in RPMI 1640 medium containing 10% FCS and 2 ng / ml mouse IL-3 (MD Systems).

[Example 2] Analysis of isolated cDNA clone

63 out of 71 integrants contained the 5 'sequence of the cDNA for 28 known proteins, all of which contained the signal sequence (Table 1).

4 Table 1

No. Identity Number of clones Secreted protein (all) 50

Extracellular matrix and related proteins (all) 28

1 Procollagen alpha-2 (I) 5

2 Collagen alpha- 1 (ΙΠ) 2

3 Collagen alpha-1 (VI) 2

4 Collagen alpha-2 (IV) 2

5 Collagen alpha- 1 (IV) 1

6 Collagen alpha- 1 (XV) 1

7 Procollagen C-proteinase enhancer protein 3

8 Cysteine-rich glycoprotein SPARC 4

9 Extracellular matrix associated protein (Sc l) 3

10 Entactin (ENT) 2

11 Fibulin-2 1

12 Lysyl oxidase 1

13 Dystroglycan (DAG1) 1 Other secreted proteins (all) 22

14 Adipocyte complement-related protein of 30kDa (Acrp30) 10

15 Sulfated glycoprotein (Sgpl) 4

16 Lipoprotein lipase 2

17 Cy statin C 2

. 18 FK506-binding protein (FKBP23) 1

19 Epithelin 1

20 Disulfide isomerase- related protein (ERp72) 1

21 Interferon receptor soluble isoform (EFNAR2) 1 Membrane protein (all) 13

22 Amyloid precursor-like protein 2 (APLP2) 4

23 Amyloid beta protein precursor 1

24 Syndecan-1 3

25 Lysosomal membrane glycoprotein-typeA 2

26 Rat ribophorin I homologue 1

2 / Tissue factor Cf-3 1

28 Putatative transmembrane receptor (frizzled 7) 1

(Total) 63 Of the 63 proteins identified, 50 (79%) were secreted proteins, and 28 of 50 secreted proteins were extracellular matrix (ECM) proteins or related proteins. Thirteen were membrane proteins. The remaining eight clones represented seven independent novel proteins with signal peptides (Table 2).

Table 2 Clones Similarity Number of clones

101 No similarity to database sequences

102 No similarity to database sequences

103 No similarity to database sequences

104 No similarity to database sequences

105 Cell surface protein MCAR

106 Collagen alpha 1 (XI) 2 107 No similarity to database sequences 1 Five proteins did not show similarity to known proteins. One is similar to the cell surface protein MCAR (mouse oxpecker virus and adenovirus receptor) (Bergelson, JM et al. (1998) J. Virol. 72, 415-419) and the other is collagen And similarities.

[Example 3] Expression of a novel adipocyte gene during differentiation

Northern blot analysis examined the expression patterns of seven novel genes, including clone # 101-107, during differentiation (Figure 1). Poly (A) + MA was prepared in the same manner as in Example 1 from proliferating 3T3-L1 cells, confluent 3T3-L1 cells, and 3T3-L1 cells induced to differentiate into adipocytes.

1 μg of poly (A) + RNA is electrophoresed on a 1.0% agarose gel containing 2% formaldehyde, and then transferred to Hybond-N-nylon membrane (Amersham Pharmacia-Biotech). did. Membrane is incubated at 42 ° C in hybridization buffer (50% formamide, 10X Denhardt's reagent, 5x SSC, 0.1% SDS, 200 μg / ml denatured salmon sperm DNA) at 42 ° C. the ^{3 2} P- labeled DNA fragments derived from examined as a probe. One of the membranes was used as a loading control to evaluate the amount of RNA in each lane. The cDNA for hydrogenase (GAPDH) was probed. After hybridization, the filter was washed in 0.1 × SSC, 0.1% SDS at 42 ° C. and subjected to autoradiography.

As a result, mRNA expression changed with differentiation in all clones except clone # 102. The mRNA of clones # 101, # 104, # 106, and # 107 increased after differentiation. Clone # 105 showed two bands. When the cells differentiated into adipocytes, low molecular mass mA was significantly expressed. For clone # 103, the only increase in mRNA was sufficient for 3T3-L1 cells to grow to confluence.

[Example 4] Northern blot analysis of multiple mouse tissues

The expression profile of the novel adipocyte-derived gene in multiple mouse tissues was examined by Northern plot analysis (FIG. 2). Mouse multi-tissue Northern plots were purchased from Clontech (Palo Alto, CA). In Example 3, six genes excluding clone # 102, whose expression was shown to be significantly increased in adipocytes, were selected and examined.

As a result, expression of clone # 101 was extremely high in testis, but mMA was weakly detected in heart, spleen, and liver. Clone # 103 was specifically expressed in heart and lung, but low molecular weight bands were weakly detected in testis. Clone # 104 expression was extremely high in heart and liver, but weak detection of mMA in brain, lung and kidney. Clone # 105 was transcribed only in the heart and brain. It is noteworthy that only high molecular weight bands were detected in the brain. Expression of clone # 106 was detected only in the heart. Clone face mRNA levels in heart, liver, kidney and testis were higher than others. Thus, it was found that there is a remarkable difference in the expression of the novel adipocyte-derived gene in tissues.

[Example 5] Daily change of mRNA Northern blot analysis was performed by the usual method (samples in each lane were 3T3-Ll in log phase, 3T3-Ll in confluent state, and 1 to 9 days after induction of adipocyte differentiation) This is 3T3-L1 of the eye, and # 103 to # 106, GAPDH was used as the probe.

As a result, as shown in Fig. 3, interestingly, in # 103, the expression level increased in the confluent, but the expression level decreased during the first 1 to 3 days after the initiation of differentiation induction, but was hardly observed. On the fourth day, the expression level increased again. In # 105, a high molecular weight band was observed until the second day after the differentiation induction, but only a low molecular weight band was observed from the third day. In # 104 and # 106, strong expression was observed up to 6 days after induction of differentiation, but the expression level decreased after 7 days.

[Example 6] Effect of adding insulin

Northern plot analysis was performed by the usual method. The samples in each lane are control adipocytes, adipocytes supplemented with 25 nM and 250 nM insulin, respectively. # 105, GAPDH was used as a probe.

As a result, as shown in FIG. 4, expression of # 105 was suppressed in adipocytes to which 250 nM insulin was added.

[Example 7] Expression of # 104 in cancer cell line

Northern plot analysis was performed by the usual method. The samples in each lane used the cell lines shown in FIG. 5, respectively. # 104 was used as a probe.

As a result, as shown in Fig. 5, some cancer cell lines (cancer cell lines) showed low molecular weight bands and some did not. Industrial applicability

According to the present invention, a gene encoding a novel protein derived from an adipocyte and having a signal sequence is provided. These genes may be important molecules involved in adipocyte differentiation due to their expression characteristics. The protein of the present invention is a target for drug development The compound that regulates the function of the protein of the present invention is expected to be applied as a pharmaceutical.

Claims

The scope of the claims

1. DNA according to any one of (a) to (f) below.

(a) DNA encoding a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 8 to 11

(b) DNA containing the coding region of the nucleotide sequence of any one of SEQ ID NOs: 1 to 7.

(c) one or more amino acids in the amino acid sequence of any one of SEQ ID NOs: 8 to 11 having substitution, deletion, insertion, and Z or added amino acid sequences, and SEQ ID NOs: 8 to 11 A DNA encoding a protein functionally equivalent to the protein comprising the amino acid sequence of any one of the above.

(d) one or more amino acids are substituted, deleted, inserted, or substituted in the amino acid sequence of the protein encoded by the full-length DNA corresponding to the DNA comprising the nucleotide sequence of any of SEQ ID NOs: 1, 2, and 7; And / or a DNA having an added amino acid sequence and encoding a protein functionally equivalent to the protein encoded by the full-length DNA.

(e) hybridizes with a DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 3 to 6 under stringent conditions, and functions as a protein comprising the amino acid sequence of SEQ ID NOs: 8 to L1. MA that encodes proteins that are equivalent to each other.

(f) hybridizes with a DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1, 2, and 7 under stringent conditions, and functionally interacts with a protein encoded by a full-length DNA corresponding to the DNA. DNA that codes for an equivalent protein.

2. A DNA encoding a partial peptide of a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 8 to 11.

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

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

5. A host cell carrying the DNA according to claim 1 or 2 or the vector according to claim 4.

6. The method for producing a protein or peptide according to claim 3, comprising a step of culturing the host cell according to claim 5, and recovering the expressed protein from the host cell or a culture supernatant thereof.

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

8. SEQ ID NO: A polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of any one of 1 to 7 or a complementary strand thereof.

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

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

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

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