JP2000037190A - Tissue-specific biologically active protein derived from mammal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA which codes for a tissue-specific biologically active protein derived from a mammal having a specific amino acid sequence or a part of it, is involved in the obesity and the appearance of complications accompanied with the 'internal organ'-type obesity, and can be used for treating and diagnosing these diseases. SOLUTION: This is a new DNA which codes for a tissue-specific biologically active protein derived from a mammal having the amino acid sequence shown by the formula or a part of it, is produced by an adipocyte which constitutes an adipose tissue which is deeply related to an onset of obesity and/or the appearance of complications accompanied with the obesity, particularly the 'internal organ'-type obesity (e.g. diabetes, hyperlipidemia, hypertension, arteriosclerosis, hyperuricemia caused by excess synthesis of uric acid, and sleep apnea syndrome), and is used, for example, for the production of proteins which are useful, for example, for treating and diagnosing these diseases. This DNA is obtained by extracting mRNA from the internal organ adipose tissue abscised from a patient with stomach cancer, preparing a cDNA library from the obtained tissue by the conventional method, followed by screening the obtained library using a partial sequence of a gene derived from the human internal organ adipose tissue as a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel physiologically active protein derived from mammals or a part thereof, a DNA encoding the protein or a part thereof, an expression vector containing the DNA, and a trait transformed with the vector. Transformed cells,
An antibody or a part thereof reactive to the protein or a part thereof, a cell producing the antibody, a pharmaceutical composition comprising the protein or a part thereof, a pharmaceutical composition comprising the antibody or a part thereof And transgenic non-human mammals.

[0002]

2. Description of the Related Art When mammals such as humans ingest more energy than their living bodies need for maintaining their lives, the surplus energy is stored in the body for a certain period of time. It has a life support mechanism that can be overcome if it is deficient. Adipose tissue plays the most important role as this energy storage organ, which is indispensable for the maintenance of life. However, in the over-nutrition state where food is overflowing as in modern advanced countries, the state of excessive storage of energy in adipose tissue occurs chronically, resulting in a state called so-called obesity.

[0003] Adipose tissue constituting adipose tissue is roughly classified into white adipose tissue composed of mature monocytic adipocytes and mainly responsible for storing nutrients (energy), and brown adipose tissue involved in heat production. Obesity is a condition in which the volume and number of white adipocytes increase, that is, the hypertrophy and hyperplasia of adipocytes exceed the normal range. In this monocytic adipocyte, the cytoplasm is occupied by a single lipid droplet, and the nucleus and organelles are pushed to the margins of the cell, completing the differentiation just storing lipid droplets. Although quiescent cells lost their ability to proliferate, they were supposed to be cells that have cell proliferation ability by constantly performing active metabolism while maintaining the balance between lipolysis and synthesis ( Differ
entiation, Vol. 31, p. 42-49, 1986; J. Lipid Res., Vo
l.29, p.1038-1045, 1987). In addition, recent studies at the molecular level of fat cells have revealed that fat cells produce and secrete various bioactive proteins (Science, Vol. 237, p. 405, 1987). Modern medicine, Vol.29, p.985-992, 1996).

On the other hand, obesity is not only a physical phenomenon but also an adult disease such as diabetes, hyperlipidemia, hypertension and / or arteriosclerosis or a common disease.
It is generally accepted that various diseases collectively referred to as se occur. However, studies on the etiology of obesity and its relationship to the disease state have not been sufficient so far. It was only captured on the simple quantitative side that it would improve if it did, and it was far from elucidating the causal relationship between obesity and various diseases.

However, studies of the scientific aspects of obesity in recent years have shown that the relationship between obesity and the pathology is not the quantitative involvement of fat accumulation, but rather the accumulation of abdominal visceral fat ("visceral fat obesity"). Has also been suggested to be strongly involved in the complications of various complications associated with obesity, and in 1995 Friedman
Obesity gene expressed by fat cells (ob gene)
And the discovery of leptin as a product,
The study of obesity and the mechanism of the development of various obesity-dependent pathological conditions has considered fat cells as secretory organs of physiologically active substances.
In particular, bioactive substances that are significantly expressed and produced in visceral adipose tissue will be identified and elucidated at the molecular level, and the relationship between the molecules and pathological conditions will be analyzed (Phar
ma Medica, Vol.15, No.9, p.11-12, 1997).

[0006] Physiologically active substances produced and secreted from adipocytes are sometimes collectively referred to as adipocytokines. From previous studies, adipocytes are a product of the aforementioned ob gene and Leptin responsible for regulation (Nature, Vol. 372, p. 425, 1994; Science,
Vol.269, p.543-546, 1995), Type-I plasminogen activator inhibitor (PAI-I) involved in the control of local growth of adipose tissue (Progress in Obesity Research,
p.197-200, 1995), tumor necrosis factor α (TNFα) involved in insulin resistance (Science, Vol.259, p.87-91, 19)
93) and the complement factor D, adipsin (J. B.
iol. Chem., Vol. 258, p. 10083, 1983; Science, Vol. 2
37, p.402-404, 1987).

[0007] Recent human genome analysis has revealed that expression of genes encoding various other protein molecules is observed in adipocytes. For example, ap
M1 (Adipose Most Abundant Gene Transcript-1) (Bio
chem. Biopys. Res. Commun., Vol. 221, p. 286-289, 19
96), apolipoprotein-J (apolipoprotein-
J), retinal epithelial differentiation factor (PDEF) involved in neural retina differentiation and development
r), complement component C1r, junD, apolipoprotein D (apol
ipoprotein-D), lecithin-cholesterol acyltransfe
rase), insulin-like growth factor binding protein (insulin-
like growth factor binding protein-3 (IGFBP-3), heparin binding EGF-like
growth factor) and carboxypeptidase E (carbo)
xypeptidase-E) has been confirmed to be expressed in genes encoding known proteins (Ayumi of Medicine, Vol. 184,
No. 6, p. 534-538, 1998).

[0008] Visceral fat obesity may be deeply involved in various diseases such as diabetes, hyperlipidemia, hypertension, arteriosclerosis, hyperuricemia caused by excessive uric acid synthesis, and sleep apnea syndrome. (Ayumi of Medicine, Vol.
184, No. 6, p. 534-538, 1998) For example, in diabetes associated with obesity, hyperlipidemia, hypertriglyceridemia and arteriosclerosis, the above-mentioned TNFα produced by fat cells causes the onset. It has been reported to be deeply involved. Regarding the relationship between obesity and diabetes, for example, insulin receptor substrate-1 (one step of signal transduction via the insulin receptor of insulin, which is extremely important for promoting the uptake and utilization of sugar in cells) is described. -1; IRES
-1) Tyrosine phosphorylation is caused by TNFα secreted by adipocytes
It has been reported that cells become insulin resistant by being inhibited by serine phosphorylation and causing insulin-independent diabetes in obese patients. Insulin resistance in obese patients
It has also been suggested that it occurs by interaction with PPARγ, a nuclear transcription factor, and PC-1, which is a protein that suppresses the tyrosine kinase activity of the insulin receptor (Pharma Medica, Vol. 15, No. 9). , p.33-37, 1997).

Regarding the relationship between obesity and hyperlipidemia, hypertriglyceridemia and arteriosclerosis, for example, insulin plays an important role not only in glucose but also in lipid metabolism (increased cholesterol absorption in the intestinal tract, Delays LDL metabolism due to a decrease in body activity, delays triglyceride metabolism due to a decrease in LPL (lipoprotein lipase) activity, and increases triglyceride production in the liver). It has been reported that the action of the constituent cells on the body causes insulin resistance, resulting in abnormalities in lipid metabolism and induction of hyperlipidemia, hypertriglyceridemia, and arteriosclerosis (Pharma Medica, Vol. .15, No.9, p.39-44, 199
7).

As described above, fat cells and / or tissues in which the dynamics of fat cells are deeply involved, particularly complications associated with obesity (diabetes, hyperlipidemia, hypertension, arteriosclerosis, hyperuric acid caused by excessive uric acid synthesis) To identify the biologically active substances that are expressed and produced in visceral adipose tissue, which are suggested to be deeply involved in the onset of blood disorders, sleep apnea syndrome, etc., and elucidate the biological functions of the biologically active substances That opens the way for the development of drugs that prevent or treat the development of obesity, which can be said to be a modern disease, and the complications associated with obesity,
The development of drugs based on the analysis of such fat cells and adipose tissue (particularly, visceral adipose tissue) at the molecular level has attracted attention as the most important issue in medicine and pharmacy toward the 21st century. As a method for identifying a gene that is significantly expressed in a certain tissue or cell, when the tissue or cell is a pathological tissue or pathological cell collected from a lesion,
Differential expression in which the expression states of various genes in the pathological tissues and cells are compared with the corresponding gene expression states in normal tissues and cells to identify those genes whose expression is different. A comparative study at the gene level called a differential display method has been used as a useful method (Nucleic Acids Research, Vol. 21, No. 18, p. 4272-4).
280, 1993; Science, Vol. 257, p. 967-971, 1992; G
enomics, Vol.36, p.316-319, 1996).

When the tissue or cell to be analyzed is a normal tissue or cell itself, such as a visceral adipose tissue or an adipocyte, the expression state of the gene in the visceral adipose tissue or the adipocyte is determined. By comparing the expression state of the gene in other normal tissues and other normal cells by the differential display method in the same manner, by identifying the gene showing a difference in gene expression between the two, the visceral fat tissue or Genes that are significantly expressed in adipocytes can be identified.

[0012]

SUMMARY OF THE INVENTION Genes having significant expression in visceral adipose tissue or adipocytes and protein molecules derived from these genes are known to be obese (including visceral obesity) and / or diabetic atherosclerosis associated with obesity. It may be closely related to various diseases such as illness. The present invention provides a method for identifying obesity and / or obesity, particularly complications associated with obesity, particularly visceral obesity (diabetes, hyperlipidemia,
Hypertension, arteriosclerosis, hyperuricemia caused by excessive uric acid synthesis, sleep apnea syndrome, etc.).

[0013]

Means for Solving the Problems The present inventors have conducted intensive studies on the analysis of genes that are significantly expressed in human visceral adipose tissue.
Human gene encoding two protein molecules (clone AG
1102, AA3901 and AA3401), and completed the present invention. The genes encoding the three novel proteins of the present invention have been identified from adipose tissue such as human visceral adipose tissue, and each have a unique tissue specificity. Complications (diabetes,
It is considered to be a gene encoding a protein molecule associated with the onset and progression of hyperlipidemia, hypertension, arteriosclerosis, hyperuricemia due to excessive uric acid synthesis, sleep apnea syndrome, and the like.

Clone AG1102 has the following features. (1) Significant expression is observed in female organ tissues such as uterus and ovarian mammary gland, mammary gland, and visceral adipose tissue. (2) Northern Blotting
As a result, in the human tissue, a band of about 3.3 kb
RNA expression is observed. (3) The open reading frame (ORF) is 2,097
Has a base sequence of SEQ ID NO: 1 (SEQ ID NO: 1)
F encodes an amino acid sequence composed of 699 amino acids as a whole including a signal peptide composed of 16 amino acids, and has a molecular weight of about 79.8 kDa (calculated value) (SEQ ID NO: 2). (4) The amino acid sequence of the coding protein contains the following characteristic structure. An important sequence in cell-cell adhesion, a characteristic common to collagen, fibronectin, fibrinogen, vitronectin, etc., integrin, an adhesion molecule, and extracellular matrix (ECM) molecules, which play an important role in cell-cell adhesion. Arg-Gl
y-Asp (RGD) sequence. Von Willebrand factor C (VWFC) domai
n). It is thought to be involved in various functions such as intracellular signal transduction, cell adhesion, cell differentiation, DNA repair and RNA processing, and is common to G protein-coupled receptors, ECM molecules, tyrosine kinase receptors and nerve growth factors. A structure called a leucine-rich repeat in which hydrophobic amino acids such as leucine, isoleucine and valine appear repeatedly. (5) The coding protein is mouse bone proteoglycan II precurso II
r; Proc. Natl. Acad. Sci., USA, Vol.83, p.7683-768
7, 1986), bovine keratokan (Keratokan; J. Biol. C)
hem., Vol.271, p.9759-9763, 1996) and rat decorin (Decorin; Eur. J. Cell. Biol., Vol.59, p.314).
-321, 1992) and 33.7%, 33.2% and 33.0%, respectively. (6) The genomic DNA corresponding to the clone DNA is a hereditary sensory neuropathy type I (hereditary sensory r.
adicular neuropathy type I) and infant neuropathy (infantil
e neurosis, infantile neuronophthisis) and has a position on the chromosome represented by 9q22.3 known as the causative site of diseases. From the above features (1) to (6), the protein molecule encoded by the clone AG1102 is important in the mutual recognition mechanism between cells, between cells and various ECM molecules, and between ECM molecules. It is considered to have a role to play a role.

Clone AA3901 has the following characteristics. (1) Specific expression is observed in visceral adipose tissue and pancreas, especially in pancreas. (2) Northern Blotting
MRNN as a band of about 1.8 kb in the pancreas
Expression of A is observed. (3) Open reading frame (ORF) is 1,092
Has a base sequence of SEQ ID NO: 3 (SEQ ID NO: 3)
F encodes an amino acid sequence composed of 364 amino acids and has a molecular weight of about 38.7 kDa (calculated) (SEQ ID NO: 4). (4) The amino acid sequence of the coding protein has no structural features such as a signal sequence, a known motif, and a transmembrane site. (5) The coding protein has no amino homology with any known protein molecule. (6) The genomic DNA corresponding to the clone DNA is 15q2
It has a position on the chromosome represented by 2. Clone AA390
1) Although the structural characteristics such as those of known protein molecules are not seen, the fact that significant expression is observed only in the pancreas where the islet of Langerhans, which is an insulin secreting organ, is observed is particularly medical and pharmaceutical. Of note, this tissue specificity suggests that the protein molecule encoded by clone AA3901 is a molecule involved in controlling the development of diabetes.

The clone AA3401 has the following characteristics. (1) Significant expression is observed in visceral adipose tissue. (2) Northern Blotting
As a result, mRNA expression was observed as a band of about 4.4 kb in the visceral fat tissue. (3) The open reading frame (ORF) is 2,832
Has a base sequence of SEQ ID NO: 5 (SEQ ID NO: 5)
F encodes an amino acid sequence composed of 944 amino acids and has a molecular weight of about 107.4 kDa (calculated value) (SEQ ID NO: 6). (4) The amino acid sequence of the coding protein has no structural features such as a signal sequence, a known motif, and a transmembrane site. (5) The coding protein is human endosome-assoc
iated protein (J. Biol. Chem., Vol.270, p.13503-13
511, 1995) and the chicken myosin heavy chain (myosin heav).
y chain; J. Mol. Biol., Vol. 198, p. 143-157, 1987)
Α-helix (coiled-coil structure)
It is predicted to have an α-helical structure in part because it has 21.6% and 22.6% amino homology with the portion, respectively. (6) The genomic DNA corresponding to the clone DNA is 1p12
Or it has a position on the chromosome represented by 1q21.1. Clone AA3401 has a tissue specificity in which significant expression is observed only in visceral adipose tissue, although there are no other structural features such as those of known protein molecules other than the presumed partial α-helix structure. By nature, the protein molecule encoded by clone AA3401 is associated with complications associated with visceral fat obesity, such as diabetes, hyperlipidemia, hypertension, arteriosclerosis, hyperuricemia due to hyperuric acid synthesis, sleeplessness. It is thought to be a molecule involved in controlling the onset of respiratory syndrome and the like.

That is, from the above-mentioned characteristics of the gene of the present invention and the protein molecule encoded thereby, the gene (DNA) of the present invention or a part thereof, the protein or a part thereof, and the protein or a part thereof Antibody or a part thereof is caused by visceral fat obesity or a complication associated with visceral fat obesity (eg, diabetes, hyperlipidemia, hypertension, arteriosclerosis, excess uric acid synthesis) as described below. It is useful as a drug for preventing and / or treating hyperuricemia, sleep apnea syndrome, etc.), and also as a tool for test research or drug development.

(1) When the overexpression of the protein molecule encoded by the gene (DNA) of the present invention acts on the onset and progress of the above-mentioned disease, in this case, the gene or the protein or Some of them (fragments) can be targeted for drug development for treatment and prevention of those diseases, for example, drugs that inhibit the transcription of the gene into mRNA, Design of drugs such as drugs that inhibit translation, drugs that inhibit the biological activity of the protein, drugs that suppress the production of the protein, or drugs that inhibit the binding of the protein to its receptor or interaction with other molecules , Screening or as a tool for activity measurement (assay). For example, the DNA can be used in a so-called reporter gene assay (repo), which is commonly used in screening and activity measurement (assay) of such drugs.
so-called high-throughput screening (High-throughput screening) in which screening is automatically performed using a machine (robot) based on the reporter gene assay and the reporter gene assay.
Throughput Screening) (Tissue Culture Engineering, Vol.23, No.13, p.521-524; U.S. Pat.
No. 670,113).

The reporter gene assay comprises a DNA encoding a protein molecule to be a drug target, a DNA encoding an expression control region of the DNA, and a DNA encoding a reporter protein molecule that emits fluorescence such as luciferase.
A transformant obtained by transforming a cell commonly used in the production of a recombinant protein with an expression vector into which the reporter protein molecule can be expressed in a manner dependent on the expression of the target protein. A cell is contacted with a test compound, and the amount of the target protein produced depending on the action of the compound is measured indirectly by measuring the amount of fluorescence emitted by the reporter protein produced simultaneously with the production of the target protein. This is a method for analyzing whether or not the compound affects the production of a target protein by measuring the target protein (US Pat. No. 5,436,128; US Pat. No. 5,436,128).
5,401,629).

Examples of the above-mentioned drug that inhibits the transcription of the DNA (gene) of the present invention into mRNA or the drug that inhibits the translation of the mRNA into the protein of the present invention include antisense drugs. That is, it is possible to design an antisense DNA or an antisense RNA based on the DNA sequence of the present invention or an mRNA sequence corresponding to the sequence, and the antisense DNA and the antisense RNA are the same as the antisense sequence, respectively. By binding to DNA or mRNA having a complementary sequence, it is useful as an antisense drug by a mechanism that inhibits transcription from DNA to mRNA or translation from mRNA to protein. As the above-mentioned agent for inhibiting the biological activity of the protein of the present invention, an agent for inhibiting the production of the protein, or an agent for inhibiting the binding of the protein to a receptor or the interaction with another molecule, a peptide antagonist, Examples include antibodies or low molecular weight compounds.

That is, a peptide antagonist can be designed based on the amino acid sequence of the protein of the present invention, and the peptide antagonist competes with the binding of the protein of the present invention to another molecule (eg, receptor, ligand). Is useful as a drug that inhibits the signal or secondary reaction generated by the protein of the present invention upon binding to the molecule, thereby indirectly preventing the biological function of the protein of the present invention from being exerted. It is. Further, an antibody (particularly, a monoclonal antibody) against the protein of the present invention or a part thereof is useful as an antibody drug by inhibiting (neutralizing) the biological activity of the protein by binding to the protein of the present invention. is there.

(2) The expression of a protein molecule encoded by the gene (DNA) of the present invention is increased depending on the onset and progress of the above-mentioned disease, and the protein molecule is regulated by the onset and progress of the disease. In this case, the protein of the present invention or a part thereof (for example, a biologically active region) itself is useful as a pharmaceutical. Further, the gene (DNA) of the present invention can be administered to a patient by gene therapy, and is itself useful as a pharmaceutical. Further, as in the case of the above (1), the present invention
DNA (gene) or protein or a part thereof (fragment) can be used as a target for drug development for treatment and prevention of those diseases. It is useful as a tool for drug design, screening or activity measurement (assay). For example, similar to the above (1), the DNA may be a so-called reporter gene assay commonly used in screening and activity measurement (assay) of such a drug, and a so-called automatic machine-based screening based on the reporter gene assay. Very useful in high-throughput screening.

In addition to the above (1) and (2), the gene (DNA), protein, and antibody of the present invention are used to search for proteins (receptors and ligands) that interact with the protein of the present invention, It is useful as a test and research reagent in the elucidation of functions and in drug development targeting the ligand. In addition, a transgenic animal as a model animal can be prepared by introducing a human-derived DNA, which is one of the DNA aspects of the present invention, into a mammal other than a human such as a mouse.

Similarly, based on the genetic information of DNA derived from rabbits or mice included in the embodiment of the DNA of the present invention, the corresponding endogenous gene is disrupted (inactivated) to produce a model animal ( Knockout animals). By analyzing the physical, biological, pathological and genetic characteristics of these model animals, it becomes possible to elucidate the functions of the genes and proteins according to the present invention. Furthermore, by crossing the transgenic animal with the model animal in which the endogenous gene has been disrupted in this way, it is possible to prepare a model animal having only the human-derived gene (DNA) of the present invention. By administering a drug (compound, antibody, etc.) targeting the introduced human gene to this model animal, the therapeutic effect of the drug can be evaluated.

The present invention provides, for the first time, the following DNAs, proteins, expression vectors, transformants, antibodies, pharmaceutical compositions, and transgenic non-human mammals. (1) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2 or a part thereof. (2) a DNA comprising the nucleotide sequence of nucleotides 74 to 2170 in the nucleotide sequence of SEQ ID NO: 1; (3) DNA having the nucleotide sequence of SEQ ID NO: 1
That hybridizes under highly stringent conditions
A. (4) a protein having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence, or a part thereof; (5) D according to any one of the above (1) to (3)
An expression vector containing NA. (6) A transformed cell transformed with the expression vector according to (5). (7) An antibody or a part of an antibody reactive with the protein of (4) or a part thereof. (8) The antibody or a part of the antibody according to (7), wherein the antibody is a monoclonal antibody. (9) A cell that produces a monoclonal antibody reactive with the protein according to (4) or a part thereof. (10) The cell according to (9), wherein the cell is a fusion cell obtained by fusing a non-human mammal-derived B cell capable of producing the monoclonal antibody with a mammal-derived myeloma cell. The cell according to (1). (11) a gene transformed by introducing the DNA into the cell, either the DNA encoding the heavy chain of the monoclonal antibody or the DNA encoding the light chain thereof, or both DNAs; The cell according to the above (9), which is a recombinant cell. (12) A pharmaceutical composition comprising the protein or a part thereof according to (4) and a pharmaceutically acceptable carrier. (13) A pharmaceutical composition comprising the antibody or a part of the antibody according to (7) or (8) and a pharmaceutically acceptable carrier. (14) base number 74 of the base sequence set forth in SEQ ID NO: 1
A transgenic non-human mammal, wherein a DNA comprising the nucleotide sequence of 1 to 2170 is incorporated into an endogenous gene of the non-human mammal. (15) DNA encoding a protein having the amino acid sequence of SEQ ID NO: 4 or a part thereof. (16) base number 13 of the base sequence described in SEQ ID NO: 3
DNA comprising the nucleotide sequence of 1 to 1221. (17) DN having the nucleotide sequence set forth in SEQ ID NO: 3
D that hybridizes to A under stringent conditions
NA. (18) a protein having the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence substantially identical to the amino acid sequence, or a part thereof; (19) An expression vector containing the DNA according to any one of (15) to (17). (20) A transformed cell transformed with the expression vector according to (19). (21) An antibody or a part of an antibody reactive with the protein of (18) or a part thereof. (22) The antibody or a part of the antibody according to (21), wherein the antibody is a monoclonal antibody. (23) A cell that produces a monoclonal antibody reactive with the protein or a part thereof according to (18). (24) The cell according to (23), wherein the cell is a fusion cell obtained by fusing a non-human mammal-derived B cell capable of producing the monoclonal antibody with a mammal-derived myeloma cell. The cell according to (1). (25) A gene transformed by introducing the DNA into the cell, either the DNA encoding the heavy chain of the monoclonal antibody or the DNA encoding the light chain thereof, or both DNAs. The cell according to the above (23), which is a recombinant cell. (26) A pharmaceutical composition comprising the protein according to (18) or a part thereof, and a pharmaceutically acceptable carrier. (27) A pharmaceutical composition comprising the antibody or a part of the antibody according to (21) or (22), and a pharmaceutically acceptable carrier. (28) base number 13 of the base sequence described in SEQ ID NO: 3
A transgenic non-human mammal, characterized in that a DNA comprising the nucleotide sequence of 1 to 1221 has been incorporated into an endogenous gene of the non-human mammal. (29) DNA encoding the protein having the amino acid sequence of SEQ ID NO: 6 or a part thereof. (30) base number 35 of the base sequence described in SEQ ID NO: 5
DNA having a base sequence of 1 to 3180. (31) DN having the base sequence of SEQ ID NO: 5
D that hybridizes to A under stringent conditions
NA. (32) a protein having the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence substantially identical to the amino acid sequence, or a part thereof; (33) An expression vector comprising the DNA according to any of (29) to (31). (34) A transformed cell transformed with the expression vector according to (33). (35) An antibody or a part of an antibody reactive with the protein of (32) or a part thereof. (36) The antibody or a part of the antibody according to (35), wherein the antibody is a monoclonal antibody. (37) A cell that produces a monoclonal antibody reactive with the protein according to (32) or a part thereof. (38) The cell according to (37), wherein the cell is a fusion cell obtained by fusing a non-human mammal-derived B cell capable of producing the monoclonal antibody with a mammal-derived myeloma cell. The cell according to (1). (39) A gene transformed by introducing the DNA into the cell, either the DNA encoding the heavy chain of the monoclonal antibody or the DNA encoding the light chain thereof, or both DNAs. The cell according to the above (37), which is a recombinant cell. (40) A pharmaceutical composition comprising the protein according to (32) or a part thereof, and a pharmaceutically acceptable carrier. (41) A pharmaceutical composition comprising the antibody or a part of the antibody according to (35) or (36), and a pharmaceutically acceptable carrier. (42) base number 35 of the base sequence described in SEQ ID NO: 5
A transgenic non-human mammal, characterized in that a DNA comprising a nucleotide sequence of 1 to 3180 is incorporated into an endogenous gene of the non-human mammal.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the meanings of the terms used in the present invention, and general methods for producing DNAs, proteins, antibodies, pharmaceutical compositions, transgenic non-human mammals and the like of the present invention will be clarified. Hereinafter, the present invention will be described in detail. The “protein” of the present invention includes humans, cows, pigs, goats, sheep, chickens, rabbits, rats,
It is a protein derived from mammals such as hamster, guinea pig, and mouse, preferably a protein derived from human, rabbit, rat or mouse, and particularly preferably a protein derived from human. In a particularly preferred embodiment,
(1) a protein having the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence; (2) an amino acid sequence represented by SEQ ID NO: 4 or a protein substantially identical to the amino acid sequence A protein having an amino acid sequence; and (3) a protein having the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence substantially identical to the amino acid sequence.

As used herein, "having substantially the same amino acid sequence" means that the amino acid sequence has substantially the same biological properties as the protein comprising the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. A protein having an amino acid sequence in which a plurality of amino acids in the sequence, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids are substituted, deleted and / or modified, and It is meant to include a protein having an amino acid sequence to which a plurality of amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids are added.

The “part of the protein” of the present invention means any partial sequence (fragment) in the amino acid sequence of the protein of the present invention described above, for example, 5 to 300.
A partial sequence having 5 amino acid residues, specifically a partial sequence having 5 to 200 amino acid residues, more specifically a partial sequence having 5 to 100 amino acid residues,
More specifically, a partial sequence having 5 to 50 amino acid residues is exemplified. Preferably, a partial sequence containing a site necessary for the protein of the present invention to exert its biological function or a site where the protein of the present invention binds or interacts with another protein molecule (receptor or ligand) is included. Can be mentioned.

In the specification and the drawings of the present application, three letters or one letter of the alphabet used to represent amino acids mean the following amino acids, respectively. (Gly / G) glycine, (Ala / A) alanine, (Val
/ V) valine, (Leu / L) leucine, (Ile / I) isoleucine, (Ser / S) serine, (Thr / T) threonine,
(Asp / D) aspartic acid, (Glu / E) glutamic acid,
(Asn / N) asparagine, (Glu / Q) glutamine, (Ly
(s / K) lysine, (Arg / R) arginine, (Cys / C) cysteine, (Met / M) methionine, (Phe / F) phenylalanine, (Tyr / Y) tyrosine, (Trp / W) tryptophan, (His / H) histidine, (Pro / P) proline.

The protein of the present invention or a part thereof can be obtained by a known method known in the technical field such as a chemical synthesis method or a cell culture method, or a modification method thereof, in addition to a gene recombination technique described below. Can be produced by appropriately using The DNA of the present invention is a DNA encoding the above-described protein of the present invention or a part thereof, and includes any base sequence capable of encoding the protein of the present invention or a part thereof. Preferably, it is a DNA encoding the human-derived protein of the present invention. Specific embodiments include the following DNAs.

(1) A protein having the amino acid sequence of SEQ ID NO: 2 (or a part thereof), or a plurality of amino acids, preferably 1 to 10 amino acids in the amino acid sequence of the protein (or a part thereof) Amino acids, particularly preferably 1 to 5 amino acids are substituted, deleted and / or modified, or a plurality of amino acids in the amino acid sequence, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids DNAs each encoding a bioequivalent obtained by inserting said amino acid. (2) a protein (or a part thereof) having the amino acid sequence described in SEQ ID NO: 4, or a plurality of amino acids in the amino acid sequence of the protein (or a part thereof);
Preferably 1 to 10 amino acids, particularly preferably 1 amino acid
By substitution, deletion and / or modification of from 1 to 5 amino acids, or insertion of a plurality of amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, into the amino acid sequence DNA encoding each of the resulting biological equivalents. (3) a protein (or a part thereof) having the amino acid sequence of SEQ ID NO: 6, or a plurality of amino acids in the amino acid sequence of the protein (or a part thereof);
Preferably 1 to 10 amino acids, particularly preferably 1 amino acid
By substitution, deletion and / or modification of from 1 to 5 amino acids, or insertion of a plurality of amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, into the amino acid sequence DNA encoding each of the resulting biological equivalents. (4) a DNA having the nucleotide sequence of SEQ ID NO: 1
That hybridizes under highly stringent conditions
A. (5) a DNA having the nucleotide sequence of SEQ ID NO: 3
That hybridizes under highly stringent conditions
A. (6) a DNA having the nucleotide sequence of SEQ ID NO: 5
That hybridizes under highly stringent conditions
A.

More specifically, (1) DNA containing the nucleotide sequence of base numbers 74 to 2170 of the nucleotide sequence shown in SEQ ID NO: 1, (2) base number 131 of the nucleotide sequence shown in SEQ ID NO: 3 DNA comprising the nucleotide sequence of from 1 to 1221, and (3)
Nucleotide numbers 351 to 318 of the nucleotide sequence described in SEQ ID NO: 5
It is a DNA containing a base sequence of 0. The DNA of the present invention
It includes genomic DNA or cDNA, and any of their antisense DNA. In addition, the DN
A includes DNA composed of any codons that encode the same amino acid.

Here, the “stringent conditions” include, for example, the following conditions.
For example, when hybridization is performed under 0.9% NaCl using a probe having 50 bases or more, an estimate of the temperature (Tm) at which 50% dissociation occurs is obtained from the following formula,
The hybridization temperature can be set as in the following formula. Tm = 82.3 ° C. + 0.41 × (G + C)% − 500 / n−0.61 × (formamide)% (n indicates the number of bases of the probe.) Temperature = Tm−25 ° C. and a probe of 100 bases or more When (G + C = 40 to 50%) is used, it is an indication that Tm changes as in the following (1) and (2). (1) Tm is reduced by about 1 ° C. for each 1% mismatch. (2) For every 1% of formamide, Tm is reduced by 0.6 to 0.7 ° C. Therefore, the temperature conditions in the case of a combination of perfectly complementary strands can be as follows. (A) 65 to 75 ° C (without formamide addition) (B) 35 to 45 ° C (in the presence of 50% formamide) The temperature conditions for the combination of incomplete complementary chains can be as follows: . (A) 45 to 55 ° C (no formamide added) (B) 35 to 42 ° C (in the presence of 30% formamide) The temperature condition when using a probe having 23 bases or less can be 37 ° C. The following formula can be used as a guide. Temperature = 2 ° C x (number of A + T) + 4 ° C x (number of C + G)-5 ° C

[0034] The DNA of the present invention may be obtained by any method. For example, complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA obtained by chemical synthesis, RN
It also includes all DNAs obtained by amplifying by PCR using A or DNA as a template and DNAs constructed by appropriately combining these methods. The DNA encoding the protein of the present invention can be obtained by a method of cloning cDNA from mRNA of the protein of the present invention, a method of isolating and splicing genomic DNA, a method of chemical synthesis, and the like according to a conventional method.

(1) For example, mR of the protein of the present invention
The following method is exemplified as a method for cloning cDNA from NA. First, the protein of the present invention is expressed and expressed.
An mRNA encoding the protein of the present invention is prepared from the above-mentioned tissue or cell to be produced. The mRNA is prepared, for example, by the guanidine thiocyanate method (Chirgwin et al., Biochemi.
stry), Vol. 18, p. 5294, 1979), using total RNA prepared by a known method such as the hot phenol method or the AGPC method, using oligo (dT) cellulose or poly-U.
-It can be carried out by subjecting to affinity chromatography using Sepharose or the like.

Then, using the obtained mRNA as a template, a known method, for example, using reverse transcriptase, for example, the method of Okayama et al. (Molecular Cell Biology (Mol. Cell)
l. Biol.), Volume 2, p. 161, 1982 and the same magazine.
3, 280, 1983) and Hoffma
n) et al. (Gene, 25, 263)
Page, 1983), and the like.
A is converted to double-stranded cDNA. This cDNA is incorporated into a plasmid vector or a phage vector and transformed into Escherichia coli, or after in vitro packaging, transfected into Escherichia coli to prepare a cDNA library.

The plasmid vector used here is not particularly limited as long as it can be replicated and maintained in the host, and any phage vector can be used as long as it can be propagated in the host. PUC19, λgt10, and cloning vectors used in a conventional manner.
λgt11 and the like are exemplified. However, when subjected to the immunological screening described below, a vector having a promoter capable of expressing the gene encoding the protein of the present invention in a host is preferable.

As a method of incorporating cDNA into a plasmid, for example, the method of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second e
dition), Cold Spring Harbor Laboratory, page 1.53, 19
1989). As a method for incorporating cDNA into a phage vector, the method of Hyunh et al. (DNA cloning, practical approach (DNA Cloning, a practical approach)
h), Vol. 1, p. 49, 1985).
For convenience, a commercially available cloning kit (for example, Takara Shuzo) can also be used. Recombinant plasmids and phage vectors thus obtained can be used for prokaryotic cells (for example, E. coli: HB101, DH5α or MC
1061 / P3).

The method for introducing a plasmid into a host is described in (Molecular Cloning, A Laboratory
Manual (Molecular Cloning, A Laboratory Manua
l, second edition), Cold Spring Harbor Laboratory, 1.74
1989), the calcium chloride method, the calcium chloride / rubidium chloride method, and the electroporation method. Examples of a method for introducing a phage vector into a host include a method in which phage DNA is packaged in vitro and then introduced into a grown host. In vitro packaging can be easily performed by using a commercially available in vitro packaging kit (for example, Stratagene, Amersham, etc.).

The cDNA library encoding the protein of the present invention was obtained from the cDNA library prepared by the above method.
The method of isolating A can be performed by combining general cDNA screening methods. For example, after separately chemically synthesizing oligonucleotides that are considered to correspond to the amino acid sequence of the protein of the present invention,
This was labeled with ³² P or [α- ³² P] dCTP to form a probe, and was used in a known colony hybridization method (Crunstein et al., Proceedings of National Academy of Sciences (Proc. Natl. Ac.)).
id. Sci. USA), vol. 72, p. 3961, 1975.
Years) or plaque hybridization (Molecu
lar Cloning, A Laboratory Manual, second edition,
Cold Spring Harbor Laboratory, 2.108, 198
9 years), a method for screening a clone containing the cDNA of interest, a method for preparing a PCR primer, amplifying a specific region of the protein of the present invention by the PCR method, and selecting a clone having a DNA fragment encoding the region. And the like.

When a cDNA library prepared using a vector capable of expressing cDNA (for example, λgt11 phage vector) is used, an antigen-antibody reaction using an antibody reactive with the protein of the present invention is used. Thus, a desired clone can be selected. When a large number of clones are processed, it is preferable to use a screening method utilizing a PCR method. The nucleotide sequence of the DNA thus obtained can be determined by the Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA. 74: 560, 1977) or phage M.
Method for terminating dideoxynucleotide synthesis using No. 13 (Sanger et al., Proc. Natl. Acad. Sci. US
A., Vol. 74, pp. 5463-5467, 1977)
Can be determined by The gene encoding the protein of the present invention can be obtained by cutting out all or a part of the clone obtained as described above using a restriction enzyme or the like.

(2) As a preparation method by isolating a DNA encoding the protein of the present invention from genomic DNA derived from a cell expressing the protein of the present invention as described above, for example, the following method is used. Illustrated. The cells are preferably lysed using SDS or proteinase K and the like, and the extraction with phenol is repeated to deproteinize the DNA. The RNA is digested, preferably by ribonuclease. The obtained DNA is partially digested with an appropriate restriction enzyme, and the obtained DNA fragment is amplified with an appropriate phage or cosmid to prepare a library. Then, a clone having the sequence of interest is replaced with, for example, radiolabeled DN.
A gene is detected by a method using the A probe, etc., and all or part of the gene encoding the protein of the present invention is cut out from the clone with a restriction enzyme or the like and obtained. To obtain cDNA encoding human-derived protein, a cosmid library ("Lab Manual Human Genome Mapping", edited by Masaaki Hori and Yusuke Nakamura, Maruzen Publishing) into which human genomic DNA (chromosomal DNA, genomic DNA) is further introduced. By preparing and screening the cosmid library, a positive clone containing the DNA of the coding region of the target protein is obtained, and using the coding DNA cut out from the positive clone as a probe,
It can also be prepared by screening the aforementioned cDNA library.

(3) The production of the DNA of the present invention by chemical synthesis can be carried out according to a conventional method based on the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5. The present invention further relates to a recombinant vector containing a DNA encoding the above-mentioned protein of the present invention. The recombinant vector of the present invention is not particularly limited as long as it can replicate and maintain or self-replicate in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and phage vectors.

The recombinant vector is a vector for recombination (plasmid DNA) which is conveniently available in the art.
And bacterial phage DNA) by ligation of a DNA encoding the protein of the present invention by a conventional method. Specifically, the recombination vector used is, for example, a plasmid derived from E. coli.
Examples of yeast-derived plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19 include pSH19 and pSH15, and examples of Bacillus subtilis-derived plasmids include pUB110, pTP5, and pC194. As phage, bacteriophage such as λ phage, further retrovirus,
Animal and insect viruses (pVL1393, manufactured by Invitrogen) such as vaccinia virus and nucleopolyhedrovirus are exemplified.

For the purpose of expressing the DNA encoding the protein of the present invention to produce the protein of the present invention,
Expression vectors are useful. The expression vector is not particularly limited as long as it has a function of expressing the gene encoding the protein of the present invention in various prokaryotic and / or eukaryotic host cells and producing these proteins. For example, pMAL C2, pEF-BOS (Nucleic Acid Research, Vol. 18, p. 5322, 1990, etc.) or pME18S (Experimental Medicine separate volume "Genetic Engineering Handbook", 1992, etc.) and the like can be mentioned. Can be.

When a bacterium, particularly Escherichia coli, is used as a host cell, the expression vector generally contains at least a promoter-
It is composed of an operator region, a start codon, a DNA encoding the protein of the present invention, a stop codon, a terminator region, and a replicable unit. When yeast, animal cells or insect cells are used as a host, the expression vector preferably contains at least a promoter, an initiation codon, a DNA encoding the protein of the present invention, and a stop codon. In addition, DNA encoding a signal peptide, an enhancer sequence, 5 'and 3' untranslated regions of a gene encoding the protein of the present invention, a splicing junction, a polyadenylation site, a selectable marker region or a replicable unit, etc. May be included. Further, it may contain a gene amplification gene (marker) which is generally used depending on the purpose.

A promoter-operator region for expressing the protein of the present invention in bacteria includes a promoter, an operator and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is a bacterium belonging to the genus Escherichia, preferably a Trp promoter, lac promoter, recA promoter, λP
Those containing the L promoter, lpp promoter, tac promoter and the like are exemplified. As a promoter for expressing the protein of the present invention in yeast, PH
05 promoter, PGK promoter, GAP promoter, and ADH promoter. When the host is a bacterium belonging to the genus Bacillus, examples include the SL01 promoter, SP02 promoter, and penP promoter. When the host is a eukaryotic cell such as a mammalian cell, SV
40-derived promoter, retrovirus promoter, heat shock promoter and the like. Preferably, SV-40 is a retrovirus. However, it is not particularly limited to these. Use of an enhancer is also an effective method for expression.

A preferred initiation codon is, for example, methionine codon (ATG). Examples of the stop codon include common stop codons (eg, TAG, TGA, TAA). As the terminator region, a commonly used natural or synthetic terminator can be used.

[0049] A replicable unit refers to its entire D in the host cell.
A DNA capable of replicating an NA sequence, which includes a natural plasmid, an artificially modified plasmid (a DNA fragment prepared from the natural plasmid), a synthetic plasmid, and the like. Suitable plasmids include plasmid pBR322 in E. coli,
Alternatively, an artificially modified product thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) is a yeast 2μ plasmid or a yeast chromosomal DNA in yeast.
However, in mammalian cells, plasmid pRSVneo ATCC 371
98, plasmid pSV2dhfr ATCC 37145, plasmid pdBP
V-MMTneo ATCC 37224, plasmid pSV2neo ATCC 37149
And the like.

As the enhancer sequence, polyadenylation site and splicing junction site, those commonly used by those skilled in the art, such as those derived from SV40, can be used. As the selection marker, a commonly used marker can be used by a conventional method. Examples thereof include antibiotic resistance genes such as tetracycline, ampicillin, and kanamycin.

Examples of gene amplification genes include dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phosphotransferase gene, and aspartate trans. A carbamylase gene and the like can be exemplified. The expression vector of the present invention contains at least the promoter described above,
It can be prepared by ligating the start codon, the DNA encoding the protein of the present invention, the stop codon and the terminator region to an appropriate replicable unit in a continuous and circular manner. At this time, if desired, an appropriate DNA fragment (for example, a linker, a linker, or the like) can be obtained by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.
Other restriction sites) can be used.

The transformed cell of the present invention can be prepared by introducing the above-described expression vector into a host cell. The host cell used in the present invention is not particularly limited as long as it is compatible with the above-described expression vector and can be transformed, and natural cells or artificially established cells usually used in the technical field of the present invention are used. Various cells such as recombinant cells (for example, bacteria (Escherichia, Bacillus), yeast (Saccharomyces, Pichia, etc.),
Animal cells or insect cells).

Preferred is Escherichia coli or animal cells, and specifically Escherichia coli (DH5α, TB1, HB101
Etc.), mouse-derived cells (COP, L, C127, Sp2
/ 0, NS-1 or NIH3T3), rat-derived cells, hamster-derived cells (such as BHK and CHO), monkey-derived cells (such as COS1, COS3, COS7, CV1 and Velo) and human-derived cells (Hela, diploid) Fibroblast-derived cells, HEK293 cells, myeloma cells, Namalwa, etc.).

The introduction (transformation (transfection)) of an expression vector into a host cell can be performed by a conventionally known method. For example, bacteria (E. coli, Bacillus subtilis
), The method of Cohen et al. (Proc. Natl. Ac
ad. Sci. USA., Vol. 69, p. 2110, 1972), the protoplast method (Mol. Gen. Genet., Vol. 168, p. 111, 1979) and the competent method (J. Mol. Biol., Vol.56, p.209, 197
According to 1), in the case of Saccharomyces cerevisiae, for example, the method of Hinnen et al. (Proc. Natl. Acad.
Sci. USA., Vol.75, p.1927, 1978) and the lithium method (J.B.
acteriol., Vol. 153, p. 163, 1983), in the case of animal cells, for example, the method of Graham (Virol
ogy, Vol. 52, p. 456, 1973), and in the case of insect cells, for example, the method of Summers et al. (Mol. Cell. Biol.,
Vol.3, p.2156-2165, 1983).

The protein of the present invention can be produced by culturing a transformed cell containing the expression vector prepared as described above (hereinafter, used to encompass the transfectant) in a nutrient medium. . The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose, and examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, Examples include soybean meal and potato extract.
If desired, other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be contained. . The culturing is performed by a method known in the art. Culture conditions such as temperature, pH of the medium, and culture time are appropriately selected so that the protein of the present invention is produced in large quantities.

Specific examples of the culture medium and culture conditions used according to the host cell are shown below, but the present invention is not limited thereto. When the host is a bacterium, actinomycete, yeast, or filamentous fungus, for example, a liquid medium containing the above-mentioned nutrients is suitable. Preferably, the medium has a pH of 5 to 8. When the host is E. coli, preferred mediums are LB medium and M9 medium (Miller et al., Exp. Mo.
l. Genet, Cold Spring Harbor Laboratory, p.431, 1
972). In such a case, the cultivation is usually performed at 14 to 43 ° C. and about 3 to 24
Time can be done. When the host is a bacterium belonging to the genus Bacillus, the reaction can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours, if necessary, with aeration and stirring.

When the host is yeast, the medium may be, for example, Burkholder minimum culture (Bostian, Proc.
Acad. Sci. USA, Vol. 77, p. 4505, 1980), and the pH is desirably 5 to 8. The cultivation is usually performed at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration and agitation can be performed. When the host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum (Science, Vol. 122, p. 501, 1952);
MEM medium (Virology, Vol. 8, p. 396, 1959), RPM
I1640 medium (J. Am. Med. Assoc., Vol. 199, p. 51)
9, 1967), 199 medium (proc. Soc. Exp. Biol. Med.,
Vol. 73, p. 1, 1950) can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually about 30.
The reaction is carried out at ４０40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed. When the host is an insect cell, for example, Grace's medium (Proc. Natl. Acad.
Sci. USA, Vol. 82, p. 8404, 1985), and the pH is preferably about 5 to 8. Culture is usually about 2
The reaction is performed at 0 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and stirring may be performed.

The protein of the present invention is the same as that of the aforementioned D of the present invention.
It can be produced by culturing the transformant prepared as described above using NA under the culture conditions as described above to produce and / or secrete it in the culture supernatant or in the cells. That is, when the protein of the present invention is produced so as to be secreted into the culture supernatant, the obtained culture is filtered or centrifuged to obtain a culture filtrate (supernatant). The protein of the present invention is purified and isolated according to a conventional method generally used for purifying and isolating a natural or synthetic protein from E. coli. Isolation and purification methods include, for example, a method using solubility such as salting out and a solvent precipitation method, a method using a difference in molecular weight such as dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Methods that use charge such as ion exchange chromatography and hydroxylapatite chromatography, methods that use specific affinity such as affinity chromatography, methods that use the difference in hydrophobicity such as reverse-phase high-performance liquid chromatography, A method utilizing a difference in isoelectric point such as point electrophoresis may be used.

On the other hand, when the protein of the present invention is present in the periplasm or cytoplasm of the cultured transformant, the culture is subjected to a conventional method such as filtration or centrifugation to collect the cells or cells. The membrane fraction containing the protein of the present invention is suspended in a suitable buffer solution, for example, after disrupting cell walls and / or cell membranes such as cells by a method such as ultrasonication, lysozyme, and freeze-thawing, followed by centrifugation or filtration. Get. The membrane fraction is solubilized using a surfactant such as Triton-X100 to obtain a crude solution. Then, the crude solution is isolated by using a conventional method as exemplified above,
It can be purified.

The “transgenic non-human mammal” in the present invention is defined as a DNA (cDNA or genomic DNA) encoding a human-derived protein included in the protein of the present invention, which is contained in a non-human mammal (eg, mouse). A transgenic non-human mammal that is integrated on a sex locus and expresses and secretes the protein of the present invention encoded by the DNA in the body. The transgenic non-human mammal can be prepared by a conventional method such as that usually used in the production of transgenic animals (for example, the latest manual for animal cell experiments, published by LCI, Chapter 7, 361 to 4).
08, 1990).

Specifically, for example, in the case of a transgenic mouse, an embryonic stem cell (ESC) obtained from a normal mouse blastcyst (blastcyst) is used.
ell) are transformed with an expression vector into which a gene encoding a human protein of the present invention and a marker gene (eg, a neomycin resistance gene) have been inserted so that they can be expressed. ES wherein the gene encoding the human protein of the present invention is integrated on an endogenous gene
Cells are selected by a conventional method based on the presence or absence of the marker gene. Next, the selected ES cells are microinjected into fertilized eggs (pulmonary blastocysts) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol. 77,
No. 12, pp. 7380-7384, 1980; U.S. Pat. No. 4,873,191). The blastocyst is implanted into the uterus of another normal mouse as a foster parent. Then, a founder mouse (child mouse) is born from the foster parent mouse. A heterotransgenic mouse is obtained by mating the founder mouse with a normal mouse. By crossing the heterogeneic transgenic mice, homogenic transgenic mice are obtained according to Mendel's law.

Further, based on the nucleotide sequence of a DNA encoding a protein derived from a mouse included in the present invention,
A so-called “knockout mouse” can be produced. The “knockout mouse” in the present invention is a mouse in which an endogenous gene encoding a mouse-derived protein of the present invention has been knocked out (inactivated). For example, a positive negative selection method utilizing homologous recombination (US Pat. No. 5,464,764, 5,487,992, 5,627,059, Proc. Natl.Acad.Sci.
A, Vol. 86, 8932-8935, 1989, Nature, Vol. 342, 435-4
38, 1989), and such a knockout mouse is also one embodiment of the present invention.

The "antibody" in the present invention means a polyclonal antibody (antiserum) or a monoclonal antibody, and is preferably a monoclonal antibody. Specifically, it is an antibody reactive with the above-mentioned protein of the present invention or a part (fragment) thereof. "Antibody" of the present invention
Can be used as a mouse, rat, hamster, guinea pig, rabbit, or the like, using the protein of the present invention or a part thereof (natural, recombinant, or synthetic) or cells producing and secreting the protein as an immunogen (antigen). Natural antibodies obtained by immunizing non-human mammals, chimeric antibodies and human antibodies (CDR-grafted) that can be produced using gene recombination techniques.
Antibodies), and human antibodies that can be produced using human antibody-producing transgenic animals and the like. In the case of a monoclonal antibody, IgG, IgM, IgA,
Includes monoclonal antibodies having any isotype such as IgD or IgE. Preferably, Ig
G or IgM.

The polyclonal antibody (antiserum) or monoclonal antibody referred to in the present invention can be produced by an existing general production method. That is, for example,
The immunogen (antigen) as described above is optionally administered together with Freund's adjuvant (Freund's Adjuvant).
Animal, preferably mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, goat, horse or cow, more preferably mouse, rat, hamster,
Immunize guinea pigs or rabbits. Polyclonal antibodies can be obtained from serum obtained from the immunized animal. Further, a monoclonal antibody is prepared by preparing a hybridoma from the antibody-producing cells obtained from the immunized animal and myeloma cells (myeloma cells) having no autoantibody-producing ability,
It is produced by cloning the hybridoma and selecting a clone that produces a monoclonal antibody showing specific affinity for the antigen used for immunizing the mammal.

The monoclonal antibody can be specifically produced as follows. That is, using the protein of the present invention or cells expressing the protein as described above as an immunogen, the immunogen may be used together with Freund's Adjuvant, if necessary, together with mice, rats, hamsters, and guinea pigs. Subcutaneously, intramuscularly, or intravenously of a rabbit or rabbit, preferably a mouse, rat or hamster (including transgenic animals produced to produce antibodies from other animals such as human antibody producing transgenic mice). The immunization is carried out by injecting one or several times or implanting into the inside, the foot pad or the intraperitoneal cavity. Usually, immunization is performed 1 to 4 times about every 1 to 14 days after the initial immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

The preparation of a hybridoma secreting a monoclonal antibody was carried out according to the method of Kohler and Milstein et al. (Nature, 256, 495-49).
7 (1975)) and a modification method equivalent thereto. That is, antibody producing cells contained in the spleen, lymph node, bone marrow, tonsil, etc., preferably in the spleen obtained from the mammal immunized as described above, and preferably mouse, rat, guinea pig, hamster, rabbit or human And more preferably, by cell fusion with a myeloma cell having no autoantibody-producing ability derived from a mammal, such as a mouse, a rat or a human.

Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (6
53; ATCC No. CRL1580), P3 / NSI / 1-Ag4-1 (NS-
1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp
2 / O, Sp2), PAI, F0 or BW5147, rat myeloma 210RCY3-Ag.2.3., Human myeloma U-2
66AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used. Screening of a hybridoma clone producing a monoclonal antibody is carried out by culturing the hybridoma, for example, in a microtiter plate, and determining the reactivity of the culture supernatant of the well in which proliferation has been observed with the immunizing antigen used in the mouse immunization described above. For example, the measurement can be carried out by measuring with an enzyme immunoassay such as RIA or ELISA.

For the production of monoclonal antibodies from hybridomas, the hybridomas can be produced in vitro, or in a mouse, rat, guinea pig, hamster or rabbit or the like.
Preferably, it is performed in vivo in a mouse or rat, more preferably in ascites of a mouse, or the like, and can be performed by isolation from the obtained culture supernatant or ascites of a mammal. When culturing in vitro, the hybridoma is grown, maintained and stored in accordance with various conditions such as the characteristics of the cell type to be cultured, the purpose of the test research and the culture method,
It can be carried out using a known nutrient medium such as that used for producing monoclonal antibodies in the culture supernatant or any nutrient medium derived and prepared from a known basal medium.

As the basic medium, for example, a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium and MCDB104 medium, MEM medium, D-MEM medium,
Examples include a high calcium medium such as a PMI1640 medium, an ASF104 medium, and an RD medium. The basic medium can contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances depending on the purpose.
For isolation and purification of the monoclonal antibody, the above-mentioned culture supernatant or ascites is obtained by using a saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), an anti-immunoglobulin column or It can be performed by subjecting it to affinity column chromatography such as a protein A column.

Further, a gene encoding a monoclonal antibody is cloned from the hybridoma, and a transgenic goat, goat, sheep or pig in which the antibody-encoding gene has been incorporated into an endogenous gene is prepared using a transgenic animal preparation technique. However, it is also possible to obtain a large amount of a monoclonal antibody derived from the antibody gene from the milk of the transgenic animal (Nikkei Science, April 1997, pages 78 to 84).

The “chimeric antibody” in the present invention is a monoclonal antibody produced by genetic engineering. Specifically, for example, the variable region is a variable region derived from mouse immunoglobulin and the constant region is Is a constant region derived from human immunoglobulin, and a chimeric monoclonal antibody such as a mouse / human chimeric monoclonal antibody. The constant regions from human immunoglobulins include IgG, IgM, IgA, IgD and Ig.
Although each has a unique amino acid sequence depending on the isotype such as E, the constant region of the recombinant chimeric monoclonal antibody in the present invention may be a human immunoglobulin constant region belonging to any isotype. Preferably, it is a human IgG constant region. The chimeric monoclonal antibody in the present invention can be produced, for example, as follows. However, it is needless to say that the present invention is not limited to such a manufacturing method.

For example, a mouse / human chimeric monoclonal antibody is described in Experimental Medicine (Special Issue), Vol.
0, 1988 and Japanese Patent Publication No. 3-73280. That is, a human immunoglobulin is located downstream of an active VH gene (rearranged VDJ gene encoding a heavy chain variable region) obtained from DNA encoding a mouse monoclonal antibody isolated from a hybridoma producing the mouse monoclonal antibody. And the active VL gene (encoding the light chain variable region) obtained from DNA encoding a mouse monoclonal antibody isolated from the hybridoma. Downstream from the rearranged VJ gene), the CL gene (C gene encoding the L chain constant region) obtained from the DNA encoding human immunoglobulin is sequenced so that it can be expressed, and one or separate Inserting into an expression vector, transforming a host cell with the expression vector, It can be produced by culturing cells.

Specifically, first, DNA is extracted from a mouse monoclonal antibody-producing hybridoma by a conventional method, and the DNA is then subjected to an appropriate restriction enzyme (eg, EcoRI, H
IndIII, etc.), and then subjected to electrophoresis (for example, using 0.7% agarose gel) to perform Southern blotting. The electrophoresed gel is stained with, for example, ethidium bromide or the like, and after photographing, a marker is attached, the gel is washed twice with water, and immersed in a 0.25 M HCl solution for 15 minutes. Then, 0.
Soak in 4N NaOH solution for 10 minutes while shaking gently. After being transferred to a filter by a conventional method, the filter is recovered after 4 hours and washed twice with 2 × SSC. After sufficiently drying the filter, baking (75 ° C., 3 hours) is performed. After baking is completed, the filter is 0.1 × SSC
/0.1% SDS solution and treated at 65 ° C for 30 minutes. Then, it is immersed in a 3 × SSC / 0.1% SDS solution. The obtained filter is put into a plastic bag together with the pre-hybridization solution, and treated at 65 ° C. for 3 to 4 hours.

Next, the probe DN labeled with ³² P was added thereto.
A and hybridization solution at 65 ° C for 12 hours.
Let react for about an hour. After hybridization,
Appropriate salt concentration, reaction temperature and time (eg, 2 × SS
(C-0.1% SDS solution, room temperature, 10 minutes). Put the filter in a plastic bag,
× SSC is added in a small amount, sealed, and autoradiography is performed.

The rearranged VDJ gene and VJ gene encoding the H chain and L chain of the mouse monoclonal antibody are identified by the Southern blot method described above. The region containing the identified DNA fragment was fractionated by sucrose density gradient centrifugation, and phage vectors (eg, Charon 4A, Charon 2
8, λEMBL3, λEMBL4, etc.), and use the phage vector with Escherichia coli (eg, LE392, NM539, etc.)
To make a genomic library. Using the genomic library as a suitable probe (H chain J gene, L
Chain (κ) J gene), for example, using the Benton-Davis method (Science, Vol. 196, No. 180-
182, 1977), and plaque hybridization is performed to obtain rearranged VDJ genes or positive clones each containing the VJ gene.
A restriction enzyme map of the obtained clone is prepared, the nucleotide sequence is determined, and it is confirmed that the desired rearranged gene containing the VH (VDJ) gene or the VL (VJ) gene has been obtained.

On the other hand, the human CH gene and human CL gene used for chimerization are separately isolated. For example, human IgG1
When a chimeric antibody is prepared, the Cγ1 gene as the CH gene and the Cκ gene as the CL gene are isolated. These genes use the mouse Cγ1 gene and mouse Cκ gene corresponding to the human Cγ1 gene and human Cκ gene as probes by utilizing the high homology of the nucleotide sequences of the mouse immunoglobulin gene and the human immunoglobulin gene. It can be obtained by isolation.

Specifically, for example, clone Ig146
(Procedurals National Academy of Sciences (Proc. Natl. Acad. Sci. USA), Vol. 75, No. 47)
09-47-1713, 1978).
ndIII-BamHI fragment and clone MEP10 (Proceedings National Academy of Sciences (P
Roc. Natl. Acad. Sci. USA), 78, 474-478, 1981).
Using the fragment as a probe, a human Lambda Charon 4A HaeIII-AluI genomic library (Cell (Cel
l), Volume 15, pages 1157 to 1174, 1978
Year), a DNA fragment containing the human Cκ gene and retaining the enhancer region is isolated. In addition, human Cγ
One gene is, for example, human fetal hepatocyte DNA
And then fractionated by agarose gel electrophoresis.
The 9 kb band is inserted into λ788 and isolated using the probe described above.

Using the mouse VH gene and mouse VL gene, and the human CH gene and human CL gene thus isolated, the human CH gene is located downstream of the mouse VH gene while considering the promoter region and enhancer region. And a human CL gene downstream of the mouse VL gene using an appropriate restriction enzyme and DNA ligase, and incorporated into an expression vector such as pSV2gpt or pSV2neo according to a conventional method. At this time, the mouse VH gene / human CH gene and the mouse VL gene / human CL gene chimeric gene may be simultaneously placed in one expression vector,
Each can also be arranged on a separate expression vector.

The chimeric gene insertion / expression vector thus prepared is used, for example, in P3X63 / Ag8 / 653 cells or SP3.
The cells are introduced into myeloma cells, such as 210 cells, which do not produce an antibody by a protoplast fusion method, a DEAE-dextran method, a calcium phosphate method, an electroporation method, or the like. The transformed cells are selected by culturing in a drug-containing medium corresponding to the drug resistance gene introduced into the expression vector to obtain a desired chimeric monoclonal antibody-producing cell. The desired chimeric monoclonal antibody is obtained from the culture supernatant of the antibody-producing cells thus selected.

In the present invention, “human-type antibody (CDR-grafte
d antibody) is a monoclonal antibody produced by genetic engineering, specifically, for example, a hypervariable region in which part or all of the complementarity determining region of the hypervariable region is derived from a mouse monoclonal antibody. Wherein the variable region framework region is a human immunoglobulin-derived variable region framework region, and the constant region is a human immunoglobulin-derived constant region. Antibody.

Here, the complementarity determining regions of the hypervariable regions are three regions (Complementary regions) which are present in the hypervariable region in the variable region of the antibody and which directly bind complementarily to the antigen.
arity-determining residue; CDR1, CDR2, C
DR3), and the framework region of the variable region refers to four relatively conserved regions (Framework; FR1, FR2, FR3, FR) intervening before and after the three complementarity determining regions.
4). In other words, for example, it means a monoclonal antibody in which all but a part or all of the complementarity determining region of the hypervariable region of a mouse monoclonal antibody has replaced the corresponding region of human immunoglobulin. The constant regions derived from human immunoglobulin include IgG, IgM, Ig
Although each has a unique amino acid sequence depending on the isotype such as A, IgD and IgE, the constant region of the humanized monoclonal antibody of the present invention may be a human immunoglobulin constant region belonging to any isotype.
Preferably, it is a human IgG constant region. Further, the framework region of the variable region derived from human immunoglobulin is not limited.

The humanized monoclonal antibody of the present invention can be produced, for example, as follows. However, it is needless to say that the present invention is not limited to such a manufacturing method. For example, a recombinant humanized monoclonal antibody derived from a mouse monoclonal antibody is disclosed in JP-A-4-506458 and JP-A-62-2968.
It can be prepared by genetic engineering with reference to Japanese Patent Publication No. 90 and the like. That is, from a hybridoma producing a mouse monoclonal antibody, at least one mouse H chain CD
An R gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene; H
Chain gene and human light chain C corresponding to the pre-mouse light chain CDR
The human light chain gene encoding all regions except DR is isolated.

The mouse H chain CDR gene and the human H chain gene are introduced into an appropriate expression vector so that they can be expressed, and similarly, the mouse L chain CDR gene and the human L chain gene can be expressed. Into another suitable expression vector as described above. Alternatively, the mouse H chain CDR gene / human H chain gene and the mouse L chain CDR gene / human L chain gene can be introduced so that they can be expressed in the same expression vector. By transforming host cells with the expression vector thus prepared, human-type monoclonal antibody-producing transformed cells are obtained, and the target human-type monoclonal antibody is obtained from the culture supernatant by culturing the transformed cells. obtain.

The term "human antibody" in the present invention means that all regions including the variable region of the H chain and the constant region of the H chain, and the variable region of the L chain and the constant region of the L chain, which constitute the immunoglobulin, are human immunoglobulins. Is an immunoglobulin derived from the gene encoding A human antibody is prepared by immunizing a transgenic animal prepared by incorporating at least a human immunoglobulin gene into a locus of a non-human mammal such as a mouse according to a conventional method, using an antigen. The polyclonal antibody or the monoclonal antibody can be produced in the same manner as described above. For example, transgenic mice producing human antibodies are described in Nature Genetics, Vol. 15,
p.146-156, 1997); Nature Genetics, Vol.7, p.13-2
1, 1994; Japanese Patent Publication No. 4-504365; International Application Publication WO94 / 25
No. 585; Nikkei Science, June, 40th to 50th
Page, 1995; Nature, Vol. 368, p. 856-859, 1994;
And a method described in JP-A-6-500233.

[0085] A "portion of an antibody" in the present invention, the antibody of the present invention described above, preferably means a partial region of a monoclonal antibody, specifically _{F (ab ') 2, Fab} ',
Fab, Fv (variable fragment of antibody), sFv, dsF
v (disulphide stabilised Fv) or dAb (single do
main antibody) (Exp. Opin. Ther. Patents,
Vol.6, No.5, p.441-456, 1996). Here, "F (a
b ') ₂ "and"Fab'"are produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, and are present between two H chains in the hinge region. Antibody fragment produced by digestion before and after a disulfide bond. For example, when IgG is treated with papain, an L chain consisting of VL (light chain variable region) and CL (light chain constant region) is cleaved upstream of a disulfide bond existing between two heavy chains in the hinge region; And an H chain fragment comprising VH (H chain variable region) and CHγ1 (γ1 region in the H chain constant region) is C
Two homologous antibody fragments joined by disulfide bonds at the terminal regions can be produced. These two
Each of the two homologous antibody fragments is called Fab '. Also I
When gG was treated with pepsin, two Hs in the hinge region
It is possible to produce an antibody fragment which is cleaved downstream of the interchain disulfide bond and is slightly larger than the two Fab's connected at the hinge region. This antibody fragment is called F (ab ') ₂ .

The “cell producing a monoclonal antibody reactive to a protein or a part thereof” according to the present invention includes:
It means any cell that produces the aforementioned monoclonal antibody of the present invention. Specifically, (1) As described above, the protein of the present invention, a part thereof, or a reaction with the protein of the present invention or a part thereof obtained by immunizing a non-human mammal with cells or the like producing the protein. A non-human mammal-derived monoclonal antibody-producing B cell that produces a monoclonal antibody having the above-mentioned structure; (2) the above-mentioned antibody-producing B cell obtained by cell fusion with a mammal-derived myeloma cell; Hybridoma (fused cells),
Or (3) a gene encoding the monoclonal antibody isolated from the monoclonal antibody-producing B cells or the monoclonal antibody-producing hybridoma (either a gene encoding a heavy chain or a gene encoding a light chain, or both genes) Means any of the monoclonal antibody-producing transformed cells obtained by transforming cells other than the B cells and hybridomas. Here, the transformed monoclonal antibody-producing cell according to the above (3) is a transgenic cell producing a recombinant of the monoclonal antibody produced by the B cell of the above (1) or the hybridoma of the above (2). Means The cells producing the recombinant monoclonal antibody can be produced in the same manner as the method used in the production of the chimeric monoclonal antibody and the humanized antibody described above.

The “pharmaceutical composition” of the present invention refers to a protein of the present invention or a part thereof (fragment), an antibody or a part of an antibody as defined above, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: Here, “pharmaceutically acceptable carrier” refers to an excipient, a diluent,
Examples include bulking agents, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, flavoring agents, solubilizing agents, and other additives. By using one or more of such carriers, pharmaceuticals in the form of tablets, pills, powders, granules, injections, liquids, capsules, troches, elixirs, suspensions, emulsions, syrups, etc. A composition can be prepared. These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include topical solutions, suppositories and pessaries for enteral administration which contain one or more active substances and are formulated in a conventional manner.

The dosage is determined by the patient's age, sex, weight and condition, therapeutic effect, administration method, treatment time, or the active ingredient (such as the protein or antibody) contained in the pharmaceutical composition.
Depending on the type, the dose can be generally administered to an adult in a dose of 10 μg to 1000 mg (or 10 μg to 500 mg) at a time. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be necessary. In particular, in the case of an injection, 0.1 μg antibody / ml carrier in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection is used.
It can be produced by dissolving or suspending to a concentration of mg antibody / ml carrier. The injection thus produced is suitable for human patients in need of treatment.
In a single administration, 1 μg to 100 mg per kg body weight, preferably 50 μg to 50 mg per day per day.
It can be administered once to several times. As a form of administration,
Medically suitable administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection or intraperitoneal injection can be exemplified. Preferably, it is an intravenous injection.

Injectables may be prepared as non-aqueous diluents (eg, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions and emulsions. You can also. Such injections can be sterilized by filtration sterilization through a bacteria retaining filter, blending of a bactericide or irradiation. Injectables can be manufactured in the form of preparation for use. That is, a sterile solid composition can be prepared by freeze-drying or the like, and dissolved in sterile distilled water for injection or another solvent before use. The pharmaceutical composition of the present invention may contain visceral fat obesity or a complication associated with visceral fat obesity (eg, diabetes, hyperlipidemia, hypertension, arteriosclerosis, hyperuricemia caused by excessive uric acid synthesis, and sleep) To prevent and / or treat apnea syndrome).

[0090]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to only the embodiments described in the Examples.

Example 1 Total RNA from various human tissues
Preparation of (poly (A) ⁺ RNA) From visceral adipose tissue excised during surgery for gastric cancer patients,
Using Trizol reagent (GIBCO BRL)
Obtain total RNA by the usual method, oligotex-dt30 <Super>
(Manufactured by TAKARA) was added to purify poly (A) ⁺ RNA. Also,
Various human tissues (heart, brain, placenta, lung, liver, skeletal muscle,
Kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine,
From colon, peripheral blood leukocytes, stomach, uterus, and mammary gland)
poly (A) + RNA mRNA was purchased from Clontech.

Example 2 cDN derived from various human tissues
Synthesis of fragment A Poly (A) ⁺ RNA (0.2 μg for each tissue) derived from each tissue obtained in Example 1 was combined with DEPC (diethyl pirocarbo).
onate) Dissolve in treated distilled water (10 μl), then
The following four types of anchor primer mixtures (each 1 μm)
1 and a concentration of 25 pmol / μl) to make a total volume of 11 μl, respectively, and incubated at 65 ° C. for 5 minutes. Immediately after the incubation, the plate was left on ice.

<Anchor Primer Mixture> G (T) 15AG (SEQ ID NO: 13), G (T) 15GG (SEQ ID NO: 1)
4) and mixtures with G (T) 15CG (SEQ ID NO: 15). G (T) 15AT (SEQ ID NO: 16), G (T) 15GT (SEQ ID NO: 1
7) and mixtures with G (T) 15CT (SEQ ID NO: 18). G (T) 15AA (SEQ ID NO: 19), G (T) 15GA (SEQ ID NO: 2)
0), and a mixture with G (T) 15CA (SEQ ID NO: 21). G (T) 15AC (SEQ ID NO: 22), G (T) 15GC (SEQ ID NO: 2)
3) and mixtures with G (T) 15CC (SEQ ID NO: 24).

Next, for each sample, 5 × first strand buffer (first strand buffer)
r, 4 μl, <Composition>: 0.25 M Tris-HCl (pH 7.5), 0.37
5 M potassium chloride, 0.05 M DTT and 0.015 M magnesium chloride), 0.1 M DTT (2 μl), 2.5 mM dN
TP (1 μl), RNase inhibitor (40 units / μl, TOYOBO
1) and reverse transcriptase (Superscript II, Gibco BRL)
μl, concentration 200 U / μl) to make a total volume of 20 μl. After incubating at 42 ° C for 1 hour, DEPC H2O (30
μl) to make a total volume of 50 μl, and add 4 μl for each human tissue.
Various types of cDNA fragments were synthesized. That is, for example, for human visceral adipose tissue, cDNA fragments prepared using the above primers, cDNA fragments prepared using the above primers, cDNA fragments prepared using the above primers, and Four types of cDNA fragments were prepared from the cDNA fragments prepared using the primers. Similarly, for each of the other tissues, cDNA fragment groups were prepared for each of the four types.

Example 3 Identification of Genes Significantly Expressed in Visceral Adipose Tissue The expression states of genes in human visceral tissues and various other tissues used as test samples in Example 1 were determined by differential display method (Nucleic Acids Research, Vol. 21, N
o.18, p.4272-4280, 1993; and Science, Vol.257,
p.967-971, 1992; Genomics, Vol.36, p.316-319, 199
6) and RT-PCR (Reverse transcription-polymer)
ase chain reaction; Experimental Medicine, Special Issue, "PCR and Its Applications", Vol. 8, No. 9, 1990; and "Gene Amplification PCR Method, Basic and New Developments", published by Kyoritsu Shuppan Co., Ltd., 1992) The gene was analyzed according to a conventional method, and compared with the expression state of the gene in the various tissues, a gene whose expression was significantly observed in the visceral adipose tissue was identified.

P in the differential display method
The template used in the CR was cDNA derived from each tissue prepared in Example 2 using the expression state of G3PDH (glyceraldehyde triphosphate dehydrogenase) cDNA as a control.
Was diluted 100-300 times and the concentration was adjusted to be constant throughout each sample. Distilled water (10.75 μl), 10 × EX Taq buffer (2 μl), 25 μM dNTP (1.5 μl), arbitary primer (arbitary primer,
1 μl, concentration: 25 pmol / μl) and anchor primer (identical to that used in Example 2, 1 μl, concentration: 25 pmol)
/ Μl), and EX Taq DNA polymerase (0.25μl),
And [α ³⁵ S] dATP (1.5 μl, 10 mCi / ml, manufactured by Amersham) to make the total volume 20 μl, and PCR was performed. That is, since there are four types of cDNA fragment groups for each tissue and three types of arbitrary primers are used,
Twelve PCRs were performed for each tissue.

(1) For the cDNA fragment group prepared by using the anchor primer in Example 2, TGGTAAAGGG, GCATCGTCTA or
A combination of any of ACTCGACCAG and the above primer mixture as an anchor primer. (2) A combination of any of TGGTAAAGGG, GCATCGTCTA or ACTCGACCAG as an arbitary primer and the above-mentioned primer mixture as an anchor primer for the cDNA fragment group prepared using the anchor primer in Example 2. (3) A combination of any of TGGTAAAGGG, GCATCGTCTA or ACTCGACCAG as an arbitary primer and the above-mentioned primer mixture as an anchor primer for the cDNA fragment group prepared using the anchor primer in Example 2. (4) A combination of any one of TGGTAAAGGG, GCATCGTCTA or ACTCGACCAG as an arbitary primer and the above-mentioned primer mixture as an anchor primer for the cDNA fragment group prepared using the anchor primer in Example 2.

The PCR was performed in one cycle of a reaction consisting of 94 ° C. for 3 minutes, 40 ° C. for 5 minutes, and 72 ° C. for 5 minutes.
Reaction at 40 ° C for 2 minutes and 72 ° C for 1 minute for 40 seconds.
After performing a cycle and a reaction at 72 ° C for 5 minutes, 4 ° C
Held. A reaction stop buffer (stop buffer, 5 μl, <composition>: formamide (30 ml), xylene cyanol (30 mg), bromophenol blue (10 mg), 0.5 M EDTA (200 μl, pH 8) was added to each of the obtained PCR products. .0))
After adding 3.5 μl, 6% acrylamide gel (composition: urea (240 g) in 500 ml, 10 × TBE (50 ml)
l), 40% acrylamide (75 ml, a mixture of 38% monoacrylamide and 2% bisacrylamide)) was used for sequence gel electrophoresis. The gel was then dried and transferred to photographic film. As a result, bands of three types of gene (cDNA) fragments characteristically found only in electrophoresis gels of adipose tissue samples such as visceral adipose tissue and mammary gland compared to other tissue samples were identified. The three cDNA fragments were excised from the gel and cloned into the pBluescript SK (-) vector according to a conventional method (Experimental Medicine, separate volume, "Genetic Engineering Handbook", published by Yodosha, 1992). Next, sequence analysis was performed using a DNA sequencer. SEQ ID NO: 10 (250 bp) and SEQ ID NO: 11 (230 b
p) and SEQ ID NO: 12 (764 bp). Existing gene databases (DDBJ, GenBan
k and EMBL), as a result of homology search,
The fragment was also found to have a completely novel sequence with no homology to the known base sequence. The cDNA fragments were designated as clones AG1102 (SEQ ID NO: 10), AA3901 (SEQ ID NO: 11) and AA3401 (SEQ ID NO: 12), respectively.

Example 4 Confirmation of Expression in Various Tissues of mRNA Corresponding to Nucleotide Sequence of Each cDNA Fragment The expression states of mRNAs corresponding to the nucleotide sequences of the obtained three cDNA fragments in various human tissues were obtained. Each cDNA fragment obtained was used as a probe, and Human Multiple Tissue No.
rthern Blot (Clontech, code: # 7760-1, # 7759-
It was confirmed by the Northern blotting method according to the experimental operation method and the conventional method attached to 1).

Poly (A) ⁺ used for Northern blotting
As the RNA blotting membrane, a commercial product or a separately prepared one was used. Human heart, brain, placenta, lung, liver, skeletal muscle,
Kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine,
Poly (A) ⁺ RNA blotting membranes (2 μg each of poly (A) ⁺ RNA were blotted) derived from large intestine and peripheral blood leukocytes were purchased from Clontech. Human stomach,
For uterus and mammary gland, poly purchased from Clontech
(A) ⁺ RNA (2 μg each) was subjected to agarose gel electrophoresis according to a conventional method, and blotted on a nylon membrane was used. For human visceral adipose tissue, poly (A) ⁺ RNA (2 μg) derived from visceral adipose tissue obtained in Example 1 was used.
Similarly, those subjected to agarose gel electrophoresis and blotted on a nylon membrane were used. Each of the poly (A) ⁺ RNA blotting membranes was labeled with [α- ³² P] dCTP.
After the cDNA fragments were hybridized, the membrane was washed and subjected to autoradiography (-80 ° C, 8 hours). The results are shown in FIGS. As a result, the cDNA of clone AG1102
The mRNA corresponding to the nucleotide sequence of the fragment was significantly expressed in tissues of female organs such as uterine and ovarian mammary glands, mammary glands, and visceral adipose tissue. The size was found to be about 3.3 kb (FIG. 1). The mRNA corresponding to the nucleotide sequence of the cDNA fragment of clone AA3901 showed significant expression in visceral adipose tissue and pancreas, and was particularly remarkable in pancreas. The size was recognized to be about 1.8 kb (FIG. 2). Clone AA3
MRNA corresponding to the nucleotide sequence of the 401 cDNA fragment was significantly expressed in visceral adipose tissue. Also, its size is about 4.
4 kb (FIG. 3).

Example 5 Acquisition of full-length cDNA The three human cDNA fragments obtained in Example 3 (AG1102,
AA3901, AA3401) as a probe and a plaque hybridization method (Maniatis et al.,
Molecular Cloning: A Labolatory Manual, Cold Spri
ng Harbor Laboratory, Cold Spring Harbor, New Yor
k) or by using a colony hybridization method in a conventional manner (Experimental Medicine, separate volume, “Genetic Engineering Handbook”, published by Yodosha Co., Ltd., 1992). A full-length cDNA including the sequence was obtained. In addition, clone AG11
Plaque hybridization was used for 02, and colony hybridization was used for clones AA3901 and AA3401.

<5-1> Cloning of full-length cDNA encoding AG1102 protein poly (A) ⁺ RNA purified from human mammary gland total RNA (Clontec
h, 5 μg) as a template and a ZAP-cDNA Synthesis Kit (St
using ratagene) according to the experimental procedure attached to the kit. The obtained cDNA was ligated to a Uni-ZAP XR vector (manufactured by Stratagene) according to the method described in the experimental operation manual attached to the kit. Next, GIGA PAC
After in vitro packaging using K II GOLD (manufactured by Stratagene), Escherichia coli XL-1 Blue MRF ′ (GIGA PACK II GOLD, Stratagene
) Was used as a host to prepare a cDNA library consisting of plaques containing the recombinant phage.

The screening of the cDNA library was performed as follows according to the plaque hybridization method. The cDNA library (1 × 10 ⁶ plaques) was plated on an agar plate, and a nylon membrane (Biodyne A, P
(manufactured by oll Co., Ltd.). The cDNA fragment of clone AG1102 obtained in Example 3 was labeled with [α- ³² P] dCTP,
Hybridization solution (7% PEG-8000 and 10% SDS,
The concentration was adjusted to 1 × 10 6 cpm / ml using Sigma) to obtain a probe solution for plaque hybridization. Using this probe solution, primary screening and 2
The next screening was performed to obtain 10 positive clones. After each clone was isolated with a single plaque, it was subjected to in vivo excision according to the manual of Stratagene, and 10 clones were recovered as plasmid DNA (pBluescriptSK (-)).

For each of the ten clones, the Cycle Se
The nucleotide sequence was determined by the dididoxy method using a quensing Kit (Perkin-Elmer). As a result, the longest DN
The clone with the A insert was 3,171 bp (excluding the poly (A) sequence) and contained a 2,097 bp open reading frame encoding a protein of 699 amino acids. Full length cDN
The A sequence (including the 5 ′ and 3 ′ terminal nucleotide sequences) is shown in SEQ ID NO: 1, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 2. Based on the deduced amino acid sequence of AG1102 protein, SMART program (Protein Sci., Vol. 5, p. 1991)
-1999, 1996) using PROSITE database (Nucleic
AcidsRes., Vol.25, p.217-221, 1997).
Analysis of whether the 02 protein had a known motif (characteristic structure found in known proteins) revealed that it contained the following characteristic structures (FIG. 4).

Signal sequence (amino acids 1 to 16 of SEQ ID NO: 2). An important sequence in cell-cell adhesion, a characteristic common to collagen, fibronectin, fibrinogen, vitronectin, etc., integrin, an adhesion molecule, and extracellular matrix (ECM) molecules, which play an important role in cell-cell adhesion. Arg-Gl
y-Asp (RGD) sequence (amino acids 294 to 296 of SEQ ID NO: 2)
Th). Von Willebrand factor C (VWFC) domai
n) (amino acids 121 to 157 of SEQ ID NO: 2). It is thought to be involved in various functions such as intracellular signal transduction, cell adhesion, cell differentiation, DNA repair and RNA processing, and is common to G protein-coupled receptors, ECM molecules, tyrosine kinase receptors and nerve growth factors. A structure called leucine-rich repeat in which hydrophobic amino acids such as leucine, isoleucine and valine appear repeatedly (amino acids 345 to 699 of SEQ ID NO: 2). In addition, as a result of homology search based on existing gene databases (DDBJ, GenBank and EMBL), AG1102 protein was found to be mouse bone proteoglycan II precursor (mouse bone proteoglycan II).
II precursor; Proc. Natl. Acad. Sci., USA, Vol.8
3, p.7683-7687, 1986), bovine keratocan (Keratoka)
n; J. Biol. Chem., Vol.271, p.9759-9763, 1996),
And rat decorin (Decorin; Eur. J. Cell. Bio)
l., Vol. 59, p. 314-321, 1992) with 33.7%, 33.2% and 33.0% amino acid homology, respectively.

<5-2> Cloning of full-length cDNA encoding AA3901 protein Based on poly (A) ⁺ RNA prepared from human visceral adipose tissue as a human cDNA library, an oligo-capping method (Gene, Vol. .138, p.171-174, 1994; Gene, Vol.20
0, p.149-156, 1997) to prepare a human cDNA library. Also, as in the case of the embodiment <5-1>, the embodiment 3
The cDNA fragment of the clone AA3901 obtained in the above was labeled with [α- ³² P] dCTP to obtain a probe for colony hybridization. Using this probe, the cDNA library was screened as follows in accordance with the colony hybridization method.

CDNA library (6 × 10 ⁵ plaques)
On agar plate, and nylon membrane (Biodyen A, Poll
(Manufactured by the company). Using this replica and a probe for colony hybridization,
Hybridization solution (7% PEG-8000 and 10% SDS,
Sigma). The primary screening and the secondary screening were performed to obtain two positive clones. After each clone was isolated as a single colony, it was recovered as plasmid DNA. <5-1> for the two clones
The nucleotide sequence was determined in the same manner as described above. As a result, the longest
A clone having a DNA insert was 1,577 bp (poly (A)
(Excluding the sequence) and a 1,092 bp open reading frame encoding a protein consisting of 364 amino acids. Full length cD
The NA sequence (including the 5 ′ and 3 ′ terminal base sequences) is shown in SEQ ID NO: 3, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 4. Based on the deduced amino acid sequence of AA3901 protein, the SMART program (Protein Sci., Vol. 5, p. 19
91-1999, 1996) using the PROSITE database (Nuclei
c AcidsRes., Vol.25, p.217-221, 1997)
Analysis of whether the 3901 protein had a known motif (characteristic structure found in the known protein) revealed that it did not have any known motifs, not only the signal sequence.

<5-3> Cloning of full-length cDNA encoding AA3401 protein A labeled cDNA obtained by labeling the cDNA fragment of clone AA3401 obtained in Example 3 with [α- ³² P] dCTP as a probe for colony hybridization is used. Except for the above, cloning was performed in substantially the same manner as in <5-2>. Primary screening and secondary screening by colony hybridization were performed, and 14 positive clones were obtained. Each clone was isolated as a single colony and then recovered as plasmid DNA. The nucleotide sequence of each of the 14 clones was determined in the same manner as in <5-2>. As a result, the clone with the longest DNA insert
It was 29 bp (excluding the poly (A) sequence) and contained a 2,832 bp open reading frame encoding a protein consisting of 944 amino acids. The full-length cDNA sequence (including the 5 ′ and 3 ′ terminal nucleotide sequences) is shown in SEQ ID NO: 5, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 6.

The SMART program (Protein Sci., Vol. 5, p. 1991-1) was used based on the deduced amino acid sequence of AA3401 protein.
999, 1996) using the PROSITE database (Nucleic Ac
idsRes., Vol.25, p.217-221, 1997) and AA3401
Analysis of whether the protein had a known motif (characteristic structure found in the known protein) revealed that it did not have any known motifs, not only the signal sequence. In addition, existing gene databases (DD
BJ, GenBank, and EMBL) based on homology search, AA3
401 protein is a human endosome-associated protein
(J. Biol. Chem., Vol. 270, p. 13503-13511, 1995) and chicken myosin heavy chain (JM).
ol. Biol., Vol. 198, p. 143-157, 1987) and an α-helix (coiled-coil structure) portion and 21.
It had 6% and 22.6% amino homology. Therefore, AA3
The 401 protein is partially (amino acid number 370 of SEQ ID NO: 6)
(In the region of 944944).

Example 6 Cloning of Human Genomic DNA Human AG1102 protein, AA cloned in the above Example
Using each full-length cDNA encoding 3901 protein and AA3401 protein as a hybridization probe, genomic DNA encoding human AG1102 protein, AA3901 protein and AA3401 protein was replaced with human genomic D.
Screened from NA library. In addition, human AG
For genomic DNA encoding 1102 protein,
Phage library containing human genomic DNA (Stratag
ene). In addition, human AA3901 protein and AA340
For genomic DNA encoding 1 protein, a cosmid library containing human genomic DNA prepared as described below was used.

<6-1> Preparation of Human Genomic DNA-Containing Cosmid Library The cosmid library used in the present invention is described in "Lab Manual Human Genome Mapping" (edited by Masaaki Hori and Yusuke Nakamura,
Maruzen Publishing, pp. 41-48). The method is briefly described below. Cosmid vector pWE15 (Wah
l, GM., Proc. Natl. Acad. Sci. USA., Vol. 84, 2160-
2164, 1987) was filled in using the Klenow enzyme and the oligonucleotide linker (5 '
-CCTCGAGG-3 ′) to convert to XhoI to construct a modified cosmid vector pWEX15. After digestion with XhoI, the vector was partially filled in with Klenow enzyme, dCTP and dTTP.

After digestion of human chromosomal genomic DNA with Sau3AI, fractionation was performed by sucrose concentration gradient centrifugation to obtain a genomic DNA fragment. The DNA fragment was digested with Klenow enzyme, d
Filled in using ATP and dGTP. The vector DNA (1 μg) and the genomic DNA (1 μg)
10x ligation buffer (Tris-HCl (0.5M (pH
7.5)), 100 mM magnesium chloride, 100 mM dithiothreyl, 500 μg / ml bovine serum albumin), 20 mM AT
P, 50% polyethylene glycol 8000, 1 M sodium chloride, T4 DNA ligase (Boehringer) and ligation in a mixed solution of distilled water. Next, GIGAPACK II GOLD
(Stratagene) for in vitro packaging.

<6-2> Cloning of Human Genomic DNA Human AG1102 protein, AA39 cloned in Example 5
Each of the full-length cDNAs encoding the 01 protein and the AA3401 protein is labeled with [α- ³² P] dCTP and used as a hybridization probe, and by plaque hybridization or colony hybridization,
In the same manner as in Example 5, the human genomic DNA-containing phage library or the human genomic DNA-containing cosmid library prepared in <6-1> was screened to obtain a positive clone. For screening of genomic DNA of each of the AG1102 protein, AA3901 protein and AA3401 protein, two, two, and six positive clones were obtained, respectively. The nucleotide sequence of human genomic DNA contained in each positive clone was analyzed using a DNA sequencer according to a conventional method, and the genomic DNA sequence of each clone was cloned in Example 5 into the genomic DNA sequence of AG1102 protein and AA39.
It was confirmed that a nucleotide sequence corresponding to the full-length cDNA of 01 protein or AA3401 protein was contained. From this, it was confirmed that the obtained gene was not a pseudogene.

Example 7 Analysis of Localization of Gene on Chromosome FISH Method (Fluorescence in situ hybridizatio
n; Experimental Medicine, Separate Volume, “Genetic Engineering Handbook”, published by Yodosha Co., Ltd., 1992, pp. 271 to 277), using standard methods for AG1102 genomic DNA, AA3901 genomic DNA and
The localization of each of the AA3401 genomics on the human chromosome was analyzed. AG1102 protein cloned in Example 6, AA39
Genomic DNAs encoding each of the 01 protein and the AA3401 protein were used as probes. In order to separate chromosomes by the R band method (Hum. Genet., Vol. 86, p. 14-16, 1990), thymidine synchronization method and bromodeoxyuridine release method were used. Using metaphase chromosomes (metaphase chromosom)
e) was prepared.

Prior to hybridization, metaphase human cells were stained with Hoechst-33258 (1 μg / ml) and irradiated with ultraviolet light. Each of the probes was labeled with biotin-16-UTP (manufactured by Boehringer) by a nick translation method and hybridized to the denatured metaphase cells. FITC-avidin (fluoresce
in isothiocyanate-avidin; Boehringer)
The hybridization signal was detected. An accurate measurement of the signal was made by visually confirming the duplicated R band. As a result, the AG1102 gene was
At 9q22.3 on the banded chromosome specimen, the AA3901 gene
The AA3401 gene was mapped to 1p12 and 1q21.1 on the replicated R-band chromosome specimen (Fig. 5 (a), Fig. 6 (a) and Fig. 7). The AA3401 gene is considered to be located in either one. The chromosome specimen of metaphase metaphase human cells was mapped on the R-staining karyotype (FIG. 5 (b) and FIG. 6 (b)).

[0116]

Industrial Applicability The present invention relates to obesity and / or complications associated with obesity, particularly visceral obesity (diabetes, hyperlipidemia, hypertension,
Atherosclerosis, hyperuricemia caused by excessive uric acid synthesis, sleep apnea syndrome, etc.), which are considered to be deeply involved in the onset of adipose tissue, ie, visceral adipose tissue and / or visceral adipose tissue. The present invention relates to a protein molecule produced in an adipocyte which is a main constituent cell, and a gene encoding the protein. That is, the present invention provides a protein having the following utility in drug development: a part of the protein; a gene (eg, genomic DNA, cDNA) encoding the protein or a part thereof;
A part of the gene (including antisense DNA); an antibody reactive with the protein or a part thereof (eg, a polyclonal antibody or a monoclonal antibody); an expression containing a part of the antibody, the gene or a part thereof A vector; a transformed cell transformed with the vector; a cell producing the antibody or a part thereof (eg, a B cell, a hybridoma, a recombinant cell); the gene, a part of the gene, the protein, A part of the protein, the antibody or a pharmaceutical composition comprising the part of the antibody; and a transgenic engineered to produce a human-derived protein encompassed by the protein in the body. A mouse is provided.

As described below, the gene (DNA) of the present invention or a part thereof, the protein or a part thereof, and the antibody or a part of the protein or the part thereof are visceral fat obesity or To prevent and / or treat complications associated with visceral fat obesity (eg, diabetes, hyperlipidemia, hypertension, arteriosclerosis, hyperuricemia due to excessive uric acid synthesis, and sleep apnea syndrome, etc.) It is also useful as a drug for pharmaceuticals and as a tool for trial research or drug development.

(1) When the overexpression of the protein molecule encoded by the gene (DNA) of the present invention acts on the onset and progression of the above-mentioned diseases, in this case, the gene or the protein or Some of them (fragments) can be targets for drug development for the treatment and prevention of those diseases. For example, drugs that inhibit the transcription of the gene into mRNA, and those from the mRNA to the protein of the present invention. Design of drugs such as drugs that inhibit translation, drugs that inhibit the biological activity of the protein, drugs that suppress the production of the protein, or drugs that inhibit the binding of the protein to its receptor or interaction with other molecules , Screening or as a tool for activity measurement (assay).

For example, the DNA can be used as a so-called reporter gene assay commonly used in screening and measuring activity of such drugs.
y) and so-called high-throughput screening in which screening is performed automatically using a machine based on the reporter gene assay.

[0120] Examples of the aforementioned agent for inhibiting the transcription of the DNA (gene) of the present invention into mRNA or the agent for inhibiting the translation of the mRNA into the protein of the present invention include antisense drugs. That is, it is possible to design an antisense DNA or an antisense RNA based on the DNA sequence of the present invention or an mRNA sequence corresponding to the sequence, and the antisense DNA and the antisense RNA are the same as the antisense sequence, respectively. By binding to DNA or mRNA having a complementary sequence, it is useful as an antisense drug by a mechanism that inhibits transcription from DNA to mRNA or translation from mRNA to protein.

Examples of the above-mentioned agent for inhibiting the biological activity of the protein of the present invention, an agent for inhibiting the production of the protein, or an agent for inhibiting the binding of the protein to a receptor or the interaction with another molecule include: Peptide antagonists, antibodies or low molecular weight compounds can be mentioned.
That is, a peptide antagonist can be designed based on the amino acid sequence of the protein of the present invention.
Receptor or ligand) by competitively inhibiting the binding of the protein of the present invention to the signal or secondary reaction caused by the binding to the molecule, thereby indirectly inhibiting the biological activity of the protein of the present invention. It is useful as a drug that prevents its function from being exhibited. Further, an antibody (particularly, a monoclonal antibody) against the protein of the present invention or a part thereof is useful as an antibody drug by inhibiting (neutralizing) the biological activity of the protein by binding to the protein of the present invention. It is.

(2) The expression of the protein molecule encoded by the gene (DNA) of the present invention is increased depending on the onset and progress of the above-mentioned disease, and the protein molecule is involved in the onset and progress of the disease. In this case, the protein of the present invention or a part thereof (for example, a biologically active region) itself is useful as a pharmaceutical. Further, the gene (DNA) of the present invention can be administered to a patient by gene therapy, and is itself useful as a pharmaceutical. Further, as in the case of the above (1), the present invention
DNA (gene) or protein or a part thereof (fragment) can be used as a target for drug development for treatment and prevention of those diseases. It is useful as a tool for drug design, screening or activity measurement (assay). For example, similar to the above (1), the DNA may be a so-called reporter gene assay commonly used in screening and activity measurement (assay) of such a drug, and a so-called automatic machine-based screening based on the reporter gene assay. Very useful in high-throughput screening.

In addition to the above (1) and (2), the gene (DNA), protein and antibody of the present invention are used to search for proteins (receptors and ligands) that interact with the protein of the present invention, It is useful as a test and research reagent in the elucidation of functions and in drug development targeting the ligand. In addition, a transgenic animal as a model animal can be prepared by introducing a human-derived DNA, which is one of the DNA aspects of the present invention, into a mammal other than a human such as a mouse. Similarly, a model animal (knockout animal) is obtained by destroying (inactivating) the corresponding endogenous gene based on the genetic information of rabbit or mouse-derived DNA included in the DNA embodiment of the present invention. It is possible to create By analyzing the physical, biological, pathological and genetic characteristics of these model animals, it becomes possible to elucidate the functions of the genes and proteins according to the present invention.

Further, by crossing the transgenic animal with the model animal in which the endogenous gene has been disrupted in this manner, a model animal having only the human-derived gene (DNA) of the present invention can be prepared. It is. By administering a drug (compound, antibody, etc.) targeting the introduced human gene to this model animal,
The therapeutic effect of the drug can be evaluated.

[0125]

[Sequence List] SEQUENCE LISTING <110> Japan Tobacco, Inc. <120> Tissue specific mammalian-derived physiologically active protein <130> J98-0108 <140> <141> <160> 12 <170> PatentIn Ver. 2.0 <210> 1 <211> 3211 <212> DNA <213> Homo sapiens <220> <221> 5'UTR <222> (1) .. (73) <220> <221> sig_peptide <222> (74) .. (124) <220> <221> CDS <222> (74) .. (2173) <220> <221> repeat_region <222> (1105) .. (2170) <220> <221> polyA_signal <222> (3154) .. (3159) <220> <221> 3'UTR <222> (2174) .. (3211) <400> 1 ggcacgagat gacaccaagc aaagcctact tagtttagat ctccagaaat tggctggtgg 60 aaaaaaatca aac atg aag att gca gtt ttg ttt tgt ttt ttt ctg ctt 109 Met Lys Ile Ala Val Leu Phe Cys Phe Phe Leu gut atac t1 aat gaa gaa att cct agg aag 157 Ile Ile Phe Gln Thr Asp Phe Gly Lys Asn Glu Glu Ile Pro Arg Lys 15 20 25 caa agg agg aag atc tac cac aga agg ttg agg aaa agt tca acc tca 205 Gln Arg Arg Lys Ile Tyr His Arg Arg Leu Arg Lys Ser Ser Thr Ser 30 35 40 cac aag cac aga tca aac aga cag ctt gga att cag caa aca aca gtt 253 His Lys His Arg Ser Asn Arg Gln Leu Gly Ile Gln Gln Thr Thr Val 45 50 55 60 ttt aca cca gta gca aga ctt cct att gtt aac ttt gat tat agc atg 301 Phe Thr Pro Val Ala Arg Leu Pro Ile Val Asn Phe Asp Tyr Ser Met 65 70 75 gag gaa aag ttt gaa tcc ttt tca agt ttt cct gga gta gaa tca agt 349 Glu Glu Lys Phe Glu Ser Phe Ser Ser Phe Pro Gly Val Glu Ser Ser 80 85 90 tat aat gtg tta cca gga aag aag gga cac tgt ttg gta aag ggc ata 397 Tyr Asn Val Leu Pro Gly L ys Lys Gly His Cys Leu Val Lys Gly Ile 95 100 105 acc atg tac aac aaa gct gtg tgg tcg cct gag ccc tgc act acc tgc 445 Thr Met Tyr Asn Lys Ala Val Trp Ser Pro Glu Pro Cys Thr Thr Cys 110 115 120 ctc tgc tca gat gga aga gtt ctt tgt gat gaa acc atg tgc cat ccc 493 Leu Cys Ser Asp Gly Arg Val Leu Cys Asp Glu Thr Met Cys His Pro 125 130 135 140 cag agg tgc ccc caa aca gtt ata cct gaa ggg gaa tgc tg ccg gtc 541 Gln Arg Cys Pro Gln Thr Val Ile Pro Glu Gly Glu Cys Cys Pro Val 145 150 155 tgc tcc gct act gtc tcc tat tct cta ctc agt ggt ata gca tta aat 589 Cys Ser Ala Thr Val Ser Tyr Ser Leu Leu Leu Ser Gly Ile Ala Leu Asn 160 165 170 gat aga aat gaa ttt tct ggt gat tct tca gaa caa aga gaa cct acc 637 Asp Arg Asn Glu Phe Ser Gly Asp Ser Ser Glu Gln Arg Glu Pro Thr 175 180 185 aat tta ctt cat aag caa ctg cca cct cct cag gtg gga atg gac cga 685 Asn Leu Leu His Lys Gln Leu Pro Pro Gln Val Gly Met Asp Arg 190 195 200 ata gta aga aaa gaa gca ctt caa tct gag gag gat gaa gaa gtg aaa 733 Ile Val Ly s Glu Ala Leu Gln Ser Glu Glu Asp Glu Glu Val Lys 205 210 215 220 gaa gaa gat aca gag caa aag aga gag acc cct gaa tct aga aat cag 781 Glu Glu Asp Thr Glu Gln Lys Arg Glu Thr Pro Glu Ser Arg Asn Gln 225 230 235 ggg caa ctt tac agt gag ggg gac agc aga gga gga gac aga aag cag 829 Gly Gln Leu Tyr Ser Glu Gly Asp Ser Arg Gly Gly Gly Asp Arg Lys Gln 240 245 250 agg cct gga gag gag agg agg ctg gca cac cag caa caa cgc caa gga 877 Arg Pro Gly Glu Glu Arg Arg Leu Ala His Gln Gln Gln Arg Gln Gly 255 260 265 agg gag gag gag gag gat gag gag gag gag ggt gag gag ggt gag gag 925 Arg Glu Glu Glu Glu Asp Glu Glu Glu Glu Gly Glu Glu Gly Glu Glu 270 275 280 gat gag gag gac gag gag gac ccg gta aga gga gat atg ttc cga atg 973 Asp Glu Glu Asp Glu Glu Asp Pro Val Arg Gly Asp Met Phe Arg Met 285 290 ct 300 ccc cga tcc ccg ctt cct gct cct ccc aga ggc aca ctg cgc ctg 1021 Pro Ser Arg Ser Pro Leu Pro Ala Pro Pro Arg Gly Thr Leu Arg Leu 305 310 315 cca agc ggg tgc tct ctg tcc tac agg acc atc agc tgc atc acc 1 069 Pro Ser Gly Cys Ser Leu Ser Tyr Arg Thr Ile Ser Cys Ile Asn Ala 320 325 330 atg ctt acc cag ata cca ccg ctg aca gca cca cag ata aca agt ctg 1117 Met Leu Thr Gln Ile Pro Pro Leu Thr Ala Pro Gln Ile Thr Ser Leu 335 340 345 gag ctc act ggc aat tcc atc gcc tcc atc cca gat gaa gca ttt aat 1165 Glu Leu Thr Gly Asn Ser Ile Ala Ser Ile Pro Asp Glu Ala Phe Asn 350 355 360 gga tta cca aat ttg gaa agg ctt gat ctg agt aaa aat aat atc act 1213 Gly Leu Pro Asn Leu Glu Arg Leu Asp Leu Ser Lys Asn Asn Ile Thr tct tca ggc ata ggt cca aaa gca ttc aag ctt ctg aag aag tta atg 1261 Ser Ser Gly Ile Gly Pro Lys Ala Phe Lys Leu Leu Lys Lys Leu Met 385 390 395 cgt ttg aat atg gat gga aat aat ttg ata cag att cct tca caa ttg 1309 Arg Leu Asn Met Asp Gly Asn Asn Leu Ile Gln Ile Pro Ser Gln Leu 400 405 410 ccat a tta gaa gaa ctt aaa gtc aat gag aac aat ctt cag gct 1357 Pro Ser Thr Leu Glu Glu Leu Lys Val Asn Glu Asn Asn Leu Gln Ala 415 420 425 atc gat gaa gaa agt tta tca gac tta aat cag ttg gtc acc tta a 1405 Ile Asp Glu Glu Ser Leu Ser Asp Leu Asn Gln Leu Val Thr Leu Glu 430 435 440 ttg gaa gga aac aat ctc agt gaa gcc aat gtc aat cct tta gct ttc 1453 Leu Glu Gly Asn Asn Leu Ser Glu Ala Asn Val Pro Leu Ala Phe 445 450 455 460 aaa cct ttg aag agc cta gcc tac ttg cgt ctg gga aaa aat aaa ttt 1501 Lys Pro Leu Lys Ser Leu Ala Tyr Leu Arg Leu Gly Lys Asn Lys Phe 465 470 475 aga agat at gc ca ctt cct ggt tct att gag gaa tta tac cta 1549 Arg Ile Ile Pro Gln Gly Leu Pro Gly Ser Ile Glu Glu Leu Tyr Leu 480 485 490 gaa aat aac caa att gaa gaa ata act gaa att tgt ttc aat cat Asc 15n Glun Gln Ile Glu Glu Ile Thr Glu Ile Cys Phe Asn His Thr 495 500 505 aga aag atc aat gtc att gta cta cgt tat aac aaa att gaa gaa aat 1645 Arg Lys Ile Asn Val Ile Val Leu Arg Tyr Asn Lys Ile Glu Glu Asn 510 515 520 agg att gct cct tta gcc tgg ata aat caa gaa aat cta gaa tcc att 1693 Arg Ile Ala Pro Leu Ala Trp Ile Asn Gln Glu Asn Leu Glu Ser Ile 525 530 535 535 540 gat ctc tcc tac aac aag gtc tat cac ccg tcc tat cta ccc aag 1741 Asp Leu Ser Tyr Asn Lys Leu Tyr His Val Pro Ser Tyr Leu Pro Lys 545 550 555 tcc ttg ctg cac cta gta ctc ctt ggg aac cag att gaa cgg atc cct 1789 Ser Leu Leu His Leu Val Leu Leu Gly Asn Gln Ile Glu Arg Ile Pro 560 565 570 ggc tat gtg ttt ggc cac atg gaa cca ggc ctg gaa tac ttg tac ctg 1837 Gly Tyr Val Phe Gly His Met Glu Pro Gly Leu Glu Tyr Leu Tyr Leuca 575 t aac aaa ctt gct gat gat ggc atg gac cgt gtc tcc ttc tat 1885 Ser Phe Asn Lys Leu Ala Asp Asp Gly Met Asp Arg Val Ser Phe Tyr 590 595 600 ggg gca tat cat tct ctg aga gaa tta ttt ctg gat cac ag 1933 Gly Ala Tyr His Ser Leu Arg Glu Leu Phe Leu Asp His Asn Asp Leu 605 610 615 620 aaa tct ata cca cct ggg ata caa gaa atg aaa gca cta cat ttt ctg 1981 Lys Ser Ile Pro Pro Gly Ile Gln Glu Met Lys Ala Leu His Phe Leu 625 630 635 agg ctg aac aac aac aag ata cgg aac att ctt cca gaa gaa att tgc 2029 Arg Leu Asn Asn Asn Lys Ile Arg Asn Ile Leu Pro Glu Glu Ile Cys 640 645 650 aat gct gat gag gat gac tca aat ctg gaa cat ctt cat ctt gaa 2077 Asn Ala Glu Glu Asp Asp Asp Ser Asn Leu Glu His Leu His Leu Glu 655 660 665 aac aat tat att aaa att aga gaa ata cca tct tac aca ttt tca Asgc 2125 As Tyr Ile Lys Ile Arg Glu Ile Pro Ser Tyr Thr Phe Ser Cys 670 675 680 ata aga tca tac tca agt atc gtt ctt aaa cca caa aac atc aag taa 2173 Ile Arg Ser Tyr Ser Ser Ile Val Leu Lys Pro Gln Asn Ile Lys 685 690 695 700 ttccaagttt tcctttgctg tttataaact ttactcatgt atttgtagta gctgcatttg 2233 tcattaataa gagagacata atcctcctgt tatactcagt atcattatat gctagtcaac 2293 ctgattcact aacacacaga tgaacaacca aaatatacct aaaaggtata gtctctagga 2353 gttttattaa tagtaaaggt aaaatctctc agtttcctac ctctagaaag aggccatctc 2413 actagaatag gatattatgc atactgagct agaccagaag agtctggaac aaaataaaca 2473 cagcctttat aatcaacttg aatactggtg ttagctgaga actctgtaag tccctttaaa 2533 aattatgtat cttttggttc aagattaaga agcataatga caacaaaaaa agcaggcaga 2593 atttatgaat aagtgttgtt tattattaaa acaataattt gttaatttct tataaggtcc 2653 tgtgctataa ttactg gtat aaatataact gaatattggg gtagctttca tttcttccat 2713 taattacatg tgtaaaatta aaacactact gtaatgttaa tttcctggtt ttgaaaatca 2773 tattatggtt atgtacattg ttaacattaa ggaaagctgg cagaagggtg tgcataaatt 2833 ctatactctt tttgaaactt ttctataatt ctaaaattat tttttaaagt taaacagtat 2893 attaagatta ccttcactat tcctcactca agattaagac attttttgaa aagcagtaga 2953 gtttgcttaa aatacaaatt aattattctt gactataacc ttgtaaaggt aaatctaatg 3013 tataaatttt tgaaaaattt tgcaccactg gtcatagcat ctatctcctt tgccttaatt 3073 tactgaaata catcatttta ttcggttcaa ttgaaataaa gctatgtctt tactatgtat 3133 tggccctacc aaaatcatat attaaaattt tctaacataa aaaaaaaaaa aaaaaaaaaa 3193 aaaaaaaaaa aaaaaaaa 3211 <210> 2 <211> 699 <212> PRT <213> Homo sapiens <400> 2 Met Lys Ile Ala Val Leu Phe Cys Phe Phe Leu Leu Ile Ile Phe Gln 1 5 10 15 Thr Asp Phe Gly Lys Asn Glu Glu Ile Pro Arg Lys Gln Arg Arg Lys 20 25 30 Ile Tyr His Arg Arg Leu Arg Lys Ser Ser Thr Ser His Lys His Arg 35 40 45 Ser Asn Arg Gln Leu Gly Ile Gln Gln Thr Thr Val Phe Thr Pro Val 50 55 60 Ala Arg Leu Pro Ile Val Asn Phe Asp Tyr Ser Met Glu Glu Lys Phe 65 70 75 80 Glu Ser Phe Ser Ser Phe Pro Gly Val Glu Ser Ser Tyr Asn Val Leu 85 90 95 Pro Gly Lys Lys Gly His Cys Leu Val Lys Gly Ile Thr Met Tyr Asn 100 105 110 Lys Ala Val Trp Ser Pro Glu Pro Cys Thr Thr Cys Leu Cys Ser Asp 115 120 125 Gly Arg Val Leu Cys Asp Glu Thr Met Cys His Pro Gln Arg Cys Pro 130 135 140 Gln Thr Val Ile Pro Glu Gly Glu Cys Cys Pro Val Cys Ser Ala Thr 145 150 155 160 Val Ser Tyr Ser Leu Leu Ser Gly Ile Ala Leu Asn Asp Arg Asn Glu 165 170 175 Phe Ser Gly Asp Ser Ser Glu Gln Arg Glu Pro Thr Asn Leu Leu His 180 185 190 Lys Gln Leu Pro Pro Pro Gln Val Gly Met Asp Arg Ile Val Arg Lys 195 200 205 Glu Ala Leu Gl n Ser Glu Glu Asp Glu Glu Val Lys Glu Glu Asp Thr 210 215 220 Glu Gln Lys Arg Glu Thr Pro Glu Ser Arg Asn Gln Gly Gln Leu Tyr 225 230 235 240 Ser Glu Gly Asp Ser Arg Gly Gly Asp Arg Lys Gln Arg Pro Gly Glu 245 250 255 Glu Arg Arg Leu Ala His Gln Gln Gln Arg Gln Gly Arg Glu Glu Glu 260 265 270 Glu Asp Glu Glu Glu Glu Glu Gly Glu Glu Gly Glu Glu Asp Glu Glu Asp 275 280 285 Glu Glu Asp Pro Val Arg Gly Asp Met Phe Arg Met Pro Ser Arg Ser 290 295 300 300 Pro Leu Pro Ala Pro Pro Arg Gly Thr Leu Arg Leu Pro Ser Gly Cys 305 310 315 320 Ser Leu Ser Tyr Arg Thr Ile Ser Cys Ile Asn Ala Met Leu Thr Gln 325 330 335 Ile Pro Pro Leu Thr Ala Pro Gln Ile Thr Ser Leu Glu Leu Thr Gly 340 345 350 Asn Ser Ile Ala Ser Ile Pro Asp Glu Ala Phe Asn Gly Leu Pro Asn 355 360 365 Leu Glu Arg Leu Asp Leu Ser Lys Asn Asn Ile Thr Ser Ser Gly Ile 370 375 380 Gly Pro Lys Ala Phe Lys Leu Leu Lys Lys Leu Met Arg Leu Asn Met 385 390 395 400 Asp Gly Asn Asn Leu Ile Gln Ile Pro Ser Gln Leu Pro Ser Thr Leu 405 410 415 Glu Glu Leu Ly s Val Asn Glu Asn Asn Leu Gln Ala Ile Asp Glu Glu 420 425 430 Ser Leu Ser Asp Leu Asn Gln Leu Val Thr Leu Glu Leu Glu Gly Asn 435 440 445 445 Asn Leu Ser Glu Ala Asn Val Asn Pro Leu Ala Phe Lys Pro Leu Lys 450 455 460 Ser Leu Ala Tyr Leu Arg Leu Gly Lys Asn Lys Phe Arg Ile Ile Pro 465 470 475 480 Gln Gly Leu Pro Gly Ser Ile Glu Glu Leu Tyr Leu Glu Asn Asn Gln 485 490 495 Ile Glu Glu Ile Thr Glu Ile Cys Phe Asn His Thr Arg Lys Ile Asn 500 505 510 Val Ile Val Leu Arg Tyr Asn Lys Ile Glu Glu Asn Arg Ile Ala Pro 515 520 525 Leu Ala Trp Ile Asn Gln Glu Asn Leu Glu Ser Ile Asp Leu Ser Tyr 530 535 540 Asn Lys Leu Tyr His Val Pro Ser Tyr Leu Pro Lys Ser Leu Leu His 545 550 555 560 Leu Val Leu Leu Gly Asn Gln Ile Glu Arg Ile Pro Gly Tyr Val Phe 565 570 575 Gly His Met Glu Pro Gly Leu Glu Tyr Leu Tyr Leu Ser Phe Asn Lys 580 585 590 Leu Ala Asp Asp Gly Met Asp Arg Val Ser Phe Tyr Gly Ala Tyr His 595 600 605 Ser Leu Arg Glu Leu Phe Leu Asp His Asn Asp Leu Lys Ser Ile Pro 610 615 620 Pro Gly Ile Gln Gl u Met Lys Ala Leu His Phe Leu Arg Leu Asn Asn 625 630 635 640 Asn Lys Ile Arg Asn Ile Leu Pro Glu Glu Ile Cys Asn Ala Glu Glu 645 650 655 Asp Asp Asp Ser Asn Leu Glu His Leu His Leu Glu Asn Asn Tyr Ile 660 665 670 Lys Ile Arg Glu Ile Pro Ser Tyr Thr Phe Ser Cys Ile Arg Ser Tyr 675 680 685 Ser Ser Ile Val Leu Lys Pro Gln Asn Ile Lys 690 695 <210> 3 <211> 1690 <212> DNA <213> Homo sapiens <220> <221> 5'UTR <222> (1) .. (129) <220> <221> CDS <222> (130) .. (1224) <220> <221> polyA_signal <222> (1555) .. (1560) <220> <221> 3'UTR <222> (1225) .. (1690) <400> 3 agtactgaag gtggcgcctc ggcagcgggc tagaggccac tggcactgag cttgcaacca 60 gatccagaga cactgaagaa gagagaaagg cgcacctctt cccgccactc ctagccag 120 cctccaagg atg cgg ctc gc gg gc cc gc gg cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc gc cc gc cc gc cc gc cc gc cc gc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc cc gc gc cc gc cc gc gc cc gc gc cc gc cc gc cc gc cc gc cc gc cc gc gcc cc 10 tcc gcg ccg aag ccc gcc ttc gcc aaa gtg ctc acg ccg aat cgc atc 219 Ser Ala Pro Lys Pro Ala Phe Ala Lys Val Leu Thr Pro Asn Arg Ile 15 20 25 30 ccc gaa ttc tgc atc ccg ccg ccg ctg tgc acg ctc ggg 267 Pro Glu Phe Cys Ile Pro Pro Arg Leu Pro Ala Pro Cys Thr Leu Gly 35 40 45 tct cca atc cgg gcc gcc gcc gtg ccc cgg cgc tgc gcc gct gaa agc 315 Ser Pro Ile Arg Ala Ala Ala Val Pro Arg Arg Cys Ala Ala Glu Ser 50 55 60 gac ctg tgg ccc cgc gcg gca gac gag gac gcc ggc cgc acg gac tgg 363 Asp Leu Trp Pro Arg Ala Ala Asp Glu Asp Ala Gly Arg Thr Asp Trp 65 70 75 gac ccg cgc tc cag gca gcg ctg tca ctg ccg cac ctg ccc cgt gtg 411 Asp Pro Arg Ser Gln Ala Ala Leu Ser Leu Pro His Leu Pro Arg Val 80 85 90 cgc acc acc tac ggc ttc tgc gcg ctg ctc gag agc ccg cac acg cgc 459 Arg Thr Thr Tyr Gly Phe Cys Ala Leu Leu Glu Ser Pro His Thr Arg 95 100 105 110 cgc aag gag tcg ctc ctg ctc ggg ggc cg ccc gcc 507 Arg Lys Glu Ser Leu Leu Leu Gly Gly Pro Pro Ala Pro Arg Pro Arg 115 120 125 gcc cac agc tgc ggc ggc ggc gga ggc ccg gac gcc ccc ctg ggg acc 555 Ala His Ser Cys Gly Gly Gly Gly Gly Gly Pro Asp Ala Pro Leu Gly Thr 130 135 140 ctg tgc ggc ccg cga ggt ccg ggc ccg gcc acc ccc gcg gcc ccc ggc 603 Leu Cys Gly Pro Arg Gly Pro Gly Pro Ala Thr Pro Ala Ala Pro Gly 145 150 155 ggt ccc cgc ctg ccc gac gac ctc gct gcg ggg ccc cgc cgc tgc 651 Gly Pro Arg Leu Pro Gln Asp Ala Leu Ala Ala Gly Pro Arg Arg Cys 160 165 170 cgc ctc ctg cgc gtc ccc gac ggg ctg ctg agt cgc gcg ctg cgg Pro Asp Gly Leu Leu Ser Arg Ala Leu Arg Ala 175 180 185 190 gga agg agt cgc cgc ctg gcc cgc gtc cgc tcc gac tcc agc ggg aac 747 Gly Arg Ser Arg Arg Leu Ala Arg Val Arg Ser Asp Ser Ser Gly Asn 195 200 Two 05 gag gac gag gag cgc cgc gcg gga tcc gag tcc ccg gcc cgg gcc ccc 795 Glu Asp Glu Glu Arg Arg Ala Gly Ser Glu Ser Pro Ala Arg Ala Pro 210 215 220 tcc tcg agc ccg ctg tca tcc agg gcc ccg ctt cgc ctg gag 843 Ser Ser Ser Pro Leu Ser Ser Arg Ala Pro Leu Pro Glu Arg Leu Glu 225 230 235 gcc aag ggc acc gtg gct ctg ggc cgc gcc ggc gac gcc ctg cgc ctg 891 Ala Lys Gly Thr Val Ala Leu Gly Ag Gly Asp Ala Leu Arg Leu 240 245 250 gct gct gag tac tgt ccg gga acc cgg cgt ctc cgc ctc cgg ctg ctc 939 Ala Ala Glu Tyr Cys Pro Gly Thr Arg Arg Leu Arg Leu Arg Leu Leu 255 260 265 270 cgc gg ctg ttc gga ggc gcc ccc ggg ccc cgc gcc gtc cgc 987 Arg Ala Glu Ser Leu Phe Gly Gly Ala Pro Gly Pro Arg Ala Val Arg 275 280 285 tgc cgc ctc agc ctc gtc ctg cgg ccg ccg cg cg cg cg gc cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg Arg Leu Ser Leu Val Leu Arg Pro Pro Gly Thr Ala Arg Trp Gln 290 295 300 tgc agc gct gtg gtg ggg cgc agc cgc aag gcc tcc ttt gac cag gac 1083 Cys Ser Ala Val Val Gly Arg Ser Arg Lys Ala Ser Phe Asp Gln Asp 305 310 315 ttt tgc ttc gac ggc ctc tcg gaa gac gag gtg cgc cgc ctg gcc gtt 1131 Phe Cys Phe Asp Gly Leu Ser Glu Asp Glu Val Arg Arg Leu Ala Val 320 325 330 cgc gtc aag gcc ggg gg gcgat c cgg gat cgg ggc cgc ctg 1179 Arg Val Lys Ala Arg Asp Glu Gly Arg Gly Arg Asp Arg Gly Arg Leu 335 340 345 350 ctg ggc cag ggt gag ctg tcc ctg ggc gcc ctc ctg ctg ctc Gly G12G Leu Leu Gly Ala Leu Leu Leu Leu 355 360 365 gggcccagcc ctccccgggg cgctctgccc tggagactcc ggacactgac agccgcgtgg 1284 tacagaataa acgttattta tttttttatt tatgatactg ctttattgac gttttacttc 1344 ctcaccatcc acatttgtca tcgtctattg gtaaagaaga aaaagataat gactccctgt 1404 tctgttcaca ggacccccat atctctttcc agaccatttt tgcattccaa ggaaaaatgg 1464 gttggcttgg gactgggaga gaaaggaagt acacccatct gcattgtttt taattccttc 1524 cggttttcta tcaatgttac agtttttttt aataaagcaa gttattcatt cagaaaaaaa 1584 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1644 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1690 <210> 4 <211> 364 <212> PRT <213> Homo sapiens <400> 4 Met Arg Leu Leu Glu Lys Leu Cys Ser Ser Ala Ala Gly Ser Ser Ala 1 5 10 15 Pro Lys Pro Ala Phe Ala Lys Val Leu Thr Pro Asn Arg Ile Pro Glu 20 25 30 Phe Cys Ile Pro Pro Arg Leu Pro Ala Pro Cys Thr Leu Gly Ser Pro 35 40 45 Ile Arg Ala Ala Ala Val Pro Arg Arg Cys Ala Ala Glu Ser Asp Leu 50 55 60 Trp Pro Arg Ala Ala Asp Glu Asp Ala Gly Arg Thr Asp Trp Asp Pro 65 70 75 80 Arg Ser Gln Ala Ala Leu Ser Leu Pro His Leu Pro Arg Val Arg Thr 85 90 95 Thr Tyr Gly Phe Cys Ala Leu Leu Glu Ser Pro His Thr Arg Arg Lys 100 105 110 Glu Ser Leu Leu Leu Gly Gly Pro Pro Ala Pro Arg Pro Arg Ala His 115 120 125 Ser Cys Gly Gly Gly Gly Gly Pro Asp Ala Pro Leu Gly Thr Leu Cys 130 135 140 Gly Pro Arg Gly Pro Gly Pro Ala Thr Pro Ala Ala Pro Gly Gly Pro 145 150 155 160 Arg Leu Pro Gln Asp Ala Leu Ala Ala Gly Pro Arg Arg Cys Arg Leu 165 170 175 Leu Arg Val Pro Asp Gly Leu Leu Ser Arg Ala Leu Arg Ala Gly Arg 180 185 190 Ser Arg Arg Leu Ala Arg Val Arg Ser Asp Ser Ser Gly Asn Glu Asp 195 200 205 Glu Glu Arg Ar g Ala Gly Ser Glu Ser Pro Ala Arg Ala Pro Ser Ser 210 215 220 Ser Pro Leu Ser Ser Arg Ala Pro Leu Pro Glu Arg Leu Glu Ala Lys 225 230 235 240 Gly Thr Val Ala Leu Gly Arg Ala Gly Asp Ala Leu Arg Leu Ala Ala 245 250 255 Glu Tyr Cys Pro Gly Thr Arg Arg Leu Arg Leu Arg Leu Leu Arg Ala 260 265 270 Glu Ser Leu Phe Gly Gly Ala Pro Gly Pro Arg Ala Val Arg Cys Arg 275 280 285 285 Leu Ser Leu Val Leu Arg Pro Pro Gly Thr Ala Arg Trp Gln Cys Ser 290 295 300 300 Ala Val Val Gly Arg Ser Arg Lys Ala Ser Phe Asp Gln Asp Phe Cys 305 310 315 320 Phe Asp Gly Leu Ser Glu Asp Glu Val Arg Arg Leu Ala Val Arg Val 325 330 335 Lys Ala Arg Asp Glu Gly Arg Gly Arg Asp Arg Gly Arg Leu Leu Gly 340 345 350 Gln Gly Glu Leu Ser Leu Gly Ala Leu Leu Leu Leu 355 360 <210> 5 <211> 4049 <212> DNA <213> Homo sapiens <220> <221> 5'UTR <222> (1) .. (348) <220> <221> CDS <222> (349) .. (3183) <220> <221> polyA_signal <222> (4008) .. (4014) <220> <221> 3'UTR <222> (3184) .. (4049) <400> 5 agtctgctaa aaggggagga cgttgaggac gcggcggctg gcgggagaga cagctgggga 60 gagacatgac agggtcggag cgcggcctgc gcctctgtca ctcagcatcc tcttaggcgt 120 ttccacgccc gccccctgcc cgaggggcgg ggctgacggc tctggtaccc ggagtcggcg 180 cgcggggcag gggcgcgccc ctgcagagtg gggaccccac tgggctgtgc catgctgacc 240 ggagaccacc gaggcgggag acagagcgcg gcgaagagcc attgagtggt cacccagtag 300 ccgccgccgc cgccgcctcg ggaagcttgc cacccgctag gagggaag atg aag gag 357 Met Lys Glu 1 att tgc agg atc tgt gcc cga gag ctg tgt gga aac cag cgg cgc tgg 405 Ile Cys Arg Ile Cys Ala Arg Glu Leu Cys Gly Asn Gln Arg Arg Trp 5 10 15 atc ttc cac acg gcg tcc aag ctc gat ctcgat ctg ctt tcg cac 453 Ile Phe His Thr Ala Ser Lys Leu Asn Leu Gln Val Leu Leu Ser His 20 25 30 35 gtc ttg ggc aag gat gtc ccc cgc gat ggc aaa gcc gag ttc gct tgc 501 Val Leu Gly Lys Asp Val Pro Arg Asp Gly Lys Ala Glu Phe Ala Cys 40 45 50 agc aag tgt gct ttc atg ctt gat cga atc tat cga ttc gac aca gtt 549 Ser Lys Cys Ala Phe Met Leu Asp Arg Ile Tyr Arg Phe Asp Thr Val 55 60 65 att gcc cgg att gaa gcg ctt tct att gag cgc ttg caa aag ctg cta 597 Ile Ala Arg Ile Glu Ala Leu Ser Ile Glu Arg Leu Gln Lys Leu Leu 70 75 80 ctg gag aag gat cgc ctc ag ttc gc atg tat cgg aag 645 Leu Glu Lys Asp Arg Leu Lys Phe Cys Ile Ala Ser Met Tyr Arg Lys 85 90 95 aat aac gat gac tct ggc gcg gag atc aag gcg ggg aat ggg acg gtt 693 Asn Asn Asp Asp Ser Gly Ala Glu Lys Ala Gly Asn Gly Thr Val 100 105 110 115 gac atg tcc gtc tta ccc gat gcg aga tac tct gca ctg ctc cag gag 741 Asp Met Ser Val Leu Pro Asp Ala Arg Tyr Ser Ala Leu Leu Gln Glu 120 125 130 gac ttc gcc tat tca ggg ttt gag tgc tgg gtg gag aat gag gat cag 789 Asp Phe Ala Tyr Ser Gly Phe Glu Cys Trp Val Glu Asn Glu Asp Gln 135 140 145 atc cag gag cca cac agc tgc cat ggt tca gaa ggc cct gga ac Ile Gln Glu Pro His Ser Cys His Gly Ser Glu Gly Pro Gly Asn Arg 150 155 160 ccc agg aga tgc cgt ggt tgt gcc gct ttg cgg gtt gct gat tct gac 885 Pro Arg Arg Cys Arg Gly Cys Ala Ala Leu Arg Val Ala Asp Ser Asp 165 170 175 tat gaa gcc att tgt aag gta cct cga aag gtg gcc aga agt atc tcc 933 Tyr Glu Ala Ile Cys Lys Val Pro Arg Lys Val Ala Arg Ser Ile Ser 180 185 190 195 tgc ggc cct tct agc agg tgg tcg acc att tgc act gaa gaa cca 981 Cys Gly Pro Ser Ser Arg Trp Ser Thr Ser Ile Cys Thr Glu Glu Pro 200 205 210 gcg ttg tct gag gtt ggg cca ccc gac tta gca agc aca aag gta ccc 1029 Ala Leu Ser Glu Val Gly Pro Pro Asp Leu Ala Ser Thr Lys Val Pro 215 220 225 cca gat gga gaa agc atg gag gaa gag acg cct ggt tcc tct gtg gaa 1077 Pro Asp Gly Glu Ser Met Glu Glu Glu Thr Pro Gly Ser Ser Val Glu 230 235 240 tct ttg gat gca agc gtc cag gct agc cct cca caa cag aaa gat gag 1125 Ser Leu Asp Ala Ser Val Gln Ala Ser Pro Pro Gln Gln Lys Asp Glu 245 250 255 gag act gag aga agt gca aag gaa ctt gga aag tgt gac tgt tgt tca 1173 Glu Thr Glu Arg Ser Ala Lys Glu Leu Gly Lys Cys Asp Cys Cys Ser 260 265 270 gat gat cag gct ccg cag cat ggg tgt aat cac aag ctg gaa tta gct 1221 Asp Asp Gln Ala Pro Gln His Gly Cys Asn His Lys Leu Glu Leu Ala 280 285 290 ctt agc atg att aaa ggt ctt gat tat aag ccc atc cag agc ccc cga 1269 Leu Ser Met Ile Lys Gly Leu Asp Tyr Lys Pro Ile Gln Ser Pro Arg 295 300 305 ggg agc agg ctt ccg cca gtg aaa tcc agc cta cct gga gcc aag 1317 Gly Ser Arg Leu Pro Ile Pro Val Lys Ser Ser Leu Pro Gly Ala Lys 310 315 320 cct ggc cct agc atg aca gat gga gtt agt tcc ggt ttc ctt aac agg 1365 Pro Gly Pro Ser Met Thr Asp Gly Val Ser Ser Gly Phe Leu Asn Arg 325 330 335 tct ttg aaa ccc ctt tac aag aca cct gtg agt tat ccc ttg gag ctt 1413 Ser Leu Lys Pro Leu Tyr Lys Thr Pro Val Ser Tyr Pro Leu Glu Leu 340 345 350 355 tca gac ctg cag gag ctg tgg gat gat ctc tgt gaa gat tat ttg ccg 1461 Ser Asp Leu Gln Glu Leu Trp Asp Asp Leu Cys Glu Asp Tyr Leu Pro 360 365 370 ctc cgg gtc cag ccc atg act gag aaa caa caa aag ctg 1509 Leu Arg Val Gln Pro Met Thr Glu Glu Leu Leu Lys Gln Gln Lys Leu 375 380 385 aat tca cat gag acc act ata act cag cag tct gta tct gat tcc cac 1557 Asn Ser His Glu Thr Thr Ile Thr Gln Gln Ser Val Ser Asp Ser His 390 395 400 ttg gca gaa ctc cag gaa aaa atc cag caa aca gag gcc acc aac aag 1605 Leu Ala Glu Leu Gln Glu Lys Ile Gln Gln Thr Glu Ala Thr Asn Lys 405 410 415 att ctt caa gag aaa ctt aat gaa atg agc tat gaa cta aag tgt gct 1653 Ile Leu Gln Glu Lys Leu Asn Glu Met Ser Tyr Glu Leu Lys Cys Ala 420 425 430 435 435 cag gag tcg tct caa aag caa gat ggt aca att cag aag gaa 1701 Gln Glu Ser Ser Gln Lys Gln Asp Gly Thr Ile Gln Asn Leu Lys Glu 440 445 450 act ctg aaa agc agg gaa cgt gag act gag gag ttg tac cag gta att 1749 Thr Leu Lys Ser Arg Glu Arg Glu Thr Glu Glu Leu Tyr Gln Val Ile 455 460 465 gaa ggt caa aat gac aca atg gca aag ctt cga gaa atg ctg cac caa 1797 Glu Gly Gln Asn Asp Thr Met Ala Lys Leu Arg Glu Met Leu His Gln 470 475 480 agc cag ctt gca cac agc tca gag ggt act tct cca gct cag 1845 Ser Gln Leu Gly Gln Leu His Ser Ser Glu Gly Thr Ser Pro Ala Gln 485 490 495 caa cag gta gct ctg ctt gat ctt cag agt gct tta ttc tgc agc caa 1893 Gln G ln Val Ala Leu Leu Asp Leu Gln Ser Ala Leu Phe Cys Ser Gln 500 505 510 515 ctt gaa ata cag aag ctc cag agg gtg gta cga cag aaa gag cgc caa 1941 Leu Glu Ile Gln Lys Leu Gln Arg Val Val Arg Gln Lys Arg Gln 520 525 530 ctg gct gat gcc aaa caa tgt gtg caa ttt gta gag gct gca gca cac 1989 Leu Ala Asp Ala Lys Gln Cys Val Gln Phe Val Glu Ala Ala Ala His 535 540 545 gag agt gaa cag cag ct gag gag tgg aaa cat aac cag gaa ttg 2037 Glu Ser Glu Gln Gln Lys Glu Ala Ser Trp Lys His Asn Gln Glu Leu 550 555 560 cga aaa gcc ttg cag cag cta caa gaa gaa ttg cag aat aag agc caa 2085 Arg Lys Ala Lea Leu Gln Glu Glu Leu Gln Asn Lys Ser Gln 565 570 575 cag ctt cgt gcc tgg gag gct gaa aaa tac aat gag att cga acc cag 2133 Gln Leu Arg Ala Trp Glu Ala Glu Lys Tyr Asn Glu Ile Arg Thr Gln 580 585 590 gaa caa aac atc cag cac cta aac cat agt ctg agt cac aag gag cag 2181 Glu Gln Asn Ile Gln His Leu Asn His Ser Leu Ser His Lys Glu Gln 600 605 610 ttg ctt cag gaa ttt cgg gag ctc cta cag tat cga gat aac tca gac 2229 Leu Leu Gln Glu Phe Arg Glu Leu Leu Gln Tyr Arg Asp Asn Ser Asp 615 620 625 aaa acc ctt gaa gca aat gaa atg ttg ctt gag aaa ctt cgc cag cga 2277 Lys Thr Leu Glu Ala Asn Glu Met Glu Lys Leu Arg Gln Arg 630 635 640 ata cat gat aaa gct gtt gct ctg gag cgg gct ata gat gaa aaa ttc 2325 Ile His Asp Lys Ala Val Ala Leu Glu Arg Ala Ile Asp Glu Lys Phe 645 650 655 tct gct cta ga aaa gaa aaa gaa ctg cgc cag ctt cgt ctt gct 2373 Ser Ala Leu Glu Glu Lys Glu Lys Glu Leu Arg Gln Leu Arg Leu Ala 660 665 670 675 gtg aga gag cga gat cat gac tta gag aga ctg cgc gat gtc cccccccccccccccccccccccccccccccccccccccccccccccccccccccc Arg Glu Arg Asp His Asp Leu Glu Arg Leu Arg Asp Val Leu Ser 680 685 690 tcc aat gaa gct act atg caa agt atg gag agt ctc ctg agg gcc aaa 2469 Ser Asn Glu Ala Thr Met Gln Ser Met Glu Ser Leu Leu Arg Ala Lys 695 700 705 ggc ctg gaa gtg gaa cag tta tct act acc tgt caa aac ctc cag tgg 2517 Gly Leu Glu Val Glu Gln Leu Ser Thr Thr Cys Gln Asn Leu Gln Trp 710 715 720 ctg aaa gaa gaa atg gaa accaaa ttt agc cgt tgg cag aag gaa caa 2565 Leu Lys Glu Glu Met Glu Thr Lys Phe Ser Arg Trp Gln Lys Glu Gln 725 730 730 735 gag agt atc att cag cag tta cag acg tct ctt cat gat agg aac aaa 2613 Glu Ser Ile Gle Gln Leu Gln Thr Ser Leu His Asp Arg Asn Lys 740 745 750 755 gaa gtg gag gat ctt agt gca aca ctg ctc tgc aaa ctt gga cca ggg 2661 Glu Val Glu Asp Leu Ser Ala Thr Leu Leu Cys Lys Leu Gly Pro Gly 760 765 770 cag agt gag ata gca gag gag ctg tgc cag cgt cta cag cga aag gaa 2709 Gln Ser Glu Ile Ala Glu Glu Leu Cys Gln Arg Leu Gln Arg Lys Glu 775 780 785 agg atg ctg cag gac ctt cta agt gat cat aat gaaa cga gtg ctg gaa 2757 Arg Met Leu Gln Asp Leu Leu Ser Asp Arg Asn Lys Gln Val Leu Glu 790 795 800 cat gaa atg gag att caa ggc ctg ctt cag tct gtg agc acc agg gag 2805 His Glu Met Glu Ile Gln Gly Le Ser Val Ser Thr Arg Glu 805 810 815 cag gaa agc caa gct gct gca gag aag ttg gtg caa gcc tta atg gaa 2853 Gln Glu Ser Gln Ala Ala Ala Glu Lys Leu Val Gln Ala Leu Met Glu 820 825 830 830 835 aga aat t ca gaa tta cag gcc ctg cgc caa tat tta gga ggg aga gac 2901 Arg Asn Ser Glu Leu Gln Ala Leu Arg Gln Tyr Leu Gly Gly Arg Asp 840 845 850 tcc ctg atg tcc caa gca ccc atc tct aac caa gac acctga 2949 Ser Leu Met Ser Gln Ala Pro Ile Ser Asn Gln Gln Ala Glu Val Thr 855 860 865 ccc act ggc cgt ctt gga aaa cag act gat caa ggt tca atg cag ata 2997 Pro Thr Gly Arg Leu Gly Lys Gln Thr Asp Gln Gly Ser Met Gln Ile 870 875 880 cct tcc aga gat gat agc act tca ttg act gcc aaa gag gat gtc agc 3045 Pro Ser Arg Asp Asp Ser Thr Ser Leu Thr Ala Lys Glu Asp Val Ser 885 890 895 ata ccc aga tcc aca tta gga gac ttg gac aca gtt gca ggg ctg gaa 3093 Ile Pro Arg Ser Thr Leu Gly Asp Leu Asp Thr Val Ala Gly Leu Glu 900 905 910 915 aaa aaa gaa ctg agt aat gcc aaa ggg aac ttg aac tca tgg cta aaa aag 3141 Lys Glu Leu Asn Ala Lys Gly Asn Leu Asn Ser Trp Leu Lys Lys 920 925 930 aaa gag aaa gtc aga tgg aac ttt ctg ctc tac agt cca tga 3183 Lys Glu Lys Val Arg Trp Asn Phe Leu Leu Tyr Ser Pro 935 940 940 tggctgtca ggaagaagag ctgcaggtgc aggctgctga tatggagtct ctgaccagga 3243 acatacagat taaagaagat ctcataaagg acctgcaaat gcaactggtt gatcctgaag 3303 acataccagc tatggaacgc ctgacccagg aagtcttact tcttcgggaa aaagttgctt 3363 cagtagaatc ccagggtcaa gaaatttcag gaaaccgaag acaacagttg ctgctgatgc 3423 tagaaggact agtagatgaa cggagtcggc tcaatgaggc cttacaagca gagagacagc 3483 tctatagcag tctggtgaag ttccatgccc atccagagag ctctgagaga gaccgaactc 3543 tgcaggtgga actggaaggg gctcaggtgt tacgcagtcg gctagaagaa gttcttggaa 3603 gaagcttgga gcgcttaaac aggctggaga ccctggccgc cattggaggt ggggaactgg 3663 aaagtgtgcg aattcatcac aagcatgcct actgagcact ggcgggtcag actgcagccc 3723 aggatggaaa accttgtttg cactaaccag aaagatcctt gtctgatttt ggcagaatta 3783 aacggtgact tactaatgat agaactggta caagtagcat caactacaaa gtgaaactca 3843 ctttagccta catggatctc actgtacata catatcaatc cctaaattga atggtggagg 3903 ttgcaaagtg tatttgcaca ttttaaatca ttctgtttta gtttttccac ttttattcat 3963 tcttatccct agccccttct cgttccctca tcccttctgt gggccaataa agtttattct 4023 tccccaaaaa aaaaa aaaaa aaaaaa 4049 <210> 6 <211> 944 <212> PRT <213> Homo sapiens <400> 6 Met Lys Glu Ile Cys Arg Ile Cys Ala Arg Glu Leu Cys Gly Asn Gln 1 5 10 15 Arg Arg Trp Ile Phe His Thr Ala Ser Lys Leu Asn Leu Gln Val Leu 20 25 30 Leu Ser His Val Leu Gly Lys Asp Val Pro Arg Asp Gly Lys Ala Glu 35 40 45 Phe Ala Cys Ser Lys Cys Ala Phe Met Leu Asp Arg Ile Tyr Arg Phe 50 55 60 Asp Thr Val Ile Ala Arg Ile Glu Ala Leu Ser Ile Glu Arg Leu Gln 65 70 75 80 Lys Leu Leu Leu Glu Lys Asp Arg Leu Lys Phe Cys Ile Ala Ser Met 85 90 95 Tyr Arg Lys Asn Asn Asp Asp Ser Gly Ala Glu Ile Lys Ala Gly Asn 100 105 110 Gly Thr Val Asp Met Ser Val Leu Pro Asp Ala Arg Tyr Ser Ala Leu 115 120 125 Leu Gln Glu Asp Phe Ala Tyr Ser Gly Phe Glu Cys Trp Val Glu Asn 130 135 140 Glu Asp Gln Ile Gln Glu Pro His Ser Cys His Gly Ser Glu Gly Pro 145 150 155 160 Gly Asn Arg Pro Arg Arg Cys Arg Gly Cys Ala Ala Leu Arg Val Ala 165 170 175 Asp Ser Asp Tyr Glu Ala Ile Cys Lys Val Pro Arg Lys Val Ala Arg 180 185 190 Ser Ile Ser Cys Gly Pro Ser Ser Arg Trp Ser Thr Ser Ile Cys Thr 195 200 205 Glu Glu Pro Al a Leu Ser Glu Val Gly Pro Pro Asp Leu Ala Ser Thr 210 215 220 Lys Val Pro Pro Asp Gly Glu Ser Met Glu Glu Glu Thr Pro Gly Ser 225 230 235 240 Ser Val Glu Ser Leu Asp Ala Ser Val Gln Ala Ser Pro Pro Gln Gln 245 250 255 Lys Asp Glu Glu Thr Glu Arg Ser Ala Lys Glu Leu Gly Lys Cys Asp 260 265 270 Cys Cys Ser Asp Asp Gln Ala Pro Gln His Gly Cys Asn His Lys Leu 275 280 285 285 Glu Leu Ala Leu Ser Met Ile Lys Gly Leu Asp Tyr Lys Pro Ile Gln 290 295 300 Ser Pro Arg Gly Ser Arg Leu Pro Ile Pro Val Lys Ser Ser Leu Pro 305 310 315 320 Gly Ala Lys Pro Gly Pro Ser Met Thr Asp Gly Val Ser Ser Gly Phe 325 330 335 Leu Asn Arg Ser Leu Lys Pro Leu Tyr Lys Thr Pro Val Ser Tyr Pro 340 345 350 Leu Glu Leu Ser Asp Leu Gln Glu Leu Trp Asp Asp Leu Cys Glu Asp 355 360 365 Tyr Leu Pro Leu Arg Val Gln Pro Met Thr Glu Glu Leu Leu Lys Gln 370 375 380 Gln Lys Leu Asn Ser His Glu Thr Thr Ile Thr Gln Gln Ser Val Ser 385 390 395 400 400 Asp Ser His Leu Ala Glu Leu Gln Glu Lys Ile Gln Gln Thr Glu Ala 405 410 415 Thr Asn Lys Il e Leu Gln Glu Lys Leu Asn Glu Met Ser Tyr Glu Leu 420 425 430 Lys Cys Ala Gln Glu Ser Ser Gln Lys Gln Asp Gly Thr Ile Gln Asn 435 440 445 Leu Lys Glu Thr Leu Lys Ser Arg Glu Arg Glu Thr Glu Glu Leu Tyr 450 455 460 Gln Val Ile Glu Gly Gln Asn Asp Thr Met Ala Lys Leu Arg Glu Met 465 470 475 480 Leu His Gln Ser Gln Leu Gly Gln Leu His Ser Ser Glu Gly Thr Ser 485 490 490 495 Pro Ala Gln Gln Gln Val Ala Leu Leu Asp Leu Gln Ser Ala Leu Phe 500 505 510 Cys Ser Gln Leu Glu Ile Gln Lys Leu Gln Arg Val Val Arg Gln Lys 515 520 525 Glu Arg Gln Leu Ala Asp Ala Lys Gln Cys Val Gln Phe Val Glu Ala 530 535 540 Ala Ala His Glu Ser Glu Gln Gln Lys Glu Ala Ser Trp Lys His Asn 545 550 555 560 Gln Glu Leu Arg Lys Ala Leu Gln Gln Leu Gln Glu Glu Leu Gln Asn 565 570 575 Lys Ser Gln Gln Leu Arg Ala Trp Glu Ala Glu Lys Tyr Asn Glu Ile 580 585 590 Arg Thr Gln Glu Gln Asn Ile Gln His Leu Asn His Ser Leu Ser His 595 600 605 Lys Glu Gln Leu Leu Gln Glu Phe Arg Glu Leu Leu Gln Tyr Arg Asp 610 615 620 620 Asn Ser Asp Lys Th r Leu Glu Ala Asn Glu Met Leu Leu Glu Lys Leu 625 630 635 640 Arg Gln Arg Ile His Asp Lys Ala Val Ala Leu Glu Arg Ala Ile Asp 645 650 655 Glu Lys Phe Ser Ala Leu Glu Glu Lys Glu Lys Glu Leu Arg Gln Leu 660 665 670 Arg Leu Ala Val Arg Glu Arg Asp His Asp Leu Glu Arg Leu Arg Asp 675 680 685 Val Leu Ser Ser Asn Glu Ala Thr Met Gln Ser Met Glu Ser Leu Leu 690 695 700 Arg Ala Lys Gly Leu Glu Val Glu Gln Leu Ser Thr Thr Cys Gln Asn 705 710 715 715 720 Leu Gln Trp Leu Lys Glu Glu Met Glu Thr Lys Phe Ser Arg Trp Gln 725 730 730 735 Lys Glu Gln Glu Ser Ile Ile Gln Gln Leu Gln Thr Ser Leu His Asp 740 745 750 Arg Asn Lys Glu Val Glu Asp Leu Ser Ala Thr Leu Leu Cys Lys Leu 755 760 765 Gly Pro Gly Gln Ser Glu Ile Ala Glu Glu Leu Cys Gln Arg Leu Gln 770 775 780 Arg Lys Glu Arg Met Leu Gln Asp Leu Leu Ser Asp Arg Asn Lys Gln 785 790 795 800 Val Leu Glu His Glu Met Glu Ile Gln Gly Leu Leu Gln Ser Val Ser 805 810 815 Thr Arg Glu Gln Glu Ser Gln Ala Ala Ala Glu Lys Leu Val Gln Ala 820 825 830 Leu Met Glu Arg As n Ser Glu Leu Gln Ala Leu Arg Gln Tyr Leu Gly 835 840 845 Gly Arg Asp Ser Leu Met Ser Gln Ala Pro Ile Ser Asn Gln Gln Ala 850 855 860 Glu Val Thr Pro Thr Gly Arg Leu Gly Lys Gln Thr Asp Gln Gly Ser 865 870 875 880 Met Gln Ile Pro Ser Arg Asp Asp Ser Thr Ser Leu Thr Ala Lys Glu 885 890 895 Asp Val Ser Ile Pro Arg Ser Thr Leu Gly Asp Leu Asp Thr Val Ala 900 905 910 Gly Leu Glu Lys Glu Leu Ser Asn Ala Lys Gly Asn Leu Asn Ser Trp 915 920 925 925 Leu Lys Lys Lys Glu Lys Val Arg Trp Asn Phe Leu Leu Tyr Ser Pro 930 935 940 <210> 7 <211> 10 <212> DNA <213> Artificial Sequence <220> <221> primer_bind <222> (1) .. (10) <220> <223> Artificially synthesized arbitary primer sequence <400> 7 tggtaaaggg 10 <210> 8 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (10) <400> 8 gcatcgtcta 10 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (10) <400> 9 actcgaccag 10 <210> 10 <211> 250 <212> DNA <213> Homo sapiens <220> <221> gene <222> (1) .. (250) <400> 10 tggtaaaggg cataaccatg tacaacaaag ctgtgtggtc gcctgagccc tgcactacct 60 gcctctgctc agatggaaga gttctttgtg atgaaaccat gtgccatccc cagaggtgcc 120 cccaaacagt tatacctgaa ggggaatgct gcccggtctg ctccgctact gtctcctatt 180 ctctactcag tggtatagca ttaaatgata gaaatgaatt ttctggtgat tcccaaaaaa 240 aaaaaaaaac 250 <210> 11 <211> 230 <212> DNA <213> Homo sapiens <220> <221> gene <222> (1) .. (230) <400> 11 gcatcgtcta ttggtaaaga agaaaaagat aatgactccc tgttctgttc acaggacccc 60 catatctctt tccagaccat ttttgcattc caaggaaaaa tgggttggct tgggactggg 120 agagaaagga agtacaccca tctgca tattattca tcattaca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca tca <210> 12 <211> 764 <212> DNA <213> Homo sapiens <220> <221> gene <222> (1) .. (764) <400> 12 actcgaccag catttgcact gaagaaccag cgttgtctga ggttgggcca cccgacttag 60 caagcacaaa ggtaccccca gatggagaaa gcatggagga agagacgcct ggttcctctg 120 tggaatcttt ggatgcaagc gtccaggcta gccctccaca acagaaagat gaggagactg 180 agagaagtgc aaaggaactt ggaaagtgtg actgttgttc agatgatcag gctccgcagc 240 atgggtgtaa tcacaagctg gaattagctc ttagcatgat taaaggtctt gattataagc 300 ccatccagag cccccgaggg agcaggcttc cgattccagt gaaatccagc ctacctggag 360 ccaagcctgg ccctagcatg acagatggag ttagttccgg tttccttaac aggtctttga 420 aaccccttta caagacacct gtgagttatc ccttggagct ttcagacctg caggagctgt 480 gggatgatct ctgtgaagat tatttgccgc tccgggtcca gcccatgact gaagagttgc 540 tgaaacaaca aaagctgaat tcacatgaga ccactataac tcagcagtct gtatctgatt 600 cccacttggc agaactccag gaaaaaatcc agcaaacaga ggccaccaac aagattcttc 660 aagagaaact taatgaaatg agctatgaac taaagtgtgc tcaggagtcg tctcaaaagc 720 aagatggtac aattcagaac ctcaagtcaa aaaaaaaaaa aaac 764 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 13 gttttttttt ttttttag 18 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 14 gttttttttt ttttttgg 18 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 15 gttttttttt ttttttcg 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 16 gttttttttt ttttttat 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 17 gttttttttt ttttttgt 18 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 18 gttttttttt ttttttct 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 19 gttttttttt ttttttaa 18 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 20 gttttttttt ttttttga 18 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 21 gttttttttt ttttttca 18 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 22 gttttttttt ttttttac 18 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 23 gttttttttt ttttttgc 18 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized arbitary primer sequence <220> <221> primer_bind <222> (1) .. (18) <400> 24 gttttttttt ttttttcc 18

[0126]

[Brief description of the drawings]

FIG. 1 is a photograph showing the expression state of human AG1102 protein mRNA in various human tissues by Northern blotting.

FIG. 2 is a photograph showing the expression state of human AA3901 protein mRNA in various human tissues by Northern blotting.

FIG. 3 is a photograph showing the expression state of human AA3401 protein mRNA in various human tissues by Northern blotting.

FIG. 4 is a diagram schematically showing primary structural features of an amino acid sequence constituting human AG1102 protein. The symbols (-, ●, #, and *) at the lower part of the amino acid represent the signal sequence (-),
The predicted positions of the RGD sequence (●), the von Willebrand factor C-like domain (#), and the leucine-rich repeat structure (*) are shown.

FIG. 5 (a) shows human A analyzed by FISH method.
The figure which shows the position on the human chromosome of G1102 gene. The two white dots (about 0.3 mm) at the end of the arrow indicate the 9q22.3 site where the human AG1102 gene is present. FIG. 5 (b) is a diagram showing mapping of chromosomes of metaphase human cells on the R-staining karyotype.

FIG. 6 (a) shows human A analyzed by FISH method.
The figure which shows the position on the human chromosome of A3901 gene. The two white dots (about 0.3 mm) at the end of the arrow indicate the 15q22 site where the human AA3901 gene is present. FIG. 6 (b) is a diagram showing mapping of chromosomes of metaphase human cells on the R-staining karyotype.

FIG. 7 shows human AA34 analyzed by the FISH method.
The figure which shows the position on the human chromosome of 01 gene. It is observed that the human AA3401 gene is present at one of the 1p12 and 1q21.1 sites.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 5/10 C12P 21/02 C 15/02 21/08 C12P 21/02 C12N 5/00 B 21/08 15/00 C // (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91) F term (reference) 4B024 AA01 BA31 BA44 CA04 DA02 DA06 FA01 GA03 GA11 GA18 GA19 GA23 HA01 4B064 AG01 AG27 CA02 CA10 CA19 CC24 CE04 CE06 CE11 CE12 CE14 DA06 DA07 4B065 AA26X AA90X AA91X AA92X AA93Y AB01 AB05 AC14 AC15 BA01 BA25 CA24 CA25 CA44 4H045 AA10 AA11 AA30 BA10 CA40 DA76 EA23 EA27 FA74 HA06

Claims (42)

[Claims] 1. A DNA encoding a protein having the amino acid sequence of SEQ ID NO: 2 or a part thereof. 2. A DNA comprising the nucleotide sequence of nucleotides 74 to 2170 in the nucleotide sequence of SEQ ID NO: 1. 3. A DNA which hybridizes to a DNA having the nucleotide sequence of SEQ ID NO: 1 under stringent conditions. 4. A protein having the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence substantially identical to the amino acid sequence, or a part thereof. An expression vector comprising the DNA according to any one of claims 1 to 3. 6. A transformed cell transformed with the expression vector according to claim 5. 7. An antibody or a part of an antibody reactive with the protein of claim 4 or a part thereof. 8. The antibody or a part of the antibody according to claim 7, wherein the antibody is a monoclonal antibody. 9. A cell that produces a monoclonal antibody reactive with the protein of claim 4 or a part thereof. 10. The cell according to claim 1, wherein the cell is a fusion cell obtained by fusing a non-human mammal-derived B cell capable of producing the monoclonal antibody with a mammal-derived myeloma cell. Item 10. The cell according to Item 9. 11. The method according to claim 8, wherein the cell is a DNA encoding the heavy chain of the monoclonal antibody or D encoding the light chain thereof.
10. The cell according to claim 9, wherein the cell is a genetically modified cell transformed by introducing one or both DNAs of NA into the cell. 12. A pharmaceutical composition comprising the protein or a part thereof according to claim 4, and a pharmaceutically acceptable carrier. 13. A pharmaceutical composition comprising the antibody or a part of the antibody according to claim 7 or 8 and a pharmaceutically acceptable carrier. 14. A transgenic non-human, characterized in that a DNA comprising the base sequence of base numbers 74 to 2170 of the base sequence set forth in SEQ ID NO: 1 has been incorporated into an endogenous gene of a non-human mammal. Mammals. 15. A DNA encoding the protein having the amino acid sequence shown in SEQ ID NO: 4 or a part thereof. 16. A DNA comprising the base sequence of base numbers 131 to 1221 of the base sequence set forth in SEQ ID NO: 3. 17. A DNA that hybridizes under stringent conditions to a DNA having the nucleotide sequence of SEQ ID NO: 3. 18. A protein having the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence substantially identical to the amino acid sequence, or a part thereof. An expression vector comprising the DNA according to any one of claims 15 to 17. 20. A transformed cell transformed with the expression vector according to claim 19. 21. An antibody or a part of an antibody reactive with the protein of claim 18 or a part thereof. 22. The antibody or part of an antibody according to claim 21, wherein the antibody is a monoclonal antibody. 23. A cell that produces a monoclonal antibody reactive with the protein of claim 18 or a part thereof. 24. The cell, wherein the cell is a fusion cell obtained by fusing a non-human mammal-derived B cell capable of producing the monoclonal antibody with a mammal-derived myeloma cell. Item 24. The cell according to Item 23. 25. The method according to claim 25, wherein the cell is a DNA encoding the heavy chain of the monoclonal antibody or D encoding the light chain thereof.
24. The cell according to claim 23, wherein the cell is a transgenic cell transformed by introducing one or both DNAs of NA into the cell. 26. A pharmaceutical composition comprising the protein according to claim 18 or a part thereof, and a pharmaceutically acceptable carrier. 27. A pharmaceutical composition comprising the antibody or a part of the antibody according to claim 21 and a pharmaceutically acceptable carrier. 28. A transgenic non-human, characterized in that a DNA comprising the base sequence of base numbers 131 to 1221 of the base sequence set forth in SEQ ID NO: 3 has been incorporated into an endogenous gene of a non-human mammal. Mammals. 29. A DNA encoding the protein having the amino acid sequence set forth in SEQ ID NO: 6 or a part thereof. 30. A DNA comprising the base sequence of base numbers 351 to 3180 of the base sequence set forth in SEQ ID NO: 5. 31. A DNA which hybridizes to a DNA having the nucleotide sequence of SEQ ID NO: 5 under stringent conditions. 32. A protein having the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence substantially identical to the amino acid sequence, or a part thereof. 33. An expression vector comprising the DNA according to any one of claims 29 to 31. 34. A transformed cell transformed with the expression vector according to claim 33. 35. An antibody or a part of an antibody reactive with the protein of claim 32 or a part thereof. 36. The antibody or part of an antibody of claim 35, wherein the antibody is a monoclonal antibody. 37. A cell that produces a monoclonal antibody reactive with the protein of claim 32 or a part thereof. 38. The cell, wherein the cell is a fusion cell obtained by fusing a non-human mammal-derived B cell capable of producing the monoclonal antibody with a mammal-derived myeloma cell. Item 38. The cell according to Item 37. 39. The method according to claim 39, wherein the cell is a DNA encoding the heavy chain of the monoclonal antibody or D encoding the light chain thereof.
38. The cell according to claim 37, wherein the cell is a genetically modified cell transformed by introducing one or both DNAs of NA into the cell. 40. A pharmaceutical composition comprising the protein according to claim 32 or a part thereof, and a pharmaceutically acceptable carrier. 41. A pharmaceutical composition comprising the antibody or a part of the antibody according to claim 35 or 36, and a pharmaceutically acceptable carrier. 42. A transgenic non-human, characterized in that a DNA comprising the nucleotide sequence of base numbers 351 to 3180 of the nucleotide sequence set forth in SEQ ID NO: 5 has been incorporated into an endogenous gene of a non-human mammal. Mammals.