JP2003052384A - New receptor protein and its dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new receptor protein useful as an excellent anticancer agent and a material for screening the same. SOLUTION: A human-derived receptor protein or its salt, a DNA encoding the protein and an antibody against the protein or its salt are provided. A method for determining the ligand for the protein is provided. A method/a kit for screening a compound capable of altering the binding properties of the ligand to the protein are provided and a compound or its salt is obtained by the screening. Thereby, the antibody, the compound capable of altering the binding properties of the ligand to the protein (an antagonist, etc.), and an antisense polynucleotide are useful as the anticancer agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel receptor protein of human origin, its DNA, its use and the like.

[0002]

BACKGROUND OF THE INVENTION The protein tyrosine kinase gene group forms the largest gene family among oncogenes and is deeply involved in cell proliferation and differentiation and morphogenesis (Harvey).
Lect. 94, 81-119, 1998-99), it has been taken up as an ideal target gene for the development of anticancer agents. Above all, receptor tyrosine kinase (RTK)
The drug development targeting the EGF receptor, HER2 protein or VEGF receptor classified into
It has been producing anti-cancer drugs at a level that is actually tested in humans, and RT is one of the protein tyrosine kinases.
K is considered to be a subfamily with a high probability of success as a drug.

Among RTKs, Eph (erythropoietin-p
roducing human hepatocellular carcinoma cell lin
e) The receptor gene family is currently composed of 14 members and has the largest scale (Cell 90,
403-404, 1997). The Ephrin gene family identified as a ligand for the Eph receptor has also been reported to consist of at least eight members, but the ligands corresponding to all Eph receptors have not been identified. There are also Eph receptors that map to receptors. Eph
The physiological functions of receptors and Ephrin ligands have been suggested to be involved in morphogenesis during developmental stage or proper projection of nerve axons from the extremely localized expression in the embryonic and brain regions (Annu. Rev. Neurosci). Volume 21,
309-345, 1998), but its role in cancer has been attracting attention in recent years. For example, upregulation of Eph receptor expression in various cancers has been reported (Oncogene 19, 5614-5619, 20.
00) has been done. Furthermore, their involvement in angiogenesis has been suggested (Genes & Dev. 13: 1055-1066, 1
999 years). That is, it is considered that the enhanced expression of the Eph receptor in cancer tissues makes it possible for cancer cells to lose tissue colonization ability and at the same time induce peripheral angiogenesis to have both explosive proliferation ability and hematogenous metastatic ability. (Oncogene Volume 19, 5614-5619, 2000).

[0004]

[Problems to be Solved by the Invention] New human-derived Eph
The receptor-like protein has an activity of regulating its activity, and enables development of a new drug useful for the prevention and treatment of various diseases based on the activity, for example, central nervous disease such as Alzheimer's disease and cancer. In addition, since it can be expected that the expression of a new human-derived Eph receptor-like protein in human cancer tissues is remarkably enhanced, the new human-derived Eph receptor-like protein itself can be used as a cancer vaccine or an antibody can be used to treat cancer. Can be used for. Therefore, in the field of the present invention, it was desired to find a novel human-derived Eph receptor-like protein and develop a method for mass production.

[0005]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found a human-derived Eph receptor-like protein having a novel nucleotide sequence. The present inventors, based on these findings,
As a result of further studies, the present invention has been completed.

That is, the present invention provides (1) SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO:
20, Ep characterized by containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 38
h receptor protein or salt thereof (2) SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO:
42, SEQ ID NO: 44 or SEQ ID NO: 46, which contains the same or substantially the same amino acid sequence as the Eph receptor protein or a salt thereof, (3) SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO:
(1) containing the amino acid sequence represented by 20, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 38
Eph receptor protein or salt thereof described (4) SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO:
No. 42, SEQ ID NO: 44 or SEQ ID NO: 46, the Eph receptor protein according to (2) above or a salt thereof, (5) the Eph receptor protein according to (1) or (2) above. Partial peptide or salt thereof (6) Sixth amino acid sequence represented by SEQ ID NO: 11
The partial peptide or a salt thereof according to (5) above, which contains the 0th to 73rd or 491st to 504th amino acid sequence, (7) the Eph receptor protein according to (1) or (2) above, or (5) A polynucleotide containing a polynucleotide encoding the partial peptide according to (5), (8) The polynucleotide according to (7) above, which is DNA, (9) SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO :.
The polynucleotide according to (12) above, which has the nucleotide sequence represented by 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 39, (10) SEQ ID NO: 15, SEQ ID NO: 41, SEQ ID NO: 43. , The polynucleotide according to (12) above, which has the nucleotide sequence represented by SEQ ID NO: 45 or SEQ ID NO: 47, (11) a recombinant vector containing the polynucleotide according to (7) above, (12) above ( A transformant transformed with the recombinant vector according to 11), (13) the transformant according to (12) above is cultured, and the Eph receptor protein according to (1) or (2) above or (5) above. Eph according to the above (1) or (2), characterized in that the partial peptide described above is produced.
Receptor protein or salt thereof or the above (5)
(14) An antibody against the Eph receptor protein or a salt thereof according to (1) or (2) or the partial peptide or a salt thereof according to (5) above, (15) a polyclonal antibody, (16) The antibody according to (14), which is a monoclonal antibody, (17) The 60th position to the 73rd position or the 491st position of the amino acid sequence represented by SEQ ID NO: 11. -An antibody according to (14) above, which specifically reacts with a peptide having the 504th amino acid sequence, (18) Neutralization that inactivates signal transduction of the Eph receptor protein according to (1) or (2) above. The antibody according to (14) above, which is an antibody, (19) a medicament comprising the antibody according to (14) above, (20) immuno (19) The drug according to the above (19) which is a toxin, (21) the drug according to the above (19) which is an anticancer agent, (22) the diagnostic agent comprising the antibody according to the above (14), (23) the above (1) Alternatively, it can be obtained by using the Eph receptor protein according to (2) or the partial peptide according to (5) above or a salt thereof.
Or (2) a ligand for the Eph receptor protein or a salt thereof, (24) a medicament comprising the ligand according to (23) above, (25) the Eph receptor protein according to (1) or (2) above, or (5) The method for determining a ligand according to (23) above, which comprises using the partial peptide or a salt thereof, (26) the ligand according to (23) and the Eph according to (1) or (2). A method for screening a compound or a salt thereof that changes the binding property to a receptor protein or a salt thereof, (27) Binding of the ligand according to (23) above to the Eph receptor protein or salt thereof according to (1) or (2) above (28) The screening kit for a compound or a salt thereof which alters the sex, (28) A ligand obtainable by using the cleaning method or the screening kit described in (27) above and E described in (1) or (2) above.
a compound or a salt thereof that changes the binding property to a ph receptor protein or a salt thereof, (29) a medicament comprising the compound or a salt thereof described in (28) above, (30) the polynucleotide described in (7) above A polynucleotide that hybridizes under high stringent conditions, (31) A diagnostic agent containing the polynucleotide according to (7) above, (32) A diagnostic agent according to (31) above which is a diagnostic agent for cancer, (33) ) A method for quantifying Eph receptor protein mRNA according to (1) or (2) above, which comprises using the polynucleotide according to (7) or a part thereof, (34) the antibody according to (14) above A method for quantifying the Eph receptor protein according to the above (1) or (2), which is used (35) above (33) or (34) The method for diagnosing a disease associated with the Eph receptor function according to (1) or (2), characterized by using the quantification method according to (1) or (36), wherein the disease is cancer (35). (37) A method for screening a compound or a salt thereof that changes the expression level of the Eph receptor protein according to (1) or (2), characterized by using the quantification method according to (33) above. ) A method for screening a compound or a salt thereof which changes the amount of Eph receptor protein in the cell membrane according to (1) or (2), characterized by using the quantification method according to (33) above, (39) above (37) Changing the expression level of the Eph receptor protein according to the above (1) or (2), which can be obtained by using the screening method described in Compound or a salt thereof, (40) above (38) above in the cell membrane which can be obtained using the screening method described (1) or (2)
The compound or salt thereof that changes the amount of Eph receptor protein described in (41), a medicine comprising the compound or salt thereof described in (39) or (40) above, (42) the above-mentioned (41) which is an anticancer agent (43) A multimer of the Eph receptor protein according to (1) or (2) above, (44) a multimer according to (43) above which is a dimer, (45) above (1) or above ( 2) Eph described
The multimer according to (44) above, which is a dimer with a receptor belonging to the receptor protein family, (46) the Eph receptor protein according to (1) or (2) above, and the receptor belonging to the Eph receptor family. And above (1)
Alternatively, the Eph receptor protein according to (2) and Ep
A method for screening a substance having an action of inhibiting multimerization with a receptor belonging to the h receptor family, (47) comprising the Eph receptor protein according to (1) or (2) above and a receptor belonging to the Eph receptor family A kit for screening a substance having an action of inhibiting multimerization of the Eph receptor protein according to (1) or (2) above and a receptor belonging to the Eph receptor family, (48) according to (46) above It has an action of inhibiting the multimerization of the Eph receptor protein according to (1) or (2) and the receptor belonging to the Eph receptor family, which can be obtained using the screening method or the screening kit according to (47) above. Material, (49) (50) A medicament comprising the substance according to (48), (50) a medicament according to (49) which is an anticancer agent, (51) a base sequence complementary to the polynucleotide according to (7) or a part thereof. (52) A medicine containing the antisense polynucleotide according to (51) above, (53) a medicine containing the Eph receptor protein according to (1) or (2) above, (54) The medicament according to (53) above, which is a vaccine preparation, (55) an antibody against a receptor having an action of inhibiting the activity of a receptor, (56) a medicament comprising the antibody according to (55) above, (57) ) A nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor having an action of inhibiting the activity of the receptor, or (58) A medicament comprising the antisense polynucleotide according to (57) above, (59) An effective amount of the antibody according to (14) above against a mammal. (60) A method for preventing / treating cancer, which comprises administering
4) Use of the antibody described above and the like.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The receptor protein of the present invention comprises SEQ ID NO: 11, SEQ ID NO: 18 and SEQ ID NO: 2.
0, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 3
8, SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 4
2, an Eph receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 46 (hereinafter sometimes abbreviated as receptor protein). The receptor protein of the present invention is, for example, a human or non-human mammal (eg, guinea pig, rat, mouse, rabbit,
Pig cells, sheep cells, bovine cells, monkey cells, etc. (for example, splenocytes, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, Fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovium Cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes or stromal cells, or progenitor cells of these cells, stem cells or cancer cells) or hematopoietic cells, or those cells Any tissue present, such as the brain, parts of the brain (eg, olfactory bulb, squamous nucleus, basal sphere, hippocampus, thalamus, hypothalamus, subthalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, lateral) Head lobe, putamen, Jokaku, brain dyeing, substantia nigra), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal, skin, muscle, lungs, digestive tract (e.g., colon,
Small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testis, testis, ovary, placenta, uterus, bone,
It may be a protein derived from a joint, skeletal muscle, or the like, or may be a synthetic protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 11 includes, for example, about 55% or more of the amino acid sequence represented by SEQ ID NO: 11,
An amino acid sequence having a homology of preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more, and the like can be mentioned. . The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 11 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 11
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 18 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70% with the amino acid sequence represented by SEQ ID NO: 18. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 18 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 18
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 20 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70%, the amino acid sequence represented by SEQ ID NO: 20. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 20 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 20, SEQ ID NO: 20
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 22 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70% of the amino acid sequence represented by SEQ ID NO: 22. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 22 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 22
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70% of the amino acid sequence represented by SEQ ID NO: 24. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 24
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 38 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70%, with the amino acid sequence represented by SEQ ID NO: 38. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 38 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 38, SEQ ID NO: 38
A protein having substantially the same activity as the amino acid sequence represented by is preferred.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14 is, for example, about 55% or more of the amino acid sequence represented by SEQ ID NO: 14,
An amino acid sequence having a homology of preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more, and the like can be mentioned. . The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 14
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 40 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70% of the amino acid sequence represented by SEQ ID NO: 40. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 40 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 40, SEQ ID NO: 40
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 42 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70% with the amino acid sequence represented by SEQ ID NO: 42. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 42 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 42, SEQ ID NO: 42
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 44 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70% of the amino acid sequence represented by SEQ ID NO: 44. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 44 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 44, SEQ ID NO: 44
A protein having substantially the same activity as the amino acid sequence represented by is preferred. The amino acid sequence which is substantially the same as the amino acid sequence represented by SEQ ID NO: 46 is, for example, about 55% or more, preferably about 60% or more, more preferably about 70% of the amino acid sequence represented by SEQ ID NO: 46. The amino acid sequences having homology of not less than 80%, more preferably not less than 80%, most preferably not less than 90%, most preferably not less than 95% are included. The protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 46 of the present invention has, for example, an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 46, SEQ ID NO: 46
A protein having substantially the same activity as the amino acid sequence represented by is preferred.

Examples of substantially the same activity include ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility regulatory activity. "Substantially the same quality" means that their activities are qualitatively the same. Therefore, the ligand binding activity,
Activities such as signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility control activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0 times). However, the quantitative factors such as the degree of activity and the molecular weight of the protein may be different. The ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity, cell motility regulatory activity and the like can be measured according to known methods. Further, it can be measured according to the ligand determination method and screening method described later.

The receptor protein of the present invention includes (1) (i) 1 or 2 or more (preferably 1 to 200, preferably 1 to 100) amino acid sequence represented by SEQ ID NO: 11. About one, preferably 1-5
About 0, preferably about 1 to 30, preferably 1
Amino acid sequence in which about 10 amino acids, more preferably several (1 to 5) amino acids are deleted, (ii) SEQ ID NO: 11
1 or 2 or more in the amino acid sequence represented by (preferably 1 to 200, preferably 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to An amino acid sequence having about 10 amino acids added, more preferably several (1 to 5) amino acids,
(Iii) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 11 (preferably about 1 to 200, preferably about 1 to 100, preferably 1 to 50, preferably 1 to 30) Amino acid sequence in which about one, preferably about 1 to 10, more preferably several (1 to 5) amino acids are replaced with other amino acids, or (i
v) 1 or 2 or more (preferably 1 to 200) in the amino acid sequence represented by (2) (i) SEQ ID NO: 18, such as a protein containing an amino acid sequence combining (i) to (iii) About 1 to 100,
An amino acid sequence in which about 1 to 50, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are deleted,
(Ii) 1 in the amino acid sequence represented by SEQ ID NO: 18
Or 2 or more (preferably about 1 to 200, preferably about 1 to 100, preferably 1 to 50,
An amino acid sequence having about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably several (1 to 5) amino acids, (iii) in the amino acid sequence represented by SEQ ID NO: 18 1 or 2 or more (preferably about 1 to 200, preferably about 1 to 100,
An amino acid sequence in which about 1 to 50, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are substituted with other amino acids, or (Iv) A protein or the like containing an amino acid sequence combining (i) to (iii), (3)
(I) 1 in the amino acid sequence represented by SEQ ID NO: 20
Or 2 or more (preferably about 1 to 200, preferably about 1 to 100, preferably 1 to 50,
An amino acid sequence in which about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably several (1 to 5) amino acids are deleted, (ii) in the amino acid sequence represented by SEQ ID NO: 20 1 or 2 or more (preferably,
1 to 200, preferably 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to 10, more preferably several (1 to
5)) amino acid sequence added, (iii)
1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 20 (preferably about 1 to 200, preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, An amino acid sequence in which about 1 to 10 amino acids, more preferably several (1 to 5) amino acids are substituted with other amino acids, or (iv) (i)
(4) (i) 1 or 2 or more (preferably 1 to 20) in the amino acid sequence represented by SEQ ID NO: 22 such as a protein containing an amino acid sequence combining (iii)
About 0, preferably about 1 to 100, preferably 1
To about 50, preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5))
Amino acid sequence from which the amino acid of (ii) SEQ ID NO:
1 or 2 or more in the amino acid sequence represented by 22 (preferably 1 to 200, preferably 1 to 10)
About 0, preferably about 1 to 50, preferably 1
About 30 amino acids, preferably about 1-10, more preferably several (1-5) amino acids, (iii) 1 or 2 in the amino acid sequence represented by SEQ ID NO: 22 Or more (preferably 1 to 200, preferably 1 to 100, preferably 1 to 50)
About 1, preferably about 1 to 30, preferably 1 to 1
A protein containing an amino acid sequence in which about 0, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence in which (iv) (i) to (iii) are combined, etc. , (5) (i) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 24 (preferably about 1 to 200, preferably about 1 to 100,
An amino acid sequence in which about 1 to 50, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are deleted,
(Ii) 1 in the amino acid sequence represented by SEQ ID NO: 24
Or 2 or more (preferably about 1 to 200, preferably about 1 to 100, preferably 1 to 50,
An amino acid sequence having 1 to 30 amino acids, preferably 1 to 10 amino acids, and more preferably several amino acids (1 to 5 amino acids) added, (iii) in the amino acid sequence represented by SEQ ID NO: 24 1 or 2 or more (preferably about 1 to 200, preferably about 1 to 100,
An amino acid sequence in which about 1 to 50, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are substituted with other amino acids, or (Iv) A protein containing an amino acid sequence obtained by combining (i) to (iii), (6)
(I) 1 in the amino acid sequence represented by SEQ ID NO: 38
Or 2 or more (preferably about 1 to 200, preferably about 1 to 100, preferably 1 to 50,
Amino acid sequence in which about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are deleted, (ii) in the amino acid sequence represented by SEQ ID NO: 38 1 or 2 or more (preferably,
1 to 200, preferably 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to 10, more preferably several (1 to
5)) amino acid sequence added, (iii)
1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 38 (preferably about 1 to 200, preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, An amino acid sequence in which about 1 to 10 amino acids, more preferably several (1 to 5) amino acids are substituted with other amino acids, or (iv) (i)
(7) 1 or 2 or more (preferably 1 to 20) in the amino acid sequence represented by (i) SEQ ID NO: 14 such as a protein containing an amino acid sequence in combination.
About 0, preferably about 1 to 100, preferably 1
To about 50, preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5))
Amino acid sequence from which the amino acid of (ii) SEQ ID NO:
1 or 2 or more in the amino acid sequence represented by 14 (preferably 1 to 200, preferably 1 to 10)
About 0, preferably about 1 to 50, preferably 1
About 30 amino acids, preferably about 1 to 10, more preferably several (1 to 5) amino acids added, (iii) 1 or 2 in the amino acid sequence represented by SEQ ID NO: 14. Or more (preferably 1 to 200, preferably 1 to 100, preferably 1 to 50)
About 1, preferably about 1 to 30, preferably 1 to 1
A protein containing an amino acid sequence in which about 0, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence in which (iv) (i) to (iii) are combined, etc. , (8) (i) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 40 (preferably about 1 to 200, preferably about 1 to 100,
An amino acid sequence in which about 1 to 50, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are deleted,
(Ii) 1 in the amino acid sequence represented by SEQ ID NO: 40
Or 2 or more (preferably about 1 to 200, preferably about 1 to 100, preferably 1 to 50,
An amino acid sequence having about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably several amino acids (1 to 5 amino acids) added, (iii) in the amino acid sequence represented by SEQ ID NO: 40 1 or 2 or more (preferably about 1 to 200, preferably about 1 to 100,
An amino acid sequence in which about 1 to 50, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are substituted with other amino acids, or (Iv) A protein containing an amino acid sequence obtained by combining (i) to (iii), (9)
(I) 1 in the amino acid sequence represented by SEQ ID NO: 42
Or 2 or more (preferably about 1 to 200, preferably about 1 to 100, preferably 1 to 50,
An amino acid sequence in which about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are deleted, (ii) in the amino acid sequence represented by SEQ ID NO: 42 1 or 2 or more (preferably,
1 to 200, preferably 1 to 100, preferably 1 to 50, preferably 1 to 30, preferably 1 to 10, more preferably several (1 to
5)) amino acid sequence added, (iii)
1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 42 (preferably 1 to 200, preferably 1 to 100, preferably 1 to 50, preferably 1 to 30) An amino acid sequence in which about 1 to 10 amino acids, more preferably several (1 to 5) amino acids are substituted with other amino acids, or (iv) (i)
(10) (i) 1 or 2 or more (preferably 1 to 2) in the amino acid sequence represented by SEQ ID NO: 44, such as a protein containing an amino acid sequence combining (iii)
00 pieces, preferably 1 to 100 pieces, preferably 1 to 50 pieces, preferably 1 to 30 pieces, preferably 1 to 10 pieces, and more preferably several pieces (1 to 5 pieces).
Amino acid sequence), wherein (1) or 2 or more (preferably 1 to 200, preferably 1 to 2 in the amino acid sequence represented by SEQ ID NO: 44).
About 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5) amino acid sequences, (iii ) 1 or 2 or more (preferably 1 to 200) in the amino acid sequence represented by SEQ ID NO: 44
About 1, preferably about 1 to 100, preferably 1 to
About 50, preferably about 1 to 30, preferably 1
A protein containing an amino acid sequence in which about 10 amino acids, more preferably several (1 to 5) amino acids are substituted with other amino acids, or an amino acid sequence in which (iv) (i) to (iii) are combined. , (11) (i) SEQ ID NO: 4
1 or 2 or more in the amino acid sequence represented by 6 (preferably about 1 to 200, preferably about 1 to 100, preferably about 1 to 50, preferably 1 to 30)
About 2, preferably about 1 to 10, more preferably several (1 to 5) amino acids, (ii) 1 or 2 in the amino acid sequence represented by SEQ ID NO: 46 Or more (preferably about 1 to 200,
Amino acids to which about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are added. Sequence, (iii) 1 or 2 or more in the amino acid sequence represented by SEQ ID NO: 46 (preferably about 1 to 200, preferably about 1 to 100,
An amino acid sequence in which about 1 to 50, preferably about 1 to 30, preferably about 1 to 10 and more preferably several (1 to 5) amino acids are substituted with other amino acids, or (Iv) A protein containing an amino acid sequence obtained by combining (i) to (iii) is also used.

In the amino acid sequence of the receptor protein in the present specification, the left end is the N-terminal (amino terminal) and the right end is the C-terminal (carboxyl terminal) according to the convention of peptide notation. Receptor protein of the present invention, C-terminal, carboxyl group (-COOH), a carboxylate (-COO ^-), may be any of an amide (-CONH ₂₎ or an ester (-COOR). Here, R in the ester is, for example, C _1-6 such as methyl, ethyl, n-propyl, isopropyl or n-butyl.
Alkyl groups such as C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl such as phenyl and α
C _6-12 aryl groups such as naphthyl, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl, or C _7-14 aralkyl such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl In addition to the group, a pivaloyloxymethyl group generally used as an ester for oral use is used. When the receptor protein of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the receptor protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Furthermore, in the receptor protein of the present invention, in the above-mentioned protein, the amino group of the N-terminal methionine residue is a protecting group (eg, formyl group, C _1-6 acyl group such as C _2-6 alkanoyl group such as acetyl, etc.). Protected by N), N
Glutamyl group produced by cleavage at the end side in vivo is pyroglutamine-oxidized, substituent on side chain of amino acid in molecule (eg, -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc. ) Is protected by an appropriate protecting group (eg, formyl group, C _1-6 acyl group such as C _2-6 alkanoyl group such as acetyl), or a complex such as a so-called glycoprotein having a sugar chain bonded thereto. It also includes proteins. Specific examples of the receptor protein of the present invention include, for example, SEQ ID NO: 1
1. A receptor protein containing the amino acid sequence represented by 1; a receptor protein containing the amino acid sequence represented by SEQ ID NO: 18; a receptor protein containing the amino acid sequence represented by SEQ ID NO: 20; an amino acid represented by SEQ ID NO: 22 Receptor protein containing sequence, receptor protein containing amino acid sequence represented by SEQ ID NO: 24, SEQ ID NO: 3
A receptor protein containing the amino acid sequence represented by 8 and a receptor protein containing the amino acid sequence represented by SEQ ID NO: 14 (amino acids 177 to 497 of the amino acid sequence represented by SEQ ID NO: 11 are deleted) ), A receptor protein containing the amino acid sequence represented by SEQ ID NO: 40, SEQ ID NO: 42
The receptor protein containing the amino acid sequence represented by, the receptor protein containing the amino acid sequence represented by SEQ ID NO: 44, the receptor protein containing the amino acid sequence represented by SEQ ID NO: 46, and the like are used.

A partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as the partial peptide of the present invention) can also be used. The partial peptide of the present invention may be any one as long as it is a partial peptide of the above-mentioned receptor protein of the present invention. For example, among the receptor protein molecules of the present invention, it is exposed outside the cell membrane. A portion or a portion located inside the cell membrane and having substantially the same activity is used. Here, the “substantially the same activity” means, for example, a ligand binding activity, a signal transduction activity, a cell death inhibitory activity, a cell adhesion activity and a cell motility regulatory activity. The ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity, cell motility regulatory activity and the like can be measured according to known methods. The amino acid number of the partial peptide of the present invention is at least 5 or more, preferably 10 or more, preferably 50 or more, more preferably 100 or more amino acid sequences among the constituent amino acid sequences of the receptor protein of the present invention described above. Peptides having the above are preferable. The substantially identical amino acid sequence means about 50% or more, preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, and particularly preferably about 90% with these amino acid sequences.
Amino acid sequences with homology of greater than or equal to%, and most preferably greater than about 95% are indicated.

The partial peptide of the present invention is (i) 1 or 2 or more (preferably 1) in the above amino acid sequence.
About 10 to about 10, more preferably several (1 to 5) amino acids are deleted, and (ii) 1 or 2 or more (preferably about 1 to 20 and more preferably 1) in the amino acid sequence. About 10 pieces, more preferably several pieces (1 to 5 pieces)
Or (iii) 1 or 2 or more (preferably about 1 to 10) in the above amino acid sequence,
(More preferably several, more preferably about 1 to 5)
The amino acid of may be substituted with another amino acid. Specific examples of the partial peptide of the present invention include: the 60th position of the amino acid sequence represented by SEQ ID NO: 11 to
A peptide containing the 73rd or 491st to 504th amino acid sequence, a peptide containing the 318th to 332nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18, represented by SEQ ID NO: 20 A peptide containing the 385th to 506th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22, a peptide containing the 128th to 142nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22 A peptide containing the 455th to 469th amino acid sequences of the amino acid sequence represented by: SEQ ID NO: 38, a peptide containing the 90th to 104th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 14, Containing the 143rd to 157th amino acid sequences of the amino acid sequence Peptide, a peptide containing the 655th to 743rd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 40, a 360th to 374th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 42 Peptide, a peptide containing the 160th to 174th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 44, a 240th to 254th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 46 Examples include peptides.

In the partial peptide of the present invention, the C-terminal has a carboxyl group (-COOH) and a carboxylate (-COH).
OO ^-), amide (-CONH ₂₎ or ester (-C
OOR) (R has the same meaning as described above).
When the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used. Further, in the partial peptide of the present invention, the amino group of the N-terminal methionine residue is protected by a protecting group, as in the above-mentioned receptor protein of the present invention,
A glutamyl group produced by cleavage at the N-terminal side in vivo is pyroglutamine-oxidized, a substituent on the side chain of an amino acid in the molecule is protected by a suitable protecting group, or a sugar chain-bonded so-called sugar Complex peptides such as peptides are also included. Examples of the salt of the receptor protein of the present invention or its partial peptide include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and salts thereof are used.

The receptor protein of the present invention or a salt thereof can be produced from the above-mentioned human or non-human mammalian cells or tissues by a known method for purifying the receptor protein, or the receptor protein of the present invention described later can be prepared. It can also be produced by culturing a transformant containing the encoding DNA. In addition, it can also be produced by a protein synthesis method described later or a method similar thereto. When producing from human or non-human mammal tissues or cells, after homogenizing human or non-human mammal tissues or cells, extraction is performed with an acid or the like, and the extract is subjected to reverse phase chromatography or ion exchange chromatography. Purification and isolation can be achieved by combining chromatography such as.

For the synthesis of the receptor protein of the present invention, its partial peptide, its salt or its amide, a commercially available resin for protein synthesis can be used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl resin. Phenylacetamide methyl resin, polyacrylamide resin, 4
Examples thereof include-(2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin and 4- (2', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the amino acid sequence of the target protein or peptide. At the end of the reaction, the protein or peptide is cleaved from the resin, at the same time various protecting groups are removed, and an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or its amide. Regarding the condensation of the above-mentioned protected amino acids, various activation reagents that can be used for protein synthesis can be used, but carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. For the activation by these, additives for suppressing racemization (for example, HOBt, H
The protected amino acid can be added directly to the resin together with OOBt) or can be added to the resin after previously performing activation of the protected amino acid as a symmetrical acid anhydride or HOBt ester or HOOBt ester.

The solvent used for activation of the protected amino acid or condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone,
Halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, methyl acetate, Esters such as ethyl acetate or an appropriate mixture thereof are used. The reaction temperature is appropriately selected from the range known to be applicable to protein bond-forming reactions, and is usually selected in the range of approximately -20 ° C to 50 ° C. The activated amino acid derivative is usually 1.5 to 4
Used in fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even after repeating the reaction, unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl oil, Fmoc and the like are used. The carboxyl group is, for example, an alkyl esterified (for example, a linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl and the like. ), Aralkyl esterification (eg benzyl ester, 4-
Nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide formation, tertiary butoxycarbonyl hydrazide formation, trityl hydrazide formation, etc. . The hydroxyl group of serine can be protected by, for example, esterification or etherification.
Examples of groups suitable for this esterification include lower alkanoyl groups such as acetyl group, aroyl groups such as benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group.
Moreover, examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. As a protective group for the phenolic hydroxyl group of tyrosine,
For example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like are used.
Examples of the protecting group for imidazole of histidine include:
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,
Boc, Trt, Fmoc, etc. are used.

The activated carboxyl group of the raw material includes, for example, corresponding acid anhydrides, azides, active esters [alcohols (for example, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinide, N-
Hydroxyphthalimide, ester with HOBt)] and the like are used. As the activated amino group of the raw material, for example, the corresponding phosphoric acid amide is used.
Examples of the method for removing (eliminating) the protective group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or anhydrous hydrogen fluoride, methanesulfonic acid, or trifluoride. Acid treatment with methanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally carried out at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole,
It is effective to add a cation trapping agent such as phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol, and 1,2-ethanedithiol. Also, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above-mentioned 1,2-ethanedithiol, 1,4-butane group. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of a functional group which should not be involved in the reaction of the starting material, the removal of the protective group and the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means. . As another method for obtaining an amide form of a protein, for example, first, the α-carboxyl group of a carboxy-terminal amino acid is amidated and protected, and then a peptide (protein) chain is extended to the amino group side to a desired chain length. , A protein from which only the protecting group of the α-amino group at the N-terminal of the peptide chain has been removed and a protein from which only the protecting group of the carboxyl group at the C-terminal has been removed, and both proteins are prepared in a mixed solvent as described above. To condense. The details of the condensation reaction are the same as above. After purifying the protected protein obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein. This crude protein can be purified by making full use of various known purification means, and the main fraction can be lyophilized to obtain the amide form of the desired protein. To obtain an ester form of a protein, for example, the α-carboxyl group of a carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then an ester form of the desired protein is obtained in the same manner as the amide form of the protein. be able to.

The partial peptide of the protein of the present invention or a salt thereof can be produced by a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can form the protein of the present invention with the remaining portion and removing the protecting group when the product has a protecting group. Examples of known condensation methods and removal of protective groups include the methods described in (i) to (v) below. (I) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) (ii) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) (iii) Nobuo Izumiya et al., Fundamentals and Experiments of Peptide Synthesis, Maruzen
(1975) (iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) (v) Supervision of Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method, and conversely, when it is obtained as a salt, it can be converted into a free form by a known method. You can

The polynucleotide encoding the receptor protein of the present invention includes a nucleotide sequence (DNA or R) encoding the above-mentioned receptor protein of the present invention.
It may be any as long as it contains NA, preferably DNA). The polynucleotide may be DN that encodes the receptor protein of the present invention.
A, RNA such as mRNA, which may be double-stranded or single-stranded. In the case of double-stranded, double-stranded DNA,
It may be double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (that is, a coding strand) or an antisense strand (that is, a non-coding strand). The DNA encoding the receptor protein of the present invention includes genomic DNA and genomic DN.
A library, cDNA derived from the above cells / tissues,
Any of the above-mentioned cell / tissue-derived cDNA library and synthetic DNA may be used. The vectors used for the library are bacteriophage, plasmid, cosmid,
It may be any such as phagemid. In addition, the reverse transcriptase polymerase can be directly used by using a total RNA or mRNA fraction prepared from the cells and tissues described above.
DNA can also be amplified by Chain Reaction (hereinafter, abbreviated as RT-PCR method).

Specifically, the DNA encoding the receptor protein of the present invention includes, for example, (1) a DNA containing the nucleotide sequence represented by SEQ ID NO: 12 or the nucleotide sequence represented by SEQ ID NO: 12. It has a DNA that hybridizes with the contained DNA under high stringent conditions and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion). DNA encoding a receptor protein having activity and cell motility controlling activity, etc. (2) DNA containing the nucleotide sequence represented by SEQ ID NO: 19 or DNA containing the nucleotide sequence represented by SEQ ID NO: 19 It has a DNA that hybridizes under high stringent conditions and has the receptor tag of the present invention. DNA encoding a receptor protein having substantially the same activity as the protein (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility regulatory activity), (3) sequence DNA having the nucleotide sequence represented by No. 21 or DNA hybridizing with the DNA having the nucleotide sequence represented by SEQ ID NO: 21 under high stringent conditions, which is substantially the receptor protein of the present invention. DNA that encodes a receptor protein having a homologous activity (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity, cell motility regulatory activity, etc.) (4) SEQ ID NO: 23 DNA containing the base sequence represented, or the base sequence represented by SEQ ID NO: 23 It has a DNA that hybridizes with the existing DNA under high stringent conditions, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity). And a DNA encoding a receptor protein having a cell motility controlling activity, etc., (5) a DNA containing the nucleotide sequence represented by SEQ ID NO: 25, or a DNA containing the nucleotide sequence represented by SEQ ID NO: 25 It has a DNA that hybridizes under stringent conditions and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility). DNA encoding a receptor protein having regulatory activity, etc. (6) A receptor protein of the present invention, which comprises a DNA containing the nucleotide sequence represented by SEQ ID NO: 39 or a DNA hybridizing with the DNA containing the nucleotide sequence represented by SEQ ID NO: 39 under high stringent conditions. DNA encoding a receptor protein having substantially the same activity (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility regulatory activity) (7) SEQ ID NO: 15 DNA having a nucleotide sequence represented by or a DNA hybridizing with the DNA having a nucleotide sequence represented by SEQ ID NO: 15 under high stringent conditions and having substantially the same quality as the receptor protein of the present invention. Activity (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity) DNA that encodes a receptor protein having cell adhesion activity and cell motility control activity, etc. (8) DNA containing the base sequence represented by SEQ ID NO: 41, or the base sequence represented by SEQ ID NO: 41 It has a DNA that hybridizes with DNA under high stringent conditions and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and DNA encoding a receptor protein having a cell motility controlling activity (9), (9) DNA containing the base sequence represented by SEQ ID NO: 43, or DNA containing the base sequence represented by SEQ ID NO: 43 and hystrin The receptor of the present invention having a DNA that hybridizes under a gentle condition. DNA encoding a receptor protein having substantially the same activity as the protein (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility regulatory activity) (10) SEQ ID NO: : 45, or a DNA containing the nucleotide sequence represented by SEQ ID NO: 45 and hybridizing with the DNA containing the nucleotide sequence represented by SEQ ID NO: 45 under high stringent conditions. Which encodes a receptor protein having a homologous activity (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity, cell motility regulatory activity, etc.) (11) represented by SEQ ID NO: 47 Containing the nucleotide sequence represented by SEQ ID NO: 47 or the nucleotide sequence represented by SEQ ID NO: 47 Having a DNA that hybridizes with a DNA containing DNA under high stringent conditions, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell Any DNA may be used as long as it encodes a receptor protein having adhesive activity and cell motility controlling activity).

Sequence number: 12, sequence number: 19, sequence number: 21, sequence number: 23, sequence number: 25, sequence number: 39, sequence number: 15, sequence number: 41, sequence number: 43, sequence number : 45 or the DNA having the nucleotide sequence represented by SEQ ID NO: 47, which hybridizes under high stringent conditions include, for example, SEQ ID NO: 12, SEQ ID NO: 19 and SEQ ID NO: 2
1, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 3
9, SEQ ID NO: 15, SEQ ID NO: 41, SEQ ID NO: 4
3, about 55% or more, preferably about 60% or more, preferably about 70% or more, preferably about 80% or more, more preferably about 90% or more with the nucleotide sequence represented by SEQ ID NO: 45 or SEQ ID NO: 47. More preferably, DNA containing a nucleotide sequence having a homology of about 95% or more is used.

Hybridization can be carried out by a known method or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al.
al., Cold Spring Harbor Lab. Press, 1989), and when using a commercially available library, the method described in the attached instruction manual can be used. More preferably, hybridization can be performed under high stringent conditions. The high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 2
At 0 mM, the temperature is about 50 to 70 ° C., preferably about 60.
The conditions of ˜65 ° C. are shown. Especially, the sodium concentration is about 19
Most preferred is mM and a temperature of about 65 ° C.

More specifically, (1) as the DNA encoding the receptor protein containing the amino acid sequence represented by SEQ ID NO: 11, the DNA containing the nucleotide sequence represented by SEQ ID NO: 12 is (2) ) As the DNA encoding the receptor protein containing the amino acid sequence represented by SEQ ID NO: 18, D containing the nucleotide sequence represented by SEQ ID NO: 19 is used.
NA is (3) a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 20, and D containing the nucleotide sequence represented by SEQ ID NO: 21.
NA is (4) as a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 22, D containing the nucleotide sequence represented by SEQ ID NO: 23.
NA is (5) as a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 24, D containing the nucleotide sequence represented by SEQ ID NO: 25.
NA is (6) as a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 38, D containing the nucleotide sequence represented by SEQ ID NO: 39.
NA is (7) as a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 14, D containing the nucleotide sequence represented by SEQ ID NO: 15.
NA is (8) as a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 40, D containing the nucleotide sequence represented by SEQ ID NO: 41
NA is (9) as a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 42, D containing the nucleotide sequence represented by SEQ ID NO: 43.
NA is (10) as a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 44, D containing the nucleotide sequence represented by SEQ ID NO: 45.
NA is (11) as a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 46, D containing the nucleotide sequence represented by SEQ ID NO: 47.
NA is mentioned.

The polynucleotide containing a part of the base sequence of the DNA encoding the receptor protein of the present invention or a part of the base sequence complementary to the DNA is
It is used not only to include the DNA encoding the partial peptide of the present invention described below, but also to include RNA. According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the Eph receptor protein gene is used as the nucleotide sequence information of the cloned or sequenced DNA encoding the Eph receptor protein. It can be designed based on and synthesized. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the Eph receptor protein gene, can inhibit the synthesis or function of the RNA, or can interact with the Eph receptor protein-related RNA through the Eph receptor protein-related RNA. The expression of the receptor protein gene can be regulated / controlled. Eph
A polynucleotide complementary to a selected sequence of a receptor protein-related RNA and a polynucleotide capable of specifically hybridizing with an Eph receptor protein-related RNA regulate the expression of the Eph receptor protein gene in vivo and in vitro. -Useful for controlling, and useful for treatment or diagnosis of diseases and the like. In addition, 5'end hairpin loop of Eph receptor protein gene, 5'end 6-base pair repeat, 5'end
Although the terminal non-translated region, the polypeptide translation stop codon, the protein coding region, the ORF translation stop codon, the 3'terminal untranslated region, the 3'terminal palindromic region, and the 3'terminal hairpin loop can be selected as preferred target regions, Any region within the Eph receptor protein gene can be selected as a subject.

The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, or the relationship between the polynucleotide capable of hybridizing with the target region is said to be "antisense". it can. Antisense polynucleotides include 2-deoxy-
Polydeoxynucleotides containing D-ribose, polydeoxynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or other polymers having a non-nucleotide backbone. (Eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that the base pairing or attachment of bases, such as found in DNA or RNA, does not occur. Containing a nucleotide having a permissible configuration) and the like. They are,
Double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded R
NA, which may further be a DNA: RNA hybrid, and further unmodified polynucleotide (or unmodified oligonucleotide), and also with known modifications, such as those with labels known in the art, caps Attached, methylated, substituted with one or more natural nucleotides by analogs, modified with an intramolecular nucleotide such as an uncharged bond (eg, methylphosphonate, phosphotriester, phospho) Those having charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, etc.) Poly-L-
Those having side chain groups such as lysine) and sugars (such as monosaccharide), those having intercurrent compounds (such as acridine and psoralen), chelate compounds (such as metal and radioactive) Metal, boron, an oxidizing metal, etc.), an alkylating agent, or a modified bond (for example, α-anomeric nucleic acid). Here, "nucleoside", "nucleotide" and "nucleic acid" not only contain purine and pyrimidine bases,
Those having other modified heterocyclic bases may be included. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
The modified nucleotide and the modified nucleotide may also have a modified sugar moiety, for example, one or more hydroxyl groups are substituted with halogen, an aliphatic group or the like, or a functional group such as ether or amine is added. It may have been converted.

The antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention has the following policies: that is, it makes the antisense nucleic acid more stable in cells, enhances the cell permeability of the antisense nucleic acid, and has a higher affinity for the target sense strand. It may be preferably designed to be greater and, if toxic, less toxic to the antisense nucleic acid. Many such modifications are known in the art, for example J. Kawakami et al., Pharm Tech Ja.
pan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992;
ST Crooke et al. Ed., Antisense Research and Ap
It is disclosed in plications, CRC Press, 1993, etc.

The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or linkages, and is provided in a special form such as liposome or microsphere, or applied by gene therapy. It may be provided or added. Thus, those used in the addition form include polycationic substances such as polylysine that act to neutralize the charge of the phosphate group skeleton,
Hydrophobic substances such as lipids (eg, phospholipids, cholesterol, etc.) that enhance the interaction with the cell membrane and increase the uptake of nucleic acid can be mentioned. Preferred lipids to add include cholesterol and its derivatives (eg cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'-end or 5'-end of the nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Examples of the other group include a capping group specifically arranged at the 3'-end or the 5'-end of a nucleic acid, which is for preventing decomposition by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl group-protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of Eph receptor protein. The nucleic acid can be applied to cells by various known methods.

Polynucleotide (eg, DNA) encoding the protein or partial peptide used in the present invention
As an antisense polynucleotide containing a base sequence complementary to, or substantially complementary to, a base sequence containing a, the polynucleotide of the present invention (eg,
DNA) containing a base sequence complementary to or substantially complementary to a base sequence containing DNA) or a part thereof and having an action capable of suppressing the expression of the polynucleotide (eg, DNA) Any antisense polynucleotide may be used, but antisense DNA is preferred.
The nucleotide sequence substantially complementary to the polynucleotide (eg, DNA) of the present invention means, for example, a nucleotide sequence complementary to the polynucleotide (eg, DNA) of the present invention (that is, complementary to the polynucleotide of the present invention). The total base sequence or partial base sequence of the chain) is about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 9%.
Examples include base sequences having a homology of 5% or more.
In particular, of the entire base sequence of the complementary strand of the polynucleotide (eg, DNA) of the present invention, the complementary strand of the base sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the base sequence near the start codon) Antisense polynucleotides having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more are suitable. Specifically, SEQ ID NO: 1
2, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 2
3, SEQ ID NO: 25, SEQ ID NO: 39, SEQ ID NO: 1
5, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45
Or D having the nucleotide sequence represented by SEQ ID NO: 47
Examples include an antisense polynucleotide having a base sequence complementary to or substantially complementary to the base sequence of NA, or a part thereof. The antisense polynucleotide is usually about 10 to 40, preferably 15 to 40.
It is composed of about 30 bases. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense polynucleotide is, for example, a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, or phosphorodithionate. May be substituted with. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

DNA encoding the partial peptide of the present invention
Any may be used as long as it contains the nucleotide sequence encoding the partial peptide of the present invention. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, RT-P can be directly used by using an mRNA fraction prepared from the cells and tissues described above.
DNA can also be amplified by the CR method. Specifically, as the DNA encoding the partial peptide of the present invention, for example, (1) SEQ ID NO: 12, SEQ ID NO: 19,
SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 39, SEQ ID NO: 15, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45 or the nucleotide sequence represented by SEQ ID NO: 47 DNA having a partial base sequence of the contained DNA, or (2) SEQ ID NO: 12, SEQ ID NO:
19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 2
5, SEQ ID NO: 39, SEQ ID NO: 15, SEQ ID NO: 4
1, SEQ ID NO: 43, SEQ ID NO: 45 or SEQ ID NO:
A protein containing a DNA that hybridizes with the DNA represented by 47 under high stringent conditions, and encodes a protein having substantially the same activity as the protein peptide of the present invention, such as a ligand binding activity and a signal transduction action. DNA having a partial base sequence of DNA
Are used.

As a means for cloning the DNA which completely encodes the receptor protein of the present invention or a partial peptide thereof (hereinafter sometimes abbreviated as the receptor protein of the present invention), the DNA encoding the receptor protein of the present invention can be used. A DNA fragment or synthetic DNA that encodes a part or the whole region of the receptor protein of the present invention is obtained by amplifying the DNA amplified by the PCR method using a synthetic DNA primer having a partial base sequence of the base sequence or incorporated into an appropriate vector. It can be selected by hybridization with the one used and labeled.

The substitution of the nucleotide sequence of DNA is carried out by using PCR or a known kit such as Mutan ^™ -superExpress Km (Takara Shuzo Co., Ltd.) or Mutan ^™ -K (Takara Shuzo Co., Ltd.).
It can be carried out according to a known method such as A-LA PCR method, gapped duplex method, Kunkel method or a method similar thereto. The cloned DNA encoding the receptor protein can be used as it is, or if desired after digestion with a restriction enzyme or addition of a linker, depending on the purpose. The DNA may have ATG as a translation initiation codon at the 5′-terminal side and TAA, TGA or TAG as a translation termination codon at the 3′-terminal side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. The DNA encoding the receptor protein of the present invention includes, for example, (a) the DNA encoding the receptor protein of the present invention, for example, a fragment of a target DNA portion is excised from cDNA, and (b) the DNA
The fragment can be incorporated into an expression vector by ligating the fragment downstream of the promoter in an appropriate expression vector.

As a vector for cloning or expression, a plasmid derived from E. coli (eg pC
R4, pCR2.1, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmid (eg, pUB110, pTP5, pC194), yeast-derived plasmid (eg, pSH19, pSH15), bacteriophage such as λ phage, retrovirus In addition to animal viruses such as vaccinia virus and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when an animal cell is used as a host, SRα promoter, S
Examples include V40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like. Among these, it is preferable to use the CMV promoter, the SRα promoter and the like. When the host is Escherichia, the trp promoter, lac promoter, recA promoter, λP _L promoter, l
When the host is a Bacillus bacterium, the pp promoter and the like are SPO1 promoter, SPO2 promoter,
When the host is yeast, such as the penP promoter,
PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. A polyhedrin promoter if the host is an insect cell,
P10 promoter and the like are preferable.

In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. Examples of the selectable marker include, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX)
Resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ^r ), neomycin resistance gene (hereinafter,
Sometimes abbreviated as Neo ^r, include G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, the target gene can be selected by a medium containing no thymidine. Also,
If necessary, a signal sequence suitable for the host can be added to the N-terminal side of the receptor protein of the present invention. When the host is Escherichia, PhoA signal sequence, OmpA signal sequence, etc., when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc., the host is yeast For MFα · signal sequence, SUC2 · signal sequence, etc., when the host is an animal cell, an insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used. A transformant can be produced using the vector containing the DNA encoding the receptor protein of the present invention thus constructed.

Examples of the host used in the present invention include Escherichia, Bacillus, yeast, insect cells, insects and animal cells. Specific examples of the Escherichia bacterium include Escherichia col.
i) K12 DH1 [Proc. Natl. Acad. Sci. USA], Volume 60, 16
0 (1968)], JM103 [Nucleic Acids Research, Volume 9, 309 (1981)],
JA221 [Journal of Molecular Biology], 120, 517
(1978)], HB101 [Journal of Molecular
Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], DH5α [Ino
ue, H., Nojima, H. and Okayama, H., Gene, 96, 23-28
(1990)], DH10B [Proc. Natl. Acad. Sci. USA], 87
Vol. 4645-4649 (1990)]. Examples of the bacterium of the genus Bacillus include Bacillus subtilis.
us subtilis) MI114 [Gene, 24, 255 (1983)], 20
7-21 [Journ of Biochemistry (Journ
al of Biochemistry), 95, 87 (1984)]. Examples of yeast include Saccharomyces cerevisiae ^{(Saccharomyces cerevisiae) AH22, AH22R -} ,
0NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC203
6, Pichia pastoris etc. are used.

Examples of insect cells include virus A
In the case of cNPV, the cell line derived from the larva of Spodoptera frugiperda (Spod
optera frugiperda cell; Sf cell), Trichoplusia n
MG1 cells from the midgut of i, from the eggs of Trichoplusia ni
High Five ^™ cells, Mamestra brassicae-derived cells, Estigmena acrea-derived cells and the like are used. When the virus is BmNPV, the silkworm-derived cell line (Bomb
yx mori N; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf
21 cells (above Vaughn, JL et al., In Vivo
ivo), 13, 213-217 (1977)). As the insect, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
As animal cells, for example, monkey cells COS-7, Ve
ro, Chinese hamster cell CHO (hereinafter, CH
O cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells),
Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

To transform Escherichia, for example, Proc. Natl. Acad. Sci. USA, Volume 69, Proc. Of the National Academy of Sciences of the USA. ， 2110 (197
2) and Gene, 17, vol. 107 (1982). To transform Bacillus, for example, Molecular & General Genetics (Molecular & General Genetics
s), Volume 168, 111 (1979) and the like. For transformation of yeast, for example, Methods in Enzymolology (Methods in Enzymol
ogy), 194, 182-187 (1991), Proc.
Of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sc
i. USA), Vol. 75, 1929 (1978) and the like. For transforming insect cells or insects, for example, Bio / Techno
logy), 6, 47-55 (1988)) and the like. To transform animal cells, see, eg, Cell Engineering Supplement 8, New Cell Engineering Experimental Protocol. 26
3-267 (1995) (published by Shujunsha), Virology
gy), 52, 456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the Eph receptor protein is obtained. When the host is culturing a transformant which is a bacterium of the genus Escherichia, a bacterium of the genus Bacillus, a liquid medium is suitable as a medium used for the culture, and among them, a carbon source necessary for the growth of the transformant, Nitrogen source,
Inorganic substances and others are included. Examples of the carbon source include glucose, dextrin, soluble starch and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal and potato extract. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa
l of Experiments in Molecular Genetics), 431-43
3, Cold Spring Harbor Laboratory, New York 1972]
Is preferred. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter in order to work it efficiently. When the host is a bacterium belonging to the genus Escherichia, the culturing is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
When the host is a bacterium of the genus Bacillus, the culture is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and aeration and agitation can be added if necessary. When culturing a transformant whose host is yeast,
Examples of the medium include Burkholde (Burkholde).
r) Minimal medium [Bostian, KL, et al., "Proc. Natl. Acad., Proc. of the National Academy of Sciences of the USA.
Sci. USA), 77, 4505 (1980) "and SD medium containing 0.5% casamino acid [Bitter, GA et al.," Procedures of the National Academy of Sciences of the. USA (Proc. Natl. Ac
ad. Sci. USA), Vol. 81, 5330 (1984) ".
The pH of the medium is preferably adjusted to about 5-8. Culturing is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and agitation are added if necessary.

When culturing a transformant whose host is an insect cell or an insect, the medium is Grace's Insect Mediu.
m (Grace, TCC, Nature, 195, 788 (1
962)) to which an additive such as immobilized 10% bovine serum is appropriately added is used. The pH of the medium is preferably adjusted to about 6.2-6.4. Culturing is usually at about 27 ° C for about 3-5
Do it for a day and add aeration and agitation as needed. When a transformant whose host is an animal cell is cultured, examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum [Science, Volume 122, 501 (1952)], DM.
EM medium [Virology, Volume 8, 396 (195
9)], RPMI1640 medium [The Journal of the American Medical Association (The Journal
of the American Medical Association) 199, 519 (1
967)], 199 medium [Procedure of the Society for the Biological Medicine (Pr.
oceeding of the Society for the Biological Medicin
e), Vol. 73, 1 (1950)]. pH is about 6
It is preferably -8. Culturing is usually performed at about 30-40 ° C.
Perform for 15 to 60 hours, add aeration and agitation as needed.
As described above, the receptor protein of the present invention can be produced inside the transformant, on the cell membrane or outside the cell.

Separation and purification of the receptor protein of the present invention from the above culture can be carried out, for example, by the following method. To extract the receptor protein of the present invention from cultured bacterial cells or cells, after culture, the bacterial cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasonic waves, lysozyme and / or freeze-thawing. A method of obtaining a crude extract of a receptor protein by centrifuging or filtering after appropriately destroying the bacterial cells or cells by, for example, is appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the receptor protein is secreted into the culture medium, after the culture is completed, the culture supernatant containing the receptor protein is collected by a known method. Purification of the receptor protein contained in the thus obtained culture supernatant or the extract can be carried out by appropriately combining known separation / purification methods. Known separation and purification methods for these are methods utilizing solubility such as salting out and solvent precipitation, methods utilizing mainly differences in molecular weight such as dialysis, ultrafiltration, and gel filtration, and ion exchange. Chromatography, etc., charge difference, affinity chromatography, etc., specific affinity, reverse phase high performance liquid chromatography, etc., hydrophobicity, isoelectric focusing, etc. The method of utilizing the difference of the isoelectric points is used.

When the receptor protein thus obtained is obtained as a free form, it can be converted into a salt by a known method or a method similar thereto, and conversely, when it is obtained as a salt, it is known. The free form or other salt can be converted by a method or a method analogous thereto. The receptor protein produced by the recombinant can be optionally modified or partially removed by reacting it with an appropriate protein-modifying enzyme before or after purification. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The activity of the receptor protein of the present invention or a salt thereof thus produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

The antibody against the receptor protein of the present invention or its partial peptide or its salt may be either a polyclonal antibody or a monoclonal antibody as long as it is an antibody capable of recognizing the receptor protein of the present invention or its partial peptide or its salt. Good. An antibody against the receptor protein of the present invention or a partial peptide thereof or a salt thereof (hereinafter sometimes abbreviated as “receptor protein of the present invention”) is a known antibody or antiserum prepared by using the receptor protein of the present invention as an antigen. It can be manufactured according to the manufacturing method.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The receptor protein or the like of the present invention is administered to a mammal at a site where antibody production is possible by itself or with a carrier or diluent. It Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of mammals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used. For preparation of monoclonal antibody-producing cells, warm-blooded animals immunized with an antigen, for example, individuals with an antibody titer were selected from mice, and 2 to 5 days after the final immunization, spleens or lymph nodes were collected and By fusing the antibody-producing cells contained therein with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum is measured, for example, by reacting a labeled receptor protein or the like described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody, or the antigen protein immobilized on the plate. And the like, and the antiserum are reacted with each other, and then the amount of the antibody bound to the antigen is detected with a labeled secondary antibody. The fusion operation is a known method, for example, the method of Kohler and Milstein [Natur (Natu
re), 256, 495 (1975)]. Examples of the fusion accelerator include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0 and the like, but P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is 10 to 10.
Cell fusion can be efficiently carried out by adding at a concentration of about 80% and incubating at about 20 to 40 ° C, preferably about 30 to 37 ° C for about 1 to 10 minutes.

Although various methods can be used for screening monoclonal antibody-producing hybridomas, for example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which an antigen such as a receptor protein is directly or adsorbed with a carrier. Then, add an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, and add the monoclonal antibody bound to the solid phase. Method for detection, hybridoma culture supernatant is added to a solid phase on which anti-immunoglobulin antibody or protein A is adsorbed, and receptor protein labeled with a radioactive substance or enzyme is added to detect the monoclonal antibody bound to the solid phase Method etc. are mentioned. The monoclonal antibody can be selected according to a known method or a method similar thereto, but usually HA
It can be performed in a medium for animal cells or the like to which T (hypoxanthine, aminopterin, thymidine) is added. As the selection and breeding medium, any medium can be used as long as the hybridoma can grow. For example, 1
RPM containing ~ 20%, preferably 10-20% fetal calf serum
I1640 medium, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.), serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) and the like can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. Cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the above antibody titer measurement in antiserum.

(B) Purification of Monoclonal Antibody The isolation and purification of the monoclonal antibody can be carried out in the same manner as the usual isolation and purification of a polyclonal antibody [eg, salting out method, alcohol precipitation method, isoelectric focusing method, Electrophoresis method, adsorption / desorption method with ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active antibody such as protein A or protein G is used to dissociate the antibody. Specific Purification Method of Obtaining Antibody to Obtain Antibody]

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be manufactured by publicly known methods or modifications thereof. For example, a complex of an immunizing antigen (an antigen such as the protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-mentioned method for producing a monoclonal antibody. It can be manufactured by collecting the antibody-containing product against and isolating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used for immunizing a mammal, the kind of the carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody can efficiently be against the hapten immunized by crosslinking with the carrier. , Whatever may be crosslinked in any proportion, for example bovine serum albumin,
Bovine thyroglobulin, keyhole, limpet, hemocyanin, etc. in a weight ratio of about 0.1 to 2 per 1 hapten.
A method of coupling at a rate of 0, preferably about 1 to 5, is used. Further, for the coupling of the hapten and the carrier, various condensing agents can be used, but glutaraldehyde, carbodiimide, maleimide active ester,
An active ester reagent containing a thiol group or a dithiopyridyl group is used. The condensation product is administered to a warm-blooded animal at a site where antibodies can be produced, by itself or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered to enhance the antibody-producing ability upon administration. Administration is usually about 2
~ Once every 6 weeks, a total of about 3 to 10 times can be performed. The polyclonal antibody can be collected from blood, ascites, etc., preferably blood of a mammal immunized by the above method. The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-mentioned antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

The receptor protein or its salt of the present invention, its partial peptide or its salt, and the polynucleotide (eg, DNA) encoding the receptor protein or its partial peptide are (1) a ligand for the receptor protein of the present invention ( Agonist) determination,
(2) a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, (3) a gene diagnostic agent, (4) a compound that changes the expression level of the receptor protein of the present invention or a partial peptide thereof Screening method, (5) prophylactic and / or therapeutic agent for various diseases containing a compound that changes the expression level of the receptor protein of the present invention or its partial peptide, (6) quantification method of ligand for the receptor protein of the present invention,
(7) A screening method for a compound (agonist, antagonist, etc.) that changes the binding property between the receptor protein of the present invention and a ligand, (8) Compound that changes the binding property between the receptor protein of the present invention and a ligand (agonist, antagonist) ) -Containing various preventive and / or therapeutic agents for diseases, (9) quantification of the receptor protein of the present invention or its partial peptide or its salt, (10) changes in the amount of the receptor protein of the present invention or its partial peptide in cell membranes (11) A preventive and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, (12) the receptor protein of the present invention or its portion Pep Of a substance having an action of inhibiting the multimerization of the receptor protein of the present invention, and (14) of a substance having an action of inhibiting the multimerization of the receptor protein of the present invention It can be used for screening, (15) production of a non-human transgenic animal having a DNA encoding the receptor protein of the present invention, (16) a drug containing an antisense polynucleotide (nucleic acid), and the like. In addition, by using a receptor binding assay system using the recombinant Eph receptor protein expression system of the present invention, Eph specific to humans and non-human mammals can be obtained.
A compound (eg, agonist, antagonist, etc.) that changes the binding property of a ligand to a receptor can be screened, and the agonist or antagonist, preferably the antagonist is used as a prophylactic / therapeutic agent for various diseases (eg, cancer) can do.
The receptor protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the receptor protein or the like of the present invention), a DNA encoding the receptor protein of the present invention or a partial peptide thereof (hereinafter abbreviated as the DNA of the present invention) The use of an antibody against the receptor protein or the like of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) will be specifically described below.

(1) Determination of Ligands for Receptor Proteins of the Present Invention The receptor proteins of the present invention or salts thereof or partial peptides of the present invention or salts thereof search or determine ligands for the receptor proteins of the present invention or salts thereof. It is useful as a reagent for That is, the present invention provides a method for determining a ligand for the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof with a test compound. As the test compound, in addition to known ligands, for example, tissue extracts of human or non-human mammals (eg, mouse, rat, pig, bovine, sheep, monkey, etc.), cell culture supernatants and the like are used. Examples of the known ligand include ephrin-A family (eg, ephrin
-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A5, e
phrin-A6), ephrin-B family (eg, ephrin-B
1, ephrin-B2, ephrin-B3, etc.) and the like. The e
The phrin-A family is usually a GPI-anchored protein, but it may be one without GPI modification. The ephrin-
The B family is usually a transmembrane protein, but may lack the transmembrane region. For example, the tissue extract, cell culture supernatant and the like are added to the receptor protein of the present invention and fractionated while measuring cell stimulating activity,
Finally a single ligand can be obtained.

Specifically, the ligand determination method of the present invention uses the receptor protein of the present invention or its partial peptide or its salt, or constructs an expression system of a recombinant receptor protein and uses the expression system. The cell-stimulating activity by binding to the receptor protein of the present invention (eg intracellular Ca ²⁺ release, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c- fo
It is a method for determining a compound (for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, etc.) or a salt thereof having s activation, activity promoting or suppressing pH lowering and the like. In the method for determining a ligand of the present invention, when the test compound is brought into contact with the receptor protein of the present invention or its partial peptide, for example, the binding amount of the test compound to the receptor protein or the partial peptide, cell stimulating activity, etc. It is characterized by measuring.

More specifically, the present invention provides a labeled test compound, which is obtained by contacting the labeled test compound with the receptor protein of the present invention or a salt thereof or a partial peptide of the present invention or a salt thereof. Or a salt thereof, or a method for determining a ligand for the receptor protein of the present invention or a salt thereof, which comprises measuring the amount of binding to the partial peptide or a salt thereof, and a labeled test compound containing the receptor protein of the present invention Determination of a ligand for the receptor protein of the present invention or a salt thereof, which comprises measuring the binding amount of a labeled test compound to the cell or the membrane fraction when contacted with the cell or the membrane fraction of the cell Method, labeled test compound is a receptor protein of the invention Characterized in that the amount of labeled test compound bound to the receptor protein or a salt thereof is measured when the transformant containing the DNA coding for is contacted with the receptor protein expressed on the cell membrane. A method for determining a ligand for the receptor protein of the present invention,

When the test compound is contacted with cells containing the receptor protein of the present invention, the cell-stimulating activity mediated by the receptor protein (eg intracellular Ca ²⁺ release, inositol phosphate production, cell membrane potential fluctuation, Phosphorylation of intracellular proteins, activation of c-fos, activity to promote or suppress pH decrease, cell death inhibitory activity, cell adhesion activity and cell motility regulatory activity etc.) The method for determining a ligand for the receptor protein of the present invention or a salt thereof, and a test compound are contacted with a receptor protein expressed on a cell membrane by culturing a transformant containing a DNA encoding the receptor protein of the present invention. Cell stimulatory activity mediated by receptor proteins (eg , Intracellular Ca ²⁺ release, inositol phosphate production, change in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, activity or inhibiting activity for promoting reduction, etc. pH, cell death suppressing activity, cell A method for determining a ligand for the receptor protein of the present invention or a salt thereof is provided, which comprises measuring adhesion activity and cell motility regulation activity). Especially above
It is preferable to carry out the above-mentioned tests (1) to (3) after confirming that the test compound binds to the receptor protein of the present invention.

First, the receptor protein used in the method for determining a ligand may be any as long as it contains the above-described receptor protein of the present invention or the partial peptide of the present invention. Receptor proteins expressed in large amounts are suitable. The above-mentioned expression method is used for producing the receptor protein of the present invention, but it is preferable to express the DNA encoding the receptor protein in mammalian cells or insect cells. CDNA is usually used as the DNA fragment encoding the target protein portion, but the DNA fragment is not necessarily limited thereto. For example, a gene fragment or synthetic DNA may be used. To introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, in order to efficiently express them, a nuclear polyhedrosis virus (nuclear polyhedrosis v
irus; NPV) polyhedrin promoter, SV40
Origin promoter, retrovirus promoter,
Metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SR
It is preferably incorporated downstream of the α promoter and the like. The amount and quality of the expressed receptor can be examined by a known method. For example, the literature [Nambi, P. Et al., The Journal of Biological Chemistry (J. Biol.
Chem.), 267, 19555-19559, 1992].

Therefore, in the method for determining the ligand of the present invention, the receptor protein of the present invention or its partial peptide or its salt contains a receptor protein or its partial peptide or its salt purified by a known method. Alternatively, cells containing the receptor protein or cell membrane fractions thereof may be used. When a cell containing the receptor protein of the present invention is used in the method for determining a ligand of the present invention, the cell may be immobilized with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a known method. The cell containing the receptor protein of the present invention refers to a host cell expressing the receptor protein of the present invention.
E. coli, Bacillus subtilis, yeast, insect cells, animal cells and the like are used. The cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after crushing cells. As a method of crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing with ultrasonic waves, and blasting cells from a thin nozzle while applying pressure with a French press etc. Examples include crushing by things. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is centrifuged at low speed (500 to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged at high speed (15000 to 30000 rpm) for usually 30 minutes to 2 hours. The obtained precipitate is used as a membrane fraction. The membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of the receptor protein in the cell containing the receptor protein or its membrane fraction is preferably 10 ³ to 10 ⁸ molecules per cell, and 10 ⁵ to 1
It is preferably 0 ⁷ molecules. It should be noted that the higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, making it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. . In order to carry out the above methods of determining the ligand for the receptor protein or salt thereof of the present invention, a suitable receptor protein fraction and a labeled test compound are required. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor fraction having an activity equivalent to that is desirable. Here, the equivalent activity refers to equivalent ligand binding activity, signal transduction action, and the like.

The labeled test compound includes [ ³ H],
Ephrin-labeled with [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc.
A family (eg, ephrin-A1, ephrin-A2, ephrin-A3,
ephrin-A4, ephrin-A5, ephrin-A6 etc.), ephrin-B family (eg ephrin-B1, ephrin-B2, ephrin-B3 etc.) and the like are suitable.

Specifically, in order to carry out the method for determining a ligand for the receptor protein of the present invention or a salt thereof, first, a cell or a membrane fraction of cells containing the receptor protein of the present invention is treated with a buffer suitable for the determination method. A receptor preparation is prepared by suspending in. Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably a pH of 6 to 8) and a Tris-hydrochloric acid buffer. For the purpose of reducing non-specific binding, surfactants such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin and deoxycholate, and various proteins such as bovine serum albumin and gelatin may be added to the buffer. it can. Further, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Laboratories), pepstatin or the like can be added for the purpose of suppressing the decomposition of the receptor and the ligand by the protease. To 0.01 to 10 ml of the receptor solution, a fixed amount (5000 to 500000 cpm) of [ ³ H], [ ¹²⁵
A test compound labeled with [I], [ ¹⁴ C], [ ³⁵ S] or the like is allowed to coexist. A reaction tube containing a large excess of unlabeled test compound is also prepared in order to know the non-specific binding amount (NSB). The reaction temperature is about 0 ° C to 50 ° C, preferably about 4 ° C to 37 ° C.
For about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter paper or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. The count (B-NSB) obtained by subtracting the nonspecific binding amount (NSB) from the total binding amount (B) is 0 cp.
Test compounds exceeding m can be selected as ligands (agonists) for the receptor protein of the present invention or salts thereof.

In order to carry out the above methods (1) to (3) for determining a ligand for the receptor protein or a salt thereof of the present invention, cell stimulating activity (eg intracellular Ca ²⁺ release, inositol phosphate production) mediated by the receptor protein is carried out. , Activity promoting or suppressing cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, etc .; cell death suppressing activity; cell adhesion activity and cell motility controlling activity etc.) It can be measured using the method described above or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like. To determine the ligand, exchange with fresh medium or an appropriate buffer that does not show toxicity to cells in advance, add a test compound etc. and incubate for a certain period of time, then extract the cells or collect the supernatant and generate The amount of the obtained product is quantified according to each method. When the production of an index of cell stimulating activity (for example, phosphoric acid of intracellular protein) is difficult to assay by the degrading enzyme contained in the cell, an inhibitor for the degrading enzyme may be added to the assay. Further, the activity of suppressing cAMP production and the like can be detected as a production suppressing action on cells in which the basic production amount of cells has been increased by forskolin or the like.

Furthermore, test compounds [eg, ephrin-A family (eg, ephrin-A1, ephrin-A2, ephrin-A3, ephrin-A3
A4, ephrin-A5, ephrin-A6, etc.), ephrin-B family (eg, ephrin-B1, ephrin-B2, ephrin-B3, etc.)] and a fusion protein with the Fc portion of human IgG, and an antibody of the present invention, Can be used to identify ligands that bind to the receptor proteins of the invention. That is, the fusion protein is mixed and bound with a cell-free extract of a human cell that endogenously contains the receptor protein of the present invention or a mammalian cell in which the receptor protein of the present invention is forcibly expressed (such as HEK293 cells). , Protein A or protein G, or a carrier bound with an anti-human IgG-Fc antibody is used to detect the presence or absence of the receptor protein of the present invention in the above fusion protein-containing fraction, It can be determined whether it is a ligand for the receptor protein or its salt. The method for detecting the receptor protein of the present invention may be any method as long as it uses the antibody of the present invention, and for example, Western blotting or the like is used. Specifically, as described in Example 22 described later, as the ligand for the receptor protein of the present invention or a salt thereof, ephrin-A2, ephrin-A3, ephrin-A2
in-A4, ephrin-A5, ephrin-A1 and ephrin-B2, preferably ephrin-A2, ephrin-A3, ephrin-A4 and ephrin-A5
Is mentioned.

The kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof comprises a receptor protein of the present invention or a salt thereof, a partial peptide of the present invention or a salt thereof, a cell containing the receptor protein of the present invention, or It contains a membrane fraction of cells containing the receptor protein of the invention. Examples of the ligand determination kit of the present invention include the following. 1. Add 0.0% to Hanks' Balanced Salt Solution (manufactured by Gibco) for measuring the ligand determination reagent and the washing buffer.
To which 5% bovine serum albumin (manufactured by Sigma) was added. It may be sterilized by filtration with a filter having a pore size of 0.45 μm and stored at 4 ° C., or may be prepared before use. Eph receptor protein preparation CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well and incubated at 37 ° C., 5%
Cultured in CO ₂ and 95% air for 2 days. Labeled test compound Commercially available compound labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like, or labeled with an appropriate method in an aqueous solution at 4 ° C or -20 Store at 0 ° C and dilute to 1 µM with measurement buffer before use. For test compounds that are poorly soluble in water, dimethylformamide,
Dissolve in DMSO, methanol, etc. Unlabeled test compound The same as the labeled compound is prepared at a concentration 100 to 1000 times higher.

2. Assay method The receptor protein-expressing CHO cells of the present invention cultured in a 12-well tissue culture plate were assayed with 1 m of the measurement buffer solution.
After washing twice with l, 490 μl of measuring buffer is added to each well. 5 μl of the labeled test compound is added, and the mixture is reacted at room temperature for 1 hour. To know the amount of non-specific binding, 5 μl of unlabeled test compound is added. The reaction solution is removed and washed 3 times with 1 ml of the washing buffer. Labeled test compound bound to cells was treated with 0.2N NaOH.
It is dissolved in -1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries). Radioactivity is measured using a liquid scintillation counter (Beckman Coulter, Inc.).

The ligand capable of binding to the receptor protein of the present invention or a salt thereof is, for example,
Examples include substances present in the brain, large intestine, small intestine, pancreas, ovary, etc. Specifically, the ephrin-A family (eg, ephrin-A family
in-A1, ephrin-A2, ephrin-A3, ephrin-A4, ephrin-A
5, ephrin-A6, etc., ephrin-B family (eg, ephrin
-B1, ephrin-B2, ephrin-B3, etc.) are used.

(2) Pharmaceutical comprising the receptor protein or DNA of the present invention When the ligand for the receptor protein of the present invention is clarified, the receptor protein of the present invention or the receptor protein of the present invention is determined according to the action of the ligand. Can be used as a medicine such as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. For example,
When there is a patient in whom the physiological action of the ligand cannot be expected because the receptor protein of the present invention is reduced in vivo (deficiency of the receptor protein), the receptor protein of the present invention is administered to the patient and By supplementing the amount, (a) administering the DNA encoding the receptor protein of the present invention to the patient to express it, or (b) DN encoding the receptor protein of the present invention in target cells.
After inserting and expressing A, the cells can be transplanted into the patient to increase the amount of the receptor protein in the patient's body and sufficiently exert the action of the ligand. That is, the DNA encoding the receptor protein of the present invention is useful as a safe and low-toxicity preventive and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention. In particular,
The receptor protein of the present invention or the DNA encoding the receptor protein is, for example, a central disease (for example, Alzheimer's disease, dementia, etc.), a metabolic disease (for example,
Diabetes, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma) ,
It is useful for prevention and / or treatment of small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.). When the receptor protein of the present invention is used as the above-mentioned preventive / therapeutic agent, it can be prepared into a pharmaceutical preparation according to a conventional method. On the other hand, DNA encoding the receptor protein of the present invention
When (hereinafter, sometimes abbreviated as the DNA of the present invention) is used as the above-mentioned preventive / therapeutic agent, the DNA of the present invention may be used alone or in a suitable retrovirus vector, adenovirus vector, adenovirus associated virus vector, or the like. After it is inserted into a suitable vector, it can be carried out by a conventional method. The DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting uptake by a gene gun or a catheter such as a hydrogel catheter. For example, (i) the receptor protein of the present invention or the DNA encoding the receptor protein is orally administered as tablets, capsules, elixirs, microcapsules and the like, optionally coated with sugar, or water or other It can be used parenterally in the form of an injectable solution such as a sterile solution or suspension with a pharmaceutically acceptable liquid of. For example, the receptor protein of the present invention or (ii) the DNA encoding the receptor protein is generally recognized together with known physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like. It can be manufactured by mixing in the unit dosage form required for carrying out the formulation.
The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. , For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate)
80 ^™ , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffer agents (eg, phosphate buffer solution, sodium acetate buffer solution), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans and non-human mammals (for example, rat, mouse, rabbit, sheep, pig,
(Cattle, cats, dogs, monkeys, etc.). The dose of the receptor protein of the present invention varies depending on the administration subject, target organ, symptom, administration method and the like, but in the case of oral administration, it is generally, for example, for cancer patients (60
(in kg), about 0.1-100 per day
mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but is usually in the form of an injection, for example, in a cancer patient (as 60 kg), About 0.01 to 30 mg per day, preferably about 0.
1 to 20 mg, more preferably about 0.1 to 10 mg
It is convenient to administer the degree intravenously. In the case of other animals, the dose converted per 60 kg can be administered. The dose of the DNA of the present invention is
Although there are differences depending on the target organs, symptoms, administration method, etc., in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to about 10 mg per day. 50 mg, more preferably about 1.0
~ 20 mg. Part 1 when administered parenterally
Although the dose may vary depending on the administration subject, target organ, symptom, administration method, etc., for example, in the form of injection, for example, in a cancer patient (60 kg), about 0.01 to 30 mg per day, Preferably about 0.1-20
It is convenient to administer about mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

Furthermore, since the receptor protein of the present invention is overexpressed in various cancers, it can be used as a cancer vaccine for activating the immune system of cancer patients. For example, so-called adoptive immunotherapy, in which strong antigen-presenting cells (eg, dendritic cells) are cultured in the presence of the receptor protein of the present invention, the receptor protein is phagocytosed, and then returned to the patient's body, is preferably applied. sell. Dendritic cells returned to the body can kill cancer cells by inducing and activating cancer antigen-specific cytotoxic T cells. Furthermore, the receptor protein of the present invention includes, for example, cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, Brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma,
It can also be administered to mammals (eg, human, monkey, mouse, rat, rabbit, pig) as a vaccine formulation for the prevention and / or treatment of malignant melanoma, thyroid cancer, bone tumor, leukemia, etc. The vaccine formulation usually contains the receptor protein of the present invention and a physiologically acceptable carrier. Examples of the carrier include water, saline (including physiological saline), buffer (eg, phosphate buffer),
Examples include liquid carriers such as alcohol (eg, ethanol). Usually, the receptor protein of the present invention is dissolved or suspended in a physiologically acceptable carrier. Also,
The receptor protein of the present invention and a physiologically acceptable carrier may be separately prepared, and they may be mixed before use. In the vaccine preparation, in addition to the receptor protein of the present invention and a physiologically acceptable carrier, for example, an adjuvant (eg, aluminum hydroxide gel, serum albumin etc.), a preservative (eg, thimerosal etc.), a soothing agent (eg. , Glucose, benzyl alcohol, etc.) and the like. In addition, for example, cytokines (eg, interleukins such as interleukin-2 and interferons such as interferon-γ) may be mixed in order to promote the production of antibodies against the receptor protein of the present invention. When used as a vaccine preparation, the receptor protein of the present invention may be used as an active substance, or the receptor protein of the present invention may be modified to enhance antigenicity. The denaturation of the receptor protein of the present invention is usually performed by heat treatment or treatment with a protein denaturing agent (eg, formalin, guanidine hydrochloride, urea). The above vaccine preparation can be prepared according to a usual method for producing a vaccine preparation. The obtained vaccine preparation has low toxicity, and may be usually administered as an injection, for example, subcutaneously, intracutaneously, intramuscularly, or locally at or near the cancer cell mass. The dose of the receptor protein of the present invention as a vaccine preparation varies depending on, for example, the target disease, the administration subject, and the administration route. For example, when the receptor protein is subcutaneously administered as an injection to an adult (body weight 60 kg) with cancer, the dose is usually about 0.1 mg to 300 mg per dose,
It is preferably about 100 mg to 300 mg. The vaccine preparation may be administered once, but the vaccine preparation may be administered 2 to 4 times at intervals of about 2 weeks to about 6 months in order to increase the amount of antibody produced. Furthermore, the receptor protein of the present invention or the DNA encoding the receptor protein can also be used as a vaccine preparation.

(3) Genetic Diagnostic Agent The DNA of the present invention can be used as a probe to form a human or non-human mammal (eg, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). Since it is possible to detect an abnormality (gene abnormality) in the DNA or mRNA encoding the receptor protein of the present invention or its partial peptide in
It is useful as a gene diagnostic agent for damaging, mutating or lowering the expression of the DNA or mRNA, and increasing or excessive expression of the DNA or mRNA. DN of the present invention
The above-mentioned gene diagnosis using A can be carried out, for example, by known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989).
,), Proceedings of the National Academy of Sciences o
f the United States of America), Vol. 86, 2766-27
P. 70 (1989)) and the like. For example, when increased expression is detected by Northern hybridization, for example, central diseases (for example, Alzheimer's disease, dementia, etc.), metabolic diseases (for example, diabetes, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer) ,
Gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain cancer, schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma, Malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.) is likely to be diagnosed. On the contrary, when decreased expression is detected or when a DNA mutation is detected by the PCR-SSCP method, for example, a central disease (for example,
Alzheimer's disease, dementia, etc., metabolic disease (eg, diabetes, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer) , Esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma,
Malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.) is likely to be diagnosed.

(4) Screening method for compounds that change the expression level of the receptor protein of the present invention or its partial peptide: The DNA of the present invention is used as a probe to
It can be used for screening a compound that changes the expression level of the receptor protein of the present invention or its partial peptide. That is, the present invention includes, for example, (i) blood of a non-human mammal, a specific organ, a tissue or cell isolated from an organ, (ii) a human cell or (iii)
Provided is a screening method for a compound that changes the expression level of the receptor protein of the present invention or its partial peptide by measuring the mRNA amount of the receptor protein of the present invention or its partial peptide contained in a transformant or the like.

The mRNA amount of the receptor protein of the present invention or its partial peptide is specifically measured as follows. (I) Normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, and more specifically, demented rat, obese mouse, arteriosclerotic rabbit, cancer-bearing) Drugs (eg anti-dementia drugs, blood pressure lowering drugs,
Anti-cancer drug, anti-obesity drug, etc.) or physical stress (eg flooding stress, electric shock, light / darkness, low temperature, etc.), and after a certain period of time, blood, or a specific organ (eg, brain, lung, large intestine, etc.) ), Or tissue or cells isolated from an organ. The mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells can be quantified, for example, by extracting mRNA from cells or the like by a conventional method and using a technique such as TaqMan PCR. Alternatively, it can be analyzed by Northern blotting by a known means. (Ii) A transformant expressing the receptor protein of the present invention or a partial peptide thereof is produced according to the above method, and the mRNA of the receptor protein of the present invention or a partial peptide thereof contained in the transformant is similarly quantified, Can be analyzed.

Screening for a compound that changes the expression level of the receptor protein of the present invention or a partial peptide thereof is carried out by (i) a method of applying a drug or physical stress to a normal or disease model non-human mammal or human cell. Before time (30 minutes to 24 hours ago, preferably 30 minutes to 12 hours ago, more preferably 1 hour to 6 hours ago) or after a certain time (30 minutes to 3 days later, preferably 1 hour later) 2 days later, more preferably 1 to 24 hours later), or the test compound is administered at the same time as the drug or physical stress, and after a lapse of a certain time (30 minutes to 3 days, preferably 1 hour) after administration. 2 days later, more preferably 1 to 24 hours later), the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the cells is quantified and analyzed. (Ii) When the transformant is cultured according to a conventional method, the test compound is mixed in the medium, and after culturing for a certain time (1 day to 7 days later, preferably 1 day to 3 days later, more preferably 2 to 3 days later),
This can be performed by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the transformant.

The compound or its salt obtained by using the screening method of the present invention is a compound having an action of changing the expression level of the receptor protein of the present invention or its partial peptide, and specifically, By increasing the expression level of the receptor protein of the present invention or a partial peptide thereof, cell stimulating activity mediated by the receptor protein of the present invention (for example, intracellular Ca ²⁺ release, inositol phosphate production, cell membrane potential fluctuation, intracellular protein Activity to promote or suppress phosphorylation, activation of c-fos, decrease in pH, etc .; cell death inhibitory activity;
A compound that promotes cell adhesion activity and cell motility regulation activity), and (b) a compound that inhibits the cell stimulating activity by decreasing the expression level of the receptor protein of the present invention or a partial peptide thereof. Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. The compound that promotes the cell stimulating activity is useful as a safe and low toxic pharmaceutical for promoting the physiological activity of the receptor protein of the present invention. (Once deleted, it was reconsidered and revived.) A compound that inhibits the cell stimulating activity is a safe and low-toxic drug for decreasing physiological activity of the receptor protein of the present invention, for example, a central disease ( (For example, Alzheimer's disease, dementia, etc.),
Metabolic disease (for example, diabetes), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, Brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.) and / or therapeutic agent, preferably anticancer agent, etc. Is useful as

When the compound or its salt obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method. For example,
Tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be prepared in the same manner as the above-mentioned pharmaceutical containing the receptor protein of the present invention. Since the preparation thus obtained is safe and has low toxicity, for example, humans and non-human mammals (for example,
Rat, mouse, rabbit, sheep, pig, cow, cat,
Dogs, monkeys, etc.). Although the dose of the compound or its salt varies depending on the administration subject, target organ, condition, administration method, etc., in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), About 0.1 to 100 mg, preferably about 1.0
~ 50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose is the subject of administration,
Depending on the target organ, symptoms, administration method, etc., for example, in the form of injection, for example, in a cancer patient (60 kg), about 0.01 to 30 mg per day,
Preferably about 0.1 to 20 mg, more preferably about 0.1
It is convenient to administer about 10 mg intravenously. In the case of other animals, the dose converted per 60 kg can be administered.

(5) Preventive and / or therapeutic agent for various diseases containing a compound that changes the expression level of the receptor protein of the present invention or its partial peptide A compound that changes the expression level of the receptor protein of the present invention or its partial peptide Is a disease associated with dysfunction or overexpression of the receptor protein of the present invention (eg, central diseases (e.g., Alzheimer's disease, dementia, etc.), metabolic diseases (e.g., diabetes, etc.), cancer (e.g., non-small cell lung cancer, Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer,
Cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer,
Endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.)] and / or therapeutic agent. Preferably, the compound that suppresses the expression of the receptor protein or its partial peptide of the present invention is, for example, a central disease (for example, Alzheimer's disease, dementia, etc.), a metabolic disease (for example, diabetes, etc.), a cancer (for example, non-small cell lung cancer). , Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer, Endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.)] and / or therapeutic agent. When the compound is used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction or overexpression of the receptor protein of the present invention, it can be formulated according to a conventional method.
For example, the compound is a tablet optionally coated with sugar,
Orally used as capsules, elixirs, microcapsules, etc., or parenterally in the form of injectable solutions such as sterile solutions with water or other pharmaceutically acceptable liquids or suspensions it can. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans and non-human mammals (for example, rat, mouse, rabbit, sheep, pig,
(Cattle, cats, dogs, monkeys, etc.). The dose of the compound or salt thereof is
Although there are differences depending on the target organs, symptoms, administration method, etc., generally, in the case of oral administration, for example, in a cancer patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day. , More preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but is usually in the form of injection, for example, in patients with cancer (as 60 kg) , About 0.01 to 30 per day
It is convenient to administer about mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(6) Method for Quantifying Ligand for Receptor Protein of the Present Invention Since the receptor protein and the like of the present invention have a binding property to the ligand, the ligand concentration in the living body can be quantified with high sensitivity. . The quantitative method of the present invention is
For example, it can be used in combination with a competitive method. That is, the concentration of the ligand in the sample can be measured by bringing the sample into contact with the receptor protein of the present invention. Specifically, for example, it can be used according to the method described in (i) or (ii) below or a method similar thereto. (I) Hiroshi Irie "Radioimmunoassay" (Kodansha, published in 1974) (ii) Hiroshi Irie "Radioimmunoassay" (Kodansha,
(Published in 1979)

(7) Screening method for compounds (agonists, antagonists, etc.) that change the binding properties between the receptor protein of the present invention and a ligand, either using the receptor protein of the present invention or expressing a recombinant receptor protein etc. And a compound that alters the binding property between the ligand and the receptor protein or the like of the present invention by using a receptor binding assay system using the expression system (for example, peptide, protein, non-peptide compound, synthetic compound, Fermentation products or the like) or salts thereof can be efficiently screened. Such compounds include (a) cell stimulating activity via the receptor protein of the present invention (eg intracellular Ca ²⁺ release, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos). (Activator for the receptor protein of the present invention), which has an activity of promoting or suppressing pH, an activity of promoting or suppressing pH decrease; a cell death inhibitory activity; a cell adhesion activity and a cell motility regulatory activity, etc. ) A compound which does not have the cell stimulating activity (so-called an antagonist to the receptor protein of the present invention), (c) a compound which enhances the binding force between the ligand and the receptor protein of the present invention, or (d) the ligand and the receptor of the present invention Compounds that decrease the binding force with proteins are included (note that the combination of (a) above Is preferably screened by the ligand determination methods described above). That is, the present invention provides (i) the receptor protein of the present invention or a partial peptide thereof or a salt thereof,
Comparison of the case of contacting with a ligand and (ii) the case of contacting the receptor protein of the present invention or its partial peptide or salt thereof with a ligand and a test compound Provided is a method for screening a compound or its salt that changes the binding property to a receptor protein or its partial peptide or its salt. In the screening method of the present invention, in the cases (i) and (ii), for example, the amount of ligand binding to the receptor protein or the like, the cell stimulating activity, etc. are measured and compared.

More specifically, the present invention relates to the case where the labeled ligand is brought into contact with the receptor protein of the present invention and the labeled ligand and the test compound are brought into contact with the receptor protein of the present invention. A method for screening a compound or a salt thereof which changes the binding property between the ligand and the receptor protein or the like of the present invention, characterized in that the amount of the labeled ligand bound to the receptor protein or the like is measured and compared. When a cell containing the receptor protein of the present invention or a membrane fraction of the cell is contacted, the labeled ligand and the test compound are added to the cell containing the receptor protein of the present invention or the membrane fraction of the cell. Binding of labeled ligand to the cells or the membrane fraction when contacted And a method for screening a compound or a salt thereof that changes the binding property between the ligand and the receptor protein or the like of the present invention, the labeled ligand being a transformant containing the DNA of the present invention. The receptor of the present invention expressed on the cell membrane by contacting with a receptor protein or the like expressed on the cell membrane by culturing, and by culturing the transformant containing the labeled ligand and the test compound containing the DNA of the present invention When contacted with protein etc.,
A method for screening a compound or a salt thereof that changes the binding property between the ligand and the receptor protein or the like of the present invention, characterized in that the amount of labeled ligand bound to the receptor protein or the like is measured and compared.

A compound that activates the receptor protein of the present invention (for example, a ligand for the receptor protein of the present invention) is brought into contact with cells containing the receptor protein of the present invention, and the receptor protein of the present invention. And the like, and a test compound are contacted with cells containing the receptor protein or the like of the present invention, receptor-mediated cell stimulating activity (eg intracellular Ca ²⁺ release, inositol phosphate production, cell membrane) Voltage fluctuation, intracellular protein phosphorylation,
The activity of c-fos, the activity of promoting or suppressing the decrease of pH; the activity of suppressing cell death; the activity of suppressing cell death; the activity of controlling cell adhesion and the activity of cell motility, etc.) are measured and compared. A method for screening a compound or a salt thereof that changes the binding property to the receptor protein or the like of the present invention, and a compound that activates the receptor protein or the like of the present invention (for example, a ligand for the receptor protein or the like of the present invention) is used as the DNA of the present invention. Containing a DNA of the present invention, and a compound and a test compound that activate the receptor protein of the present invention and the like when contacted with the receptor protein of the present invention expressed on a cell membrane by culturing a transformant containing The receptor tamper of the present invention expressed on the cell membrane by culturing the transformant In the case of contacting the quality or the like, cell-stimulating activity mediated by the receptor (e.g., intracellular Ca ²⁺ release, inositol phosphate production, change in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, pH of Activity of promoting or suppressing decrease; cell death inhibitory activity; cell adhesion activity and cell motility regulatory activity etc.) are measured and compared, and the binding property between the ligand and the receptor protein or the like of the present invention A method for screening a compound or a salt thereof that changes

The screening method of the present invention will be specifically described below. First, the receptor protein or the like of the present invention used in the screening method of the present invention may be any as long as it contains the above-described receptor protein of the present invention. The cell membrane fraction of the mammalian organ containing is preferred. However, since human-derived organs are extremely difficult to obtain, human-derived receptor proteins and the like that are expressed in large amounts using recombinants are suitable for use in screening.

The above-mentioned method is used to produce the receptor protein and the like of the present invention, but it is preferable to express the DNA of the present invention in mammalian cells or insect cells. DN encoding the protein portion of interest
Although cDNA is used for the A fragment, it is not necessarily limited thereto. For example, gene fragments and synthetic DN
A may be used. In order to introduce DNA fragments encoding the receptor protein of the present invention into host animal cells and to express them efficiently, in order to efficiently express them, a nuclear polyhedrosis virus (nu
clear polyhedrosis virus (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter,
It is preferably incorporated downstream of human heat shock promoter, cytomegalovirus promoter, SRα promoter and the like. The amount and quality of the expressed receptor can be examined by a known method. For example, the literature [Namb
i, P. Et al., The Journal of Biological Chemistry (J. Biol. Chem.), Volume 267, 19555-19559.
Page, 1992]. Therefore, in the screening method of the present invention, the receptor protein or the like of the present invention may be a receptor protein or the like purified according to a known method, or a cell containing the receptor protein or the like may be used. Alternatively, a membrane fraction of cells containing the receptor protein or the like may be used.

When cells containing the receptor protein of the present invention are used in the screening method of the present invention, the cells may be immobilized with glutaraldehyde, formalin and the like. The immobilization method can be performed according to a known method. The cells containing the receptor protein or the like of the present invention refer to host cells expressing the receptor protein or the like, and the host cells are preferably Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like. The cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after crushing cells.
As a method of crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing with ultrasonic waves, ejection of cells from a thin nozzle while applying pressure with a French press, etc. For example, crushing by allowing it to occur. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate may be fed at low speed (500-3000 rp).
m) for a short time (usually about 1 to 10 minutes), and the supernatant is spun at a higher speed (15000 to 30000 rpm) for 30 times.
Centrifuge for minutes to 2 hours, and use the resulting precipitate as a membrane fraction. The membrane fraction is rich in expressed receptor proteins and other membrane components such as cell-derived phospholipids and membrane proteins. The amount of the receptor protein in the cell or the membrane fraction containing the receptor protein is 10 ³ per cell.
It is preferably 10 ⁸ molecules, and more preferably 10 ⁵ 10 ⁷ molecules. In addition, the higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which not only enables the construction of a highly sensitive screening system,
It becomes possible to measure a large number of samples in the same lot.

In order to carry out the above steps (1) to (3) for screening a compound that alters the binding property between the ligand and the receptor protein or the like of the present invention, for example, an appropriate receptor protein fraction and a labeled ligand are required. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent to that is desirable. Here, the equivalent activity refers to equivalent ligand binding activity, signal transduction action, and the like. As the labeled ligand, a labeled ligand, a labeled ligand analog compound or the like is used. For example, [ ³ H],
A ligand labeled with [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like is used. Specifically, in order to screen a compound that changes the binding property between a ligand and the receptor protein or the like of the present invention, first, a cell containing the receptor protein or the like of the present invention or a membrane fraction of the cell is suitable for screening. A receptor protein preparation is prepared by suspending in a buffer. Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably a pH of 6 to 8) and a Tris-hydrochloric acid buffer. Further, for the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin and deoxycholate may be added to the buffer. Further, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Laboratories), pepstatin or the like can be added for the purpose of suppressing the decomposition of the receptor or the ligand by the protease. A fixed amount (5000 to 500000 cpm) of the labeled ligand is added to 0.01 to 10 ml of the receptor solution, and 10 ^-4 to 10 ^-10 M of the test compound is allowed to coexist at the same time. A reaction tube containing a large excess of unlabeled ligand is also prepared in order to know the amount of non-specific binding (NSB).
The reaction is carried out at about 0 ° C to 50 ° C, preferably about 4 ° C to 37 ° C,
It is performed for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
After the reaction, the mixture is filtered through a glass fiber filter paper or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or a γ-counter. When the count (B ₀ -NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) in the absence of an antagonistic substance is taken as 100%, the specific binding amount (B
-NSB), for example, a test compound having 50% or less can be selected as a candidate substance capable of competitive inhibition.

In order to carry out the above-mentioned methods (1) to (3) for screening a compound that changes the binding property between the ligand and the receptor protein or the like of the present invention, for example, the cell-stimulating activity mediated by the receptor protein (eg intracellular Ca ²⁺ Activity to promote or suppress release, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, etc .; cell death inhibitory activity; cell adhesion activity and cell motility The regulatory activity etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein or the like of the present invention are cultured in a multiwell plate or the like. In screening, the medium was exchanged with a fresh medium or an appropriate buffer that did not show toxicity to cells in advance, and after adding a test compound or the like and incubating for a certain period of time, cells were extracted or supernatant was collected to generate The product is quantified according to the respective method. When the production of an index of cell stimulating activity (for example, phosphoric acid of intracellular protein) is difficult to assay by the degrading enzyme contained in the cell, an inhibitor for the degrading enzyme may be added to the assay. In order to measure the cell stimulating activity and perform screening, cells expressing an appropriate receptor protein are required. Cells expressing the receptor protein or the like of the present invention include:
A cell line having the naturally occurring receptor protein of the present invention, a cell line expressing the above-mentioned recombinant receptor protein and the like are preferable. As the test compound, for example,
Peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like are used, and these compounds may be novel compounds or known compounds. It may be. For example, ephrin
-A2, ephrin-A3, ephrin-A4, ephrin-A5, ephrin-A1 and ephrin-B2, preferably ephrin-A2, ephrin-A3, eph
rin-A4 and ephrin-A5 are used.

A kit for screening a compound or a salt thereof that alters the binding property between a ligand and the receptor protein or the like of the present invention comprises a cell containing the receptor protein or the like of the present invention,
Alternatively, it contains a membrane fraction of cells containing the receptor protein or the like of the present invention. Examples of the screening kit of the present invention include the following. 1. Add 0.0% to the screening reagent measuring buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco).
To which 5% bovine serum albumin (manufactured by Sigma) was added. It may be sterilized by filtration with a filter having a pore size of 0.45 μm and stored at 4 ° C., or may be prepared before use. Eph receptor preparation CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well at 37 ° C., 5%
Cultured in CO ₂ and 95% air for 2 days. Labeled ligand Commercially available ligands labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc., at 4 ° C. or −2
Store at 0 ° C and dilute to 1 μM with assay buffer before use. Ligand standard solution The ligand is dissolved in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) to a concentration of 1 mM and stored at -20 ° C.

2. Assay method CHO cells expressing the receptor protein of the present invention cultured in a 12-well tissue culture plate were assayed with 1 m of the measurement buffer solution.
After washing twice with l, 490 μl of measuring buffer is added to each well. After adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M, 5 μl of a labeled ligand is added, and the mixture is reacted at room temperature for 1 hour. To determine the amount of non-specific binding, 5 μl of 10 ⁻³ M ligand was added instead of the test compound. The reaction solution is removed and washed 3 times with 1 ml of the washing buffer. Labeled ligand bound to cells was 0.2N NaOH-
Dissolve in 1% SDS, 4 ml of liquid scintillator A
Mix with (Wako Pure Chemical Industries). Radioactivity was measured using a liquid scintillation counter (manufactured by Beckman Coulter), and Percent Maximu
m Binding (PMB) is calculated by the following formula. PMB = [(B-NSB) / (B 0 -NSB)] × 100 PMB: Percent Maximum Binding B: a value NSB when adding the sample: Non-specific Binding (nonspecific binding) B _0: maximum binding amount

The compound or its salt obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between the ligand and the receptor protein or the like of the present invention, and specifically, (A) Cell stimulating activity via the receptor protein of the present invention (eg intracellular Ca ²⁺ release, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease) And the like (cell death inhibitory activity; cell adhesion activity and cell motility regulatory activity, etc.) (so-called agonist for the receptor protein of the present invention), (b) the cell stimulating activity. Compound (so-called antagonist for the receptor protein of the present invention), (c) Compound that potentiates the binding affinity of the receptor protein of the command and the present invention, or (d) is a compound that reduces the binding affinity of the receptor protein of the ligand with the present invention. Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. The agonist for the receptor protein or the like of the present invention has the same physiological activity as that of the ligand for the receptor protein or the like of the present invention, and is therefore useful as a safe and low-toxic pharmaceutical according to the ligand activity. The antagonist for the receptor protein etc. of the present invention can suppress the physiological activity of the ligand for the receptor protein etc. of the present invention, and therefore a safe and low-toxic drug that suppresses the ligand activity, for example, a central disease (for example, Alzheimer's disease, dementia, etc., metabolic diseases (for example,
Diabetes, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma) ,
It is useful as a preventive and / or therapeutic agent for small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc., preferably an anticancer agent. The compound that enhances the binding force between the ligand and the receptor protein of the present invention is useful as a safe and low-toxic pharmaceutical agent for enhancing the physiological activity of the ligand for the receptor protein of the present invention. The compound that decreases the binding force between the ligand and the receptor protein of the present invention is useful as a safe and low-toxic pharmaceutical agent for reducing the physiological activity of the ligand for the receptor protein of the present invention.

When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional means. For example, tablets, capsules, elixirs, microcapsules, similar to the drug containing the receptor protein of the present invention described above,
It can be a sterile solution, suspension or the like. Since the preparation thus obtained is safe and has low toxicity, for example, humans and non-human mammals (for example, rat, rabbit,
Sheep, pigs, cows, cats, dogs, monkeys, etc.). The dose of the compound or its salt varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), About 0.1-100
mg, preferably about 1.0-50 mg, more preferably about 1.
It is 0 to 20 mg. Part 1 when administered parenterally
Although the dose may vary depending on the administration subject, target organ, symptom, administration method, etc., for example, in the form of injection, for example, in a cancer patient (60 kg), about 0.01 to 30 mg per day, It is convenient to administer about 0.1 to 20 mg, preferably about 0.1 to 10 mg by intravenous injection. 60k for other animals
An amount converted per g can be administered.

(8) Preventive and / or therapeutic agent for various diseases containing a compound (agonist, antagonist) which changes the binding property between the receptor protein of the present invention and a ligand. The compound (agonist, antagonist) or the ligand for the receptor protein of the present invention that changes can be used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention.
In a preferred embodiment, the antagonist to the receptor protein of the present invention is, for example, a central disease (for example, Alzheimer's disease, dementia, etc.), metabolic disease (for example, diabetes, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate). Cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant Lymphoma,
It can be used as a preventive and / or therapeutic agent for malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.). When the compound or ligand is used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to a conventional method. For example, the compound or ligand may be orally administered as a sugar-coated tablet, capsule, elixir, microcapsule, etc., or aseptic with water or other pharmaceutically acceptable liquid. It can be used parenterally in the form of injection such as solution or suspension.
For example, a known physiologically acceptable carrier for the compound,
It can be prepared by mixing with a flavoring agent, an excipient, a vehicle, a preservative, a stabilizer, a binder and the like in a unit dose form generally required for the implementation of the formulation. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. , For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate)
80 ^™ , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Furthermore, the above-mentioned prophylactic / therapeutic agents can be used in combination with an appropriate agent as a DDS preparation specifically targeting an organ or tissue in which the receptor protein of the present invention is highly expressed. Since the preparation thus obtained is safe and has low toxicity, for example, administration to humans and non-human mammals (eg, rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) can do. The dose of the compound or its salt varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), The amount is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose is the administration subject, target organ, symptom,
Although it depends on the administration method, for example, in the form of injection, for example, in a cancer patient (60 kg), about 0.01 to 30 mg per day, preferably about 0.1 mg per day.
It is convenient to administer about 20 mg, more preferably about 0.1-10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(9) Quantification of Receptor Protein of the Present Invention or Partial Peptide or Salt thereof Since the antibody of the present invention can specifically recognize the receptor protein of the present invention, etc., the present invention in a test solution Can be used for the quantification of the receptor protein, etc., especially by the sandwich immunoassay. That is, the present invention provides, for example, (i) the antibody of the present invention is allowed to react competitively with a test solution, a labeled receptor protein, etc., and the ratio of the labeled receptor protein etc. bound to the antibody is measured. A method for quantifying the receptor protein or the like of the present invention in a test liquid, characterized in that (ii) the test liquid and the antibody of the present invention insolubilized on a carrier and the labeled antibody of the present invention are simultaneously or successively The method for quantifying the receptor protein or the like of the present invention in a test liquid is characterized by measuring the activity of the labeling agent on the insolubilized carrier after the reaction with the above. In the above (ii), it is preferable that one antibody recognizes the N-terminal portion of the receptor protein of the present invention and the other antibody reacts with the C-terminal portion of the receptor protein of the present invention. .

For the receptor protein etc. of the present invention
Monoclonal antibody (hereinafter referred to as the monoclonal antibody of the present invention
(Sometimes referred to as the body)
In addition to measuring protein quality, detection by tissue staining etc.
You can also do For these purposes, the antibody molecule
Alternatively, the antibody molecule F (a
b ') ₂, Fab ', or Fab fractions may be used.
Yes. Uses an antibody against the receptor protein or the like of the present invention
The measurement method used is not particularly limited and
The amount of antigen (eg, amount of receptor protein) in the constant solution
Quantify corresponding antibody, antigen or antibody-antigen complex
Detected by biological or physical means, and
Calculate from a standard curve prepared using a standard solution containing raw material
Any measuring method may be used as long as it is a measuring method. example
For example, nephrometry, competitive method, immunometric method and
The sandwich method is preferably used, but the sensitivity and specificity
In this respect, it is particularly preferable to use the sandwich method described later.
Good A labeling agent used in a measurement method using a labeling substance
For example, radioactive isotopes, enzymes, fluorescent substances,
A light substance or the like is used. Examples of radioactive isotopes
For example, [¹²⁵I], [¹³¹I], [³H], [¹⁴C] etc.
Is used. As the above enzyme, it is stable and has a large specific activity.
Preferred are, for example, β-galactosidase, β
-Glucosidase, alkaline phosphatase, pero
Xidase, malate dehydrogenase, etc. are used. firefly
Examples of the light substance include fluorescamine and fluorescein.
For example, sussen isothiocyanate is used. Luminescent substance
Are, for example, luminol, luminol derivatives,
Ciferin, lucigenin, etc. are used. In addition,
Biotin-avidin for binding between body or antigen and labeling agent
A system can also be used.

For the insolubilization of an antigen or an antibody, physical adsorption may be used, or a method using a chemical bond which is usually used for insolubilizing or immobilizing a protein or an enzyme may be used. As the carrier, for example, agarose,
Insoluble polysaccharides such as dextran and cellulose, synthetic resins such as polystyrene, polyacrylamide and silicone,
Alternatively, glass or the like is used. In the sandwich method, the insolubilized monoclonal antibody of the present invention is reacted with a test solution (first reaction), and further the labeled monoclonal antibody of the present invention is reacted (secondary reaction). By measuring the activity, the amount of the receptor protein of the present invention in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in reverse order, may be performed simultaneously, or may be performed at different times. The labeling agent and the method of insolubilization can be the same as those described above. In the immunoassay by the sandwich method, the antibody used as the solid phase antibody or the labeling antibody does not necessarily have to be one type, and a mixture of two or more types of antibodies may be used for the purpose of improving the measurement sensitivity. May be. In the method for measuring a receptor protein and the like by the sandwich method of the present invention, the monoclonal antibodies of the present invention used in the primary reaction and the secondary reaction are preferably antibodies having different binding sites for the receptor protein and the like. That is, when the antibody used in the secondary reaction recognizes the C-terminal portion of the receptor protein, the antibody used in the primary reaction is preferably other than the C-terminal portion. For example, an antibody that recognizes the N-terminal portion is used.

The monoclonal antibody of the present invention can be used in a measuring system other than the sandwich method, for example, a competitive method, an immunometric method or nephrometry. In the competitive method, the antigen in the test liquid and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B
/ F separation), the labeled amount of either B or F is measured to quantify the amount of antigen in the test liquid. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed by polyethylene glycol, a liquid phase method using a second antibody or the like against the above antibody, and a solid phase antibody is used as the first antibody, or The first antibody is soluble and the second antibody is a solid-phased antibody. In the immunometric method, the antigen in the test liquid and the solid-phased antigen are allowed to undergo a competitive reaction with a fixed amount of the labeled antibody, and then the solid phase and the liquid phase are separated, or
Alternatively, the antigen in the test liquid is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. To do. Next, the labeled amount of either phase is measured to quantify the amount of antigen in the test liquid. In nephrometry, the amount of insoluble precipitate generated as a result of the antigen-antibody reaction in the gel or in the solution is measured. Even when the amount of antigen in the test liquid is small and only a small amount of precipitate can be obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological assay methods to the assay method of the present invention, it is not necessary to set special conditions, operations and the like. The assay system for the receptor protein of the present invention or a salt thereof may be constructed by adding ordinary technical consideration to those skilled in the art to the ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, textbooks, etc. [eg,
Hiroshi Irie "Radioimmunoassay" (Kodansha, 1974)
Issue), edited by Hiroshi Irie, "Continued Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al., "Enzyme Immunoassay" (medical school, published in 1978), Eiji Ishikawa et al., "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982),
Eiji Ishikawa et al. “Enzyme Immunoassay” (3rd edition) (Medical Shoin, published in 1987), “Methods in ENZYMOLOGY” Vol.70 (Immunochem)
ical Techniques (Part A)), ibid Vol. 73 (Immunoch
emicalTechniques (Part B)), ibid Vol. 74 (Immunoc
hemical Techniques (Part C), ibid Vol. 84 (Immuno
chemical Techniques (Part D: Selected Immunoassay
s)), ibid Vol. 92 (Immunochemical Techniques (Par
t E: Monoclonal Antibodies and General Immunoassay
Methods)), ibid Vol. 121 (Immunochemical Techniqu
es (Part I: Hybridoma TechnoloGy and Monoclonal Ant
ibodies)) (Published by Academic Press, Inc.) etc.]. As described above, the receptor protein of the present invention or its salt can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, by quantifying the receptor protein of the present invention or a salt thereof in vivo using the antibody of the present invention, various diseases associated with dysfunction of the receptor protein of the present invention can be diagnosed. Further, the antibody of the present invention can be used for specifically detecting the receptor protein or the like of the present invention present in a subject such as body fluid or tissue. In addition, preparation of an antibody column used for purifying the receptor protein of the present invention, detection of the receptor protein of the present invention in each fraction during purification, of the behavior of the receptor protein of the present invention in a test cell It can be used for analysis etc.

(10) Method for screening compounds that alter the amount of the receptor protein of the present invention or its partial peptide in the cell membrane The antibody of the present invention specifically recognizes the receptor protein of the present invention or its partial peptide or its salt. Therefore, it can be used for screening a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. That is, the present invention includes, for example, (i) the blood contained in a non-human mammal, a specific organ, a tissue or cell isolated from the organ, and the like, the cell membrane fraction is isolated, and the present invention is included in the cell membrane fraction. Method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by quantifying the receptor protein of the present invention or its partial peptide, (ii) a trait expressing the receptor protein of the present invention or its partial peptide After disrupting the transformant etc., the cell membrane fraction is isolated, and the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is determined by quantifying the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction. A method for screening a compound to be changed, (ii
i) Quantify the degree of staining of the receptor protein on the cell surface by immunostaining after sectioning blood, specific organs, tissues or cells isolated from organs of non-human mammals This provides a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane. (Iv) After sectioning a transformant or the like that expresses the receptor protein of the present invention or a partial peptide thereof, by using an immunostaining method to quantify the degree of staining of the receptor protein on the cell surface, Provided is a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane.

Specifically, the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is quantified as follows. (I) Normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, and more specifically, demented rat, obese mouse, arteriosclerotic rabbit, cancer-bearing) Drugs (eg anti-dementia drugs, blood pressure lowering drugs,
Anti-cancer drug, anti-obesity drug, etc.) or physical stress (eg flooding stress, electric shock, light / darkness, low temperature, etc.), and after a certain period of time, blood, or a specific organ (eg, brain, lung, large intestine, etc.) ), Or tissue or cells isolated from an organ. The obtained organs, tissues or cells are suspended in, for example, an appropriate buffer solution (eg Tris-HCl buffer solution, phosphate buffer solution, Hepes buffer solution, etc.) to destroy the organs, tissues or cells, and A cell membrane fraction is obtained by using an activator (for example, Triton X-100 ^™ , Tween-20 ^™, etc.) and a technique such as centrifugation, filtration or column fractionation.

As the cell membrane fraction, after crushing the cells,
It means a fraction containing a large amount of cell membranes obtained by a known method. As a method of crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or polytron (Kinematica), crushing with ultrasonic waves, ejection of cells from a thin nozzle while applying pressure with a French press, etc. For example, crushing by allowing it to occur. For the fractionation of the cell membrane, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate may be spun at low speed (500-3000).
(rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15000 to 30000 rpm) for usually 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction.
The membrane fraction is rich in expressed receptor proteins and other membrane components such as cell-derived phospholipids and membrane proteins.

The receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis and the like. Such sandwich immunoassay can be performed in the same manner as the above method, and Western blotting can be performed by a known means.

(Ii) A transformant expressing the receptor protein of the present invention or its partial peptide can be prepared according to the above method to quantify the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction. .

Screening for a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane gives (i) a drug or physical stress to a normal or disease model non-human mammal or human cell. Before a fixed time (30 minutes to 24 hours ago, preferably 30 minutes to 12 hours ago, more preferably 1 hour to 6 hours ago) or a fixed time (30 minutes to 3 days later, preferably 1 hour later) ~ 2 days later, more preferably 1 hour to 24 hours later), or the test compound is administered at the same time as the drug or physical stress, and after a lapse of a fixed time (30 minutes to 3 days, preferably 1 hour). ~ 2 days, more preferably 1 to 24 hours later), the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is quantified. Can be done by
(Ii) When the transformant is cultured according to a conventional method, the test compound is mixed in the medium and cultured for a certain period of time (1 day to 7 days later, preferably 1 day to 3 days later, more preferably 2 days to 3 days). Days later), the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be quantified.

Specifically, the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is confirmed as follows. (Iii) normal or disease model non-human mammal (for example, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc., more specifically, demented rat, obese mouse, arteriosclerotic rabbit,
Drugs (eg, anti-dementia drugs, blood pressure lowering drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flood stress, electric shock, light / darkness, low temperature, etc.) against human cells After a certain period of time, blood, a specific organ (for example, heart, placenta, lung, etc.), or a tissue or cell isolated from the organ is obtained. The obtained organs, tissues, cells or the like are cut into tissue sections according to a conventional method and immunostained with the antibody of the present invention. The amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be confirmed quantitatively or qualitatively by confirming the protein on the cell membrane by quantifying the degree of staining of the receptor protein on the cell surface. can do. (Iv) It can also be confirmed by the same procedure using a transformant expressing the receptor protein of the present invention or its partial peptide.

The compound or its salt obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, and specifically, (a)
By increasing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, cell stimulating activity mediated by the receptor protein of the present invention (for example,
Intracellular Ca ²⁺ release, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease; cell death suppressing activity; cell adhesion Activity and cell motility controlling activity), and (b) a compound that reduces the cell stimulating activity by decreasing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. These compounds may be novel compounds or known compounds. The compound that enhances the cell stimulating activity is useful as a safe and low toxic pharmaceutical for enhancing the physiological activity of the receptor protein of the present invention. The compound that attenuates the cell stimulating activity is useful as a safe and low toxic pharmaceutical for reducing the physiological activity of the receptor protein of the present invention.

When the compound or its salt obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method. For example,
Tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like can be prepared in the same manner as the above-mentioned pharmaceutical containing the receptor protein of the present invention. Since the preparation thus obtained is safe and has low toxicity, for example, humans and non-human mammals (for example,
Rat, mouse, rabbit, sheep, pig, cow, cat,
Dogs, monkeys, etc.). The dose of the compound or its salt varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), About 0.1 to 100 mg, preferably about 1.0 to
It is 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but usually in the form of injection, for example, in cancer patients (as 60 kg) , About 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1
It is convenient to administer about 0 mg intravenously. In the case of other animals, the dose converted per 60 kg can be administered.

(11) A preventive and / or therapeutic agent for various diseases containing a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. Changes in the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. The compound can be used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction or overexpression of the receptor protein of the present invention. Preferably, a compound that reduces the amount of the receptor protein of the present invention or a partial peptide thereof in the cell membrane is used as a disease associated with overexpression of the receptor protein of the present invention [eg, cancer (eg, non-small cell lung cancer, ovarian cancer, prostate Cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant (Lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.)
When used as a preventive and / or therapeutic agent, etc.], it can be formulated according to a conventional method. For example, the compound is orally as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or a sterile solution with water or another pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injection such as suspension. For example, admixing the compound with a known physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a unit dose form generally required for the implementation of the formulation. Can be manufactured by. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. , For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate)
80 ^™ , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans and non-human mammals (for example, rat, mouse, rabbit, sheep, pig,
(Cattle, cats, dogs, monkeys, etc.). The dose of the compound or salt thereof is
Although there are differences depending on the target organs, symptoms, administration method, etc., generally, in the case of oral administration, for example, in a cancer patient (as 60 kg), about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day. , More preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but, for example, in the form of injection, it is usually used, for example, in cancer patients (60
(as kg) about 0.01-30 mg per day
Conveniently, about 0.1 to 20 mg, more preferably about 0.1 to 10 mg, is administered by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(12) Pharmaceutical comprising the antibody of the present invention Since the expression of the receptor protein of the present invention is elevated in cancer, the antibody of the present invention can be used in, for example, cancer (eg non-small cell lung cancer, Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer,
Cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer,
Endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.) can be used as a medicament such as a prophylactic and / or therapeutic agent. When the antibody of the present invention has neutralizing activity (activity that inactivates the signal transduction function involving the receptor protein of the present invention), the signal transduction involving the receptor protein of the present invention, for example, via the receptor protein Cell stimulating activity (eg, intracellular Ca ²⁺ release, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH decrease; Death inhibitory activity; cell adhesion activity and cell motility regulatory activity, etc.) can be inactivated.

Hereinafter, the antibody of the present invention and an antibody partial peptide having an antigen-binding site (eg, Fv, F (ab ′) ₂ ,
Fab fragment, single chain antibody, CDR, etc.) may be collectively referred to as an antibody. Since the receptor protein of the present invention has increased expression in various cancers,
The antibody against the receptor protein of the present invention or its partial peptide, and the immunotoxin containing the antibody against the receptor protein of the present invention or its partial peptide are useful as anticancer agents. When the antibody itself is used alone, it can attack cancer cells by its complement-dependent cytotoxic activity or antibody-dependent cytotoxic activity. Alternatively, the antibody can be administered to other chemotherapeutic agents (eg, Ifosfamide, Adriamyci).
n), Peplomycin, Cisplatin (C
isplatin), Carboplatin, Cyclophosphamide, 5-FU, UF
T, Methotrexate, Mitomycin C, Mitoxantron
e), Irinotecan, Topotecan
ecan), paclitaxel (Paclitaxel), docetaxel (Docetaxel), gemcitabine (Gemcitabine), Gleevec, Arimidex (Arimidex), etc., and synergistic antitumor effect can be shown (Breast Cancer Res. . and Treatmen
t, 29, 127-138, 1994). Furthermore, the antibody can also be used as a bispecific antibody. That is, an antibody having both an antigen-binding site for the receptor protein of the present invention or a partial peptide thereof and a site that specifically binds to cells having antitumor activity may be prepared by a chemical method or a genetic engineering method and used. it can. The cell having an antitumor effect is not limited as long as it exhibits an antitumor effect, and for example, cytotoxic T cells, natural killer cells, macrophages and the like are preferably used. The immunotoxin containing the antibody of the present invention is a substance having an activity of killing cancer cells, or a substance having an activity of suppressing the growth of cancer cells, by covalent bond to the antibody of the present invention, directly or through a linker. , Radioisotopes, chemotherapeutic agents, toxins, etc.) and the like. Specifically, known methods such as USP 4,
It can be manufactured according to the method described in 671,958, USP 4,741,900, USP 4,867,973 or the like. Examples of the above radioactive isotopes include yttrium 90 and iodine 131, and examples of chemotherapeutic agents include calichemicin and maytansine.
As the toxin, for example, ricin A, Pseudomonas aeruginosa exotoxin, diphtheria toxin and the like are used. When a chemotherapeutic agent is used, for example, the method described in USP 5,208,020 and the like are preferably used. For example, the antibody of the present invention can be treated with N-succin according to a known method, for example, the method described in Biochem. J. 173, 723-737, 1978 or a method analogous thereto.
Treated with imidyl-3- (2-pyridyldithio) propionate and treated with 2-py
Introduce ridyl disulfide group. Furthermore, the modified antibody and the Maytansine derivative May-SH (Cancer Res. 5
(2, pp. 127-131, 1992), the conjugate obtained by mixing
An immunotoxin fraction is obtained by removing May-SH. When a toxin is used, for example, a DNA encoding the variable region of an immunoglobulin molecule is obtained from a hybridoma producing the antibody of the present invention, and a fusion protein with ricin A or Pseudomonas aeruginosa exotoxin is obtained according to a known recombinant DNA technique. Can be used as an immunotoxin (Adv Cancer Res. 81, 93-124, 2001).

The pharmaceuticals for the above diseases containing the antibody of the present invention have low toxicity and can be used as a liquid formulation as it is or as a pharmaceutical composition in an appropriate dosage form, for human or non-human mammals (eg,
(Rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) orally or parenterally. The dose varies depending on the administration subject, target disease, symptom, administration route, etc., but when it is used for treating adult cancer, for example, the antibody of the present invention is usually used as a single dose of 0.01. ~ 20 mg / kg body weight, preferably 0.1-10 mg / kg body weight, more preferably 0.1-5 mg / kg body weight about 1-5 times a day, preferably about 1-3 times a day , Conveniently administered by intravenous injection. In the case of other parenteral administration and oral administration, an amount similar to this can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a composition is provided as a dosage form suitable for oral or parenteral administration. That is, for example, as a composition for oral administration, a solid or liquid dosage form, specifically, tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (soft capsules Including), syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets. As the composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, etc. Includes. Such an injection is prepared according to a known method, for example, by dissolving, suspending or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like are used. Suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg polysorbate 8
0, HCO-50 (polyoxyethylene (50mol) adduct
of hydrogenatedcastor oil)] and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is usually filled in a suitable ampoule. The suppository used for rectal administration is prepared by mixing the above antibody or a salt thereof with an ordinary suppository base. The oral or parenteral pharmaceutical compositions described above are conveniently prepared in dosage unit form for administration of the active ingredient. Examples of dosage forms of such dosage units include tablets, pills, capsules, injections (ampoules), suppositories, and the like.
Usually 5 to 500 mg per dosage unit form, especially 5 to 100 mg for injections, 1 for other dosage forms
It is preferable that the above antibody is contained in an amount of 0 to 250 mg. Each composition described above may contain other active ingredients as long as the composition does not cause an unfavorable interaction with the antibody.

(13) Pharmaceutical Agent Containing Antisense Polynucleotide The present invention is capable of binding complementarily to the polynucleotide (eg, DNA) of the present invention and suppressing the expression of the polynucleotide (eg, DNA). The antisense polynucleotide has low toxicity, and the receptor protein of the present invention or the polynucleotide of the present invention (eg, DN
Since the function of A) can be suppressed, for example, diseases associated with overexpression of the receptor protein of the present invention [eg, central diseases (eg Alzheimer's disease, dementia, etc.), metabolic diseases (eg diabetes, etc.), Cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer,
Cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer,
Endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.)] and / or the like. When the above antisense polynucleotide is used as the above medicine, it can be formulated and administered according to a known method. For example, when the antisense polynucleotide is used, the antisense polynucleotide may be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector, according to a conventional method. Human mammals (eg, rat, rabbit,
Can be orally or parenterally administered to sheep, pigs, cows, cats, dogs, monkeys, etc.). The antisense polynucleotide can be administered as it is or in the form of a preparation with a physiologically acceptable carrier such as an auxiliary agent for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter. The dose of the antisense polynucleotide may be a target disease, an administration subject,
Although there are differences depending on the administration route, for example, when the antisense polynucleotide of the present invention is locally administered to an organ (eg, liver, lung, heart, kidney, etc.) for the purpose of treating cancer, an adult (body weight 60 kg) is generally used. ), The antisense polynucleotide is added in an amount of about 0.1 to 10 per day.
Administer 0 mg. Furthermore, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for investigating the presence of the polynucleotide (eg, DNA) of the present invention in tissues or cells and the expression state thereof.

The present invention further relates to a double-stranded RNA containing a part of RNA encoding the receptor protein of the present invention, a medicament comprising the double-stranded RNA, and an RNA encoding the receptor protein of the present invention. A ribozyme containing a part thereof, and a medicine containing the ribozyme are provided. These double-stranded RNA (RNAi; RNA interference method),
Ribozymes and the like can suppress the expression of the polynucleotide (eg, DNA) of the present invention in the same manner as the above antisense polynucleotides, and thus the peptide of the present invention or the polynucleotide (eg, DNA) of the present invention in vivo.
Since it can inhibit the activity or function of, for example, central diseases (for example, Alzheimer's disease, dementia, etc.), metabolic diseases (for example, diabetes, etc.), cancer (for example, non-small cell lung cancer,
Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer,
Colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor,
Use as a drug such as a prophylactic and / or therapeutic agent for diseases such as schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia) can do. Double-stranded RNA is
It can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001). Ribozymes can be prepared by known methods (eg, TRENDS in Molecular Medicine, 7: 221, 20).
2001), and can be designed and manufactured based on the sequence of the polynucleotide of the present invention. For example, it can be produced by linking a known ribozyme to a part of RNA encoding the receptor protein of the present invention. RNA encoding the receptor protein of the present invention
Part of the RNA of the present invention that can be cleaved by known ribozymes (RNA
Fragment). When the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned preventive / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.

(14) Preparation of Transgenic Animal Having DNA Encoding Receptor Protein of the Present Invention Using the DNA of the present invention, a transgenic animal expressing the receptor protein of the present invention can be prepared. Animals include mammals (eg, rat,
Mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) (hereinafter sometimes abbreviated as animal) and the like, and particularly mouse and the like are preferable. When the DNA of the present invention is introduced into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter that can be expressed in animal cells. For example, when the mouse-derived DNA of the present invention is introduced, a gene construct in which the DNA of the present invention highly homologous thereto is linked to the downstream of various promoters capable of expressing in animal cells is used, for example, in a mouse fertilized egg. By microinjection into DNA, a DNA-introduced animal that highly produces the receptor protein of the present invention can be produced. As this promoter, for example, a ubiquitous expression promoter such as a virus-derived promoter or a metallothionein promoter is used.

The introduction of the DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal. The presence of the receptor protein or the like of the present invention in the germ cells of the created animal after the introduction of DNA means that all the progeny of the created animal have the receptor protein or the like of the present invention in all of their germ cells and somatic cells. The offspring of this type of animal that has inherited the gene has the receptor protein of the present invention in all of its germ cells and somatic cells. The DNA-introduced animal of the present invention was confirmed to have a stable gene by mating, and
As the A-carrying animal, breeding passage can be performed in a normal breeding environment. Furthermore, a male and female animal carrying the target DNA is mated to obtain a homozygous animal having the transgene on both homologous chromosomes. By mating the male and female animals, all offspring are made to have the DNA. Can be bred and passaged. The animal into which the DNA of the present invention has been introduced has high expression of the receptor protein of the present invention, and therefore is useful as an animal for screening agonists or antagonists of the receptor protein of the present invention. The DNA-introduced animal of the present invention can also be used as a cell source for tissue culture. For example, the receptor protein or the like of the present invention can be analyzed by directly analyzing DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or by analyzing the tissue in which the receptor protein of the present invention expressed by a gene is present. can do. Cells of a tissue having the receptor protein or the like of the present invention are cultured by a standard tissue culture technique, and using these, for example,
The function of cells from tissues that are generally difficult to culture, such as those from the brain and peripheral tissues, can be studied. Further, by using the cells, for example, it is possible to select a drug that enhances the functions of various tissues. Further, if there is a highly expressing cell line, the receptor protein of the present invention can be isolated and purified from the cell line.

(15) Multimer of Receptor Protein of the Present Invention The receptor protein of the present invention forms a multimer (eg, homodimer, heterodimer, etc.). As the multimer, for example, a receptor belonging to the Eph receptor family [eg, EphA1 (Science, 238, 1717-1720
P., 1987), EphA2 (Mol. Cell. Biol., 10 volumes,
6316-6324, 1990, EphA3 (J. Biol. Chem. 2
67, 3262-3267, 1992), EphA4, EphA
5, EphA7 (both Oncogene, Volume 10, 897-905,
1995), EphA8 (Oncogene, Vol. 6, 1057-1061,
1991) and such as hetero-multimers. The receptor proteins of the present invention are compatible with other E proteins such as EphA7.
Since it is considered to be a dominant negative of the ph receptor family, substances having an action of inhibiting multimerization include, for example, central diseases (for example, Alzheimer's disease, dementia, etc.), metabolic diseases (for example, diabetes, etc.), cancer (for example, , Non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer,
Breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma, malignant melanoma, thyroid It is useful as a drug such as a prophylactic and / or therapeutic agent for diseases such as cancer, bone tumor, leukemia, etc., preferably anticancer agent. The substance blocks the information signal of the multimer.

Multimerization of the Receptor Proteins of the Invention
A method for screening a substance having an action of inhibiting
It will be described below. For example, the receptor protein of the present invention
And other receptors belonging to the Eph receptor family
And a co-expression system with the
By using a receptor binding assay system using
And screening for substances that inhibit multimerization
You can Specifically, (i) the receptor protein of the present invention
Protein and other members of the Eph receptor family
When culturing cells containing the receptor, (ii) test
In the presence of the test substance, the receptor protein of the present invention and E
Includes other receptors in the ph receptor family
Inhibits multimerization compared to the case of culturing cells with
Screen for substances that have the effect of Above screen
In case of (i) and (ii) in the learning method
For example, cell stimulating activity (eg, arachidonic acid migration
Detachment, acetylcholine release, intracellular Ca ²⁺Free, intracellular c
AMP production, intracellular cAMP production suppression, intracellular cGMP
Production, inositol phosphate production, cell membrane potential fluctuation, cell
Phosphorylation of internal proteins, activation of c-fos, pH
Decrease, activity or suppression that promotes GTPγS binding activity, etc.
Control activity; cell death suppression activity; cell adhesion activity and cells
Activity control activity, etc.), known methods or commercially available measurements
Use a kit for measurement and compare. As a test item,
For example, peptides, proteins, non-peptidic compounds,
Synthetic compounds, fermentation products, etc. are used. Multimerization
As the substance having an inhibitory action, the antibody of the present invention,
Invention antisense polynucleotide, ribozyme, book
Promoter activity of the gene of the receptor protein of the invention
Examples include substances that inhibit sex. Specifically,
Ming receptor protein and Eph receptor protein
Lacti containing other receptor proteins belonging to Milly
Mammalian cells (eg, various human cancer cell lines, the receptor of the present invention)
HEK2 with forced expression of protein and human EphA7
93 cells, etc.) on a tissue culture plate and tested on the medium
After adding substances, prepare cell-free extract according to known methods
To do. The receptor protein of the present invention and a heteromultimer
Specific for the Eph receptor protein that forms
Antibodies and responsible for binding to the Fc portion of immunoglobulins
Body (eg, with protein G or anti-IgG-Fc antibody, etc.)
Bound agarose, etc.) to the cell-free extract and
After stirring overnight at ℃, the fraction bound to the carrier is separated and collected.
Whether or not the receptor protein of the present invention is contained.
Heteromultimer formation was confirmed by investigating
Or not. Similar operation without addition of test substance
Compared with the results of the test, the test substance formed heteromultimers.
It is determined whether or not The receptor tan of the present invention
The detection of protein is not particularly limited, and for example, known methods or
May be carried out in accordance with it, and as a specific example, the
Western blotting using antibodies is used.
It

A substance having an action of inhibiting the multimerization of the receptor protein of the present invention is, for example, a central disease (eg Alzheimer's disease, dementia etc.), metabolic disease (eg diabetes etc.), cancer (eg non-small size). Cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney Cancer, endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.)
When used as a preventive and / or therapeutic agent, etc.], it can be formulated according to a conventional method. For example, the substance is orally as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injection such as suspension. For example, a known physiologically acceptable carrier, flavoring agent, excipient, vehicle,
It can be prepared by admixture with preservatives, stabilizers, binders and the like in a unit dose form generally required for the practice of the formulation. The amount of active ingredient in these preparations is such that a suitable dose within the indicated range can be obtained.

Additives that can be mixed with tablets, capsules and the like include, for example, binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid. Puffing agents such as and the like, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry, and the like are used. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. , For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate)
80 ^™ , HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The above-mentioned prophylactic / therapeutic agents include, for example, buffer agents (eg, phosphate buffer solution, sodium acetate buffer solution), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers ( For example, human serum albumin, polyethylene glycol, etc.), preservatives (eg,
(Benzyl alcohol, phenol, etc.), antioxidant, etc. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, humans and non-human mammals (for example, rat, mouse, rabbit, sheep, pig,
(Cattle, cats, dogs, monkeys, etc.). The dose of the substance or its salt varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the case of oral administration, generally, for example, in a cancer patient (as 60 kg), The amount is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc., but, for example, in the form of injection, it is usually used, for example, in cancer patients (60
(as kg) about 0.01-30 mg per day
Conveniently, about 0.1 to 20 mg, more preferably about 0.1 to 10 mg, is administered by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

(16) Antibodies to Receptors Having the Inhibitory Activity of Receptors Antibodies to the receptors having an inhibitory activity of the receptors include the receptor activities (eg, arachidonic acid release, acetylcholine release, intracellular Ca ^{2 +} Release, intracellular cAMP production, intracellular cAMP production suppression, intracellular c
GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, activity promoting or suppressing GTPγS binding activity, etc .; cell death suppressing activity; cell adhesion Activity and cell motility regulatory activity, etc.) may be any antibody to a receptor having an action of inhibiting, for example,
The antibody or the like of the present invention is used. The antibody is, for example, a central disease (for example, Alzheimer's disease, dementia, etc.), metabolic disease (for example, diabetes, etc.), cancer (for example, non-small cell lung cancer,
Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer,
Colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, brain tumor,
Schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.] and the like. . The formulation method and the like may be performed according to the antibody formulation method and the like of the present invention.

(17) Action of inhibiting the activity of an antisense polynucleotide receptor containing a base sequence complementary to a polynucleotide containing a polynucleotide encoding the receptor having an action of inhibiting the activity of the receptor or a part thereof. As an antisense polynucleotide containing a nucleotide sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor having the or a portion thereof, the receptor activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cAMP production suppression, intracellular cGMP production, inositol phosphate production,
Cell membrane potential fluctuation, intracellular protein phosphorylation, cf
Polynucleotide encoding a receptor having an activity of inhibiting os activation, pH decrease, GTPγS binding activity, or the like; cell death inhibitory activity; cell adhesion activity, cell motility regulatory activity, etc. A polynucleotide containing (eg, DNA) (eg, DN
Any antisense polynucleotide containing a base sequence complementary to A) or a part thereof may be used, and for example, the antisense polynucleotide of the present invention is used. The antisense polynucleotide, for example, central diseases (for example, Alzheimer's disease, dementia, etc.),
Metabolic disease (for example, diabetes), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, pancreatic cancer, esophageal cancer, duodenal cancer, Brain tumor, schwannoma, small cell lung cancer, liver cancer, kidney cancer, endometrial cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, leukemia, etc.) and / or the like it can. The formulation method and the like may be performed according to the formulation method and the like of the antisense polynucleotide of the present invention.

In the present specification and drawings, when bases, amino acids, etc. are indicated by abbreviations, the indication is IUPAC-IUB.
It is based on the abbreviations by Commission on Biochemical Nomenclature or the abbreviations commonly used in this field.
An example is shown below. When amino acids may have optical isomers, the L form is shown unless otherwise specified.

Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine pGlu: pyroglutamic acid Me: Methyl group Et: ethyl group Bu: Butyl group Ph: Phenyl group TC: thiazolidine-4 (R) -carboxamide group

Further, substituents, protecting groups and reagents commonly used in the present specification are represented by the following symbols. Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-Butoxycarbonyl DNP: Dinitrophenol Trt: Trityl Bum: t-Butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-Hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy -4-
Oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,
3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing of the present specification show the following sequences. SEQ ID NO: 1 This shows a part of the nucleotide sequence (5 'side) of the cDNA encoding the human brain-derived novel receptor protein of the present invention. SEQ ID NO: 2 Shows the base sequence of primer 1 used in the PCR reaction in Example 1 below. SEQ ID NO: 3 Shows the base sequence of primer 2 used in the PCR reaction in Example 1 below. SEQ ID NO: 4 This shows a part (central part) of the nucleotide sequence of cDNA encoding the novel human brain-derived receptor protein of the present invention. SEQ ID NO: 5 This shows the base sequence of primer 3 used in the PCR reaction of Example 2 below. SEQ ID NO: 6 This shows the base sequence of primer 4 used in the PCR reaction of Example 2 below. SEQ ID NO: 7 This shows a part of the nucleotide sequence (3 'side) of the cDNA encoding the human brain-derived novel receptor protein of the present invention. SEQ ID NO: 8 This shows the base sequence of primer 5 used in the PCR reaction of Example 3 below. SEQ ID NO: 9 This shows the base sequence of primer 6 used in the PCR reaction of Example 3 below. SEQ ID NO: 10 Shows the base sequence of primer 7 used in the PCR reaction in Example 3 below. SEQ ID NO: 11 This shows the amino acid sequence encoding the novel human brain-derived receptor protein NEPHA of the present invention obtained in Example 5. SEQ ID NO: 12 Human brain-derived novel receptor protein NEPH of the present invention
The entire base sequence of the cDNA encoding A is shown. SEQ ID NO: 13 This shows the base sequence of cDNA encoding the human brain-derived novel receptor protein of the present invention. SEQ ID NO: 14 This shows the amino acid sequence encoding the novel human ovarian cancer-derived receptor protein of the present invention. SEQ ID NO: 15 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 14. SEQ ID NO: 16 This shows the base sequence of primer 8 used in the PCR reactions in Example 6 and Example 7 below. SEQ ID NO: 17 This shows the base sequence of primer 9 used in the PCR reaction in Examples 6 and 7 below. SEQ ID NO: 18 This shows the amino acid sequence encoding the novel human ovarian cancer-derived novel receptor protein of the present invention obtained in Example 7. SEQ ID NO: 19 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 18. SEQ ID NO: 20 This shows the amino acid sequence encoding the novel human ovarian cancer-derived novel receptor protein of the present invention obtained in Example 7. SEQ ID NO: 21 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 20. SEQ ID NO: 22 This shows the amino acid sequence encoding the novel human brain-derived receptor protein of the present invention obtained in Example 7. SEQ ID NO: 23 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 22. SEQ ID NO: 24 This shows the amino acid sequence encoding the novel human brain-derived receptor protein of the present invention obtained in Example 7. SEQ ID NO: 25 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 24. SEQ ID NO: 26 This shows the base sequence of primer 10 used in the PCR reactions in Examples 8 to 12 below. SEQ ID NO: 27 This shows the base sequence of primer 11 used in the PCR reactions in Examples 8 to 12 below. SEQ ID NO: 28 This shows the base sequence of probe 1 used in the PCR reactions in Examples 8 to 12 below. SEQ ID NO: 29 This shows the base sequence of primer 12 used in the PCR reaction of Example 8 below. SEQ ID NO: 30 This shows the base sequence of primer 13 used in the PCR reaction in Example 8 below. SEQ ID NO: 31 This shows the base sequence of probe 2 used in the PCR reaction in Example 8 below. SEQ ID NO: 32 This shows the base sequence of primer 14 used in the PCR reactions in Example 13 and Example 26 below. SEQ ID NO: 33 This shows the base sequence of primer 15 used in the PCR reactions in Example 13 and Example 26 below. SEQ ID NO: 34 This shows the base sequence of primer 16 used in the PCR reaction of Example 15 described below. SEQ ID NO: 35 This shows the base sequence of primer 17 used in the PCR reaction of Example 15 described below. SEQ ID NO: 36 This shows the amino acid sequence of peptide 1 used for antibody production in Example 18 below. SEQ ID NO: 37 This shows the amino acid sequence of peptide 2 used for antibody production in Example 18 below. SEQ ID NO: 38 This shows the amino acid sequence encoding the novel human ovarian cancer-derived receptor protein of the present invention obtained in Example 6. SEQ ID NO: 39 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 38. SEQ ID NO: 40 This shows the amino acid sequence encoding the novel receptor protein derived from human ovarian cancer of the present invention. SEQ ID NO: 41 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 40. SEQ ID NO: 42 This shows the amino acid sequence encoding the novel receptor protein derived from human ovarian cancer of the present invention. SEQ ID NO: 43 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 42. SEQ ID NO: 44 This shows the amino acid sequence encoding the novel human brain-derived receptor protein of the present invention. SEQ ID NO: 45 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 44. SEQ ID NO: 46 This shows the amino acid sequence encoding the human brain-derived novel receptor protein of the present invention. SEQ ID NO: 47 This shows the base sequence of cDNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 46. SEQ ID NO: 48 This shows the base sequence of the antisense oligonucleotide of the present invention. SEQ ID NO: 49 This shows the base sequence of the antisense oligonucleotide of the present invention. SEQ ID NO: 50 This shows the base sequence of the oligonucleotide used in EXAMPLE 28 below. SEQ ID NO: 51 This shows the base sequence of the oligonucleotide used in EXAMPLE 28 below. SEQ ID NO: 52 This shows the base sequence of the primer used in the PCR reaction of Example 28 below. SEQ ID NO: 53 This shows the base sequence of the primer used in the PCR reaction of Example 28 below. SEQ ID NO: 54 This shows the base sequence of the probe used in the PCR reaction in Example 28 below.

[0128] Escherichia coli TOP10 / pTB223 obtained in Example 5 described below.
0 is from May 24, 2001, 1-1, Higashi 1-1-chome, Tsukuba-shi, Ibaraki, Japan, Chuo No. 6 (zip code 305-85)
66), the National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center, under the deposit number FERM BP-7606 and the deposit number IFO 166 from April 24, 2001.
As 23, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka
It has been deposited with the Fermentation Research Institute (IFO) of (zip code 532-8686). Escherichia coli TOP10 / pTB2 obtained in Example 6 described later
259 has been in operation from March 28, 2002, 1-chome, 1-chome, 1-chome, Tsukuba-shi, Ibaraki, Japan, Chuo No. 6 (zip code 305-
8566), the National Institute of Advanced Industrial Science and Technology, Patent and Bio-Chemical Depository, under the deposit number FERM BP-7986 and the deposit number IFO 167 from March 12, 2002.
68 as 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka
It has been deposited with the Fermentation Research Institute (IFO) of (zip code 532-8686). Escherichia coli TOP10 / pTB2 obtained in Example 7 described later
260 has been in operation from March 28, 2002, 1-1, Higashi 1-1-chome, Tsukuba, Ibaraki, Japan, Chuo No. 6 (zip code 305-
8566) under the deposit number FERM BP-7987 of the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, and the deposit number IFO 167 from March 12, 2002.
69 as 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka
It has been deposited with the Fermentation Research Institute (IFO) of (zip code 532-8686). Escherichia coli TOP10 / pTB2 obtained in Example 7 described later
From March 28, 2002, 261 is Chuo No. 6 (Postal code 305-
8566) under the deposit number FERM BP-7988 of the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, and the deposit number IFO 167 from March 12, 2002.
70 as 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka
It has been deposited with the Fermentation Research Institute (IFO) of (zip code 532-8686). Escherichia coli TOP10 / pTB2 obtained in Example 7 described later
262 is Chuo No. 6 (zip code 305-
8566) under the deposit number FERM BP-7989 of the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, and deposit number IFO 167 from March 12, 2002.
As 71, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka-shi, Osaka
It has been deposited with the Fermentation Research Institute (IFO) of (zip code 532-8686). Escherichia coli TOP10 / pTB2 obtained in Example 7 described later
263 is Chuo No. 6 (postal code 305- from 1-chome, Higashi 1-1-chome, Tsukuba-shi, Ibaraki, Japan since March 28, 2002)
8566) under the deposit number FERM BP-7990 of the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, and deposit number IFO 167 from March 12, 2002.
As 72, 2-17-85, Jusohon-cho, Yodogawa-ku, Osaka-shi, Osaka
It has been deposited with the Fermentation Research Institute (IFO) of (zip code 532-8686).

[0129]

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention.

Example 1 cD encoding a novel receptor protein derived from human brain
Determination of 5'partial nucleotide sequence of NA Human brain cDNA (CL contained in MTC Panel I)
A PCR reaction was carried out using 2 primers, Primer 1 (SEQ ID NO: 2) and Primer 2 (SEQ ID NO: 3), using ONTECH as a template. The composition of the reaction solution in the reaction was such that 0.5 μl of the above-mentioned cDNA was used as a template and Pfu Turbo DNA Polymer was used.
se (STRATAGENE) 1U, primer 1
(SEQ ID NO: 2) and primer 2 (SEQ ID NO: 3) each at 1 μM, dNTPs at 200 μM, and 2xGC B
10 μl of user I (Takara Shuzo) was added to make a volume of 20 μl. PCR reaction at 96 ° C, 1 minute later, 96 ° C,
The cycle of 1 minute, 60 ° C., 1 minute, 72 ° C., 1 minute was repeated 40 times. Next, the P
After purifying the CR reaction product, it was purified using Zero Blun.
t TOPO PCR Cloning Kit (Invit
plasmid vector pCR according to the prescription of Rogen.
-BluntII-TOPO (Invitrogen
Company). E. coli TOP10
And selected on LB agar containing kanamycin. As a result of analyzing the sequences of the individual clones, a base sequence (SEQ ID NO: 1) of a cDNA encoding a part of the novel receptor protein was obtained.

Example 2 cD encoding a novel receptor protein derived from human brain
Determination of the central base sequence of NA Human brain cDNA (CLONTECH MTC Pan)
elI) as a template, two primers, primer 3
PCR reaction was performed using (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6). The composition of the reaction solution in the reaction was such that 1 μl of the above cDNA was used as a template and Pfu was used.
Turbo DNA Polymerase (STRA
2.5U, primer 3 (SEQ ID NO:
5) and primer 4 (SEQ ID NO: 6) at 1 μM each, d
200 μM of NTPs, and 2XGC Buffer I
(Takara Shuzo) was added in an amount of 25 μl to give a liquid volume of 50 μl. P
CR reaction is 96 ° C, 1 minute, 96 ° C, 1 minute, 60
The cycle of 1 minute at 72 ° C and 2 minutes at 72 ° C was repeated 40 times. Next, after purifying the PCR reaction product by agarose gel electrophoresis, it was used for Zero Blunt TOP
O PCR Cloning Kit (Invitroge
n company) according to the prescription of plasmid vector pCR-Blue
It was subcloned into ntII-TOPO (Invitrogen). This was introduced into E. coli TOP10,
Selection was done on LB agar containing kanamycin. As a result of analyzing the sequences of the individual clones, a nucleotide sequence (SEQ ID NO: 4) of a cDNA encoding a part of the novel receptor protein was obtained.

Example 3 cD encoding a novel receptor protein derived from human brain
Determination of 3'partial base sequence of NA Human brain cDNA (CLONTECH MTC Pan
elI) as a template, two primers, primer 5
PCR reaction was performed using (SEQ ID NO: 8) and primer 6 (SEQ ID NO: 9). The composition of the reaction solution in the reaction was such that 1 μl of the above cDNA was used as a template and Pfu was used.
Turbo DNA Polymerase (STRA
2.5U of TAGENE, primer 5 (SEQ ID NO:
8) and primer 6 (SEQ ID NO: 9) at 1 μM each, d
200 μM of NTPs, and 2XGC Buffer I
(Takara Shuzo) was added in an amount of 25 μl to give a liquid volume of 50 μl. P
CR reaction is 96 ° C, 1 minute, 96 ° C, 1 minute, 60
The cycle of 1 minute at 72 ° C and 2 minutes at 72 ° C was repeated 40 times. Next, using 5 μl of the PCR reaction product as a template, Pfu Turbo DNA Polymers was used.
e (STRATAGENE) 2.5U, primer 5
(SEQ ID NO: 8) and primer 7 (SEQ ID NO: 10)
1 μM each, dNTPs 200 μM, and 2XGC
Buffer I (Takara Shuzo) was added in an amount of 25 μl to give a liquid volume of 50 μl. PCR reaction was performed at 96 ° C. for 1 minute and then 96
℃, 1 minute, 60 ℃, 1 minute, 72 ℃, 2 minutes cycle 4
Repeated 5 times. Next, after purifying the PCR reaction product by agarose gel electrophoresis, this was purified with Zero Bl.
unt TOPO PCR Cloning Kit (In
plasmid vector pCR-BluntII-TOPO (Invitrog) according to the prescription of Vitrogen.
en). E. coli TOP
Introduced into 10 and selected in LB agar containing kanamycin. As a result of analyzing the sequences of the individual clones, a base sequence (SEQ ID NO: 7) of a cDNA encoding a part of the novel receptor protein was obtained.

Example 4 Determination of Base Sequence of Full Length cDNA Encoding Novel Receptor Protein Derived from Human Brain Base sequence of partial cDNA encoding novel receptor protein obtained in Example 1, Example 2 and Example 3 (sequence No.:1 and SEQ ID NO: 4 and SEQ ID NO: 7)
Based on this, the base sequence of the full-length cDNA (SEQ ID NO: 12) was determined. SEQ ID NO: 12 corresponds to the sequence from 71st to 789th of SEQ ID NO: 1, the sequence from 284th to 1134th of SEQ ID NO: 4, and the sequence from 473th to 1926th of SEQ ID NO: 7. Full length cDNA
The amino acid sequence of the protein predicted from the base sequence of A (SEQ ID NO: 12) is shown in SEQ ID NO: 11. The protein containing the amino acid sequence represented by SEQ ID NO: 11 was named NEPHA (novelEphA). The receptor protein NEPHA of the present invention and known Ephr
The homology with the in receptor (Eph receptor) family is shown in FIGS. 1 to 5. The receptor protein of the present invention and the human Ephrin receptor EphA7 have a homology of 50.7% at the amino acid level. The gene encoding the full length is obtained by cleaving the cDNA fragments obtained in Examples 1, 2 and 3 with an appropriate restriction enzyme and then ligating with T4 ligase. Alternatively,
A primer that amplifies the cDNA encoding the full length is designed based on the nucleotide sequences clarified in Examples 1 and 3 and is obtained by the PCR method.

Example 5 Construction of Plasmid Having Full-Length cDNA Nucleotide Sequence Encoding Novel Receptor Protein Derived from Human Brain Nucleotide sequences of the cDNAs obtained in Examples 2 and 3 (SEQ ID NO: 4 and SEQ ID NO: 7) The plasmid containing) was cleaved with a restriction enzyme. The composition of the reaction solution is SEQ ID NO:
1 μg of a plasmid containing the nucleotide sequence of the cDNA represented by 4 or a plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 7, XhoI (Takara Shuzo) 5U, and 1
Add 5 μl of 0xH Buffer (Takara Shuzo) and add 50 μl
Of the liquid. The restriction enzyme reaction was performed at 37 ° C. for 1 hour, and 5 U of BstEII (Takara Shuzo) was added to the reaction product, and the reaction was performed at 60 ° C. for 1 hour. Next, SEQ ID NO: 4
After purifying the reaction product corresponding to 941 bp from the plasmid containing the cDNA nucleotide sequence represented by and the plasmid corresponding to 5273 bp from the plasmid containing the cDNA nucleotide sequence represented by SEQ ID NO: 7, by agarose electrophoresis, DNA Ligation Kit Ver. They were combined according to the prescription of 2 (Takara Shuzo). E. coli TOP1
0 introduced and selected in LB agar containing kanamycin. As a result of analyzing the sequences of the individual clones, the base sequence of the cDNA represented by SEQ ID NO: 13 was obtained. Furthermore, the plasmid containing the nucleotide sequence of cDNA (SEQ ID NO: 1) obtained in Example 1 and the plasmid containing the nucleotide sequence of cDNA represented by SEQ ID NO: 13 were cleaved with a restriction enzyme. The composition of the reaction solution was 1 μg of a plasmid containing the cDNA nucleotide sequence represented by SEQ ID NO: 1 or a plasmid containing the cDNA nucleotide sequence represented by SEQ ID NO: 13, PvuI (Takara Shuzo) 10U, and 10xK.
Buffer (Takara Shuzo) was added to make a liquid volume of 50 μl.
The restriction enzyme reaction was performed at 37 ° C. for 1 hour. Next, from the plasmid containing the nucleotide sequence of cDNA represented by SEQ ID NO: 1, 743 bp and the cDNA represented by SEQ ID NO: 13
The reaction product corresponding to 5977 bp was purified from the plasmid containing the nucleotide sequence of A by agarose gel electrophoresis, and then the DNA ligation kit Ve
r. They were combined according to the prescription of 2 (Takara Shuzo). This was introduced into E. coli TOP10 and selected in LB agar medium containing kanamycin. As a result of analyzing the sequences of the individual clones, a plasmid having the base sequence (SEQ ID NO: 12) of the full-length cDNA encoding the human brain-derived novel receptor protein was obtained. The plasmid having SEQ ID NO: 12 obtained here was named pTB2230, and the obtained transformant was designated as Escherichia coli TOP10 / pT.
It was named B2230.

Example 6 Determination of cDNA Encoding Novel Receptor Protein Derived from Human Ovarian Cancer Using human ovarian cancer (GI-102) cDNA (CLONTECH) contained in MTC Panel Tumor as a template, two primers, a primer 8 (SEQ ID NO:
PCR reaction was performed using 16) and primer 9 (SEQ ID NO: 17). The composition of the reaction solution in the reaction was 50X Adv using 1 μl of the above cDNA as a template.
event-GC 2 Polymerase Mix
(CLONTECH) 0.4 μl, primer 8 (SEQ ID NO: 16) and primer 9 (SEQ ID NO: 17)
0.4 μM each, dNTPs 200 μM, GC-Me
lt (CLONTECH) 1M, 5xGC 2 P
4 μl of CR Buffer (CLONTECH) was added to make a liquid volume of 20 μl. PCR reaction at 94 ° C, 3
After 3 minutes, 3 cycles of 94 ° C, 30 seconds, 68 ° C, 4 minutes
Repeated 5 times. Next, after purifying the PCR reaction product by agarose gel electrophoresis, this was purified by TOPO XL.
PCR Cloning Kit (Invitrogen
Plasmid vector pCR-XL-T
It was subcloned into OPO (Invitrogen). This was introduced into E. coli TOP10 and selected in LB agar medium containing kanamycin. As a result of analyzing the sequences of the individual clones, a cDNA sequence (SEQ ID NO: 39) encoding a novel receptor protein was obtained. The amino acid sequence encoded by the base sequence represented by SEQ ID NO: 39 is shown in SEQ ID NO: 38. Sequence number obtained here:
The plasmid containing 39 was designated as pTB2259, and the obtained transformant was designated as Escherichia coli TO.
It was named P10 / pTB2259. SEQ ID NO: 38
A protein containing an amino acid sequence represented by NEP
It was named HA-SV1. In the nucleotide sequence represented by SEQ ID NO: 39, the 380th G is A, the 606th T is C,
911 A is G, 1081 G is A, 11
22nd T is C, 1250th G is A, 128
3rd A to G, 1323th G to A, 1553
The 1st C is T, the 1690th G is A, the 1719th T is C, the 1780th G is A, the 1812th C is G, the 1853th A is G, and the 2036th G is The base sequence in which G is replaced with A is shown in SEQ ID NO: 15. The amino acid sequence encoded by the base sequence represented by SEQ ID NO: 15 is shown in SEQ ID NO: 14. The protein containing the amino acid sequence represented by SEQ ID NO: 14 is NEPHA.
-Has similar activity to SV1. No. 1 to No. 1 of the amino acid sequence represented by SEQ ID NO: 14
The 76th sequence corresponds to the 1st to 176th sequences of the amino acid sequence represented by SEQ ID NO: 11, and the 177th to 687th sequence of the amino acid sequence represented by SEQ ID NO: 14 The 498th amino acid sequence represented by SEQ ID NO: 11
It is the same as the sequence from No. to No. 1008.

Example 7 (1) Determination of cDNA sequence encoding novel receptor protein derived from human ovarian cancer Human Tumor MTC ^™ Panel (CL
Human ovarian cancer cell line GI- included in ONTECH)
A PCR reaction was performed using the cDNA derived from 102 as a template and using primer 8 (SEQ ID NO: 16) and primer 9 (SEQ ID NO: 17). The composition of the reaction solution in the reaction was 50x using 1 μl of the above cDNA as a template.
Advantage-GC 2 Polymerase
Mix (CLONTECH) 0.4 μl, primer 8 (SEQ ID NO: 16) and primer 9 (SEQ ID NO: 17) 0.4 μM each, dNTPs 200 μM,
GC-Melt (CLONTECH) 1M, 5xG
C 2 PCR Buffer (CLONTECH)
Was added to make a liquid volume of 20 μl. The PCR reaction is
After 94 ° C. for 3 minutes, a cycle of 94 ° C. for 30 seconds and 68 ° C. for 4 minutes was repeated 35 times. After the PCR products were separated by agarose gel electrophoresis, Gel Extraction K was used.
The PCR product was purified using it (Qiagen) and was purified using the TOPO XL PCR cloning kit (In
Subcloning into the plasmid vector pCR-XL-TOPO (Invitrogen) according to the prescription of Vitrogen). This was introduced into E. coli TOP10 and selected in LB agar medium containing kanamycin. As a result of analyzing the sequences of the individual clones, the nucleotide sequences (SEQ ID NO: 19 and SEQ ID NO: 21) of the cDNAs encoding the two novel receptor proteins (SEQ ID NO: 18 and SEQ ID NO: 20) were determined. Sequence number obtained here:
The plasmids having 19 and SEQ ID NO: 21 were designated as pTB2260 and pTB2261, respectively, and the resulting transformants were designated as Escherichia coli T.
OP10 / pTB2260 and E. coli (Escherichia co
li) It was named TOP10 / pTB2261. The protein containing the amino acid sequence represented by SEQ ID NO: 18 is NEPHA-SV2, and the protein containing the amino acid sequence represented by SEQ ID NO: 20 is NEPHA-SV2.
They were named SV3 respectively. In the base sequence represented by SEQ ID NO: 19, the 155th T is C, the 273nd T is C, the 822nd G is A,
1091st G is A and 1216th T is C
264th G is A, 1277th T is C, 18
62nd G is T, 1909th A is G, 193
The nucleotide sequence in which the 8th A has been replaced with G is SEQ ID NO: 41.
Shown in. In the nucleotide sequence represented by SEQ ID NO: 21, G at the 1039th position is replaced with A, and T at the 1446th position is replaced with C, 11
The 54th C was deleted, and a new 1262 bp nucleotide sequence was added to the 3'end (from SEQ ID NO: 43, 1520 to 27).
The base sequence in which the 81st base sequence corresponds to this sequence is shown in SEQ ID NO: 43. The amino acid sequence encoded by the base sequence represented by SEQ ID NO: 41 is shown in SEQ ID NO: 40. The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 43 is SEQ ID NO: 4.
2 shows. The protein containing the amino acid sequence represented by SEQ ID NO: 40 or SEQ ID NO: 42 is NEPHA-S.
It has the same activity as V2 and NEPHA-SV3.

(2) Determination of cDNA sequence encoding human brain-derived novel receptor protein Human MTC ^™ Panel I (CLONTEC
Using a human brain-derived cDNA contained in H company) as a template, a PCR reaction was carried out using primer 8 (SEQ ID NO: 16) and primer 9 (SEQ ID NO: 17). The composition of the reaction solution in the reaction was 50 x Advantage-GC 2 Po using 1 μl of the above-mentioned cDNA as a template.
Lymerase Mix (CLONTECH) 0.
0.4 μM each of 4 μl, primer 8 (SEQ ID NO: 16) and primer 9 (SEQ ID NO: 17), dNTPs
200 μM, GC-Melt (CLONTECH)
1M, 5xGC 2 PCR Buffer (CLO
4 μl of NTECH) was added to make a liquid volume of 20 μl.
PCR reaction is 94 ° C for 3 minutes, 94 ° C for 30 seconds, 68 ° C
The 4-minute cycle was repeated 35 times. After the PCR products were separated by agarose gel electrophoresis, Gel Extra was used.
The PCR product was purified using the action kit (Qiagen) and the plasmid vector pCR-XL-TOPO (Invitr) was purified according to the prescription of TOPO XL PCR cloning kit (Invitrogen).
(Ogen). E. coli T
Selected in LB agar containing OP10 and containing kanamycin. As a result of analyzing the sequences of the individual clones, a cDNA sequence (SEQ ID NO: 23) encoding a novel receptor protein (SEQ ID NO: 22 and SEQ ID NO: 24)
And SEQ ID NO: 25) were determined. The plasmids having SEQ ID NO: 23 and SEQ ID NO: 25 obtained here were designated as pTB2262 and pTB2263, respectively, and the obtained transformants were transformed into Escherichia co
li) TOP10 / pTB2262 and Escherichia coli
hia coli) TOP10 / pTB2263. The protein containing the amino acid sequence represented by SEQ ID NO: 22 is NEPHA-SV4, and the protein containing the amino acid sequence represented by SEQ ID NO: 24 is NEPHA-.
They were named SV5 respectively. The 240th A of the nucleotide sequence represented by SEQ ID NO: 23 is G, the 740th G is A, the 937th C is A,
The 1243rd C is T and the 1396th T is C, 2
The nucleotide sequence in which T at the 051st position was replaced with C and G at the 2171st position was replaced with A is shown in SEQ ID NO: 45. The 12th T of the nucleotide sequence represented by SEQ ID NO: 25 is C, and the 78th G is
Is the A, the 96th T is the C, the 189th T is the C,
194th G is A, 1045th G is A, 11
The nucleotide sequence in which A at the 05th position is replaced with T and A at the 2150th position is replaced with G is shown in SEQ ID NO: 47. The amino acid sequence encoded by the base sequence represented by SEQ ID NO: 45 is shown in SEQ ID NO: 44. The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 47 is SEQ ID NO: 4.
6 shows. The protein containing the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 46 is NEPHA-S.
It has the same activity as V4 and NEPHA-SV5. NE
The homology of PHA and NEPHA-SV1-5 is shown in FIGS.

Example 8 Examination of Gene Expression Levels in Human Normal Tissue and Cancer Cell Tumor Tissue Using human normal tissue, human fetal tissue, and human cancer cell tumor tissue-derived MTC ^™ Panels (CLONTECH) as a template, Primer pair and FAM labeled T
aqMan probe 1 (Applied Biosys)
by carrying out a PCR reaction using tems) to compare the expression levels of genes encoding NEPHA protein or its variants NEPHA-SV1-5 in cancer tissues and normal tissues. The composition of the reaction solution in the reaction was such that 1 μl of the above cDNA was used as a template and Taq was used.
Man universal PCR master
mix (Applied Biosystems) 1
2.5 μl, primer 10 (SEQ ID NO: 26) and primer 11 (SEQ ID NO: 27) 0.5 μM each,
TaqMan probe 1 (SEQ ID NO: 28) was added to 100
The solution was added to make nM, and the volume was adjusted to 25 μl. PCR reaction: 50 ° C for 2 minutes, 95 ° C for 10 minutes, 95 ° C for 15 seconds,
The cycle of 60 ° C. for 1 minute was repeated 40 times. as a result,
The expression level of the whole gene encoding NEPHA protein or all its variants NEPHA-SV1-5 in 1 μl of the above-mentioned cDNA was as follows: brain, heart, liver, lung, kidney, pancreas, skeletal muscle, placenta, spleen, thymus, small intestine, In the large intestine, testis, prostate, ovary, and white blood cells, 6.6, 0,
0, 0, 0, 27, 0, 36, 0.38, 2, 11, 3
9, 1100, 0.47, 2, 0.16 copies, fetal brain,
In fetal heart, fetal liver, fetal lung, fetal kidney, fetal spleen, fetal thymus, and fetal skeletal muscle, 110, 0, 6.7, respectively.
0, 0, 0, 0, 10 copies, lung cancer (GI-11
7), lung cancer (LX-1), colon cancer (GI-11
2), colon cancer (CX-1), breast cancer (GI-101),
Prostate cancer (PC-3), pancreatic cancer (GI-103) and ovarian cancer (GI-102) are 16, 75 and 5, respectively.
3, 520, 180, 41, 140, 870 copies, showing high expression in the testis, fetal brain, and cancer tissue. In addition, cDNA derived from human brain contained in the above MTC ^™ Panels and Marat derived from human fetal brain
hon-ready cDNA (CLONTECH)
Using 1 μl as a template, primer 12 (SEQ ID NO: 2
9), primer 13 (SEQ ID NO: 30), and FAM
Labeled TaqMan probe 2 (Applied B)
The expression level was examined at the same time using (Biosystems) (SEQ ID NO: 31). The reaction conditions were the same as above. As a result, the copy number of the whole gene encoding the NEPHA protein or all of its variants NEPHA-SV1-5 was 0.7 copies in human brain and 56 copies in human fetal brain,
Even when the expression levels were measured using the primer 12, the primer 13, and the TaqMan probe 2, it was revealed that the expression was high in the human fetal brain.

Example 9 Examination of Gene Expression Level in Human Cancer Tissue (Part 1) Tumor / Derived from Human Cancer Patient Tissue (Breast Cancer, Lung Cancer, Colorectal Cancer, Rectal Cancer, Ovarian Cancer, Prostate Cancer) nor
mal matched cDNA pair (CLON
TECH) as a template and the following primer pair and F
By carrying out PCR reaction using AM-labeled TaqMan probe, NEPH in cancer tissue and normal tissue
A protein or its variant NEPHA-S
The expression levels of genes encoding all of V1 to V5 were compared. The composition of the reaction solution in the reaction is the above cDNA 1
Using μl as template, TaqMan univer
salPCR master mix (Applied
Biosystems) 12.5 μl, primer 1
0 (SEQ ID NO: 26) and primer 11 (SEQ ID NO: 27) at 0.5 μM each, TaqMan probe 1
(SEQ ID NO: 28) was added to 100 nM and 2
The volume was 5 μl. PCR reaction is 50 ° C for 2 minutes, 95
After 10 minutes at 0 ° C, a cycle of 95 ° C for 15 seconds and 60 ° C for 1 minute was repeated 40 times. As a result, NEPHA protein,
Alternatively, the total amount of genes encoding all of the variants NEPHA-SV1 to 5 was about 8 times, 4 times, 14 times as much as that of the peripheral normal tissue in all of the 4 human breast cancer tissues, respectively.
Approximately 5 times, in all 3 cases of human lung cancer tissue, about 34 times, 12 times, 3 times, respectively, in all 3 cases, and in 5 cases in 7 cases of human colon cancer tissue, approximately 5 times, respectively, in comparison with peripheral normal tissue , 1 out of 3 human rectal cancer tissues compared with 2 normal tissues
Approximately 150 times, 36 times, 36 times, 4 times, 2 times respectively in 4 out of 5 human ovarian cancer tissues, and approximately 1 in 2 out of 2 cases of human prostate cancer tissue, respectively. 7
The expression level was doubled, and a remarkable increase in expression was observed in cancer tissues.

Example 10 Examination of gene expression level in human cancer tissue (Part 2) tu derived from human cancer tissue (breast cancer, lung cancer, colon cancer)
mor multi-sample cDNA panel
(Biochain) and normal human tissues (mammary gland, lung,
Marathon-ready cDNA derived from large intestine)
(CLONTECH) as a template and NEPHA protein in cancer tissue and normal tissue in the same manner as in Example 9,
Alternatively, the expression levels of genes encoding all of the variants NEPHA-SV1-5 were compared. Regarding the composition of the reaction solution in the reaction, 1 μl of the above cDNA was used as a template, and PCR reaction was performed under the same conditions as in Example 9. In parallel, TaqMan Human β-actin cont
roll reagent (Applied Biosy
Stems) was used to calculate the copy number of β-actin gene contained in 1 μl of the above cDNA, which was used as an internal standard. The concentrations of the primer and probe were adjusted to the concentrations described in Example 9. When standardized by β-actin gene expression level, the expression level of the whole gene encoding NEPHA protein or all of its variants NEPHA-SV1-5 was 4 in 10 out of 10 human lung cancer tissues compared with that in normal lung tissue. 31 times, 6 times, 195 times, and 9 times
It was revealed that the number of human breast cancer tissues increased about 4-fold in 1 of 7 cases compared with the normal breast tissue.

Example 11 Examination of Gene Expression Level in Human Lung Cancer Cell Line In Examples 8, 9 and 10, NEPHA protein or its variant NEP in human cancer tissues was examined.
Since it was revealed that an increase in the expression of genes encoding all of HA-SV1 to 5 was observed, the same study was performed in the cultured cell lines. Human small cell lung cancer cell lines NCI-H187, NCI-H378, NCI-N41
7, NCI-H526, NCI-H889, NCI-H1
417, NCI-H1672, NCI-H1836, NC
I-H2227, NCI-H1963, human non-small cell lung cancer cell line NCI-H522, NCI-H1395, NCI
-H1435, NCI-H1581, NCI-H165
1, NCI-H1793, NCI-H2073, NCI
-H2085, NCI-H2228, NCI-H234
2, NCI-H2347 (above, purchased from ATCC),
Human normal small airway epithelial cells SAEC (Clonetics)
, Respectively, at a cell number of 10 ⁶ to 10 ⁷ and RNea
Total RNA was prepared using a symmini kit (Qiagen). By performing reverse transcription reaction on this total RNA to form cDNA, and performing PCR reaction, N
EPHA protein or variant NEPHA thereof
-The expression levels of genes encoding all of SV1 to 5 were examined. Regarding the composition of the reaction solution in the reaction, cDNA obtained from 1 ng of the above total RNA was used as a template, and the reaction composition and PCR reaction conditions were the same as in Example 9. Β- contained in 1 ng of total RNA in parallel
The actin gene copy number was calculated in the same manner as in Example 10 and used as an internal standard. As a result, expression was not observed in SAEC, but human small cell lung cancer cell line NCI-
H187, NCI-H378, NCI-N417, NC
I-H526, NCI-H889, NCI-H141
7, NCI-H1672, NCI-H1836, NCI
-H2227, NCI-H1963, human non-small cell lung cancer cell line NCI-H522, NCI-H1395, NCI
-H1435, NCI-H1581, NCI-H165
1, NCI-H1793, NCI-H2073, NCI-
H2085, NCI-H2228, NCI-H2342,
In NCI-H2347, the expression level of β-actin gene was 0.61%, 0.51%, 0.49%, 3.0%, 1.
9%, 0.93%, 0.53%, 1.4%, 0.84%,
2.00%, 0.17%, 0.18%, 0.16%, 0.3
7%, 0.09%, 0.08%, 0.05%, 0.01%,
It was expressed in the proportions of 0.05%, 0.14% and 0.16%, and in particular, a marked increase in expression of NEPHA protein or its variants NEPHA-SV1-5 was observed in small cell lung cancer cell lines. It was

Example 12 Examination of gene expression level in human cancer cell lines In Examples 8, 9, 10 and 11, all of NEPHA protein or its variants NEPHA-SV1 to 5 in human cancer tissues and lung cancer cell lines. Since it was revealed that an increase in the expression of the entire gene encoding the gene was observed, the same study was performed in other cultured cell lines. Human breast cancer cell line, HCC70, HCC114
3, ZR-75-1, MCF-7, MDA-MB-43
5, MDA-MB-231, MDA-MB-175-VI
I, T-47D, AU565, human prostate cancer cell line,
PC-3, LNCaP, DU145, human testicular cancer cell line, Tera-1, Tera-2, human uterine cancer cell line,
KLE, RL95-2, human ovarian cancer cell line, PA-1,
Human choriocarcinoma cell line, JAR, JEG-3, BeWo,
Human neuroepithelial cancer cell line, SK-N-MC, human pancreatic cancer cell line, AsPC-1, MIA PaCa-2, human bronchoalveolar adenocarcinoma cell line NCI-H358, human colon cancer cell line WiDr , SW620, LS123, COLO20
5, LoVo (above, purchased from ATCC), human normal prostate epithelial cell PrEC, human normal mammary epithelial cell HMEC
Total RNA was prepared from the above (purchased from Clonetics), and reverse transcription reaction was performed on this total RNA to obtain cDNA. The cDNA and cDNA derived from colon cancer cell line SW480, lung cancer cell line A549, and prostate cancer cell line MRI-H-1579 (above, Marathon-ready of CLONTECH)
by performing a PCR reaction using
PHA protein or variant NEPHA-
The expression levels of genes encoding all of SV1-5 were examined. The composition of the reaction solution in the reaction is RNA 1
cDNA obtained from ng and Marathon-re
Using 1 μl of ady cDNA as a template, the gene expression level was measured by the same method as in Examples 9, 10 and 11.
The β-actin gene expression level was also measured in parallel. As a result, expression was not observed in PrEC or HMEC, but cancer cell lines HCC1143, ZR-75-1, MCF
-7, MDA-MB-435, MDA-MB-175-VI
I, T-47D, LNCaP, DU145, KLE, R
L95-2, BeWo, AsPC-1, NCI-H35
8, WiDr, SW620, LS123, COLO20
5, LoVo, SW480, A549, MRI-H-1
In 579, an increase in the expression of the entire gene encoding the NEPHA protein or all of its variants NEPHA-SV1-5 was observed.

Example 13 Construction of Recombinant Full-Length Protein Expression Vector for Animal Cells A vector for expressing the NEPHA protein in animal cells was constructed in two stages, N-terminal side and C-terminal side. First, using the pTB2230 obtained in Example 5 as a template, a partial sequence on the N-terminal side was obtained by PCR reaction using a primer 14 (SEQ ID NO: 32) and a primer 15 (SEQ ID NO: 33) to obtain pcDNA3.1 (+). Plasmid (Invitr
(Ogen). The composition of the reaction solution in this reaction was 10 ng of pTB2230 obtained in Example 5 as a template, and Pfu Turbo DNA Polym was used.
erase (STRATAGENE) 2.5 uni
t, primer 14 (SEQ ID NO: 32) and primer 15 (SEQ ID NO: 33) at 1 μM each, and dNTPs at 20
25 μl of 0 μM and 2 × GC Buffer I (Takara Shuzo) were added to make a liquid volume of 50 μl. PCR reaction: 96 ° C for 1 minute, 96 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C
The 2-minute cycle was repeated 30 times. After separating the PCR product by agarose gel electrophoresis, Gel Extr
purification using the action Kit (Qiagen),
This was used as a plasmid vector pCR-Blunt II-TOP according to the prescription of Zero Blunt TOPO PCR cloning kit (Invitrogen).
It was subcloned into O (Invitrogen). This was introduced into E. coli TOP10 and selected in LB agar medium containing kanamycin. As a result of analyzing the sequences of the individual clones, a plasmid pCR-Blun containing a cDNA sequence encoding the N-terminal side of the NEPHA protein was obtained.
tII-N was obtained. pCR-BluntII-N and p
cDNA 3.1 (+) (Invitrogen) was treated with the restriction enzymes BamHI and EcoRI, separated by agarose gel electrophoresis, and the insert containing the N-terminal partial sequence and the DNA fragment corresponding to pcDNA3.1 (+) were separated. It collect | recovered and refined using Gel Extraction Kit (Qiagen). DNA of each DNA fragment
ligation kit ver. After performing a ligation reaction using 2 (Takara Shuzo), E. coli TOP10
And were selected in LB agar medium containing ampicillin. As a result of analyzing the sequences of individual clones, NEPHA
A plasmid pcDNA3.1 (+)-N having a cDNA sequence encoding the N-terminal side of the protein was obtained. pTB
The C-terminal partial sequence was excised from the 2230 plasmid by EcoRI and BstPI treatment, and at the same time, p
The cDNA 3.1 (+)-N plasmid was digested with EcoRI and BstPI, and subjected to electrophoresis, purification, and ligation reaction as described above to ligate the N-terminal side and the C-terminal side at the BstPI site. Escherichia coli TOP10 was transformed with the plasmid thus prepared and selected in LB agar medium containing ampicillin. As a result of analyzing the sequences of individual clones, an expression vector for animal cells having a full-length cDNA sequence encoding the NEPHA protein
pcDNA3.1 (+)-NEPHA was obtained.

Example 14 Establishment of stable expression cell line of recombinant full-length protein A cell line constitutively expressing NEPHA protein was established using the human embryonic kidney-derived cell line HEK293. 2 × 10 ⁵ HEK293 cells were suspended in 2 ml of Dulbecco's modified Eagle's minimal medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and seeded in one well of a 6-well plate. After culturing overnight at 37 ° C. under 5% carbon dioxide gas flow, the medium was exchanged again with 2 ml of the same medium, and 7.5 μl of FuGENE6 transfection reagent (Roche Diagnostics) and OPTI-MEM I (Invitrogen) 9
PcD mixed with 1.4 μl and left at room temperature for 15 minutes
NA3.1 (+)-NEPHA (2.5 μg) was added and the culture was continued under the same conditions. Two days later, trypsin-EDTA (I
nvitrogen) to recover the cells,
The above medium (G418 selective medium) containing 0.75 mg / ml G418 (Promega) was diluted 6-fold and the culture was continued for further 4 days. Continue culturing in G418 selective medium to increase the number of cells to 3.2 × 10 ^6, and then add 1 cell per well.
Two 96-well plates seeded at a rate of 0.1 (medium volume 0.1 ml) were prepared, and further cultured in G418 selective medium. 3 from G418 resistant cell line obtained from 64 wells
Strains were selected and recloned by the same operation to complete cloning.

Example 15 Construction of Escherichia coli Expression Vector of Recombinant Extracellular Domain Partial Protein Expression of a partial protein corresponding to the extracellular domain of NEPHA protein was carried out in Escherichia coli. That is, using the pTB2230 obtained in Example 5 as a template, the primer 16
(SEQ ID NO: 34) and primer 17 (SEQ ID NO:
PCR reaction was carried out using 35). The composition of the reaction solution in the reaction was 5 ng of the above plasmid as a template, and Pfu Turbo DNA Polymers was used.
e (STRATAGENE) 2.5 unit, primer 16 (SEQ ID NO: 34) and primer 17
(SEQ ID NO: 35) each 1 μM, dNTPs 200 μ
25 M and 2x GC Buffer I (Takara Shuzo)
μl was added to give a volume of 50 μl. PCR reaction is 96 ℃
After 1 minute, 96 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1 minute 3
After repeating the 0 second cycle 30 times, an extension reaction at 72 ° C. for 5 minutes was performed. Ex-Taq (Takara Shuzo) 2.5 unit was added to the PCR product and reacted at 72 ° C. for 10 minutes, and then MinElute ^™ PCR Purifica.
The PCR product was purified using a Tion Kit (Qiagen). According to the prescription of pCRT7 / CT TOPO TA cloning kit (Invitrogen), the plasmid vector pCRT7 / CT-TOPO (I
nvitrogen), and E. coli TOP10
F'was transformed. A clone having a plasmid containing a PCR amplification product was selected from this Escherichia coli, and the sequence of the plasmid extracted from each clone was analyzed to obtain a plasmid (pCRT7-NEPHA) having no error in the nucleotide sequence.

Example 16 Expression and Purification of Recombinant Extracellular Domain Partial Protein in Escherichia coli Plasmid pCRT7-NEPH obtained in Example 15
E. coli BL21 (DE3) pLys S was transformed with A and used for expression. Expression was induced with 1 mM isopropylthiogalactopyranoside and purified with Chelatin
g Sepharose ^™ Fast Flow (Ame
The attached manual was followed using rsham-Pharmacia. As a result, the target recombinant protein of about 66 kD was 20 mM sodium phosphate (pH
4.0), 0.5M sodium chloride, 8M urea. The obtained eluate was concentrated at 4 ° C using an ultra-concentration membrane with a molecular weight cut-off of 10,000 (Millipore), and then a dialysis membrane with a molecular weight cut-off of 6,000 to 8,000 (Spectrum Medical). ) Using 4 ℃
Dialyzed against buffer [20 mM Tris-HCl (pH 7.4) and 0.5 M L-arginine]. In this way, 1.6 mg of recombinant protein was obtained from 2.6 L of culture solution.

Example 17 Preparation and Purification of Rabbit Polyclonal Antibody Using the recombinant protein prepared in Example 16, a rabbit polyclonal antibody was prepared. One male rabbit KBL: JW (11-week-old, Oriental yeast) was used as an immunized animal, and sensitization was performed by subcutaneous injection on the back, and repeated every 14 days for 3 times. The amount of recombinant protein used for the first sensitization was 0.536 mg, the first sensitization was a complete Freund's adjuvant (Difco) suspension, and the second and subsequent ones were incomplete Freund's adjuvant (Difco) suspension. Was used. On the 38th day after the first sensitization, blood was collected from the carotid artery under anesthesia,
About 58 ml of serum was obtained. The whole amount of the serum thus obtained was concentrated by the ammonium sulfate salting-out method, and the whole amount of the crude IgG fraction obtained was purified by a protein A affinity column (Amersham-Pharmacia), and about 189 mg of the purified IgG fraction was a polyclonal antibody. It was AS-2231.

Example 18 Preparation and Purification of Peptide Antibody Two types of partial peptides consisting of 15 amino acids were synthesized based on the amino acid sequence of NEPHA protein. Peptide 1 is a sequence in which Cys is added to the C-terminus of the 60th to 73rd amino acid sequence of SEQ ID NO: 11, and Peptide 2 is added to the C-terminus of the 491st to 504th amino acid sequence of SEQ ID NO: 11, respectively. Is. Peptide 1: Glu-Ile-Ser-Gly-Val-Asp-Glu-His-Asp-Ar
g-Pro-Ile-Arg-Thr-Cys [SEQ ID NO: 36] Peptide 2: Glu-Ile-Arg-Tyr-Tyr-Glu-Lys-Gly-Gln-Se
r-Glu-Gln-Thr-Tyr-Cys [SEQ ID NO: 37] Keyhole limpet hemocyanin (KLH) was bound to each peptide as a carrier protein to serve as an antigen. The antigen sensitization was according to the method of Example 17 described above. 0.5 mg of antigen was used for one sensitization, and this was repeated 4 times every 14 days. 52 days after the first sensitization, whole blood was collected and about 60 ml of serum was obtained for each antigen. The same operation as in Example 17 was performed on the whole amount of serum, and about 585 mg and 440 mg of purified IgG for each peptide 1 and 2.
The fraction was acquired. Of these, about 290 mg and 220 m
g was used for purification of each peptide on an immobilized column.
For immobilization, cysteine at the carboxyl terminal of each peptide was used, and a borate buffer was used for coupling to a Sepharose column (Amersham-Pharmacia). Elution from the column used 8 M urea / phosphate buffered saline (PBS). Eluate is PBS
After dialysis against urea to remove urea, peptide 1 and peptide 2 were obtained by ultraconcentration and filter sterilization.
Affinity purified antibodies AS-2141, AS-
The amount of 2142 obtained was about 44 mg and 19 mg, respectively.

Example 19 Preparation and purification of antibody F (ab ′) ₂ fragment AS-of the antibodies prepared in Examples 17 and 18
2141 and AS-2142 are 15 mg, AS-223
1 was digested with immobilized pepsin using 30 mg, and the flow-through fraction of the digested solution on the protein A column was F (a
b ′) ₂ fragment. In this way AS-214
F (ab ′) ₂ fragment derived from 1 antibody was 7.5 mg, A
The F (ab ′) ₂ fragment derived from the S-2142 antibody was 7.8.
mg, 10.4 mg of F (ab ′) ₂ fragment derived from the AS-2231 antibody was obtained.

Example 20 Western Blotting Using Rabbit Polyclonal Antibody and Peptide Antibody Detection of NEPHA protein was performed using the rabbit polyclonal antibody and peptide antibody prepared in Examples 17 and 18. 2 × 10 ⁶ HEK293 cells derived from human fetal kidney were added to 10% fetal bovine serum (Invitro
(Denbecco) modified Dulbecco's modified Eagle's minimum medium (Invitrogen) 10 ml in diameter and suspended.
cm seedlings. After overnight culture at 37 ° C. in a 5% carbon dioxide gas flow, 18 μl of FuGENE6 transfection reagent (Roche Diag was previously prepared.
nosis) and OPTI-MEM I (Invit
rogen) and left at room temperature for 15 minutes
6 μg of pcDNA3.1 (+)-NEPHA was added and the culture was continued under the same conditions. After 2 days, the cells were washed with PBS and then ice-cooled RIPA buffer [50 mM Tris-hydrochloric acid, pH 7.5, 150 mM sodium chloride, 1% Tr was added.
iton X-100, 0.1% sodium dodecyl sulfate, 1% deoxycholic acid, Complete ^™ tablets (Roche Diagnostics), Phos
phase Cocktail Inhibito
r 2 (SIGMA)] 800 μl and added at 4 ° C.
Leave for 5 minutes. This RIPA buffer was recovered and
The supernatant obtained by centrifugation at 00 rpm for 20 minutes was used as a cell-free extract, and 20 μl of the supernatant was subjected to SDS-PAGE on a 7.5% acrylamide gel. The proteins separated by electrophoresis were transferred to a clear blot P membrane (ATTO) according to a conventional method, and then a blocking solution (Tris buffered saline,
1 in 0.1% Tween-20, 5% skim milk)
It was left at room temperature for an hour. Next, the rabbit polyclonal antibody AS-2231, the peptide antibodies AS-2141 and AS-2142 prepared in Examples 17 and 18 were respectively used in 4.
Incubate overnight at 4 ° C. in blocking solution added to give concentrations of 3, 3.4 and 4.0 μg / ml,
Then, an anti-rabbit IgG-HRP labeled antibody (Amersh
am-Pharmacia) with blocking solution 1
The solution was left at room temperature for 1 hour in a secondary antibody solution diluted by 0.00000 times. Detection is ECL plus (Amersham-
(Pharmacia) was used and the attached manual was followed. A specific band derived from the NEPHA protein was observed at a position near a molecular weight of 110 kD using any of the rabbit polyclonal antibody AS-2231 and the peptide antibodies AS-2141 and AS-2142.

Example 21 Immunoprecipitation Using Peptide Antibody Using the peptide antibody prepared in Example 18, NEP
Immunoprecipitation against HA protein was performed in a non-denaturing state. 1 ml of cell-free extract prepared in the same manner as in Example 20,
Protein G-Sepharose 4FF (A
mersham-Pharmacia) suspension 50μ
Rabbit IgG in 1 (50% suspension of RIPA buffer)
(Chemicon) 30 μg or the peptide antibodies AS-2141 and AS-2142 prepared in Example 18.
30 μg of any of the above was added and stirred at 4 ° C. overnight.
The protein G sepharose co-precipitated fraction was washed with RIPA buffer, and then with 50 μl of sample buffer at 95 ° C.
After heating for 5 minutes, 20 μl was subjected to SDS-PAGE on a 7.5% acrylamide gel. Peptide antibody AS-2
The detection was carried out by the method described in Example 20 using 142. A molecular weight of 110 was obtained when immunoprecipitation was carried out using either of the peptide antibodies AS-2141 and AS-2142.
A specific band derived from the NEPHA protein of about kD was observed, and the peptide antibodies AS-2141 and AS-2
It was revealed that 142 binds to the NEPHA protein under a non-denaturing state.

Example 22 Identification of Ligand The identification of the ligand of NEPHA protein was carried out as follows. 1 ml of the cell-free extract prepared in the same manner as in Example 20 described above, Protein G-Se described in Example 21.
pharose 4FF (Amersham-Phar
50 μl of suspension of Ephrin and human Ig
A fusion protein with the Fc part of G, ie, EphrinA1 /
Fc, EphrinA2 / Fc, EphrinA3 / Fc, EphrinA4 / Fc, EphrinA5 / Fc, EphrinB1 / F
c, ephrinB2 / Fc, ephrinB3 / Fc (above R &
D Systems) or human IgG Fc alone (A
eithers Research and Technology) 5 μg
Was added and stirred overnight at 4 ° C. The protein G sepharose coprecipitated fraction was washed with RIPA buffer, and then heated with 50 μl of SDS-PAGE sample buffer at 95 ° C. for 5 minutes, and 20 μl of S on 7.5% acrylamide gel.
It was subjected to DS-PAGE. AS- produced in Example 18
2142 was used for detection by the method described in Example 20. As a result, ephrin A2 / Fc, ephrin A3 /
When Fc, EphrinA4 / Fc and EphrinA5 / Fc were used, a specific band derived from the NEPHA protein was observed, indicating that EphrinA1 / Fc and EphrinB2 / F
When c was used, a small band having the same size was observed. From these, it became clear that the NEPHA protein mainly uses ephrin A2, ephrin A3, ephrin A4 and ephrin A5 as ligands, and ephrin A1 and ephrin B2 as weak ligands.

Example 23 Detection of Autophosphorylation of Tyrosine Residue Autophosphorylation of NEPHA was detected. pcDNA
3.1 (+)-NEPHA 6μg or human Eph
PcDNA3.1 with inserted gene encoding A7
(+) (Invitrogen) [hereinafter, pcDNA
3.1 (+)-EphA7 is named] 6 μg was used for transfection in the same manner as in Example 20.
After a day, EphrinA5 / Fc or human Fc (ICN) was added to the culture supernatant to a concentration of 1 μg / ml, and ligand stimulation was performed at 37 ° C. for 30 minutes. Then, 1 ml of the cell-free extract was prepared in the same manner as in Example 20, and NE
Peptide antibody AS-2141 3 μg and anti-rabbit IgG antibody agarose (SIGMA) suspension 50 were added to 1 ml of cell-free extract of HEK293 cells forcibly expressing PHA.
μl (50% suspension of RIPA buffer) was added, and the mixture was stirred at 4 ° C. overnight. In addition, goat anti-Ep was added to 1 ml of a cell-free extract of HEK293 cells in which EphA7 was forcibly expressed.
3 μg of hA7 antibody (R & D Systems) and 50 μ of suspension of anti-goat IgG antibody agarose (SIGMA)
1 (50% suspension of RIPA buffer) was added, and the mixture was stirred overnight at 4 ° C. After washing each antibody agarose co-precipitated fraction with RIPA buffer, 50 μl of SDS-PA
Add GE sample buffer and heat at 95 ° C for 5 minutes.
0 μl of SDS-PA on 7.5% acrylamide gel
It was subjected to GE. Detection of NEPHA is AS-2141, E
For detection of phA7, anti-EphA7 antibody (R & D
Systems) as the primary antibody, 1 μg / m
After binding overnight at 4 ° C. for 1 hour, the anti-goat IgG-HRP labeled antibody (ICN) was allowed to stand for 1 hour at room temperature in a secondary antibody solution diluted 4,000 times to bind the secondary antibody. . In addition, the detection of phosphorylated tyrosine is carried out using an anti-phosphotyrosine antibody (Upstate Biotechnology).
Co., Ltd.) at 2 μg / ml at 4 ° C. overnight, and then an anti-mouse IgG-HRP labeled antibody (Amersham-Pharm)
Aca) was allowed to stand for 1 hour at room temperature in a secondary antibody solution diluted 50,000 times to bind the secondary antibody. Detection was according to the method described in Example 20. As a result, it was revealed that EphA7 is phosphorylated in response to ephrinA5 / Fc stimulation, whereas NEPHA is not phosphorylated.

Example 24 Detection of NEPHA / EphA7 heterooligomer It was investigated whether NEPHA forms a heterooligomer with EphA7. pcDNA3.1 (+)-NEPHA 3
μg and pcDNA3.1 (+)-EphA7 3 μg,
Or pcDNA3.1 (+) 3 μg and pcDNA3.1
3 μg of (+)-EphA7 was co-transfected into HEK293 cells, and 1 ml of cell-free extract was prepared in the same manner as in Example 20. Pr described in Example 21
otain G-Sepharose 4FF suspension 5
0 μl and AS-21413 μg were added, and the mixture was stirred at 4 ° C. overnight. Protein G Sepharose co-precipitated fraction is RI
After washing with PA buffer, 50 μl of SDS-PAGE sample buffer was added and heated at 95 ° C. for 5 minutes to give 20 μl.
Was subjected to SDS-PAGE on a 7.5% acrylamide gel. The method described in Example 23 was followed for the detection of EphA7. As a result, an EphA7-specific band was detected only in the presence of the NEPHA protein,
It was revealed that HA and EphA7 form a hetero-oligomer.

Example 25 Preparation and Evaluation of Immunotoxins AS-2141 and AS-21 prepared in Example 19 for the purpose of creating an antibody drug against human cancer cells highly expressing the NEPHA protein or a variant thereof.
Immunotoxin was prepared using F (ab ′) ₂ fragment derived from 42 or AS-2231 antibody. Regarding the preparation method, the method of Chari, RVJ, et al. (Cancer
Res. 52, 127-p131, 1992; USP 5,208,020). F (ab ′) ₂ fragment obtained in Example 19 1
Starting from mg, N-succinimidyl-
3- (2-pyridyldithio) propiona
te to F (ab ') ₂ fragment with dithiopyri
A dyl group was introduced, and a maytansine derivative was further added by SS cross-linking to form an immunotoxin. ChromoP as immunotoxin for negative control
ure ^™ non-immunized rabbit IgG F (ab ′) ₂ fragment (Jackson Immunoresearch)
A complex with a maytansine derivative was similarly prepared using 1 mg. When the number of bound maytansine derivative molecules per molecule of immunotoxin was calculated based on the absorbances at 280 nm and 252 nm, it was 3.0 molecules to 4.2.
It was found to be molecularly bonded. Using the immunotoxin thus produced, Example 11 and Example 1
Human lung cancer cell line NCI-H526, NC found in 2.
When the cytotoxic activity against I-N417 and human breast cancer cell line ZR-75-1 was examined, AS-2141, A
S-2142, and F (ab ') ₂ derived from AS-2231
A fragment-specific growth inhibitory activity was observed. That is, in NCI-H526, 60 nM of immunotoxin showed 3% inhibition and almost no growth inhibition in the negative control, whereas AS-2141-derived immunotoxin inhibited 43% and AS-2142-derived immunotoxin. 59% inhibition, AS-2231-derived immunotoxin showed 37% inhibition. In NCI-N417, 30 nM immunotoxin showed 7% inhibition and almost no growth inhibition in the negative control, whereas AS-2141-derived immunotoxin inhibited 32% and AS-2142-derived immunotoxin inhibited 46%. ,
The immunotoxin derived from AS-2231 showed 27% inhibition. The 60 nM immunotoxin in ZR-75-1 showed 4% inhibition and almost no growth inhibition in the negative control, whereas AS-2141-derived immunotoxin inhibited 19% and AS-2142-derived immunotoxin produced 26% inhibition. % Inhibition, AS-2231-derived immunotoxin showed 16% inhibition. From this, it was shown that the NEPHA protein and its variants or their partial peptides were useful as antigens for immunotoxins.

Example 26 Construction of Expression Vector of Recombinant Extracellular Domain Partial Protein for Animal Cells An expression vector for expressing a partial protein corresponding to the extracellular domain of NEPHA protein in animal cells was constructed. PcDNA3.1 (+)-obtained in Example 13
Primer 14 (SEQ ID NO: 3) using NEPHA as a template
2) and P15 using primer 15 (SEQ ID NO: 33)
A CR reaction was performed. The composition of the reaction solution in the reaction was 10 ng of the above plasmid as a template, and Pfu Tu
rbo DNA Polymerase (STRAT
AGENE) 2.5 unit, primer 14 (SEQ ID NO: 32) and primer 15 (SEQ ID NO: 33)
1 μM each, dNTPs 200 μM, and 2xGC
Add 25 μl of Buffer I (Takara Shuzo) to 50 μl
Of the liquid. PCR reaction is 96 ° C for 1 minute, then 96 ° C
A cycle of 30 seconds, 60 ° C. for 20 seconds, 72 ° C. for 2 minutes was repeated 25 times, and LA-Taq (Takara Shuzo) 2.5 uni
After adding t, 96 ° C for 1 minute, 96 ° C for 20 seconds, 60 ° C for 15 seconds
The cycle of 72 ° C. for 2 minutes was repeated twice. Next, the PCR reaction product was purified using PCR Purification Kit (Qiagen), and DNA Lig was used.
ation Kit ver.2 (Takara Shuzo)
It was inserted into an ARGET vector (Promega) and transformed into E. coli JM109. A clone having a plasmid containing a PCR amplification product was selected from the transformants that appeared, and a part of the NEPHA protein (SEQ ID NO: 11) was selected.
To obtain a plasmid vector pTARGET-NEPHA encoding the 1st to 550th of the above.

Example 27 Construction of cell line stably expressing recombinant extracellular region partial protein Plasmid vector pTARG obtained from Example 26
Using ET-NEPHA, a cell line that stably expresses a part of the NEPHA protein (1st to 550th of SEQ ID NO: 11) was prepared. Hamster cell line CHO-K1
1 x10 ⁵ or 10% fetal bovine serum (Invitro
Gen-containing α-MEM medium (Invitroge)
(Company n), and the mixture was suspended in 2 ml and seeded on a Petri dish having a diameter of 3.5 cm. After culturing overnight at 37 ° C. in a 5% carbon dioxide gas stream, 6 μl of FuGENE6 transfection reagent (Roche Diagnostics) and O were previously prepared.
PTARGET-NE mixed with PTI-MEM I (Invitrogen) and left at room temperature for 15 minutes
PHA 2 μg was added and 0.5 mg / ml G418
Culturing was continued in the presence of (Promega). Transfected cells were trypsin-EDTA 10 days later.
(Invitrogen) recovered by processing, flat bottom 9
A 6-well plate was seeded. G418-resistant cells that had grown in a colony after 2-3 weeks were separately obtained from 145 wells, and they were seeded on a flat bottom 6-well, 12-well, 24-well plate, or a Petri dish with a diameter of 10 cm, The culture was continued. When the cell density reached 90% or more, the medium was replaced with OPTI-MEM I medium (I
nvitrogen) and cultured for 48 hours, and then the culture supernatant was collected and sent to Centricon (Millipor).
(Company e) was used to perform a concentration operation of about 5 to 10 times. 20 μl of the concentrated solution was subjected to S on 10% acrylamide gel.
Separation was performed by DS-PAGE, and detection was performed using the peptide antibody AS-2141 according to the method of Example 20. As a result, from the culture supernatants recovered from clones 1A-4, 2B-3 and 2B-5, NEPH was found at a position near the molecular weight of 70 kD.
A specific band derived from the A protein fragment was observed. Further, clones 1A-4 and 2B-5 were recloned using a flat bottom 96-well plate, and 12 and 14 clones were reexamined. The amount of expressed protein was measured by the same procedure for the culture supernatants collected from these clones, and converted into the concentration in the culture supernatant to obtain clone 1A-4 # 10 showing an expression level of 200-300 ng / ml.

Example 28 In order to analyze the influence of the NEPHA gene on apoptosis, an antisense oligonucleotide introduction experiment was conducted. First, antisense to the base sequence represented by SEQ ID NO: 12 (SEQ ID NO: 48 and SEQ ID NO: 4
After designing 9), a phosphorothioate oligonucleotide was synthesized, purified by HPLC and used in the introduction experiment. As the control oligonucleotide, the reverse sequence of the nucleotide sequence represented by SEQ ID NO: 48 (SEQ ID NO: 50) and the reverse sequence of the nucleotide sequence represented by SEQ ID NO: 49 (SEQ ID NO: 51) are similarly phosphorothioated,
It was used after being purified by HPLC. Breast cancer cell line ZR-75-1 (Cancer Res, 38 (10), 3352-3364 as test cells
(Page, 1978, purchased from ATCC), 2 × 10 ⁴ cells were seeded in a 24-well plate (Falcon) on the day before the introduction of the oligonucleotide. OligofectAMINE (manufactured by Invitrogen) was used for oligonucleotide introduction, and the protocol was followed. 20 hours after introduction RNeasy mini
RNA was extracted according to the protocol of the kit (Qiagen), and c was obtained using the TaqMan RT kit (Perkin-Elmer).
DNA was prepared. At the same time, the same reaction was carried out without adding reverse transcriptase to serve as a non-reverse transcription control. Two
Species primers (SEQ ID NO: 52 and SEQ ID NO: 5
3) and NEP using the probe (SEQ ID NO: 54)
TaqMan β-ac for correction of HA gene expression level
β using tin Control Reagents (Perkin-Elmer)
Let's look at the expression level of actin.
PCR was performed according to the manual (manufactured by er). NEPH
The expression amount of the A gene was subtracted from the data obtained by using the cDNA using the non-reverse transcription control, and then corrected by the β-actin expression amount and compared. On the other hand, by introducing antisense oligonucleotide, NEP
In order to see the effect on the apoptosis when the expression of the HA gene was suppressed, the cells were compared with the control oligonucleotide-introduced sample using Cell death detection ELISA (Roche) 3 days after the introduction of the antisense oligonucleotide. As a result, when antisense to the NEPHA gene (SEQ ID NO: 48) was used, the expression level of the NEPHA gene was 53% 20 hours after the introduction of the antisense oligonucleotide, compared with the control oligonucleotide (SEQ ID NO: 50). , And when using antisense (SEQ ID NO: 49),
The control oligonucleotide (SEQ ID NO: 5
Compared to 1), the expression level of NEPHA gene was reduced to 57%. On the third day after the introduction of the antisense oligonucleotide, the apoptosis was 224% when the antisense oligonucleotide (SEQ ID NO: 48) was introduced, and the control oligonucleotide ( When SEQ ID NO: 51) was set to 100%, it increased to 185% when the antisense oligonucleotide (SEQ ID NO: 49) was introduced. From these, it was confirmed that cancer cells undergo apoptosis by suppressing the expression of NEPHA gene expressed in cancer cells.
It was revealed that the A gene is involved in cancer cell proliferation and apoptosis.

[0159]

INDUSTRIAL APPLICABILITY The receptor protein of the present invention or a partial peptide thereof or a salt thereof, a polynucleotide (eg, DNA, RNA and derivatives thereof) encoding the receptor protein or a partial peptide thereof is
Determination of ligand (agonist), acquisition of antibody and antiserum, construction of recombinant receptor protein expression system, development of receptor binding assay system using the expression system and screening of drug candidate compounds, structurally similar ligands It can be used as a drug for carrying out drug design based on comparison with a receptor, preparation of a probe or PCR primer in gene diagnosis, preparation of transgenic animals, or a drug such as gene prophylactic / therapeutic agent. The antibody of the present invention and the antagonist of the present invention are useful as an excellent anticancer agent with low toxicity.

[0160]

[Sequence list]                              SEQUENCE LISTING <110> Takeda Chemical Industries, Ltd. <120> Novel Receptor Protein and its DNA <130> P02-0047 <150> JP2001-109053 <151> 2001-04-06 <150> JP2001-168820 <151> 2001-06-04 <160> 54 <210> 1 <211> 789 <212> DNA <213> Human <400> 1 ggcattgctc agcggtgcta ggctggcgcg gcttgagccg ccgccggact gacagctcgg 60 tctgcggacc atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat 120 gcagctctgt ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat 180 cctcctggat tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg 240 gtgggaggag atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg 300 caatgtgctg gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg 360 cgggcagcgc atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg 420 cgccgcgggt acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct 480 gggccgtggg cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc 540 ggacgagagc ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt 600 gcgcgagatc ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc 660 atgcgtggcg cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg 720 cctggccacg ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc 780 cggaacgtg 789 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding 5'-Region of the Novel Receptor <400> 2 ggcattgctc agcggtgcta ggct 24 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding 5'-Region of the Novel Receptor <400> 3 cacgttccgg ccacttccac cag 23 <210> 4 <211> 1134 <212> DNA <213> Human <400> 4 gcagccggcc ccgcaaaatc gacacgatcg cggcggacga gagcttcacg cagggcgacc 60 tgggtgagcg caagatgaag ctgaacacag aggtgcgcga gatcggaccg ctcagccggc 120 ggggtttcca cctggccttt caggacgtgg gcgcatgcgt ggcgcttgtc tcggtgcgcg 180 tctactacaa gcagtgccgc gccaccgtgc ggggcctggc cacgttccca gccaccgcag 240 ccgagagcgc cttctccaca ctggtggaag tggccggaac gtgcgtggcg cactcggaag 300 gggagcctgg cagcccccca cgcatgcact gcggcgccga cggcgagtgg ctggtgcctg 360 tgggccgctg cagctgcagc gcgggattcc aggagcgtgg tgacttctgc gaagcctgtc 420 ccccagggtt ttacaaggtg tccccgcggc ggcccctctg ctcaccgtgc ccagagcaca 480 gccgggccct ggaaaacgcc tccaccttct gcgtgtgcca ggacagctat gcgcgctcac 540 ccaccgaccc gccctcggct tcctgcaccc ggccgccgtc ggcgccgcgg gacctgcagt 600 acagcctgag ccgctcgccg ctggtgctgc gactgcgctg gctgccgccg gccgactcgg 660 gaggccgctc ggacgtcacc tactcgctgc tgtgcctgcg ctgcggccgc gagggcccgg 720 cgggcgcctg cgagccgtgc gggccgcgcg tggccttcct accgcgccag gcagggctgc 780 gggagcgagc cgccacgctg ctgcacctgc ggcccggcgc gcgctacacc gtgcgcgtgg 840 ccgcgctcaa cggcgtctcg ggcccggcgg ccgccgcggg aaccacctac gcgcaggtca 900 ccgtctccac cgggcccggg gcgccctggg aggaggatga gatccgcagg gaccgagtgg 960 aaccccagag cgtgtccctg tcgtggcggg agcccatccc tgccggagcc cctggggcca 1020 atgacacgga gtacgagatc cgatactacg agaagggtca gagtgagcag acttactcca 1080 tggtgaagac aggggcgccc acagtcaccg tcaccaacct gaagccggct accc 1134 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding Central Re gion of the Novel Receptor <400> 5 gcagccggcc ccgcaaaatc 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding Central Re gion of the Novel Receptor <400> 6 gggtagccgg cttcaggttg gtg 23 <210> 7 <211> 2033 <212> DNA <213> Human <400> 7 ggccgctcgg acgtcaccta ctcgctgctg tgcctgcgct gcggccgcga gggcccggcg 60 ggcgcctgcg agccgtgcgg gccgcgcgtg gccttcctac cgcgccaggc agggctgcgg 120 gagcgagccg ccacgctgct gcacctgcgg cccggcgcgc gctacaccgt gcgcgtggcc 180 gcgctcaacg gcgtctcggg cccggcggcc gccgcgggaa ccacctacgc gcaggtcacc 240 gtctccaccg ggcccggggc gccctgggag gaggatgaga tccgcaggga ccgagtggaa 300 ccccagagcg tgtccctgtc gtggcgggag cccatccctg ccggagcccc tggggccaat 360 gacacggagt acgagatccg atactacgag aagggtcaga gtgagcagac ttactccatg 420 gtgaagacag gggcgcccac agtcaccgtc accaacctga agccggctac ccgctacgtc 480 tttcagatcc gggccgcttc cccggggcca tcctgggagg cccagagttt taaccccagc 540 attgaagtac agaccctggg ggaggctgcc tcagggtcca gggaccagag ccccgccatt 600 gtcgtcaccg tagtgaccat ctcggccctc ctcgtcctgg gctccgtgat gagtgtgctg 660 gccatttgga ggaggccctg cagctatggc aaaggaggag gggatgccca tgatgaagag 720 gagctgtatt tccacttcaa agtcccaaca cgtcgcacat tcctggaccc ccagagctgt 780 ggggacctgc tgcaggctgt gcatctgttc gccaaggaac tggatgcgaa aagcgtcacg 840 ctggagagga gccttggagg agggcggttt ggggagctgt gctgtggctg cttgcagctc 900 cccggtcgcc aggagctgct cgtagccgtg catatgctga gggacagcgc ctccgactca 960 cagaggctcg gcttcctggc cgaggccctc acgctgggcc agtttgacca tagccacatc 1020 gtgcggctgg agggcgttgt tacccgagga agcaccttga tgattgtcac cgagtacatg 1080 agccatgggg ccctggacgg cttcctcagg cggcacgagg ggcagctggt ggctgggcaa 1140 ctgatggggt tgctgcctgg gctggcatca gccatgaagt atctgtcaga gatgggctac 1200 gttcaccggg gcctggcagc tcgccatgtg ctggtcagca gcgaccttgt ctgcaagatc 1260 tctggcttcg ggcggggccc ccgggaccga tcagaggctg tctacaccac tatgagtggc 1320 cggagcccag cgctatgggc cgctcccgag acacttcagt ttggccactt cagctctgcc 1380 agtgacgtgt ggagcttcgg catcatcatg tgggaggtga tggcctttgg ggagcggcct 1440 tactgggaca tgtctggcca agacgtgatc aaggctgtgg aggatggctt ccggctgcca 1500 ccccccagga actgtcctaa ccttctgcac cgactaatgc tcgactgctg gcagaaggac 1560 ccaggtgagc ggcccaggtt ctcccagatc cacagcatcc tgagcaagat ggtgcaggac 1620 ccagagcccc ccaagtgtgc cctgactacc tgtcccaggc ctcccacccc actagcggac 1680 cgtgccttct ccaccttccc ctcctttggc tctgtgggcg cgtggctgga ggccctggac 1740 ctgtgccgct acaaggacag cttcgcggct gctggctatg ggagcctgga ggccgtggcc 1800 gagatgactg cccaggacct ggtgagccta ggcatctctt tggctgaaca tcgagaggcc 1860 ctcctcagcg ggatcagcgc cctgcaggca cgagtgctcc agctgcaggg ccagggggtg 1920 caggtgtgag tggaccccat tcttccaagg caggactccg gtgggggtcc agtcccccag 1980 ccctgcccaa ggaccgtggc aagctgcgct ccagcagtgt gggagggagc gct 2033 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding 3'-Region of the Novel Receptor <400> 8 ggccgctcgg acgtcaccta ctc 23 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding 3'-Region of the Novel Receptor <400> 9 tcgtgaaaat gggaggggca ggcagtgaaa 30 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify DNA encoding 3'-Region of the Novel Receptor <400> 10 agcgctccct cccacactgc 20 <210> 11 <211> 1008 <212> PRT <213> Human <400> 11 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Cys Pro Pro Gly         275 280 285 Phe Tyr Lys Val Ser Pro Arg Arg Pro Leu Cys Ser Pro Cys Pro Glu     290 295 300 His Ser Arg Ala Leu Glu Asn Ala Ser Thr Phe Cys Val Cys Gln Asp 305 310 315 320 Ser Tyr Ala Arg Ser Pro Thr Asp Pro Pro Ser Ala Ser Cys Thr Arg                 325 330 335 Pro Pro Ser Ala Pro Arg Asp Leu Gln Tyr Ser Leu Ser Arg Ser Pro             340 345 350 Leu Val Leu Arg Leu Arg Trp Leu Pro Pro Ala Asp Ser Gly Gly Arg         355 360 365 Ser Asp Val Thr Tyr Ser Leu Leu Cys Leu Arg Cys Gly Arg Glu Gly     370 375 380 Pro Ala Gly Ala Cys Glu Pro Cys Gly Pro Arg Val Ala Phe Leu Pro 385 390 395 400 Arg Gln Ala Gly Leu Arg Glu Arg Ala Ala Thr Leu Leu His Leu Arg                 405 410 415 Pro Gly Ala Arg Tyr Thr Val Arg Val Ala Ala Leu Asn Gly Val Ser             420 425 430 Gly Pro Ala Ala Ala Ala Gly Thr Thr Tyr Ala Gln Val Thr Val Ser         435 440 445 Thr Gly Pro Gly Ala Pro Trp Glu Glu Asp Glu Ile Arg Arg Asp Arg     450 455 460 Val Glu Pro Gln Ser Val Ser Leu Ser Trp Arg Glu Pro Ile Pro Ala 465 470 475 480 Gly Ala Pro Gly Ala Asn Asp Thr Glu Tyr Glu Ile Arg Tyr Tyr Glu                 485 490 495 Lys Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro             500 505 510 Thr Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln         515 520 525 Ile Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn     530 535 540 Pro Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg 545 550 555 560 Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu                 565 570 575 Leu Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro             580 585 590 Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu         595 600 605 Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro Gln     610 615 620 Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu Leu 625 630 635 640 Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg Phe                 645 650 655 Gly Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu Leu             660 665 670 Leu Val Ala Val His Met Leu Arg Asp Ser Ala Ser Asp Ser Gln Arg         675 680 685 Leu Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His Ser     690 695 700 His Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Ser Thr Leu Met 705 710 715 720 Ile Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu Arg                 725 730 735 Arg His Glu Gly Gln Leu Val Ala Gly Gln Leu Met Gly Leu Leu Pro             740 745 750 Gly Leu Ala Ser Ala Met Lys Tyr Leu Ser Glu Met Gly Tyr Val His         755 760 765 Arg Gly Leu Ala Ala Arg His Val Leu Val Ser Ser Asp Leu Val Cys     770 775 780 Lys Ile Ser Gly Phe Gly Arg Gly Pro Arg Asp Arg Ser Glu Ala Val 785 790 795 800 Tyr Thr Thr Met Ser Gly Arg Ser Pro Ala Leu Trp Ala Ala Pro Glu                 805 810 815 Thr Leu Gln Phe Gly His Phe Ser Ser Ala Ser Asp Val Trp Ser Phe             820 825 830 Gly Ile Ile Met Trp Glu Val Met Ala Phe Gly Glu Arg Pro Tyr Trp         835 840 845 Asp Met Ser Gly Gln Asp Val Ile Lys Ala Val Glu Asp Gly Phe Arg     850 855 860 Leu Pro Pro Pro Arg Asn Cys Pro Asn Leu Leu His Arg Leu Met Leu 865 870 875 880 Asp Cys Trp Gln Lys Asp Pro Gly Glu Arg Pro Arg Phe Ser Gln Ile                 885 890 895 His Ser Ile Leu Ser Lys Met Val Gln Asp Pro Glu Pro Pro Lys Cys             900 905 910 Ala Leu Thr Thr Cys Pro Arg Pro Pro Thr Pro Leu Ala Asp Arg Ala         915 920 925 Phe Ser Thr Phe Pro Ser Phe Gly Ser Val Gly Ala Trp Leu Glu Ala     930 935 940 Leu Asp Leu Cys Arg Tyr Lys Asp Ser Phe Ala Ala Ala Gly Tyr Gly 945 950 955 960 Ser Leu Glu Ala Val Ala Glu Met Thr Ala Gln Asp Leu Val Ser Leu                 965 970 975 Gly Ile Ser Leu Ala Glu His Arg Glu Ala Leu Leu Ser Gly Ile Ser             980 985 990 Ala Leu Gln Ala Arg Val Leu Gln Leu Gln Gly Gln Gly Val Gln Val         995 1000 1005 <210> 12 <211> 3024 <212> DNA <213> Human <400> 12 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag cctgtccccc agggttttac aaggtgtccc cgcggcggcc cctctgctca 900 ccgtgcccag agcacagccg ggccctggaa aacgcctcca ccttctgcgt gtgccaggac 960 agctatgcgc gctcacccac cgacccgccc tcggcttcct gcacccggcc gccgtcggcg 1020 ccgcgggacc tgcagtacag cctgagccgc tcgccgctgg tgctgcgact gcgctggctg 1080 ccgccggccg actcgggagg ccgctcggac gtcacctact cgctgctgtg cctgcgctgc 1140 ggccgcgagg gcccggcggg cgcctgcgag ccgtgcgggc cgcgcgtggc cttcctaccg 1200 cgccaggcag ggctgcggga gcgagccgcc acgctgctgc acctgcggcc cggcgcgcgc 1260 tacaccgtgc gcgtggccgc gctcaacggc gtctcgggcc cggcggccgc cgcgggaacc 1320 acctacgcgc aggtcaccgt ctccaccggg cccggggcgc cctgggagga ggatgagatc 1380 cgcagggacc gagtggaacc ccagagcgtg tccctgtcgt ggcgggagcc catccctgcc 1440 ggagcccctg gggccaatga cacggagtac gagatccgat actacgagaa gggtcagagt 1500 gagcagactt actccatggt gaagacaggg gcgcccacag tcaccgtcac caacctgaag 1560 ccggctaccc gctacgtctt tcagatccgg gccgcttccc cggggccatc ctgggaggcc 1620 cagagtttta accccagcat tgaagtacag accctggggg aggctgcctc agggtccagg 1680 gaccagagcc ccgccattgt cgtcaccgta gtgaccatct cggccctcct cgtcctgggc 1740 tccgtgatga gtgtgctggc catttggagg aggccctgca gctatggcaa aggaggaggg 1800 gatgcccatg atgaagagga gctgtatttc cacttcaaag tcccaacacg tcgcacattc 1860 ctggaccccc agagctgtgg ggacctgctg caggctgtgc atctgttcgc caaggaactg 1920 gatgcgaaaa gcgtcacgct ggagaggagc cttggaggag ggcggtttgg ggagctgtgc 1980 tgtggctgct tgcagctccc cggtcgccag gagctgctcg tagccgtgca tatgctgagg 2040 gacagcgcct ccgactcaca gaggctcggc ttcctggccg aggccctcac gctgggccag 2100 tttgaccata gccacatcgt gcggctggag ggcgttgtta cccgaggaag caccttgatg 2160 attgtcaccg agtacatgag ccatggggcc ctggacggct tcctcaggcg gcacgagggg 2220 cagctggtgg ctgggcaact gatggggttg ctgcctgggc tggcatcagc catgaagtat 2280 ctgtcagaga tgggctacgt tcaccggggc ctggcagctc gccatgtgct ggtcagcagc 2340 gaccttgtct gcaagatctc tggcttcggg cggggccccc gggaccgatc agaggctgtc 2400 tacaccacta tgagtggccg gagcccagcg ctatgggccg ctcccgagac acttcagttt 2460 ggccacttca gctctgccag tgacgtgtgg agcttcggca tcatcatgtg ggaggtgatg 2520 gcctttgggg agcggcctta ctgggacatg tctggccaag acgtgatcaa ggctgtggag 2580 gatggcttcc ggctgccacc ccccaggaac tgtcctaacc ttctgcaccg actaatgctc 2640 gactgctggc agaaggaccc aggtgagcgg cccaggttct cccagatcca cagcatcctg 2700 agcaagatgg tgcaggaccc agagcccccc aagtgtgccc tgactacctg tcccaggcct 2760 cccaccccac tagcggaccg tgccttctcc accttcccct cctttggctc tgtgggcgcg 2820 tggctggagg ccctggacct gtgccgctac aaggacagct tcgcggctgc tggctatggg 2880 agcctggagg ccgtggccga gatgactgcc caggacctgg tgagcctagg catctctttg 2940 gctgaacatc gagaggccct cctcagcggg atcagcgccc tgcaggcacg agtgctccag 3000 ctgcagggcc agggggtgca ggtg 3024 <210> 13 <211> 2695 <212> DNA <213> Human <400> 13 gcagccggcc ccgcaaaatc gacacgatcg cggcggacga gagcttcacg cagggcgacc 60 tgggtgagcg caagatgaag ctgaacacag aggtgcgcga gatcggaccg ctcagccggc 120 ggggtttcca cctggccttt caggacgtgg gcgcatgcgt ggcgcttgtc tcggtgcgcg 180 tctactacaa gcagtgccgc gccaccgtgc ggggcctggc cacgttccca gccaccgcag 240 ccgagagcgc cttctccaca ctggtggaag tggccggaac gtgcgtggcg cactcggaag 300 gggagcctgg cagcccccca cgcatgcact gcggcgccga cggcgagtgg ctggtgcctg 360 tgggccgctg cagctgcagc gcgggattcc aggagcgtgg tgacttctgc gaagcctgtc 420 ccccagggtt ttacaaggtg tccccgcggc ggcccctctg ctcaccgtgc ccagagcaca 480 gccgggccct ggaaaacgcc tccaccttct gcgtgtgcca ggacagctat gcgcgctcac 540 ccaccgaccc gccctcggct tcctgcaccc ggccgccgtc ggcgccgcgg gacctgcagt 600 acagcctgag ccgctcgccg ctggtgctgc gactgcgctg gctgccgccg gccgactcgg 660 gaggccgctc ggacgtcacc tactcgctgc tgtgcctgcg ctgcggccgc gagggcccgg 720 cgggcgcctg cgagccgtgc gggccgcgcg tggccttcct accgcgccag gcagggctgc 780 gggagcgagc cgccacgctg ctgcacctgc ggcccggcgc gcgctacacc gtgcgcgtgg 840 ccgcgctcaa cggcgtctcg ggcccggcgg ccgccgcggg aaccacctac gcgcaggtca 900 ccgtctccac cgggcccggg gcgccctggg aggaggatga gatccgcagg gaccgagtgg 960 aaccccagag cgtgtccctg tcgtggcggg agcccatccc tgccggagcc cctggggcca 1020 atgacacgga gtacgagatc cgatactacg agaagggtca gagtgagcag acttactcca 1080 tggtgaagac aggggcgccc acagtcaccg tcaccaacct gaagccggct acccgctacg 1140 tctttcagat ccgggccgct tccccggggc catcctggga ggcccagagt tttaacccca 1200 gcattgaagt acagaccctg ggggaggctg cctcagggtc cagggaccag agccccgcca 1260 ttgtcgtcac cgtagtgacc atctcggccc tcctcgtcct gggctccgtg atgagtgtgc 1320 tggccatttg gaggaggccc tgcagctatg gcaaaggagg aggggatgcc catgatgaag 1380 aggagctgta tttccacttc aaagtcccaa cacgtcgcac attcctggac ccccagagct 1440 gtggggacct gctgcaggct gtgcatctgt tcgccaagga actggatgcg aaaagcgtca 1500 cgctggagag gagccttgga ggagggcggt ttggggagct gtgctgtggc tgcttgcagc 1560 tccccggtcg ccaggagctg ctcgtagccg tgcatatgct gagggacagc gcctccgact 1620 cacagaggct cggcttcctg gccgaggccc tcacgctggg ccagtttgac catagccaca 1680 tcgtgcggct ggagggcgtt gttacccgag gaagcacctt gatgattgtc accgagtaca 1740 tgagccatgg ggccctggac ggcttcctca ggcggcacga ggggcagctg gtggctgggc 1800 aactgatggg gttgctgcct gggctggcat cagccatgaa gtatctgtca gagatgggct 1860 acgttcaccg gggcctggca gctcgccatg tgctggtcag cagcgacctt gtctgcaaga 1920 tctctggctt cgggcggggc ccccgggacc gatcagaggc tgtctacacc actatgagtg 1980 gccggagccc agcgctatgg gccgctcccg agacacttca gtttggccac ttcagctctg 2040 ccagtgacgt gtggagcttc ggcatcatca tgtgggaggt gatggccttt ggggagcggc 2100 cttactggga catgtctggc caagacgtga tcaaggctgt ggaggatggc ttccggctgc 2160 caccccccag gaactgtcct aaccttctgc accgactaat gctcgactgc tggcagaagg 2220 acccaggtga gcggcccagg ttctcccaga tccacagcat cctgagcaag atggtgcagg 2280 acccagagcc ccccaagtgt gccctgacta cctgtcccag gcctcccacc ccactagcgg 2340 accgtgcctt ctccaccttc ccctcctttg gctctgtggg cgcgtggctg gaggccctgg 2400 acctgtgccg ctacaaggac agcttcgcgg ctgctggcta tgggagcctg gaggccgtgg 2460 ccgagatgac tgcccaggac ctggtgagcc taggcatctc tttggctgaa catcgagagg 2520 ccctcctcag cgggatcagc gccctgcagg cacgagtgct ccagctgcag ggccaggggg 2580 tgcaggtgtg agtggacccc attcttccaa ggcaggactc cggtgggggt ccagtccccc 2640 agccctgccc aaggaccgtg gcaagctgcg ctccagcagt gtgggaggga gcgct 2695 <210> 14 <211> 687 <212> PRT <213> Human <400> 14 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro Thr             180 185 190 Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln Ile         195 200 205 Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn Pro     210 215 220 Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg Asp 225 230 235 240 Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu Leu                 245 250 255 Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro Cys             260 265 270 Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu Tyr         275 280 285 Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro Gln Ser     290 295 300 Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu Leu Asp 305 310 315 320 Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg Phe Gly                 325 330 335 Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu Leu Leu             340 345 350 Val Ala Val His Met Leu Arg Asp Ser Ala Ser Asp Ser Gln Arg Leu         355 360 365 Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His Ser His     370 375 380 Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Ser Thr Leu Met Ile 385 390 395 400 Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu Arg Arg                 405 410 415 His Glu Gly Gln Leu Val Ala Gly Gln Leu Met Gly Leu Leu Pro Gly             420 425 430 Leu Ala Ser Ala Met Lys Tyr Leu Ser Glu Met Gly Tyr Val His Arg         435 440 445 Gly Leu Ala Ala Arg His Val Leu Val Ser Ser Asp Leu Val Cys Lys     450 455 460 Ile Ser Gly Phe Gly Arg Gly Pro Arg Asp Arg Ser Glu Ala Val Tyr 465 470 475 480 Thr Thr Met Ser Gly Arg Ser Pro Ala Leu Trp Ala Ala Pro Glu Thr                 485 490 495 Leu Gln Phe Gly His Phe Ser Ser Ala Ser Asp Val Trp Ser Phe Gly             500 505 510 Ile Ile Met Trp Glu Val Met Ala Phe Gly Glu Arg Pro Tyr Trp Asp         515 520 525 Met Ser Gly Gln Asp Val Ile Lys Ala Val Glu Asp Gly Phe Arg Leu     530 535 540 Pro Pro Pro Arg Asn Cys Pro Asn Leu Leu His Arg Leu Met Leu Asp 545 550 555 560 Cys Trp Gln Lys Asp Pro Gly Glu Arg Pro Arg Phe Ser Gln Ile His                 565 570 575 Ser Ile Leu Ser Lys Met Val Gln Asp Pro Glu Pro Pro Lys Cys Ala             580 585 590 Leu Thr Thr Cys Pro Arg Pro Pro Thr Pro Leu Ala Asp Arg Ala Phe         595 600 605 Ser Thr Phe Pro Ser Phe Gly Ser Val Gly Ala Trp Leu Glu Ala Leu     610 615 620 Asp Leu Cys Arg Tyr Lys Asp Ser Phe Ala Ala Ala Gly Tyr Gly Ser 625 630 635 640 Leu Glu Ala Val Ala Glu Met Thr Ala Gln Asp Leu Val Ser Leu Gly                 645 650 655 Ile Ser Leu Ala Glu His Arg Glu Ala Leu Leu Ser Gly Ile Ser Ala             660 665 670 Leu Gln Ala Arg Val Leu Gln Leu Gln Gly Gln Gly Val Gln Val         675 680 685 <210> 15 <211> 2061 <212> DNA <213> Human <400> 15 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggg tcagagtgag 540 cagacttact ccatggtgaa gacaggggcg cccacagtca ccgtcaccaa cctgaagccg 600 gctacccgct acgtctttca gatccgggcc gcttccccgg ggccatcctg ggaggcccag 660 agttttaacc ccagcattga agtacagacc ctgggggagg ctgcctcagg gtccagggac 720 cagagccccg ccattgtcgt caccgtagtg accatctcgg ccctcctcgt cctgggctcc 780 gtgatgagtg tgctggccat ttggaggagg ccctgcagct atggcaaagg aggaggggat 840 gcccatgatg aagaggagct gtatttccac ttcaaagtcc caacacgtcg cacattcctg 900 gacccccaga gctgtgggga cctgctgcag gctgtgcatc tgttcgccaa ggaactggat 960 gcgaaaagcg tcacgctgga gaggagcctt ggaggagggc ggtttgggga gctgtgctgt 1020 ggctgcttgc agctccccgg tcgccaggag ctgctcgtag ccgtgcatat gctgagggac 1080 agcgcctccg actcacagag gctcggcttc ctggccgagg ccctcacgct gggccagttt 1140 gaccatagcc acatcgtgcg gctggagggc gttgttaccc gaggaagcac cttgatgatt 1200 gtcaccgagt acatgagcca tggggccctg gacggcttcc tcaggcggca cgaggggcag 1260 ctggtggctg ggcaactgat ggggttgctg cctgggctgg catcagccat gaagtatctg 1320 tcagagatgg gctacgttca ccggggcctg gcagctcgcc atgtgctggt cagcagcgac 1380 cttgtctgca agatctctgg cttcgggcgg ggcccccggg accgatcaga ggctgtctac 1440 accactatga gtggccggag cccagcgcta tgggccgctc ccgagacact tcagtttggc 1500 cacttcagct ctgccagtga cgtgtggagc ttcggcatca tcatgtggga ggtgatggcc 1560 tttggggagc ggccttactg ggacatgtct ggccaagacg tgatcaaggc tgtggaggat 1620 ggcttccggc tgccaccccc caggaactgt cctaaccttc tgcaccgact aatgctcgac 1680 tgctggcaga aggacccagg tgagcggccc aggttctccc agatccacag catcctgagc 1740 aagatggtgc aggacccaga gccccccaag tgtgccctga ctacctgtcc caggcctccc 1800 accccactag cggaccgtgc cttctccacc ttcccctcct ttggctctgt gggcgcgtgg 1860 ctggaggccc tggacctgtg ccgctacaag gacagcttcg cggctgctgg ctatgggagc 1920 ctggaggccg tggccgagat gactgcccag gacctggtga gcctaggcat ctctttggct 1980 gaacatcgag aggccctcct cagcgggatc agcgccctgc aggcacgagt gctccagctg 2040 cagggccagg gggtgcaggt g 2061 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify the Novel Receptor <400> 16 cggtgctagg ctggcgcggc ttgag 25 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer to amplify the Novel Receptor <400> 17 agcgctccct cccacactgc 20 <210> 18 <211> 743 <212> PRT <213> Human <400> 18 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Val Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Cys Pro Pro Gly         275 280 285 Phe Tyr Lys Val Ser Pro Arg Arg Pro Leu Cys Ser Pro Cys Pro Glu     290 295 300 His Ser Arg Ala Leu Glu Asn Ala Ser Thr Phe Cys Val Cys Gln Asp 305 310 315 320 Ser Tyr Ala Arg Ser Pro Thr Asp Pro Pro Ser Ala Ser Cys Thr Arg                 325 330 335 Pro Pro Ser Ala Pro Arg Asp Leu Gln Tyr Ser Leu Ser Arg Ser Pro             340 345 350 Leu Val Leu Arg Leu Arg Trp Leu Pro Pro Ala Gly Ser Gly Gly Arg         355 360 365 Ser Asp Val Thr Tyr Ser Leu Leu Cys Leu Arg Cys Gly Arg Glu Gly     370 375 380 Pro Ala Gly Ala Cys Glu Pro Cys Gly Pro Arg Val Ala Phe Leu Pro 385 390 395 400 Arg Gln Ala Gly Leu Trp Glu Arg Ala Ala Thr Leu Leu His Leu Arg                 405 410 415 Pro Gly Ala Arg Tyr Ala Val Arg Val Val Ala Leu Asn Gly Val Ser             420 425 430 Gly Pro Ala Ala Ala Ala Gly Thr Thr Tyr Ala Gln Val Thr Val Ser         435 440 445 Thr Gly Pro Gly Ala Pro Trp Glu Glu Asp Glu Ile Arg Arg Asp Arg     450 455 460 Val Glu Pro Gln Ser Val Ser Leu Ser Trp Arg Glu Pro Ile Pro Ala 465 470 475 480 Gly Ala Pro Gly Ala Asn Asp Thr Glu Tyr Glu Ile Arg Tyr Tyr Glu                 485 490 495 Lys Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro             500 505 510 Thr Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln         515 520 525 Ile Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn     530 535 540 Pro Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg 545 550 555 560 Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu                 565 570 575 Leu Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro             580 585 590 Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu         595 600 605 Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Arg Asp Pro Gln     610 615 620 Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Thr Lys Glu Leu 625 630 635 640 Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Leu Cys                 645 650 655 Ser Cys Leu Glu Ala Leu Pro Pro His Gln Ala His Pro Pro Pro Arg             660 665 670 Lys Ser Leu Ala Cys Leu Ile Ile Gly Lys Arg Met Ala Glu Gly Ser         675 680 685 Val Thr Leu Ser Trp Ser Ser Arg Ala Val Trp Gly Ala Val Leu Trp     690 695 700 Leu Leu Ala Ala Pro Arg Ser Pro Gly Ala Ala Arg Ser Arg Ala Tyr 705 710 715 720 Ala Glu Gly Gln Arg Leu Arg Leu Thr Glu Ala Arg Leu Pro Gly Arg                 725 730 735 Gly Pro His Ala Gly Pro Val             740 <210> 19 <211> 2229 <212> DNA <213> Human <400> 19 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgtactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggttggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg ggttccagga gcgtggtgac 840 ttctgcgaag cctgtccccc agggttttac aaggtgtccc cgcggcggcc cctctgctca 900 ccgtgcccag agcacagccg ggccctggaa aacgcctcca ccttctgcgt gtgccaggac 960 agctatgcgc gctcacccac cgacccgccc tcggcttcct gcacccggcc gccgtcggcg 1020 ccgcgggacc tgcagtacag cctgagccgc tcgccgctgg tgctgcgact gcgctggctg 1080 ccgccggccg gctcgggagg ccgctcggac gtcacctact cgctgctgtg cctgcgctgc 1140 ggccgcgagg gcccggcggg cgcctgcgag ccgtgcgggc cgcgcgtggc cttcctaccg 1200 cgccaggcag ggctgtggga gcgagccgcc acgctgctgc acctgcggcc cggcgcgcgc 1260 tacgccgtgc gcgtggtcgc gctcaacggc gtctcgggcc cggcggccgc cgcgggaacc 1320 acctacgcgc aggtcaccgt ctccaccggg cccggggcgc cctgggagga ggatgagatc 1380 cgcagggacc gagtggaacc ccagagcgtg tccctgtcgt ggcgggagcc catccctgcc 1440 ggagcccctg gggccaatga cacggagtac gagatccgat actacgagaa gggtcagagt 1500 gagcagactt actccatggt gaagacaggg gcgcccacag tcaccgtcac caacctgaag 1560 ccggctaccc gctacgtctt tcagatccgg gccgcttccc cggggccatc ctgggaggcc 1620 cagagtttta accccagcat tgaagtacag accctggggg aggctgcctc agggtccagg 1680 gaccagagcc ccgccattgt cgtcaccgta gtgaccatct cggccctcct cgtcctgggc 1740 tccgtgatga gtgtgctggc catttggagg aggccctgca gctatggcaa aggaggaggg 1800 gatgcccatg atgaagagga gctgtatttc cacttcaaag tcccaacacg tcgcacattc 1860 cgggaccccc agagctgtgg ggacctgctg caggctgtgc atctgttcac caaggaactg 1920 gatgcgaaaa gcgtcacact ggagaggagc cttggaggag gtctctgctc gtgtctggaa 1980 gccctgcctc cacaccaagc ccaccctcca cccagaaagt ccctggcttg cctgattata 2040 gggaagagga tggcagaagg gtctgtgacc ctctcctggt cctcaagggc ggtttgggga 2100 gctgtgctgt ggctgcttgc agctccccgg tcgccaggag ctgctcgtag ccgtgcatat 2160 gctgagggac agcgcctccg actcacagag gctcggcttc ctggccgagg ccctcacgct 2220 gggccagtt 2229 <210> 20 <211> 506 <212> PRT <213> Human <400> 20 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Cys Pro Pro Gly         275 280 285 Phe Tyr Lys Val Ser Pro Arg Arg Pro Leu Cys Ser Pro Cys Pro Glu     290 295 300 His Ser Arg Ala Leu Glu Asn Ala Ser Thr Phe Cys Val Cys Gln Asp 305 310 315 320 Ser Tyr Ala Arg Ser Pro Thr Asp Pro Pro Ser Ala Ser Cys Thr Arg                 325 330 335 Pro Pro Ser Ala Pro Arg Asp Leu Gln Tyr Gly Leu Ser Arg Ser Pro             340 345 350 Leu Val Leu Arg Leu Arg Trp Leu Pro Pro Ala Asp Ser Gly Gly Arg         355 360 365 Ser Asp Val Thr Tyr Ser Leu Leu Cys Leu Arg Cys Gly Arg Glu Gly     370 375 380 Arg Arg Ala Pro Ala Ser Arg Ala Gly Arg Ala Trp Pro Ser Tyr Arg 385 390 395 400 Ala Arg Gln Gly Cys Gly Ser Glu Pro Pro Arg Cys Cys Thr Cys Gly                 405 410 415 Pro Ala Arg Ala Thr Pro Cys Ala Trp Pro Arg Ser Thr Ala Ser Arg             420 425 430 Ala Arg Arg Pro Pro Arg Glu Pro Pro Thr Arg Arg Ser Pro Ser Pro         435 440 445 Pro Gly Pro Gly Arg Pro Gly Arg Arg Met Arg Ser Ala Gly Thr Glu     450 455 460 Trp Asn Pro Arg Ala Cys Pro Cys Arg Gly Gly Ser Pro Ser Leu Pro 465 470 475 480 Glu Pro Leu Gly Pro Met Thr Arg Ser Thr Arg Ser Asp Thr Thr Arg                 485 490 495 Arg Val Arg Val Ser Arg Leu Thr Pro Trp             500 505 <210> 21 <211> 1518 <212> DNA <213> Human <400> 21 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag cctgtccccc agggttttac aaggtgtccc cgcggcggcc cctctgctca 900 ccgtgcccag agcacagccg ggccctggaa aacgcctcca ccttctgcgt gtgccaggac 960 agctatgcgc gctcacccac cgacccgccc tcggcttcct gcacccggcc gccgtcggcg 1020 ccgcgggacc tgcagtacgg cctgagccgc tcgccgctgg tgctgcgact gcgctggctg 1080 ccgccggccg actcgggagg ccgctcggac gtcacctact cgctgctgtg cctgcgctgc 1140 ggccgcgagg gccggcgggc gcctgcgagc cgtgcgggcc gcgcgtggcc ttcctaccgc 1200 gccaggcagg gctgcgggag cgagccgcca cgctgctgca cctgcggccc ggcgcgcgct 1260 acaccgtgcg cgtggccgcg ctcaacggcg tctcgggccc ggcggccgcc gcgggaacca 1320 cctacgcgca ggtcaccgtc tccaccgggc ccggggcgcc ctgggaggag gatgagatcc 1380 gcagggaccg agtggaaccc cagagcgtgt ccctgtcgtg gcgggagccc atccctgccg 1440 gagcctctgg ggccaatgac acggagtacg agatccgata ctacgagaag ggtcagagtg 1500 agcagactta ctccatgg 1518 <210> 22 <211> 737 <212> PRT <213> Human <400> 22 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Gly Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Ala Ser Gly Ser         275 280 285 Arg Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala     290 295 300 Leu Leu Val Leu Gly Ser Val Met Gly Val Leu Ala Ile Trp Arg Arg 305 310 315 320 Pro Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu                 325 330 335 Leu Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro             340 345 350 Gln Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu         355 360 365 Leu Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg     370 375 380 Phe Gly Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu 385 390 395 400 Leu Leu Val Ala Val His Met Leu Arg Asp Ser Ala Ser Asp Pro Gln                 405 410 415 Arg Leu Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His             420 425 430 Ser His Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Ser Thr Leu         435 440 445 Met Ile Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu     450 455 460 Arg Trp His Glu Gly Gln Leu Val Ala Gly Gln Leu Met Gly Leu Leu 465 470 475 480 Pro Gly Leu Ala Ser Ala Met Lys Tyr Leu Ser Glu Met Gly Tyr Val                 485 490 495 His Arg Gly Leu Ala Ala Arg His Val Leu Val Ser Ser Asp Leu Val             500 505 510 Cys Lys Ile Ser Gly Phe Gly Arg Gly Pro Arg Asp Arg Ser Glu Ala         515 520 525 Val Tyr Thr Thr Met Ser Gly Arg Ser Pro Ala Leu Trp Ala Ala Pro     530 535 540 Glu Thr Leu Gln Phe Gly His Phe Ser Ser Ala Ser Asp Val Trp Ser 545 550 555 560 Phe Gly Ile Ile Met Trp Glu Val Met Ala Phe Gly Glu Arg Pro Tyr                 565 570 575 Trp Asp Met Ser Gly Gln Asp Val Ile Lys Ala Val Glu Asp Gly Phe             580 585 590 Arg Leu Pro Pro Pro Arg Asn Cys Pro Asn Leu Leu His Arg Leu Met         595 600 605 Leu Asp Cys Trp Gln Lys Asp Pro Gly Glu Arg Pro Arg Phe Ser Gln     610 615 620 Ile His Ser Ile Leu Ser Lys Met Val Gln Asp Pro Glu Pro Pro Lys 625 630 635 640 Cys Ala Leu Thr Thr Cys Pro Arg Pro Pro Thr Pro Leu Ala Asp Arg                 645 650 655 Ala Phe Ser Thr Phe Pro Ser Phe Gly Ser Val Gly Ala Trp Leu Glu             660 665 670 Ala Leu Asp Leu Cys Arg Tyr Lys Asp Ser Phe Val Ala Ala Gly Tyr         675 680 685 Gly Ser Leu Glu Ala Val Ala Glu Met Thr Ala Gln Asp Leu Val Ser     690 695 700 Leu Gly Ile Ser Leu Ala Glu His Arg Glu Ala Leu Leu Ser Gly Ile 705 710 715 720 Ser Ala Leu Arg Ala Arg Val Leu Gln Leu Gln Gly Gln Gly Val Gln                 725 730 735 Val <210> 23 <211> 2211 <212> DNA <213> Human <400> 23 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgcta 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaaggggg gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag ctgcctcagg gtccagggac cagagccccg ccattgtcgt caccgtagtg 900 accatctcgg ccctcctcgt cctgggctcc gtgatgggtg tgctggccat ttggaggagg 960 ccctgcagct atggcaaagg aggaggggat gcccatgatg aagaggagct gtatttccac 1020 ttcaaagtcc caacacgtcg cacattcctg gacccccaga gctgtgggga cctgctgcag 1080 gctgtgcatc tgttcgccaa ggaactggat gcgaaaagcg tcacgctgga gaggagcctt 1140 ggaggagggc ggtttgggga gctgtgctgt ggctgcttgc agctccccgg tcgccaggag 1200 ctgctcgtag ccgtgcatat gctgagggac agcgcctccg acccacagag gctcggcttc 1260 ctggccgagg ccctcacgct gggccagttt gaccatagcc acatcgtgcg gctggagggc 1320 gttgttaccc gaggaagcac cttgatgatt gtcaccgagt acatgagcca tggggccctg 1380 gacggcttcc tcaggtggca cgaggggcag ctggtggctg ggcaactgat ggggttgctg 1440 cctgggctgg catcagccat gaagtatctg tcagagatgg gctacgttca ccggggcctg 1500 gcagctcgcc atgtgctggt cagcagcgac cttgtctgca agatctctgg cttcgggcgg 1560 ggcccccggg accgatcaga ggctgtctac accactatga gtggccggag cccagcgcta 1620 tgggccgctc ccgagacact tcagtttggc cacttcagct ctgccagtga cgtgtggagc 1680 ttcggcatca tcatgtggga ggtgatggcc tttggggagc ggccttactg ggacatgtct 1740 ggccaagacg tgatcaaggc tgtggaggat ggcttccggc tgccaccccc caggaactgt 1800 cctaaccttc tgcaccgact aatgctcgac tgctggcaga aggacccagg tgagcggccc 1860 aggttctccc agatccacag catcctgagc aagatggtgc aggacccaga gccccccaag 1920 tgtgccctga ctacctgtcc caggcctccc accccactag cggaccgtgc cttctccacc 1980 ttcccctcct ttggctctgt gggcgcgtgg ctggaggccc tggacctgtg ccgctacaag 2040 gacagcttcg tggctgctgg ctatgggagc ctggaggccg tggccgagat gactgcccag 2100 gacctggtga gcctaggcat ctctttggct gaacatcgag aggccctcct cagcgggatc 2160 agcgccctgc gggcacgagt gctccagctg cagggccagg gggtgcaggt g 2211 <210> 24 <211> 749 <212> PRT <213> Human <400> 24 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Gly Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Cys Pro Pro Gly         275 280 285 Phe Tyr Lys Val Ser Pro Arg Arg Pro Leu Cys Ser Pro Cys Pro Glu     290 295 300 His Ser Arg Ala Leu Glu Asn Ala Ser Thr Phe Cys Val Cys Gln Asp 305 310 315 320 Ser Tyr Ala Arg Ser Pro Thr Asp Pro Pro Ser Ala Ser Cys Thr Arg                 325 330 335 Pro Pro Ser Ala Pro Arg Asp Leu Gln Tyr Ser Leu Gly Arg Ser Pro             340 345 350 Leu Val Leu Arg Leu Arg Trp Leu Pro Pro Ala Asp Ser Gly Gly Arg         355 360 365 Thr Asp Val Thr Tyr Ser Leu Leu Cys Leu Arg Cys Gly Arg Glu Gly     370 375 380 Pro Ala Gly Ala Cys Glu Pro Cys Gly Pro Arg Val Ala Phe Leu Pro 385 390 395 400 Arg Gln Ala Gly Leu Arg Glu Arg Ala Ala Thr Leu Leu His Leu Arg                 405 410 415 Pro Gly Ala Arg Tyr Thr Val Arg Val Ala Ala Leu Asn Gly Val Ser             420 425 430 Gly Pro Ala Ala Ala Ala Gly Thr Thr Tyr Ala Gln Val Thr Val Ser         435 440 445 Thr Gly Pro Gly Ala Pro Trp Glu Glu Asp Glu Ile Arg Arg Asp Arg     450 455 460 Val Glu Pro Gln Ser Val Ser Leu Ser Trp Arg Glu Pro Ile Pro Ala 465 470 475 480 Gly Ala Pro Gly Ala Asn Asp Thr Glu Tyr Glu Ile Arg Tyr Tyr Glu                 485 490 495 Lys Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro             500 505 510 Thr Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln         515 520 525 Ile Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn     530 535 540 Pro Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg 545 550 555 560 Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu                 565 570 575 Leu Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro             580 585 590 Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu         595 600 605 Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro Gln     610 615 620 Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu Leu 625 630 635 640 Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg Phe                 645 650 655 Gly Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu Leu             660 665 670 Leu Val Ala Val His Met Leu Arg Asp Ser Ala Ser Asp Ser Gln Arg         675 680 685 Leu Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His Ser     690 695 700 His Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Asn Thr Leu Met 705 710 715 720 Ile Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu Arg                 725 730 735 Val Ser Gly Pro Gly Ala Ala Asn Asp His Phe Ile Leu             740 745 <210> 25 <211> 2247 <212> DNA <213> Human <400> 25 atggagacct gtgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttggggcc ctggcggcct gggactgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggtg tgggtgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag cctgtccccc agggttttac aaggtgtccc cgcggcggcc cctctgctca 900 ccgtgcccag agcacagccg ggccctggaa aacgcctcca ccttctgcgt gtgccaggac 960 agctatgcgc gctcacccac cgacccgccc tcggcttcct gcacccggcc gccgtcggcg 1020 ccgcgggacc tgcagtacag cctgggccgc tcgccgctgg tgctgcgact gcgctggctg 1080 ccgccggccg actcgggagg ccgcacggac gtcacctact cgctgctgtg cctgcgctgc 1140 ggccgcgagg gcccggcggg cgcctgcgag ccgtgcgggc cgcgcgtggc cttcctaccg 1200 cgccaggcag ggctgcggga gcgagccgcc acgctgctgc acctgcggcc cggcgcgcgc 1260 tacaccgtgc gcgtggccgc gctcaacggc gtctcgggcc cggcggccgc cgcgggaacc 1320 acctacgcgc aggtcaccgt ctccaccggg cccggggcgc cctgggagga ggatgagatc 1380 cgcagggacc gagtggaacc ccagagcgtg tccctgtcgt ggcgggagcc catccctgcc 1440 ggagcccctg gggccaatga cacggagtac gagatccgat actacgagaa gggtcagagt 1500 gagcagactt actccatggt gaagacaggg gcgcccacag tcaccgtcac caacctgaag 1560 ccggctaccc gctacgtctt tcagatccgg gccgcttccc cggggccatc ctgggaggcc 1620 cagagtttta accccagcat tgaagtacag accctggggg aggctgcctc agggtccagg 1680 gaccagagcc ccgccattgt cgtcaccgta gtgaccatct cggccctcct cgtcctgggc 1740 tccgtgatga gtgtgctggc catttggagg aggccctgca gctatggcaa aggaggaggg 1800 gatgcccatg atgaagagga gctgtatttc cacttcaaag tcccaacacg tcgcacattc 1860 ctggaccccc agagctgtgg ggacctgctg caggctgtgc atctgttcgc caaggaactg 1920 gatgcgaaaa gcgtcacgct ggagaggagc cttggaggag ggcggtttgg ggagctgtgc 1980 tgtggctgct tgcagctccc cggtcgccag gagctgctcg tagccgtgca tatgctgagg 2040 gacagcgcct ccgactcaca gaggctcggc ttcctggccg aggccctcac gctgggccag 2100 tttgaccata gccacatcgt gcggctggag ggcgttgtta cccgaggaaa caccttgatg 2160 attgtcaccg agtacatgag ccatggggcc ctggacggct tcctcagggt gagtggccct 2220 ggggctgcca atgaccactt tattctg 2247 <210> 26 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 26 gccgtggccg agatga 16 <210> 27 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 27 cctctcgatg ttcagccaaa g 21 <210> 28 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 28 tgcccaggac ctggtgagcc tagg 24 <210> 29 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 29 ctgcactgcc aagtaatggg t 21 <210> 30 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 30 acgtgcggat gggacg 16 <210> 31 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 31 aggagatcag cggcgtggat gaac 24 <210> 32 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 32 aattggatcc atggagacct gcgccggt 28 <210> 33 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 33 aattctcgag tcactgtact tcaatgctgg 30 <210> 34 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 34 atggaggaag ttatcctcct ggattcc 27 <210> 35 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 35 ctgtacttca atgctggggt taaaac 26 <210> 36 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synsthetic peptide base on the aminoacid Sequence of NEPHA <400> 36 Glu Ile Ser Gly Val Asp Glu His Asp Arg Pro Ile Arg Thr Cys                  5 10 15 <210> 37 <211> 15 <212> PRT <213> Aritificial Sequence <220> <223> synsthetic peptide base on the aminoacid Sequence of NEPHA <400> 37 Glu Ile Arg Tyr Tyr Glu Lys Gly Gln Ser Glu Gln Thr Tyr Cys                  5 10 15 <210> 38 <211> 687 <212> PRT <213> Human <400> 38 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Ser Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro Thr             180 185 190 Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln Ile         195 200 205 Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn Pro     210 215 220 Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg Asp 225 230 235 240 Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu Leu                 245 250 255 Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro Cys             260 265 270 Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu Tyr         275 280 285 Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro Gln Asn     290 295 300 Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu Leu Asp 305 310 315 320 Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg Phe Gly                 325 330 335 Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu Leu Leu             340 345 350 Val Ala Val His Met Leu Arg Asp Gly Ala Ser Asp Ser Gln Arg Leu         355 360 365 Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His Ser His     370 375 380 Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Ser Thr Leu Met Ile 385 390 395 400 Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu Arg Arg                 405 410 415 Arg Glu Gly Gln Leu Val Ala Gly Gln Leu Met Glu Leu Leu Pro Gly             420 425 430 Leu Ala Ser Ala Met Lys Tyr Leu Ser Glu Met Gly Tyr Val His Arg         435 440 445 Gly Leu Ala Ala Arg His Val Leu Val Ser Ser Asp Leu Val Cys Lys     450 455 460 Ile Ser Gly Phe Gly Arg Gly Pro Arg Asp Arg Ser Glu Ala Val Tyr 465 470 475 480 Thr Thr Met Ser Gly Arg Ser Pro Ala Leu Trp Ala Ala Pro Glu Thr                 485 490 495 Leu Gln Phe Gly His Phe Ser Ser Ala Ser Asp Val Trp Ser Phe Gly             500 505 510 Ile Ile Met Trp Glu Ala Met Ala Phe Gly Glu Arg Pro Tyr Trp Asp         515 520 525 Met Ser Gly Gln Asp Val Ile Lys Ala Val Glu Asp Gly Phe Arg Leu     530 535 540 Pro Pro Pro Arg Asn Cys Pro Asn Leu Leu His Arg Leu Met Leu Asp 545 550 555 560 Cys Trp Gln Glu Asp Pro Gly Glu Arg Pro Arg Phe Ser Gln Ile His                 565 570 575 Ser Ile Leu Ser Lys Met Val Gln Asp Pro Glu Pro Pro Lys Cys Ala             580 585 590 Leu Ala Thr Cys Pro Arg Pro Pro Thr Pro Leu Ala Asp Arg Ala Phe         595 600 605 Ser Thr Phe Pro Ser Phe Gly Ser Val Asp Ala Trp Leu Glu Ala Leu     610 615 620 Asp Leu Cys Arg Tyr Lys Asp Ser Phe Ala Ala Ala Gly Tyr Gly Ser 625 630 635 640 Leu Glu Ala Val Ala Glu Met Thr Ala Gln Asp Leu Val Ser Leu Gly                 645 650 655 Ile Ser Leu Ala Glu His Arg Glu Ala Leu Leu Ser Gly Ile Ser Ala             660 665 670 Leu Gln Ala Arg Val Leu Arg Leu Gln Gly Gln Gly Val Gln Val         675 680 685 <210> 39 <211> 2061 <212> DNA <213> Human <400> 39 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcag cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggg tcagagtgag 540 cagacttact ccatggtgaa gacaggggcg cccacagtca ccgtcaccaa cctgaagccg 600 gctactcgct acgtctttca gatccgggcc gcttccccgg ggccatcctg ggaggcccag 660 agttttaacc ccagcattga agtacagacc ctgggggagg ctgcctcagg gtccagggac 720 cagagccccg ccattgtcgt caccgtagtg accatctcgg ccctcctcgt cctgggctcc 780 gtgatgagtg tgctggccat ttggaggagg ccctgcagct atggcaaagg aggaggggat 840 gcccatgatg aagaggagct gtatttccac ttcaaagtcc caacacgtcg cacattcctg 900 gacccccaga actgtgggga cctgctgcag gctgtgcatc tgttcgccaa ggaactggat 960 gcgaaaagcg tcacgctgga gaggagcctt ggaggagggc ggtttgggga gctgtgctgt 1020 ggctgcttgc agctccccgg tcgccaggag ctgctcgtag ccgtgcatat gctgagggac 1080 ggcgcctccg actcacagag gctcggcttc ctggccgagg ctctcacgct gggccagttt 1140 gaccatagcc acatcgtgcg gctggagggc gttgttaccc gaggaagcac cttgatgatt 1200 gtcaccgagt acatgagcca tggggccctg gacggcttcc tcaggcggcg cgaggggcag 1260 ctggtggctg ggcaactgat ggagttgctg cctgggctgg catcagccat gaagtatctg 1320 tcggagatgg gctacgttca ccggggcctg gcagctcgcc atgtgctggt cagcagcgac 1380 cttgtctgca agatctctgg cttcgggcgg ggcccccggg accgatcaga ggctgtctac 1440 accactatga gtggccggag cccagcgcta tgggccgctc ccgagacact tcagtttggc 1500 cacttcagct ctgccagtga cgtgtggagc ttcggcatca tcatgtggga ggcgatggcc 1560 tttggggagc ggccttactg ggacatgtct ggccaagacg tgatcaaggc tgtggaggat 1620 ggcttccggc tgccaccccc caggaactgt cctaaccttc tgcaccgact aatgctcgac 1680 tgctggcagg aggacccagg tgagcggccc aggttctctc agatccacag catcctgagc 1740 aagatggtgc aggacccaga gccccccaag tgtgccctgg ctacctgtcc caggcctccc 1800 accccactag ccgaccgtgc cttctccacc ttcccctcct ttggctctgt ggacgcgtgg 1860 ctggaggccc tggacctgtg ccgctacaag gacagcttcg cggctgctgg ctatgggagc 1920 ctggaggccg tggccgagat gactgcccag gacctggtga gcctaggcat ctctttggct 1980 gaacatcgag aggccctcct cagcgggatc agcgccctgc aggcacgagt gctccggctg 2040 cagggccagg gggtgcaggt g 2061 <210> 40 <211> 743 <212> PRT <213> Human <400> 40 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Cys Pro Pro Gly         275 280 285 Phe Tyr Lys Val Ser Pro Arg Arg Pro Leu Cys Ser Pro Cys Pro Glu     290 295 300 His Ser Arg Ala Leu Glu Asn Ala Ser Thr Phe Cys Val Cys Gln Asp 305 310 315 320 Ser Tyr Ala Arg Ser Pro Thr Asp Pro Pro Ser Ala Ser Cys Thr Arg                 325 330 335 Pro Pro Ser Ala Pro Arg Asp Leu Gln Tyr Ser Leu Ser Arg Ser Pro             340 345 350 Leu Val Leu Arg Leu Arg Trp Leu Pro Pro Ala Asp Ser Gly Gly Arg         355 360 365 Ser Asp Val Thr Tyr Ser Leu Leu Cys Leu Arg Cys Gly Arg Glu Gly     370 375 380 Pro Ala Gly Ala Cys Glu Pro Cys Gly Pro Arg Val Ala Phe Leu Pro 385 390 395 400 Arg Gln Ala Gly Leu Arg Glu Arg Ala Ala Thr Leu Leu His Leu Arg                 405 410 415 Pro Gly Ala Arg Tyr Thr Val Arg Val Ala Ala Leu Asn Gly Val Ser             420 425 430 Gly Pro Ala Ala Ala Ala Gly Thr Thr Tyr Ala Gln Val Thr Val Ser         435 440 445 Thr Gly Pro Gly Ala Pro Trp Glu Glu Asp Glu Ile Arg Arg Asp Arg     450 455 460 Val Glu Pro Gln Ser Val Ser Leu Ser Trp Arg Glu Pro Ile Pro Ala 465 470 475 480 Gly Ala Pro Gly Ala Asn Asp Thr Glu Tyr Glu Ile Arg Tyr Tyr Glu                 485 490 495 Lys Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro             500 505 510 Thr Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln         515 520 525 Ile Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn     530 535 540 Pro Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg 545 550 555 560 Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu                 565 570 575 Leu Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro             580 585 590 Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu         595 600 605 Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro Gln     610 615 620 Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu Leu 625 630 635 640 Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Leu Cys                 645 650 655 Ser Cys Leu Glu Ala Leu Pro Pro His Gln Ala His Pro Pro Pro Arg             660 665 670 Lys Ser Leu Ala Cys Leu Ile Ile Gly Lys Arg Met Ala Glu Gly Ser         675 680 685 Val Thr Leu Ser Trp Ser Ser Arg Ala Val Trp Gly Ala Val Leu Trp     690 695 700 Leu Leu Ala Ala Pro Arg Ser Pro Gly Ala Ala Arg Ser Arg Ala Tyr 705 710 715 720 Ala Glu Gly Gln Arg Leu Arg Leu Thr Glu Ala Arg Leu Pro Gly Arg                 725 730 735 Gly Pro His Ala Gly Pro Val             740 <210> 41 <211> 2229 <212> DNA <213> Human <400> 41 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag cctgtccccc agggttttac aaggtgtccc cgcggcggcc cctctgctca 900 ccgtgcccag agcacagccg ggccctggaa aacgcctcca ccttctgcgt gtgccaggac 960 agctatgcgc gctcacccac cgacccgccc tcggcttcct gcacccggcc gccgtcggcg 1020 ccgcgggacc tgcagtacag cctgagccgc tcgccgctgg tgctgcgact gcgctggctg 1080 ccgccggccg actcgggagg ccgctcggac gtcacctact cgctgctgtg cctgcgctgc 1140 ggccgcgagg gcccggcggg cgcctgcgag ccgtgcgggc cgcgcgtggc cttcctaccg 1200 cgccaggcag ggctgcggga gcgagccgcc acgctgctgc acctgcggcc cggcgcgcgc 1260 tacaccgtgc gcgtggccgc gctcaacggc gtctcgggcc cggcggccgc cgcgggaacc 1320 acctacgcgc aggtcaccgt ctccaccggg cccggggcgc cctgggagga ggatgagatc 1380 cgcagggacc gagtggaacc ccagagcgtg tccctgtcgt ggcgggagcc catccctgcc 1440 ggagcccctg gggccaatga cacggagtac gagatccgat actacgagaa gggtcagagt 1500 gagcagactt actccatggt gaagacaggg gcgcccacag tcaccgtcac caacctgaag 1560 ccggctaccc gctacgtctt tcagatccgg gccgcttccc cggggccatc ctgggaggcc 1620 cagagtttta accccagcat tgaagtacag accctggggg aggctgcctc agggtccagg 1680 gaccagagcc ccgccattgt cgtcaccgta gtgaccatct cggccctcct cgtcctgggc 1740 tccgtgatga gtgtgctggc catttggagg aggccctgca gctatggcaa aggaggaggg 1800 gatgcccatg atgaagagga gctgtatttc cacttcaaag tcccaacacg tcgcacattc 1860 ctggaccccc agagctgtgg ggacctgctg caggctgtgc atctgttcgc caaggaactg 1920 gatgcgaaaa gcgtcacgct ggagaggagc cttggaggag gtctctgctc gtgtctggaa 1980 gccctgcctc cacaccaagc ccaccctcca cccagaaagt ccctggcttg cctgattata 2040 gggaagagga tggcagaagg gtctgtgacc ctctcctggt cctcaagggc ggtttgggga 2100 gctgtgctgt ggctgcttgc agctccccgg tcgccaggag ctgctcgtag ccgtgcatat 2160 gctgagggac agcgcctccg actcacagag gctcggcttc ctggccgagg ccctcacgct 2220 gggccagtt 2229 <210> 42 <211> 927 <212> PRT <213> Human <400> 42 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Cys Pro Pro Gly         275 280 285 Phe Tyr Lys Val Ser Pro Arg Arg Pro Leu Cys Ser Pro Cys Pro Glu     290 295 300 His Ser Arg Ala Leu Glu Asn Ala Ser Thr Phe Cys Val Cys Gln Asp 305 310 315 320 Ser Tyr Ala Arg Ser Pro Thr Asp Pro Pro Ser Ala Ser Cys Thr Arg                 325 330 335 Pro Pro Ser Ala Pro Arg Asp Leu Gln Tyr Ser Leu Ser Arg Ser Pro             340 345 350 Leu Val Leu Arg Leu Arg Trp Leu Pro Pro Ala Asp Ser Gly Gly Arg         355 360 365 Ser Asp Val Thr Tyr Ser Leu Leu Cys Leu Arg Cys Gly Arg Glu Gly     370 375 380 Pro Ala Gly Ala Cys Glu Pro Cys Gly Pro Arg Val Ala Phe Leu Pro 385 390 395 400 Arg Gln Ala Gly Leu Arg Glu Arg Ala Ala Thr Leu Leu His Leu Arg                 405 410 415 Pro Gly Ala Arg Tyr Thr Val Arg Val Ala Ala Leu Asn Gly Val Ser             420 425 430 Gly Pro Ala Ala Ala Ala Gly Thr Thr Tyr Ala Gln Val Thr Val Ser         435 440 445 Thr Gly Pro Gly Ala Pro Trp Glu Glu Asp Glu Ile Arg Arg Asp Arg     450 455 460 Val Glu Pro Gln Ser Val Ser Leu Ser Trp Arg Glu Pro Ile Pro Ala 465 470 475 480 Gly Ala Pro Gly Ala Asn Asp Thr Glu Tyr Glu Ile Arg Tyr Tyr Glu                 485 490 495 Lys Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro             500 505 510 Thr Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln         515 520 525 Ile Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn     530 535 540 Pro Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg 545 550 555 560 Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu                 565 570 575 Leu Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro             580 585 590 Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu         595 600 605 Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro Gln     610 615 620 Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu Leu 625 630 635 640 Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg Phe                 645 650 655 Gly Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu Leu             660 665 670 Leu Val Ala Val His Met Leu Arg Asp Ser Ala Ser Asp Ser Gln Arg         675 680 685 Leu Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His Ser     690 695 700 His Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Ser Thr Leu Met 705 710 715 720 Ile Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu Arg                 725 730 735 Arg His Glu Gly Gln Leu Val Ala Gly Gln Leu Met Gly Leu Leu Pro             740 745 750 Gly Leu Ala Ser Ala Met Lys Tyr Leu Ser Glu Met Gly Tyr Val His         755 760 765 Arg Gly Leu Ala Ala Arg His Val Leu Val Ser Ser Asp Leu Val Cys     770 775 780 Lys Ile Ser Gly Phe Gly Arg Gly Pro Arg Asp Arg Ser Glu Ala Val 785 790 795 800 Tyr Thr Thr Met Ser Gly Arg Ser Pro Ala Leu Trp Ala Ala Pro Glu                 805 810 815 Thr Leu Gln Phe Gly His Phe Ser Ser Ala Ser Asp Val Trp Ser Phe             820 825 830 Gly Ile Ile Met Trp Glu Val Met Ala Phe Gly Glu Arg Pro Tyr Trp         835 840 845 Asp Met Ser Gly Gln Asp Val Ile Lys Ala Val Glu Asp Gly Phe Arg     850 855 860 Leu Pro Pro Pro Arg Asn Cys Pro Asn Leu Leu His Arg Leu Met Leu 865 870 875 880 Asp Cys Trp Gln Lys Asp Pro Gly Glu Arg Pro Arg Phe Ser Gln Ile                 885 890 895 His Ser Ile Leu Ser Lys Met Val Gln Asp Pro Glu Pro Pro Lys Cys             900 905 910 Ala Leu Thr Thr Cys Pro Arg Leu Phe Arg Ile Arg Gln Tyr Leu         915 920 925 <210> 43 <211> 2781 <212> DNA <213> Human <400> 43 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag cctgtccccc agggttttac aaggtgtccc cgcggcggcc cctctgctca 900 ccgtgcccag agcacagccg ggccctggaa aacgcctcca ccttctgcgt gtgccaggac 960 agctatgcgc gctcacccac cgacccgccc tcggcttcct gcacccggcc gccgtcggcg 1020 ccgcgggacc tgcagtacag cctgagccgc tcgccgctgg tgctgcgact gcgctggctg 1080 ccgccggccg actcgggagg ccgctcggac gtcacctact cgctgctgtg cctgcgctgc 1140 ggccgcgagg gcccggcggg cgcctgcgag ccgtgcgggc cgcgcgtggc cttcctaccg 1200 cgccaggcag ggctgcggga gcgagccgcc acgctgctgc acctgcggcc cggcgcgcgc 1260 tacaccgtgc gcgtggccgc gctcaacggc gtctcgggcc cggcggccgc cgcgggaacc 1320 acctacgcgc aggtcaccgt ctccaccggg cccggggcgc cctgggagga ggatgagatc 1380 cgcagggacc gagtggaacc ccagagcgtg tccctgtcgt ggcgggagcc catccctgcc 1440 ggagcccctg gggccaatga cacggagtac gagatccgat actacgagaa gggtcagagt 1500 gagcagactt actccatggt gaagacaggg gcgcccacag tcaccgtcac caacctgaag 1560 ccggctaccc gctacgtctt tcagatccgg gccgcttccc cggggccatc ctgggaggcc 1620 cagagtttta accccagcat tgaagtacag accctggggg aggctgcctc agggtccagg 1680 gaccagagcc ccgccattgt cgtcaccgta gtgaccatct cggccctcct cgtcctgggc 1740 tccgtgatga gtgtgctggc catttggagg aggccctgca gctatggcaa aggaggaggg 1800 gatgcccatg atgaagagga gctgtatttc cacttcaaag tcccaacacg tcgcacattc 1860 ctggaccccc agagctgtgg ggacctgctg caggctgtgc atctgttcgc caaggaactg 1920 gatgcgaaaa gcgtcacgct ggagaggagc cttggaggag ggcggtttgg ggagctgtgc 1980 tgtggctgct tgcagctccc cggtcgccag gagctgctcg tagccgtgca tatgctgagg 2040 gacagcgcct ccgactcaca gaggctcggc ttcctggccg aggccctcac gctgggccag 2100 tttgaccata gccacatcgt gcggctggag ggcgttgtta cccgaggaag caccttgatg 2160 attgtcaccg agtacatgag ccatggggcc ctggacggct tcctcaggcg gcacgagggg 2220 cagctggtgg ctgggcaact gatggggttg ctgcctgggc tggcatcagc catgaagtat 2280 ctgtcagaga tgggctacgt tcaccggggc ctggcagctc gccatgtgct ggtcagcagc 2340 gaccttgtct gcaagatctc tggcttcggg cggggccccc gggaccgatc agaggctgtc 2400 tacaccacta tgagtggccg gagcccagcg ctatgggccg ctcccgagac acttcagttt 2460 ggccacttca gctctgccag tgacgtgtgg agcttcggca tcatcatgtg ggaggtgatg 2520 gcctttgggg agcggcctta ctgggacatg tctggccaag acgtgatcaa ggctgtggag 2580 gatggcttcc ggctgccacc ccccaggaac tgtcctaacc ttctgcaccg actaatgctc 2640 gactgctggc agaaggaccc aggtgagcgg cccaggttct cccagatcca cagcatcctg 2700 agcaagatgg tgcaggaccc agagcccccc aagtgtgccc tgactacctg tcccagactg 2760 ttccggatac gccaatacct t 2781 <210> 44 <211> 737 <212> PRT <213> Human <400> 44 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Ala Ser Gly Ser         275 280 285 Arg Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala     290 295 300 Leu Leu Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg 305 310 315 320 Pro Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu                 325 330 335 Leu Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro             340 345 350 Gln Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu         355 360 365 Leu Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg     370 375 380 Phe Gly Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu 385 390 395 400 Leu Leu Val Ala Val His Met Leu Arg Asp Ser Ala Ser Asp Ser Gln                 405 410 415 Arg Leu Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His             420 425 430 Ser His Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Ser Thr Leu         435 440 445 Met Ile Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu     450 455 460 Arg Arg His Glu Gly Gln Leu Val Ala Gly Gln Leu Met Gly Leu Leu 465 470 475 480 Pro Gly Leu Ala Ser Ala Met Lys Tyr Leu Ser Glu Met Gly Tyr Val                 485 490 495 His Arg Gly Leu Ala Ala Arg His Val Leu Val Ser Ser Asp Leu Val             500 505 510 Cys Lys Ile Ser Gly Phe Gly Arg Gly Pro Arg Asp Arg Ser Glu Ala         515 520 525 Val Tyr Thr Thr Met Ser Gly Arg Ser Pro Ala Leu Trp Ala Ala Pro     530 535 540 Glu Thr Leu Gln Phe Gly His Phe Ser Ser Ala Ser Asp Val Trp Ser 545 550 555 560 Phe Gly Ile Ile Met Trp Glu Val Met Ala Phe Gly Glu Arg Pro Tyr                 565 570 575 Trp Asp Met Ser Gly Gln Asp Val Ile Lys Ala Val Glu Asp Gly Phe             580 585 590 Arg Leu Pro Pro Pro Arg Asn Cys Pro Asn Leu Leu His Arg Leu Met         595 600 605 Leu Asp Cys Trp Gln Lys Asp Pro Gly Glu Arg Pro Arg Phe Ser Gln     610 615 620 Ile His Ser Ile Leu Ser Lys Met Val Gln Asp Pro Glu Pro Pro Lys 625 630 635 640 Cys Ala Leu Thr Thr Cys Pro Arg Pro Pro Thr Pro Leu Ala Asp Arg                 645 650 655 Ala Phe Ser Thr Phe Pro Ser Phe Gly Ser Val Gly Ala Trp Leu Glu             660 665 670 Ala Leu Asp Leu Cys Arg Tyr Lys Asp Ser Phe Ala Ala Ala Gly Tyr         675 680 685 Gly Ser Leu Glu Ala Val Ala Glu Met Thr Ala Gln Asp Leu Val Ser     690 695 700 Leu Gly Ile Ser Leu Ala Glu His Arg Glu Ala Leu Leu Ser Gly Ile 705 710 715 720 Ser Ala Leu Gln Ala Arg Val Leu Gln Leu Gln Gly Gln Gly Val Gln                 725 730 735 Val <210> 45 <211> 2211 <212> DNA <213> Human <400> 45 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag ctgcctcagg gtccagggac cagagccccg ccattgtcgt caccgtagtg 900 accatctcgg ccctcctcgt cctgggctcc gtgatgagtg tgctggccat ttggaggagg 960 ccctgcagct atggcaaagg aggaggggat gcccatgatg aagaggagct gtatttccac 1020 ttcaaagtcc caacacgtcg cacattcctg gacccccaga gctgtgggga cctgctgcag 1080 gctgtgcatc tgttcgccaa ggaactggat gcgaaaagcg tcacgctgga gaggagcctt 1140 ggaggagggc ggtttgggga gctgtgctgt ggctgcttgc agctccccgg tcgccaggag 1200 ctgctcgtag ccgtgcatat gctgagggac agcgcctccg actcacagag gctcggcttc 1260 ctggccgagg ccctcacgct gggccagttt gaccatagcc acatcgtgcg gctggagggc 1320 gttgttaccc gaggaagcac cttgatgatt gtcaccgagt acatgagcca tggggccctg 1380 gacggcttcc tcaggcggca cgaggggcag ctggtggctg ggcaactgat ggggttgctg 1440 cctgggctgg catcagccat gaagtatctg tcagagatgg gctacgttca ccggggcctg 1500 gcagctcgcc atgtgctggt cagcagcgac cttgtctgca agatctctgg cttcgggcgg 1560 ggcccccggg accgatcaga ggctgtctac accactatga gtggccggag cccagcgcta 1620 tgggccgctc ccgagacact tcagtttggc cacttcagct ctgccagtga cgtgtggagc 1680 ttcggcatca tcatgtggga ggtgatggcc tttggggagc ggccttactg ggacatgtct 1740 ggccaagacg tgatcaaggc tgtggaggat ggcttccggc tgccaccccc caggaactgt 1800 cctaaccttc tgcaccgact aatgctcgac tgctggcaga aggacccagg tgagcggccc 1860 aggttctccc agatccacag catcctgagc aagatggtgc aggacccaga gccccccaag 1920 tgtgccctga ctacctgtcc caggcctccc accccactag cggaccgtgc cttctccacc 1980 ttcccctcct ttggctctgt gggcgcgtgg ctggaggccc tggacctgtg ccgctacaag 2040 gacagcttcg cggctgctgg ctatgggagc ctggaggccg tggccgagat gactgcccag 2100 gacctggtga gcctaggcat ctctttggct gaacatcgag aggccctcct cagcgggatc 2160 agcgccctgc aggcacgagt gctccagctg cagggccagg gggtgcaggt g 2211 <210> 46 <211> 749 <212> PRT <213> Human <400> 46 Met Glu Thr Cys Ala Gly Pro His Pro Leu Arg Leu Phe Leu Cys Arg                  5 10 15 Met Gln Leu Cys Leu Ala Leu Leu Leu Gly Pro Trp Arg Pro Gly Thr              20 25 30 Ala Glu Glu Val Ile Leu Leu Asp Ser Lys Ala Ser Gln Ala Glu Leu          35 40 45 Gly Trp Thr Ala Leu Pro Ser Asn Gly Trp Glu Glu Ile Ser Gly Val      50 55 60 Asp Glu His Asp Arg Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Leu  65 70 75 80 Glu Pro Asn Gln Asp Asn Trp Leu Gln Thr Gly Trp Ile Ser Arg Gly                  85 90 95 Arg Gly Gln Arg Ile Phe Val Glu Leu Gln Phe Thr Leu Arg Asp Cys             100 105 110 Ser Ser Ile Pro Gly Ala Ala Gly Thr Cys Lys Glu Thr Phe Asn Val         115 120 125 Tyr Tyr Leu Glu Thr Glu Ala Asp Leu Gly Arg Gly Arg Pro Arg Leu     130 135 140 Gly Gly Ser Arg Pro Arg Lys Ile Asp Thr Ile Ala Ala Asp Glu Ser 145 150 155 160 Phe Thr Gln Gly Asp Leu Gly Glu Arg Lys Met Lys Leu Asn Thr Glu                 165 170 175 Val Arg Glu Ile Gly Pro Leu Ser Arg Arg Gly Phe His Leu Ala Phe             180 185 190 Gln Asp Val Gly Ala Cys Val Ala Leu Val Ser Val Arg Val Tyr Tyr         195 200 205 Lys Gln Cys Arg Ala Thr Val Arg Gly Leu Ala Thr Phe Pro Ala Thr     210 215 220 Ala Ala Glu Ser Ala Phe Ser Thr Leu Val Glu Val Ala Gly Thr Cys 225 230 235 240 Val Ala His Ser Glu Gly Glu Pro Gly Ser Pro Pro Arg Met His Cys                 245 250 255 Gly Ala Asp Gly Glu Trp Leu Val Pro Val Gly Arg Cys Ser Cys Ser             260 265 270 Ala Gly Phe Gln Glu Arg Gly Asp Phe Cys Glu Ala Cys Pro Pro Gly         275 280 285 Phe Tyr Lys Val Ser Pro Arg Arg Pro Leu Cys Ser Pro Cys Pro Glu     290 295 300 His Ser Arg Ala Leu Glu Asn Ala Ser Thr Phe Cys Val Cys Gln Asp 305 310 315 320 Ser Tyr Ala Arg Ser Pro Thr Asp Pro Pro Ser Ala Ser Cys Thr Arg                 325 330 335 Pro Pro Ser Ala Pro Arg Asp Leu Gln Tyr Ser Leu Ser Arg Ser Pro             340 345 350 Leu Val Leu Arg Leu Arg Trp Leu Pro Pro Ala Asp Ser Gly Gly Arg         355 360 365 Ser Asp Val Thr Tyr Ser Leu Leu Cys Leu Arg Cys Gly Arg Glu Gly     370 375 380 Pro Ala Gly Ala Cys Glu Pro Cys Gly Pro Arg Val Ala Phe Leu Pro 385 390 395 400 Arg Gln Ala Gly Leu Arg Glu Arg Ala Ala Thr Leu Leu His Leu Arg                 405 410 415 Pro Gly Ala Arg Tyr Thr Val Arg Val Ala Ala Leu Asn Gly Val Ser             420 425 430 Gly Pro Ala Ala Ala Ala Gly Thr Thr Tyr Ala Gln Val Thr Val Ser         435 440 445 Thr Gly Pro Gly Ala Pro Trp Glu Glu Asp Glu Ile Arg Arg Asp Arg     450 455 460 Val Glu Pro Gln Ser Val Ser Leu Ser Trp Arg Glu Pro Ile Pro Ala 465 470 475 480 Gly Ala Pro Gly Ala Asn Asp Thr Glu Tyr Glu Ile Arg Tyr Tyr Glu                 485 490 495 Lys Gly Gln Ser Glu Gln Thr Tyr Ser Met Val Lys Thr Gly Ala Pro             500 505 510 Thr Val Thr Val Thr Asn Leu Lys Pro Ala Thr Arg Tyr Val Phe Gln         515 520 525 Ile Arg Ala Ala Ser Pro Gly Pro Ser Trp Glu Ala Gln Ser Phe Asn     530 535 540 Pro Ser Ile Glu Val Gln Thr Leu Gly Glu Ala Ala Ser Gly Ser Arg 545 550 555 560 Asp Gln Ser Pro Ala Ile Val Val Thr Val Val Thr Ile Ser Ala Leu                 565 570 575 Leu Val Leu Gly Ser Val Met Ser Val Leu Ala Ile Trp Arg Arg Pro             580 585 590 Cys Ser Tyr Gly Lys Gly Gly Gly Asp Ala His Asp Glu Glu Glu Leu         595 600 605 Tyr Phe His Phe Lys Val Pro Thr Arg Arg Thr Phe Leu Asp Pro Gln     610 615 620 Ser Cys Gly Asp Leu Leu Gln Ala Val His Leu Phe Ala Lys Glu Leu 625 630 635 640 Asp Ala Lys Ser Val Thr Leu Glu Arg Ser Leu Gly Gly Gly Arg Phe                 645 650 655 Gly Glu Leu Cys Cys Gly Cys Leu Gln Leu Pro Gly Arg Gln Glu Leu             660 665 670 Leu Val Ala Val His Met Leu Arg Asp Ser Ala Ser Asp Ser Gln Arg         675 680 685 Leu Gly Phe Leu Ala Glu Ala Leu Thr Leu Gly Gln Phe Asp His Ser     690 695 700 His Ile Val Arg Leu Glu Gly Val Val Thr Arg Gly Ser Thr Leu Met 705 710 715 720 Ile Val Thr Glu Tyr Met Ser His Gly Ala Leu Asp Gly Phe Leu Arg                 725 730 735 Val Ser Gly Pro Gly Ala Ala Asn Asp His Phe Ile Leu             740 745 <210> 47 <211> 2247 <212> DNA <213> Human <400> 47 atggagacct gcgccggtcc acacccgctg cgcctcttcc tctgccggat gcagctctgt 60 ctcgcgctgc ttttgggacc ctggcggcct gggaccgccg aggaagttat cctcctggat 120 tccaaagcct cccaggccga gctgggctgg actgcactgc caagtaatgg gtgggaggag 180 atcagcggcg tggatgaaca cgaccgtccc atccgcacgt accaagtgtg caatgtgctg 240 gagcccaacc aggacaactg gctgcagact ggctggataa gccgtggccg cgggcagcgc 300 atcttcgtgg aactgcagtt cacactccgt gactgcagca gcatccctgg cgccgcgggt 360 acctgcaagg agaccttcaa cgtctactac ctggaaactg aggccgacct gggccgtggg 420 cgtccccgcc taggcggcag ccggccccgc aaaatcgaca cgatcgcggc ggacgagagc 480 ttcacgcagg gcgacctggg tgagcgcaag atgaagctga acacagaggt gcgcgagatc 540 ggaccgctca gccggcgggg tttccacctg gcctttcagg acgtgggcgc atgcgtggcg 600 cttgtctcgg tgcgcgtcta ctacaagcag tgccgcgcca ccgtgcgggg cctggccacg 660 ttcccagcca ccgcagccga gagcgccttc tccacactgg tggaagtggc cggaacgtgc 720 gtggcgcact cggaagggga gcctggcagc cccccacgca tgcactgcgg cgccgacggc 780 gagtggctgg tgcctgtggg ccgctgcagc tgcagcgcgg gattccagga gcgtggtgac 840 ttctgcgaag cctgtccccc agggttttac aaggtgtccc cgcggcggcc cctctgctca 900 ccgtgcccag agcacagccg ggccctggaa aacgcctcca ccttctgcgt gtgccaggac 960 agctatgcgc gctcacccac cgacccgccc tcggcttcct gcacccggcc gccgtcggcg 1020 ccgcgggacc tgcagtacag cctgagccgc tcgccgctgg tgctgcgact gcgctggctg 1080 ccgccggccg actcgggagg ccgctcggac gtcacctact cgctgctgtg cctgcgctgc 1140 ggccgcgagg gcccggcggg cgcctgcgag ccgtgcgggc cgcgcgtggc cttcctaccg 1200 cgccaggcag ggctgcggga gcgagccgcc acgctgctgc acctgcggcc cggcgcgcgc 1260 tacaccgtgc gcgtggccgc gctcaacggc gtctcgggcc cggcggccgc cgcgggaacc 1320 acctacgcgc aggtcaccgt ctccaccggg cccggggcgc cctgggagga ggatgagatc 1380 cgcagggacc gagtggaacc ccagagcgtg tccctgtcgt ggcgggagcc catccctgcc 1440 ggagcccctg gggccaatga cacggagtac gagatccgat actacgagaa gggtcagagt 1500 gagcagactt actccatggt gaagacaggg gcgcccacag tcaccgtcac caacctgaag 1560 ccggctaccc gctacgtctt tcagatccgg gccgcttccc cggggccatc ctgggaggcc 1620 cagagtttta accccagcat tgaagtacag accctggggg aggctgcctc agggtccagg 1680 gaccagagcc ccgccattgt cgtcaccgta gtgaccatct cggccctcct cgtcctgggc 1740 tccgtgatga gtgtgctggc catttggagg aggccctgca gctatggcaa aggaggaggg 1800 gatgcccatg atgaagagga gctgtatttc cacttcaaag tcccaacacg tcgcacattc 1860 ctggaccccc agagctgtgg ggacctgctg caggctgtgc atctgttcgc caaggaactg 1920 gatgcgaaaa gcgtcacgct ggagaggagc cttggaggag ggcggtttgg ggagctgtgc 1980 tgtggctgct tgcagctccc cggtcgccag gagctgctcg tagccgtgca tatgctgagg 2040 gacagcgcct ccgactcaca gaggctcggc ttcctggccg aggccctcac gctgggccag 2100 tttgaccata gccacatcgt gcggctggag ggcgttgtta cccgaggaag caccttgatg 2160 attgtcaccg agtacatgag ccatggggcc ctggacggct tcctcagggt gagtggccct 2220 ggggctgcca atgaccactt tattctg 2247 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisence oligonucleotide <400> 48 cacctcccac atgatgatgc 20 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisence oligonucleotide <400> 49 catcctccac agccttgatc 20 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisence oligonucleotide <400> 50 cgtagtagta caccctccac 20 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisence oligonucleotide <400> 51 ctagttccga cacctcctac 20 <210> 52 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 52 gccgtggccg agatga 16 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 53 cctctcgatg ttcagccaaa g 21 <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe <400> 54 tgcccaggac ctggtgagcc tagg 24

[Brief description of drawings]

FIG. 1 Receptor protein of the present invention and human Eph
It shows homology in amino acid sequence with the receptor family. In the figure, NEPHA.PRO is the receptor protein NEPHA of the present invention and EphA7.PRO is the human Eph receptor.
EphA7, EphA1.PRO, human Eph receptor EphA1, EpA
hA2.PRO is the human Eph receptor EphA2, EphA3.PRO is
Human Eph receptor EphA3, EphA4.PRO, human Eph receptor EphA4, EphA5.PRO, human Eph receptor EphA5
And EphA8.PRO represent human Eph receptor EphA8, respectively.

FIG. 2 shows the amino acid sequence homology between the receptor protein NEPHA of the present invention and the human Eph receptor family (continuation of FIG. 1).

FIG. 3 shows the amino acid sequence homology between the receptor protein NEPHA of the present invention and the human Eph receptor family (continuation of FIG. 2).

FIG. 4 shows the amino acid sequence homology between the receptor protein NEPHA of the present invention and the human Eph receptor family (continuation of FIG. 3).

FIG. 5 shows the amino acid sequence homology between the receptor protein NEPHA of the present invention and the human Eph receptor family (continuation of FIG. 4).

FIG. 6 shows the homology in the amino acid sequence between NEPHA and NEPHA-SV1-5. NEPHA.pro is N
EPHA, NEPHA-SV1.PRO, NEPHA-SV1
NEPHA-SV2.PRO, NEPHA-SV2, NEPHA-S
V3.PRO is NEPHA-SV3, NEPHA-SV4.PRO is
NEPHA-SV4, NEPHA-SV5.PRO is NEPHA
-SV5 is shown respectively.

FIG. 7 shows the homology in the amino acid sequences of NEPHA and NEPHA-SV1-5 (continuation of FIG. 6).

FIG. 8 shows the homology in the amino acid sequences of NEPHA and NEPHA-SV1-5 (continuation of FIG. 7).

FIG. 9 shows the homology in the amino acid sequence between NEPHA and NEPHA-SV1-5 (continuation of FIG. 8).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 45/00 A61P 3/00 4C084 48/00 3/10 4C085 A61P 3/00 25/00 101 4C086 3 / 10 25/28 4H045 25/00 101 35/00 25/28 C07K 14/705 35/00 16/28 C07K 14/705 C12N 1/15 16/28 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 21/08 5/10 C12Q 1/02 C12P 21/02 1/68 A 21/08 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/574 A G01N 33 / 15 Z 33/50 33/577 B 33/574 C12N 15/00 ZNAA 5/00 A 33/577 A61K 37/02 (72) Inventor Naoki Ishii 4-16-1 Namiki, Tsukuba City, Ibaraki Prefecture (72) Inventor Eiji Sunahara 5351-5F, Ami, Ami-machi, Inashiki-gun, Ibaraki Prefecture (reference) 2G045 AA 26 AA29 AA40 BA13 BB03 BB20 CB17 CB21 DA12 DA13 DA14 DA36 DA78 FB02 FB03 FB04 FB06 FB07 4B024 AA01 AA12 BA63 CA04 CA09 CA12 CA20 DA02 DA03 DA06 EA04 GA13 GA18 GA19 HA03 HA11 HA12 HA17 4B063 QA01 QA05 QA19 QQ21 QQ41 QQ53 QQ61 QQ89 QR08 QR32 QR35 QR40 QR42 QR56 QR62 QR77 QR80 QS16 QS25 QS34 QS36 QX02 4B064 AG20 AG27 CA02 CA10 CA19 CA20 CC01 CC24 CE06 CE12 DA05 DA14 4B065 AA26X AA58X AA72X AA90X AA93X AA93Y AB01 AC14 BA02 BA05 BA25 BB01 BC03 BC07 BD14 BD16 BD17 CA24 CA45 CA46 4C084 AA02 AA06 AA07 AA13 AA17 BA01 BA08 BA22 BA23 MA01 NA14 ZA022 ZA152 ZA162 ZB262 ZC352 ZC412 4C085 AA03 AA13 AA14 CC32 EE01 GG01 4C086 AA01 AA02 AA03 AA04 EA16 MA01 MA04 NA14 ZA02 ZA15 ZA16 ZB26 ZC35 ZC41 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA75 DA76 EA20 EA51 FA71 FA72 FA74 GA01 GA26

Claims (60)

[Claims] 1. An amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 38. An Eph receptor protein or a salt thereof, which comprises: 2. It contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46. A characteristic Eph receptor protein or a salt thereof. 3. The Eph receptor according to claim 1, which contains the amino acid sequence represented by SEQ ID NO: 11, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24 or SEQ ID NO: 38. Protein or its salt. 4. The Eph receptor protein or a salt thereof according to claim 2, which contains the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46. 5. A partial peptide of the Eph receptor protein according to claim 1 or 2, or a salt thereof. 6. The 60th position to the 73rd position or the 491st position to the 50th position of the amino acid sequence represented by SEQ ID NO: 11.
The partial peptide according to claim 5, which comprises the 4th amino acid sequence, or a salt thereof. 7. A polynucleotide containing a polynucleotide encoding the Eph receptor protein according to claim 1 or claim 2 or the partial peptide according to claim 5. 8. The polynucleotide according to claim 7, which is DNA. 9. The polynucleotide according to claim 12, which has a nucleotide sequence represented by SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 39. 10. The polynucleotide according to claim 12, which has a base sequence represented by SEQ ID NO: 15, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45 or SEQ ID NO: 47. 11. A recombinant vector containing the polynucleotide according to claim 7. 12. A transformant transformed with the recombinant vector according to claim 11. 13. The transformant according to claim 12 is cultured to produce the Eph receptor protein according to claim 1 or 2, or the partial peptide according to claim 5, wherein the transformant is produced. E of item 2
A method for producing a ph receptor protein or a salt thereof or the partial peptide according to claim 5 or a salt thereof. 14. The Eph according to claim 1 or 2.
An antibody against the receptor protein or a salt thereof or the partial peptide according to claim 5 or a salt thereof. 15. The method according to claim 14, which is a polyclonal antibody.
The described antibody. 16. The method according to claim 14, which is a monoclonal antibody.
The described antibody. 17. The 60th to 73rd positions or the 491st to 5th positions of the amino acid sequence represented by SEQ ID NO: 11.
The antibody according to claim 14, which specifically reacts with a peptide having the 04th amino acid sequence. 18. The Eph according to claim 1 or 2.
The antibody according to claim 14, which is a neutralizing antibody that inactivates signal transduction of a receptor protein. 19. A medicine comprising the antibody according to claim 14. 20. The medicine according to claim 19, which is an immunotoxin. 21. The medicine according to claim 19, which is an anticancer agent. 22. A diagnostic agent comprising the antibody according to claim 14. 23. The Eph according to claim 1 or 2.
A ligand for the Eph receptor protein or a salt thereof according to claim 1 or 2, which can be obtained by using the receptor protein or the partial peptide according to claim 5 or a salt thereof. 24. A medicine comprising the ligand according to claim 23. 25. The Eph according to claim 1 or 2.
24. A receptor protein, the partial peptide according to claim 5, or a salt thereof is used.
A method for determining the described ligand. 26. The ligand according to claim 23 and claim 1.
Alternatively, a method for screening a compound or a salt thereof that changes the binding property to the Eph receptor protein or the salt thereof according to claim 2. 27. The ligand according to claim 23 and claim 1.
A kit for screening a compound or a salt thereof, which changes the binding property to the Eph receptor protein or the salt thereof according to claim 2. 28. The binding property between a ligand obtainable by using the screening method according to claim 26 or the screening kit according to claim 27 and the Eph receptor protein according to claim 1 or 2 or a salt thereof is changed. A compound or a salt thereof. 29. A medicine comprising the compound according to claim 28 or a salt thereof. 30. A polynucleotide which hybridizes with the polynucleotide according to claim 7 under high stringent conditions. 31. A diagnostic agent containing the polynucleotide according to claim 7. 32. The diagnostic agent according to claim 31, which is a diagnostic agent for cancer. 33. The method for quantifying Eph receptor protein mRNA according to claim 1 or 2, wherein the polynucleotide according to claim 7 or a part thereof is used. 34. The method for quantifying the Eph receptor protein according to claim 1 or 2, wherein the antibody according to claim 14 is used. 35. The method for diagnosing a disease associated with the Eph receptor function according to claim 1 or 2, wherein the quantification method according to claim 33 or 34 is used. 36. The diagnostic method according to claim 35, wherein the disease is cancer. 37. A screening method for a compound or a salt thereof which changes the expression level of the Eph receptor protein according to claim 1 or 2, wherein the quantification method according to claim 33 is used. 38. The cell membrane according to claim 1 or 2, wherein the quantification method according to claim 33 is used.
A method for screening a compound or a salt thereof, which changes the amount of Eph receptor protein described above. 39. The Ep according to claim 1 or 2, which can be obtained by using the screening method according to claim 37.
A compound or a salt thereof that changes the expression level of h receptor protein. 40. A compound or a salt thereof which alters the amount of Eph receptor protein according to claim 1 or 2 in a cell membrane obtainable by using the screening method according to claim 38. (41) A pharmaceutical comprising the compound according to (39) or (40) or a salt thereof. 42. The medicine according to claim 41, which is an anticancer agent. 43. The Eph according to claim 1 or claim 2.
A multimer of receptor proteins. 44. The multimer according to claim 43, which is a dimer. 45. A dimer of the Eph receptor protein according to claim 1 or 2 and a receptor belonging to the Eph receptor protein family.
The described multimer. 46. The Eph according to claim 1 or claim 2.
Screening for a substance having an action of inhibiting the multimerization of the Eph receptor protein according to claim 1 or 2 and the receptor belonging to the Eph receptor family, characterized in that a receptor protein and a receptor belonging to the Eph receptor family are used. Method. 47. The Eph according to claim 1 or claim 2.
A substance having an action of inhibiting the multimerization of the Eph receptor protein according to claim 1 or 2 and the receptor belonging to the Eph receptor family, which comprises a receptor protein and a receptor belonging to the Eph receptor family. Screening kit. 48. A large amount of the Eph receptor protein according to claim 1 or 2 and a receptor belonging to the Eph receptor family, which can be obtained using the screening method according to claim 46 or the screening kit according to claim 47. Substances that have the effect of inhibiting somatization. 49. A medicine comprising the substance according to claim 48. 50. The medicine according to claim 49, which is an anticancer agent. 51. An antisense polynucleotide containing a base sequence complementary to the polynucleotide according to claim 7, or a part thereof. 52. A medicine comprising the antisense polynucleotide according to claim 51. 53. The Eph according to claim 1 or claim 2.
A medicament containing a receptor protein. 54. The medicine according to claim 53, which is a vaccine preparation. 55. An antibody against a receptor having an action of inhibiting the activity of the receptor. 56. A medicine comprising the antibody according to claim 55. 57. An antisense polynucleotide containing a base sequence complementary to a polynucleotide containing a polynucleotide encoding a receptor having an action of inhibiting the activity of a receptor, or a part thereof. 58. A medicine comprising the antisense polynucleotide according to claim 57. 59. A method for preventing / treating cancer, which comprises administering an effective amount of the antibody of claim 14 to a mammal. 60. Use of the antibody according to claim 14 for producing a prophylactic / therapeutic agent for cancer.