JP2001238685A - New gene and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein that is useful as a medicine for preventing and treating a disease such as hypoglycemia, and to provide a DNA encoding the protein, wherein the protein and the DNA are useful as agents for screening a compound inhibiting the binding of the protein with IRAP (insulin responsive aminopeptidase) or GLUT4 (glucose transporter 4), wherein the compound is useful as a medicine for preventing and treating a disease such as melitemia and diabetes. SOLUTION: A new protein, a DNA encoding the protein, a method for producing the protein, a medicine containing the protein, an antibody against the protein, a method for screening a compound inhibiting the binding of the protein with IRAP, and a compound obtained by the screening, are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel gene and its use. More specifically, the present invention relates to a novel insulin responsive aminopeptidase binding protein (IRAP-BP) gene and its use.

[0002]

BACKGROUND OF THE INVENTION Blood glucose levels are regulated by the action of insulin and by the uptake of glucose in skeletal muscle and adipose tissue. In diabetes, this decrease in action results from sustained hyperglycemia. The uptake of glucose into cells requires the intervention of a membrane protein called a glucose transporter (sugar transport carrier). Currently, seven types of glucose transporters, GLUT1 to GLUT7, are known (Bell et al., J. Biol. Chem., 26
8, 3352-3356, 1993; Olson & Pessin, Annu. Rev. Nut
r. 16, 235-256, 1996). Among them, glucose transporter 4 (GLUT4) whose expression in skeletal muscle and adipose tissue is mainly involved in the glucose transport activity by insulin
(Fukumoto et al., Proc. Natl. Acad. Sci. US
A., 85, 5434-5438, 1988; Birnbaum et al., Cell, 5
7, 305-315, 1989). Normally, GLUT4 is present in intracellular vesicles called GLUT4 endoplasmic reticulum, but when blood glucose rises, it is thought that insulin uptake promotes glucose uptake by translocation to the cell membrane by the action of insulin. (Bell et al., Diabetes Car
e, 13, 198-208, 1990; Czech et al., Trend.Bioche
m. Sci., 17, 197-201, 1992). In order to elucidate the molecular mechanism of GLUT4 ER transport, identification of not only GLUT4 itself but also other proteins constituting GLUT4 ER has been performed. At present, VAMPs (vesicle-assosiated membrane p
roteins; Cain et al., J. Biol. Chem., 267, 11681-1
1634, 1992), SCAMPs (secretory component-associate
d membrane proteins; Thoidis et al, J. Biol. Chem.,
268, 11691-11696, 1993; Laurie et al., J. Biol. C
hem, 268, 19110-19117, 1993), phosphatidylinositol
4-kinase (Del Vacchio & Pilch, J. Biol. Chem, 266,
13278-13283, 1991), small GTP-binding protein Rab4 (Cor
mont et al., J. Biol. Chem., 268, 19491-19497, 199
3) In addition to IRAP (insulin-responsive aminopeptida
se; Kandror & Pilch, Proc. Natl. Acad. Sci. USA, 9
1, 8017-8021, 1994, Kandror et al., J. Biol. Chem.
269, 30777-30780, 1994, Keller et al, J. Biol. Che
m., 270, 23612-23618, 1995). IRAP
It is a one-time transmembrane membrane protein also called gp160, and is localized in the GLUT4 endoplasmic reticulum in cells. In terms of protein structure, the amino-terminal (N-terminal) 109 amino acids are a cytoplasmic domain, followed by a 22-amino acid transmembrane domain and a carboxy-terminal (C-terminal) 785 amino acid extracellular domain ( Kandror & Pilch, Proc. Natl. Acad.
Sci. USA, 91, 8017-8021, 1994; Keller et al, J. B
iol. Chem., 270, 23612-23618, 1995). The extracellular domain is a zinc-dependent protease (aminopeptidase) whose activity has been confirmed (Kandror et al., J. Bi
ol. Chem. 269, 30777-30780, 1994). When a peptide corresponding to the N-terminal domain (cytoplasmic domain) of these domains is injected into cells, GLUT4 endoplasmic reticulum is translocated to the cell surface. Is expected to be a protein that binds to IRAP (Waters et al., J. Biol. Chem, 2
72, 23323-23327, 1997). In addition, the cDNA and deduced amino acid sequence of the FHOS gene are known (Westendorfet a
l., Gene, 232, 173-182, 1999; Genbank Accession N
o. AF113615), it has not been reported that FHOS binds to molecules constituting the GLUT4 endoplasmic reticulum such as IRAP.

[0003]

SUMMARY OF THE INVENTION The present invention relates to a novel IRAP-B
An object of the present invention is to provide P, its gene, a method for screening a compound having a blood glucose lowering effect using the same, a compound obtained by the screening method, and the like.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, the yeast two-hybrid
brid method (Fields & Strenglanz, Trends Genet., 10, 28
6-292, 1994; Brent & Finley, Annu. Rev. Genet., 31,
663-704, 1997), a new IRAP-BP gene was successfully cloned from a cDNA library derived from human skeletal muscle. The present inventors have further studied and completed the present invention.

That is, the present invention relates to (1) SEQ ID NO: 1.
A protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by
(2) a protein or salt thereof according to the above (1), wherein the substantially identical amino acid sequence is the amino acid sequence represented by SEQ ID NO: 15, and (3) a DNA encoding the protein according to the above (1). (4) The above (1)
The DNA encoding the protein described in the above is a DNA containing the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 16.
(5) The DNA according to the above (3), which is NA.
(6) a transformant transformed with the recombinant vector according to the above (5), and (7) culturing the transformant according to the above (6). )) Producing, accumulating, and collecting the protein described in (1), (8) the method for producing the protein or salt thereof according to (1), (8) the protein or salt thereof, or (2) A) a medicament comprising the DNA of the above); A diagnostic agent comprising the antibody of the above (10), (12) SEQ ID NO: 2
Or a protein containing the amino acid sequence represented by SEQ ID NO: 17 or a salt thereof, (13) a DNA containing a DNA encoding the protein of (12),
(14) D encoding the protein of (12) above
The DNA according to the above (13), wherein NA is a DNA containing the base sequence represented by SEQ ID NO: 4 or SEQ ID NO: 18.
NA, (15) a recombinant vector containing the DNA of (13), (16) a transformant transformed with the recombinant vector of (15), (17) a transformant of (1)
(6) a method for producing the protein or the salt thereof according to (12), wherein the transformant according to (6) is cultured to produce and accumulate the protein according to (12), and collecting the protein; A medicament comprising the protein or salt thereof according to the above (12) or the DNA according to the above (13); (19) the above (1) which is a preventive or therapeutic agent for hypoglycemia;
8) the medicine according to the above, (20) an antibody against the protein or a salt thereof according to the above (12), (21) a diagnostic agent comprising the antibody according to the above (20), (22) SEQ ID NO: 1.
A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, a partial peptide of a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: : A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by 2, SEQ ID NO:
2. A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by 2, or a salt thereof, wherein the amino acid sequence is the same as or substantially the same as the amino acid sequence represented by SEQ ID NO: 1. A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 2, a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or A method for screening a compound or a salt thereof that inhibits the binding of the salt to insulin-responsive aminopeptidase or glucose transporter 4, (2)
3) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1,
A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A protein or a cell having the ability to produce a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2; A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a compound that inhibits the binding of a salt thereof to insulin-responsive aminopeptidase or glucose transporter 4; A method for screening a salt thereof, (24) a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, or a protein identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Partial peptide of a protein having the same amino acid sequence, SEQ ID NO:
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 2, a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 containing a salt thereof; an amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Partial peptide of a protein having a sequence, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, identical or substantially the same as the amino acid sequence represented by SEQ ID NO: 2 Partial peptide of a protein having the amino acid sequence of, or a salt thereof and insulin responsive Minopepuchidaze or kit for screening a compound or its salt that inhibits the binding of glucose transporter 4, (25) SEQ ID NO:
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by No. 1, a partial peptide or a sequence of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 A portion of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a portion of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 SEQ ID NO: containing cells having the ability to produce the peptide
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by No. 1, a partial peptide or a sequence of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO:
2. A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by 2, or a compound or a salt thereof, which inhibits the binding between insulin responsive aminopeptidase or glucose transporter 4 and a salt thereof. A screening kit, (26) the screening method according to the above (22),
The screening method according to the above (23),
A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 obtained by using the screening kit described in the above or the screening kit described in the above (25);
A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by 1, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a sequence A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by No. 2, or a compound thereof which inhibits binding between insulin responsive aminopeptidase or glucose transporter 4 or a salt thereof Salt, (27) a medicament comprising the compound according to (26) or a salt thereof, and (28) a prophylactic / therapeutic agent for hyperglycemia or diabetes comprising the compound according to (26) or a salt thereof. ,
(29) a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 Partial peptide of the protein, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 (30) a drug comprising a compound that inhibits the binding of a partial peptide of a protein having, or a salt thereof, and insulin-responsive aminopeptidase or glucose transporter 4, or a salt thereof, (30) represented by SEQ ID NO: 1. A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence , A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 Inhibits the binding of a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a salt thereof to insulin-responsive aminopeptidase or glucose transporter 4 (31) a protein or sequence containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1; Identical or substantially identical to the amino acid sequence represented by No. 2 (32) a medicine comprising a compound or a salt thereof that promotes or suppresses the expression of a protein containing a amino acid sequence, (32) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Prevention and treatment of hyperglycemia or diabetes comprising a compound or a salt thereof that suppresses the expression of a protein or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 (33) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A prophylactic / therapeutic agent for hypoglycemia, comprising a compound or a salt thereof that promotes the expression of a protein containing (1) The protein or a salt thereof, the DNA of the above (3), the protein of the above (12) or a salt thereof, or the above (1) for producing a medicine comprising the DNA of the above (13). )) The use of the protein described in (3) above, the DNA described in (12) above or the salt thereof or the DNA described in (13) above, (35) the protein described in (1) above or a salt thereof. The DNA according to (3) above, (12)
A method for preventing or treating hypoglycemia, which comprises administering the protein or the salt thereof or the DNA of the above (13) to a mammal; (36) containing the compound or the salt thereof of the above (26). Or a salt thereof for the manufacture of a medicament comprising: (37) administering a compound or a salt thereof according to the above (26) to a mammal; It provides a method for preventing and treating diabetes.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the present invention (hereinafter referred to as protein I of the present invention)
) Or a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 (hereinafter sometimes referred to as protein II) used in the present invention is a warm-blooded animal Cells (eg, human, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells) , Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, Cells or stromal cells, or precursors of these cells, stem cells, or cancer cells) or any tissue in which these cells are present, for example, the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus) , Thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum),
Spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland , Peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joints, proteins derived from skeletal muscle, etc.,
It may be a synthetic protein. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes about 98% of the amino acid sequence represented by SEQ ID NO: 1.
As mentioned above, preferably, the amino acid sequence has about 99% or more homology.

The protein of the present invention having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 has, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1. However, a protein having substantially the same properties as the protein having the amino acid sequence represented by SEQ ID NO: 1 is preferred. As substantially the same property, for example, IRAP or
GLUT4 binding activity and the like. Substantially the same means that those properties are qualitatively the same. Therefore, it is preferable that the binding activity to IRAP or GLUT4 is equivalent, but quantitative factors such as the degree of these properties and the molecular weight of the protein may be different. The protein I of the present invention includes, for example, SEQ ID NO: 1
1 or 2 or more in the amino acid sequence represented by (preferably about 1 to 25, preferably about 1 to 10,
More preferably, an amino acid sequence in which a number (1 to 5) amino acids have been deleted, and 1 or 2 or more (preferably about 1 to 25, preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequence to which about (more preferably number (1 to 5)) amino acids have been added,
One or more (preferably about 1 to 25, preferably 1 to 1) amino acid sequences represented by SEQ ID NO: 1
About 0, more preferably number (1-5) amino acids inserted, 1 or 2 or more (preferably, in the amino acid sequence represented by SEQ ID NO: 1)
A protein containing an amino acid sequence in which about 1 to 25, preferably about 1 to 10, and more preferably number (1 to 5) amino acids have been substituted with another amino acid, or an amino acid sequence obtained by combining them; So-called muteins are also included. More specifically, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes, for example, the amino acid sequence represented by SEQ ID NO: 15. Examples of the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 include an amino acid sequence having about 98% or more, preferably about 99% or more homology with the amino acid sequence represented by SEQ ID NO: 2. Is raised. Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 include, for example,
A protein having substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 and having substantially the same properties as the protein having the amino acid sequence represented by SEQ ID NO: 2 is preferred. Substantially the same properties include, for example, the binding activity to IRAP or GLUT4. Substantially the same means that those properties are qualitatively the same. So IRAP or GLUT
Preferably, the binding activities to 4 are the same, but the quantitative factors such as the degree of these properties and the molecular weight of the protein may be different. Further, as the protein II, for example, 1 in the amino acid sequence represented by SEQ ID NO: 2
Or two or more (preferably about 1 to 25, preferably about 1 to 10, more preferably a number (1 to 5)
Amino acids), SEQ ID NO:
2 or more (preferably about 1 to 25, preferably about 1 to 10,
More preferably, an amino acid sequence to which number (1 to 5) amino acids are added, 1 or 2 or more (preferably about 1 to 25, preferably 1 to 10) to the amino acid sequence represented by SEQ ID NO: 2 Degree, more preferably number (1-5) amino acids inserted,
1 or 2 or more (preferably about 1 to 25, preferably 1 to 2) in the amino acid sequence represented by SEQ ID NO: 2
A so-called mutein such as a protein containing an amino acid sequence in which about 10 to about 10 amino acids are substituted with another amino acid, and more preferably a number (1 to 5) of amino acids, or an amino acid sequence obtained by combining them is also included.

[0008] More specifically, the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 includes, for example, the amino acid sequence represented by SEQ ID NO: 17. According to the protein labeling convention, the left end is the N-terminal (amino terminal) and the right end is the C
Terminal (carboxyl terminal). The protein I or protein II of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, usually has a carboxyl group (-CO
OH) or carboxylate (—COO ⁻ ),
C-terminal amide (-CONH ₂₎ or ester (-C
OOR). Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl and n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl And C _6-12 aryl groups such as α-naphthyl, for example, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl.
_{7-14 An} aralkyl group, a pivaloyloxymethyl group and the like are used. Protein I or protein II of the present invention
Has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the protein I or protein II of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Furthermore, in the protein I or protein II of the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) has a protecting group (for example, C _1-6 alkanoyl such as formyl group and acetyl group). _1-6 acyl group), N-terminal glutamine residue formed by cleavage in vivo, pyroglutamine oxidation, substituent on the side chain of amino acid in the molecule (eg, -O
H, -SH, amino group, imidazole group, indole group, guanidino group, etc. are suitable protecting groups (for example, C _1-6 acyl groups such as C _1-6 alkanoyl groups such as formyl group and acetyl group). Protected proteins and complex proteins such as so-called glycoproteins to which sugar chains are bound are also included. Specific examples of the protein I of the present invention include, for example, a protein derived from human skeletal muscle having the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 1
And a mouse-derived protein represented by No. 5. Further, specific examples of protein II include, for example,
Human spleen-derived protein having the amino acid sequence represented by SEQ ID NO: 2, FHOS protein described in the literature (Westendorf et al., Gene, 232, 173-182, 1999; Gen)
bank Accession No. AF113615), a mouse-derived protein represented by SEQ ID NO: 17, and the like.

The partial peptide of the protein I of the present invention (hereinafter sometimes abbreviated as the partial peptide I of the present invention) is the partial peptide of the protein I of the present invention described above, and is preferably Inventive protein I
Any material may be used as long as it has the same properties as described above. For example, at least 20 or more, preferably 50 or more, of the constituent amino acid sequences of the protein I of the present invention,
More preferably, a peptide having an amino acid sequence of 70 or more, more preferably 100 or more, and most preferably 200 or more is used. Particularly preferably, 200 or more 1 continuous from the C-terminus of the protein I of the present invention.
Less than 190 (more preferably, 200 or more and 400
Peptides having an amino acid sequence consisting of (less than) amino acid residues. Further, the partial peptide I of the present invention
Is 1 or 2 or more (preferably about 1 to 10, more preferably number (1 to 5)
Amino acids), or one or more (preferably about 1 to 10, more preferably number (1 to 5)) amino acids are added to the amino acid sequence, or One or more (preferably about 1 to 10, more preferably number (1 to 5)) amino acid sequences
Amino acids), or one or more (preferably about 1 to 10)
(More preferably, about (1 to 5) amino acids) may be substituted with another amino acid. In the partial peptide I of the present invention, the C-terminus usually has a carboxyl group (—COO).
H) or carboxylate (—COO ⁻ ),
As the protein I of the present invention described above, C-terminal, it may be an amide (-CONH ₂₎ or ester (-COOR). When the partial peptide I of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the partial peptide I of the present invention includes a carboxyl group amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Further, the partial peptide I of the present invention includes, as in the case of the above-mentioned protein I of the present invention, those in which the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group, Glutamine residue generated by cleavage of the side in vivo is pyroglutamine-oxidized, substituent on the side chain of amino acid in the molecule is protected by an appropriate protecting group, or so-called glycopeptide to which a sugar chain is bound And the like. The partial peptide I of the present invention can be used as an antigen for preparing an antibody, and can also be used for screening a compound that inhibits the binding between the protein I of the present invention and IRAP or GLUT4.

The partial peptide of protein II (hereinafter sometimes abbreviated as partial peptide II) is a partial peptide of protein II described above, and preferably has the same properties as protein II described above. Any of them may be used. For example, protein I
A peptide having an amino acid sequence of at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more of the constituent amino acid sequences of I is used.
Particularly preferably, a peptide having an amino acid sequence consisting of 200 or more and less than 1164 (more preferably 200 or more and less than 400) amino acid residues consecutive from the C-terminus of the protein I of the present invention is used. Also,
Partial peptide II has 1 or 2 in its amino acid sequence.
Or more (preferably about 1 to 10, more preferably number (1 to 5)) amino acids are deleted, or 1 or 2 or more (preferably 1 to 1)
About 0, more preferably number (1 to 5) amino acids are added, or one or more (preferably about 1 to 10, more preferably number (1 to 5)) amino acids are added to the amino acid sequence. )) Amino acids, or one or more (preferably 1 to
About 10 (preferably about 1 to 5) amino acids may be substituted with another amino acid. Also,
The partial peptide II has a carboxyl group (-C
OOH) or carboxylate (—COO ⁻ ), but the C-terminal may be an amide (—CONH ₂ ) or an ester (—COOR), as in Protein II described above. When the partial peptide II of the present invention has a carboxyl group (or a carboxylate) other than the C-terminus, the carboxyl group amidated or esterified is also included in the partial peptide II of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Further, the partial peptide II has the same structure as the protein II of the present invention described above, except that the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected with a protecting group, and the N-terminal side is in vivo. Pyroglutamine-oxidized glutamine residues generated by cleavage in the above, those in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, or those in which a sugar chain is bonded to a so-called glycopeptide Peptides are also included. The partial peptide II can also be used as an antigen for preparing an antibody, and the protein II of the present invention and IRAP or GL
It can also be used to screen for compounds that inhibit binding to UT4. Protein I or partial peptide I or protein II or partial peptide I of the present invention
As the salt of I, a salt with a physiologically acceptable acid (eg, an inorganic acid or an organic acid) or a base (eg, an alkali metal salt) is used. Particularly, a physiologically acceptable acid addition salt is used. preferable. Such salts include, for example, 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) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. Protein I, partial peptide I, protein II, partial peptide I of the present invention
I or a salt thereof can be produced from a cell or tissue of a warm-blooded animal by a known method for purifying a protein, or culturing a transformant containing DNA encoding the protein or peptide. Can also be manufactured. Further, it can also be produced according to the peptide synthesis method described later. When produced from mammalian tissues or cells, the homogenized mammalian tissues or cells are extracted with an acid or the like, and the extract is subjected to a combination of chromatography such as reverse phase chromatography and ion exchange chromatography. It can be purified and isolated.

The protein I of the present invention, the partial peptide I,
For the synthesis of protein II, partial peptide II or its amide, or a salt thereof, a commercially available resin for protein synthesis can be usually used. Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,
-Benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-
(Hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin and the like. Using such a resin, α-
An amino acid having an amino group and a side chain functional group appropriately protected is condensed on a resin according to the sequence of a target protein or peptide according to various known condensation methods. At the end of the reaction, a protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein, peptide or an amide thereof. Regarding the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used,
Particularly, carbodiimides are preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. Activation by these involves adding the protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt) or using a symmetrical anhydride or HO
The protected amino acid can be added as a Bt ester or HOOBt ester to the resin after activation of the protected amino acid in advance. The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol,
Sulfoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond forming reaction, and is usually appropriately selected from a range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-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 by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2
-Nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used. The carboxyl group is
For example, alkyl esterification (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl,
Cyclohexyl, cycloheptyl, cyclooctyl, 2
Linear, branched or cyclic alkyl esterification such as adamantyl), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification) , Phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like. The hydroxyl group of serine is
For example, it can be protected by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, and groups derived from carbonic acid such as benzyloxycarbonyl groups and ethoxycarbonyl groups. Used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. Examples of the protecting group for the phenolic hydroxyl group of tyrosine include, for example, Bzl, Cl ₂ -Bzl,
Nitrobenzyl, Br-Z, t-butyl and the like are used.
As a protecting group for imidazole of histidine, for example,
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc
Are used. Examples of the activated carboxyl group of the raw material include, for example, the corresponding acid anhydride, azide,
Activated ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt ) And the like) are used.
As the activated amino group of the raw material, for example,
The corresponding phosphoric amide is used. As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon,
Also, acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia Also used. The elimination reaction by the above acid treatment is generally performed at a temperature of about -20 ° C to 40 ° C,
In the acid treatment, for example, anisole, phenol,
It is effective to add a cation scavenger such as thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like. In addition, 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 1,2-ethanedithiol, 1,4-butane described above. 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. Protection of functional groups that should not participate in the reaction of the raw materials, as well as protecting groups,
The elimination of the protecting group and the activation of the functional group involved in the reaction can be appropriately selected from known groups or known means.

As another method for obtaining an amide of the target protein or peptide, for example, after protecting the α-carboxyl group of the carboxy terminal amino acid by amidation, a peptide (protein) chain is attached to the amino group side. After extending to a desired chain length, a protein or peptide from which only the protecting group for the N-terminal α-amino group of the peptide chain is removed and a protein or peptide from which only the protecting group for the C-terminal carboxyl group is removed are produced. The two proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein or peptide. This crude protein or peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein or peptide. In order to obtain an ester of the desired protein or peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired amino acid ester is prepared in the same manner as in the amide of the protein or peptide. Can be obtained. The partial peptide I, the partial peptide II or a salt thereof of the present invention can be produced according to a peptide synthesis method known per se, or by cleaving the protein I or the protein II of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, a partial peptide or amino acid that can constitute the partial peptide of the present invention is condensed with the remaining portion (peptide or amino acid), and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide. can do. Examples of the known condensation method and elimination of the protecting group include the methods described in (1) to (4) below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, NewYork (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten

After the reaction, the protein or peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the protein or peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the protein or peptide obtained by a salt is a known method or It can be converted to a free form or another salt by an analogous method. D encoding protein I or protein II of the present invention
The NA may be any as long as it contains the nucleotide sequence encoding the above-described protein I or protein II of the present invention. Genome DN
A, any of the genomic DNA library, the cDNA derived from the cells / tissues described above, the cDNA library derived from the cells / tissues described above, and the synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, Reverse Transcription was directly performed using a total RNA or mRNA fraction prepared from the cells and tissues described above.
scriptase Polymerase Chain Reaction (RT-P
It can also be amplified by the CR method). As the DNA encoding the protein I of the present invention, for example, a DNA containing the base sequence represented by SEQ ID NO: 3
(DNA containing the base sequence of base numbers 19 to 3588 in the base sequence represented by SEQ ID NO: 23), DNA containing the base sequence represented by SEQ ID NO: 23, base sequence represented by SEQ ID NO: 16 DNA containing the base sequence represented by SEQ ID NO: 26, DNA containing the base sequence represented by SEQ ID NO: 3 (the base sequence represented by SEQ ID NO: 23
A base sequence that hybridizes under high stringent conditions with a DNA containing the base sequence of 3588), a base sequence that hybridizes under high stringent conditions with a DNA containing the base sequence represented by SEQ ID NO: 23, DN containing the base sequence represented by SEQ ID NO: 16
A, a nucleotide sequence that hybridizes under stringent conditions with A, a DNA comprising the nucleotide sequence represented by SEQ ID NO: 26, and a nucleotide sequence that hybridizes under highly stringent conditions with the DNA of the present invention; DN encoding a protein having substantially the same properties as I
Any one may be used as long as it is A. Examples of the DNA encoding the protein II include a DNA having a base sequence represented by SEQ ID NO: 4 (a DNA having a base sequence of base numbers 19 to 3510 in the base sequence represented by SEQ ID NO: 24), DNA containing the base sequence represented by SEQ ID NO: 24, DNA containing the base sequence represented by SEQ ID NO: 18, DNA containing the base sequence represented by SEQ ID NO: 27, SEQ ID NO: 4 A nucleotide sequence that hybridizes under high stringent conditions to a DNA containing the nucleotide sequence represented by SEQ ID NO: 24 (a DNA containing the nucleotide sequence of nucleotides 19 to 3510 in the nucleotide sequence represented by SEQ ID NO: 24); : A nucleotide sequence that hybridizes under high stringent conditions with DNA containing the nucleotide sequence represented by 24, SEQ ID NO: 1 Or a base that hybridizes under high stringent conditions with a DNA containing the base sequence represented by SEQ ID NO: 27 or with a DNA containing the base sequence represented by SEQ ID NO: 27 Any DNA may be used as long as it has a sequence and encodes a protein having substantially the same properties as protein II.

DNA containing the nucleotide sequence represented by SEQ ID NO: 3 (DN having the nucleotide sequence of nucleotide numbers 19 to 3588 in the nucleotide sequence represented by SEQ ID NO: 23)
A) DNA containing the base sequence represented by SEQ ID NO: 23, DNA containing the base sequence represented by SEQ ID NO: 16 or DNA containing the base sequence represented by SEQ ID NO: 26, and high stringency Examples of the DNA that can hybridize under the following conditions include the nucleotide sequence represented by SEQ ID NO: 3, the nucleotide sequence represented by SEQ ID NO: 23, the nucleotide sequence represented by SEQ ID NO: 16 or the nucleotide sequence represented by SEQ ID NO: 26. D containing a nucleotide sequence having about 98% or more, preferably about 99% or more homology with the represented nucleotide sequence.
NA or the like is used. A DNA containing the base sequence represented by SEQ ID NO: 4 (a DNA containing the base sequence of base numbers 19 to 3510 in the base sequence represented by SEQ ID NO: 24), and a base sequence represented by SEQ ID NO: 24 Examples of the DNA that can hybridize with the DNA containing the DNA, the DNA containing the nucleotide sequence represented by SEQ ID NO: 18 or the DNA containing the nucleotide sequence represented by SEQ ID NO: 27 under high stringent conditions include: The base sequence represented by SEQ ID NO: 4, the base sequence represented by SEQ ID NO: 24, the base sequence represented by SEQ ID NO: 18 or the base sequence represented by SEQ ID NO: 27 is about 98% or more, preferably DNA containing a base sequence having about 99% or more homology is used. Hybridization can be performed by a method known per se or a method analogous thereto, for example, molecular cloning (Molecular Clonin).
g) 2nd (J. Sambrook et al., Cold Spring Harbor La
b. Press, 1989). When using a commercially available library,
It can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions. High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM,
It is preferably about 19-20 mM and the temperature is about 50-70.
C, preferably about 60-65C. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable. More specifically, a DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 1
For example, a DNA having the base sequence represented by SEQ ID NO: 3 is used. As the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 2, more specifically, a DNA having the base sequence represented by SEQ ID NO: 4 is used. SEQ ID NO: 15
More specifically, the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 16
DNA having the base sequence represented by

As the DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 17, more specifically, a DNA having the base sequence represented by SEQ ID NO: 18 is used. Partial peptide I of the present invention
Alternatively, as DNA encoding partial peptide II,
Partial peptide I or partial peptide I of the present invention described above
Any nucleotide may be used as long as it contains the nucleotide sequence encoding I. Genomic DNA, Genome D
NA library, cell / tissue-derived cDN
A, a cDNA library derived from the cells and tissues described above,
Any of synthetic DNAs may be used. Partial peptide I of the present invention
Are, for example, a DNA having a part of the base sequence represented by SEQ ID NO: 3, a DNA having a part of the base sequence represented by SEQ ID NO: 16, or a base sequence represented by SEQ ID NO: 3 DNA that has
Alternatively, D having the base sequence represented by SEQ ID NO: 16
A DNA having a base sequence that hybridizes with NA under high stringent conditions and having a part of a DNA encoding a protein having substantially the same activity as the protein I of the present invention is used. DNA capable of hybridizing with the DNA having the nucleotide sequence represented by SEQ ID NO: 3 or the nucleotide sequence represented by SEQ ID NO: 16
Is as defined above. Examples of the DNA encoding the partial peptide II include a DNA having a part of the base sequence represented by SEQ ID NO: 4, a DNA having a part of the base sequence represented by SEQ ID NO: 18, or SEQ ID NO: 4. A DNA encoding a protein having a nucleotide sequence that hybridizes under high stringent conditions with a DNA having the nucleotide sequence represented by the nucleotide sequence or SEQ ID NO: 18 and having substantially the same activity as protein II; A DNA having a part is used. The DNA hybridizable to the DNA having the nucleotide sequence represented by SEQ ID NO: 4 or the nucleotide sequence represented by SEQ ID NO: 18 has the same significance as described above. The same hybridization method and high stringency conditions as described above are used. As a means for cloning a DNA encoding the protein I, the partial peptide I, the protein II or the partial peptide II of the present invention (hereinafter, these may be simply abbreviated as the protein of the present invention), a part of the protein of the present invention may be used. DNA amplified by the PCR method using a synthetic DNA primer having a base sequence, or incorporated into an appropriate vector
Can be selected by hybridization with a DNA fragment encoding a part or the entire region of the protein of the present invention or a DNA fragment labeled with a synthetic DNA. Hybridization methods include, for example, molecular cloning (Molecular Cloning) 2nd
(J. Sambrook et al., Cold Spring Harbor Lab. Pres
s, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted using a known kit, for example, Mutan ^™ -super ExpressKm (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), or the like.
-The method can be carried out according to a method known per se, such as the LA PCR method, the Gapped duplex method, the Kunkel method, or a method analogous thereto. The cloned DNA encoding the protein of the present invention can be used as it is depending on the purpose, or may be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The expression vector for the protein of the present invention is, for example, (a) cutting out a target DNA fragment from DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by the following. As the vector, a plasmid derived from Escherichia coli (eg, pBR322, pB
R325, pUC12, pUC13) and plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC19)
4), yeast-derived plasmids (eg, pSH19, pSH1
5), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., as well as pA1-11, pXT1, pR
c / CMV, pRc / RSV, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, the SRα promoter,
SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like. Of these, it is preferable to use a CMV (cytomegalovirus) promoter, SRα promoter, or the like. When the host is Escherichia, the trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, T7 promoter and the like are used. When the host is Bacillus,
SPO1 promoter, SPO2 promoter, pen
When the host is yeast, such as the P promoter, PHO
5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, the polyhedrin promoter, P10
Promoters and the like are preferred. In addition to those described above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like may be used as the expression vector. it can. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistant gene (hereinafter sometimes abbreviated as Amp ^r), neomycin resistant gene (Hereinafter, may be abbreviated as Neo ^r , G418 resistance) and the like. In particular, using dhfr gene-deficient Chinese hamster cells, dhfr
When a gene is used as a selection marker, the target gene can be selected using a thymidine-free medium.

[0018] If necessary, a DNA encoding a signal sequence suitable for the host is added to the 5 'terminal side of the DNA encoding the protein of the present invention. When the host is Escherichia, PhoA signal sequence, OmpA
When the host is a bacterium belonging to the genus Bacillus, the α-amylase signal sequence, the subtilisin signal sequence, and the like are used when the host is yeast.
When the host is an animal cell, such as a signal sequence or an SUC2 signal sequence, an insulin signal sequence;
Signal sequences such as α-interferon signal sequence and antibody molecule / signal sequence can be used. A transformant can be produced using the vector containing the DNA encoding the protein of the present invention thus constructed. As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of the genus Escherichia include, for example, Escherichia col
i) K12 DH1 [Progressing's of the National Academy of Sciences of
The USA (Proc. Natl. Acad. Sci. US
A), Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research,
arch), 9, 309 (1981)], JA 221 [Journal of Molecular Biology (Journal)
of Molecular Biology)], 120, 517 (197
8)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C60
0 [Genetics, 39, 440
(1954)]. As the Bacillus genus, for example, Bacillus subtilis (Bacillus subtilis)
s) MI114 [Gene, 24, 255 (198)
3)], 207-21 [Journal of Biochemistry, 95, 87]
(1984)]. Examples of yeast include, for example, Saccharomyces cerevisiae.
revisiae) ^AH22, AH22R ^-, NA87-11
A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1
913, NCYC2036, Pichia Pastoris (Pich
ia pastoris) KM71 and the like. As the insect cell, for example, when the virus is AcNPV, a cell line derived from a larva of a night roth moth (Spodoptera frugiperda cell;
Sf cells), MG1 cells from Trichoplusia ni midgut, High Five ^™ cells from Trichoplusia ni eggs, Mame
Cells derived from stra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. As the Sf cells, for example,
Sf9 cells (ATCC CRL1711), Sf21 cells (Va
ughn, JL et al., In Vivo, 13, 213-217,
(1977)).

As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315].
Vol., 592 (1985)]. As animal cells, for example,
Monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter, CH)
O (dhfr ⁻ ) cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3,
Human FL cells and the like are used. In order to transform Escherichia, for example, Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. US
A), Vol. 69, 2110 (1972) and Gene,
17, 107 (1982). To transform Bacillus sp.
For example, Molecular & General Genetics, 168
Vol. 111, (1979). To transform yeast, for example, Methods in Enzymolog
y), 194, 182-187 (1991), Processings of the National Academy of Sciences of the USA (Proc. Na)
Acad. Sci. USA), 75, 1929 (197).
8) and the like. In order to transform insect cells or insects, for example, Bio / Technology, 6, 47-55 (1988))
And the like. To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995)
(Published by Shujunsha), Virology, Volume 52,
456 (1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the protein of the present invention can be obtained. The host is Escherichia,
When culturing a transformant which is a bacterium of the genus Bacillus, a liquid medium is suitable as a medium to be used for culturing, and a carbon source, a nitrogen source, an inorganic substance, and the like necessary for growth of the transformant are included therein. It is included. As a carbon source, for example,
Glucose, dextrin, soluble starch, sucrose, etc.
As the nitrogen source, for example, inorganic or organic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and inorganic substances such as calcium chloride, diphosphate Examples include sodium hydrogen and magnesium chloride. In addition, yeast, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5 to 8. As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Mille (Mille)
r), Journal of Experimen in Molecular Genetics
ts in Molecular Genetics), 431-433, Cold S
Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
When the host is a bacterium belonging to the genus Bacillus, culturing is usually performed at about 30 to 40 ° C.
For about 6 to 24 hours, and if necessary, aeration and stirring may be added. When culturing a transformant in which the host is yeast, for example, Burk holder (Burk Holder)
holder) minimal medium [Bostian, KL et al., Processings of the National Academy of Sciences of the USA (Proc. Natl. Ac
ad. Sci. USA), 77, 4505 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA
Proc. Natl. Acad. Sci. USA, Proc. Of the National Academy of Sciences of the USA, 81, 53
30 (1984)]. The pH of the medium is about 5
Adjustment to 8 is preferred. Culture is usually about 20 ° C to 35
C. for about 24-72 hours, adding aeration and agitation as needed. When culturing a transformant in which the host is an insect cell or an insect, the medium used is Grace's Insect Medium
(Grace, TCC, Nature, 195,788 (196
Those to which an additive such as 10% bovine serum immobilized is appropriately added in 2)) are used. The pH of the medium is about 6.2-6.
Adjustment to 4 is preferred. Culture is usually performed at about 27 ° C for about 3
５5 days, with aeration and / or agitation as needed. When culturing a transformant in which the host is an animal cell, the medium may be, for example, MEM containing about 5 to 20% fetal bovine serum.
Medium [Science, Vol. 122, 501 (19)
52)], DMEM medium [Virology,
8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medica
l Association) 199, 519 (1967)], 19
9 medium [Proceeding of the Society for the Biological Medicine (Proceeding o
fthe Society for the Biological Medicine), 73
Vol., 1 (1950)]. pH is about 6-8
It is preferred that The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added.

As described above, in the cells of the transformant,
The protein of the present invention can be produced on the cell membrane or extracellularly. The protein of the present invention can be separated and purified from the culture by, for example, the following method. When the protein of the present invention is extracted from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme, and / or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein of the present invention is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. Purification of the protein of the present invention contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and the like. Method using the difference in charge, method using the difference in charge such as ion exchange chromatography,
Methods that use specific affinity such as affinity chromatography, methods that use differences in hydrophobicity such as reversed-phase high-performance liquid chromatography, and methods that use differences in isoelectric points such as isoelectric focusing Used. When the thus obtained protein of the present invention is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when the protein of the present invention is obtained in a salt, It can be converted to a free form or another salt by a method analogous thereto. The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The presence of the protein of the present invention thus produced can be measured by an enzyme immunoassay using a specific antibody, Western blotting, or the like. An antibody against the protein I, the partial peptide I, the protein II, the partial peptide II or a salt thereof of the present invention is an antibody capable of recognizing the protein I, the partial peptide I, the protein II, the partial peptide II or a salt thereof of the present invention. , Polyclonal antibodies and monoclonal antibodies. Antibodies against the protein I, the partial peptide I, the protein II, the partial peptide II or a salt thereof (hereinafter, these may be simply abbreviated as the protein of the present invention) of the present invention are prepared by using the protein of the present invention as an antigen, It can be produced according to a known antibody or antiserum production method. Examples of the protein of the present invention used as an antigen include the above-mentioned protein I of the present invention, partial peptide I,
Any protein II, partial peptide II or a salt thereof may be used, for example, Arg-Glu-Ar
g-Lys-Arg-Ser-Arg-Gly-Asn-Arg-Lys-Ser-Leu-Arg-Arg
(A partial amino acid sequence at positions 1152 to 1166 from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1), for example, Ala-Val-
Consists of an amino acid sequence represented by Gly-Asn-Phe-Leu-Asn-Gly-Ser-Gln-Ser (a partial amino acid sequence at positions 852 to 862 from the N-terminal of the amino acid sequence represented by SEQ ID NO: 1) Specific examples include partial peptides.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The protein of the present invention is administered to a warm-blooded animal at a site capable of producing an antibody by administration to itself or together with a carrier and a diluent. You. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order 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 the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used. When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in the spleen or lymph node. By fusing the antibody-producing cells thus obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled protein described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation is performed by known methods, for example, the method of Kohler and Milstein [Nature, 256, 495 (1
975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include NS-1,
Myeloma cells of warm-blooded animals such as P3U1, SP2 / 0 and AP-1 can be mentioned, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1,
PEG (preferably PEG1000-PEG6000)
Is added at a concentration of about 10 to 80%,
Preferably, cell fusion can be carried out efficiently by incubating at 30 to 37 ° C for 1 to 10 minutes. Various methods can be used for screening a monoclonal antibody-producing hybridoma. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier, and then a radioactive substance or An anti-immunoglobulin antibody labeled with an enzyme or the like (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A
A method for detecting a monoclonal antibody bound to a solid phase, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, adding a protein labeled with a radioactive substance or an enzyme, and adding A method for detecting a monoclonal antibody bound to a phase is exemplified. The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Normal H
It can be performed in a medium for animal cells to which AT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, 1
RPMI 1640 medium containing 10-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM) -101, Nissui Pharmaceutical Co., Ltd.) can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of the monoclonal antibody can be performed by a method known per se, for example, an immunoglobulin separation and purification method (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis). Method, adsorption / desorption method using an ion exchanger (eg, DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active antibody such as protein A or protein G to collect only antibody and dissociate the bond. Specific Purification Method for Obtaining Antibody]. [Preparation of polyclonal antibody] The polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, an immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting a substance and separating and purifying the antibody. With respect to the complex of the immunizing antigen and the carrier protein used to immunize a warm-blooded animal, the type of the carrier protein and the mixing ratio of the carrier and the hapten are such that the antibody is efficiently cross-linked to the hapten immunized by crosslinking the carrier If possible, any material may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like may be used in a weight ratio of about 0.1 to 20, preferably about 1 to 20, relative to hapten 1. A method of coupling at a rate of ５5 is used. Further, various coupling agents can be used for coupling the hapten and the carrier,
Glutaraldehyde, carbodiimide, maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, and the like are used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is
Usually, once every 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. The polyclonal antibody can be separated and purified according to the same method for separating and purifying immunoglobulins as in the above-described method for separating and purifying a monoclonal antibody. DNA encoding protein I, partial peptide I, protein II or partial peptide II of the present invention (hereinafter, these DNs
A may be abbreviated as the DNA of the present invention) as an antisense DNA having a base sequence complementary to or substantially complementary to the DNA of the present invention. Any antisense DNA may be used as long as it has a simple base sequence and an action capable of suppressing the expression of the DNA.

The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). And about 70% or more, preferably about 80%
% Or more, more preferably about 90% or more, and most preferably about 95% or more. In particular, of the entire base sequence of the complementary strand of the DNA of the present invention,
About 70% or more, preferably about 80% or more, more preferably about 90% or more, which is complementary to the complementary sequence of the base sequence (for example, the base sequence near the start codon) of the portion encoding the N-terminal site of the protein of the present invention; Most preferably, an antisense DNA having about 95% or more homology is suitable. These antisense DNAs can be produced using a known DNA synthesizer or the like. In addition, the antisense DNA is, for example, a protein having the amino acid sequence represented by SEQ ID NO: 1, the base sequence represented by SEQ ID NO: 3, or the protein having the amino acid sequence represented by SEQ ID NO: 2. Having a base sequence complementary to or substantially complementary to the DNA having the base sequence represented by SEQ ID NO: 4 or a partial DNA thereof,
Not only those having the effect of suppressing the expression of NA,
SEQ ID NO: 4 which encodes the protein having the amino acid sequence represented by SEQ ID NO: 3 or the nucleotide sequence represented by SEQ ID NO: 2 which encodes the protein having the amino acid sequence represented by SEQ ID NO: 1 Base sequence further upstream of the 5 'end of the DNA having the base sequence (specifically, the first to 18th base sequence of the base sequence represented by SEQ ID NO: 23, represented by SEQ ID NO: 24) A base sequence of the 1st to 18th base sequence of the base sequence or a base sequence further downstream of the 3 'end (specifically, a base sequence of the 3592th to 3853th position of the amino acid sequence represented by SEQ ID NO: 23;
Nos. 3514 to 37 of the base sequence represented by SEQ ID NO: 24
SEQ ID NO: encoding a protein having a base sequence complementary to or substantially complementary to a DNA having the 75th base sequence or the like or a partial DNA thereof and having the amino acid sequence represented by SEQ ID NO: 1 : Which also has a function of suppressing the expression of the DNA having the nucleotide sequence represented by SEQ ID NO: 4 which encodes the protein having the nucleotide sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 2 Used in a meaning. Hereinafter, protein I, partial peptide I, protein II, and partial peptide I of the present invention will be described.
I or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), DNA encoding protein I, partial peptide I, protein II or partial peptide II of the present invention (hereinafter abbreviated as DNA of the present invention) ), Antibodies against protein I, partial peptide I, protein II, partial peptide II or salts thereof of the present invention (hereinafter sometimes abbreviated as the antibodies of the present invention), and uses of antisense DNA.

(1) Agent for Prevention / Treatment of Various Diseases Involving the Protein of the Present Invention The protein of the present invention binds to GLUT by binding to IRAP.
4 Glucose by retaining the endoplasmic reticulum (ERs in which proteins such as GLUT4, IRAP, VAMPs, SCAMPs, and Rab4 are localized) and suppressing the uptake of blood sugar into muscle cells and fat cells Increase the value. Therefore, the protein of the present invention or the DNA of the present invention can be used as a medicament such as an agent for preventing or treating various diseases such as hypoglycemia. For example, when there is a patient who cannot maintain or maintain the homeostasis of the living body or the body defense mechanism sufficiently or normally because the protein or the like of the present invention is reduced or deleted in the living body, (a) the present invention By administering the DNA to the patient and expressing the protein of the present invention in vivo, (ii) inserting the DNA of the present invention into cells and expressing the protein of the present invention, and then transplanting the cells into the patient. By carrying out or (c) administering the protein of the present invention to the patient, the role of the protein of the present invention in the patient can be sufficiently or normally exerted. When the DNA of the present invention is used as the above-described prophylactic / therapeutic agent, the DNA is inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. It can be administered to warm-blooded animals. The DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter. When the protein of the present invention is used as the above prophylactic / therapeutic agent, it is at least 90%, preferably 95% or more, more preferably 98% or more, and further preferably 99% or more.
% Or more is preferably used. The protein of the present invention can be administered, for example, as a sugar-coated tablet, capsule, elixir, microcapsule, or the like, or as an aerosol in the form of an inhalant, or water or other pharmaceutical agent. It can be used parenterally in the form of an injection, such as a sterile solution with a liquid that is acceptable or a suspension. For example, the protein of the present invention is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. Can be manufactured by The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained. Examples of additives that can be mixed with tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry and the like are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical manufacturing methods such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. . Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents. For example, alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, Polysorbate 80 ^™ , HCO-50)
Etc.). Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with a preservative (for example, benzyl alcohol, phenol, etc.), an antioxidant, etc. The prepared injection is usually filled in an appropriate ampoule.

The vector into which the DNA of the present invention has been inserted is also formulated as described above, and is usually used parenterally.
The preparations obtained in this way are safe and have low toxicity, for example, warm-blooded animals (eg, humans, rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows,
Horses, cats, dogs, monkeys, chimpanzees, etc.). The dose of the protein of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the protein of the present invention is orally administered for the purpose of treating hypoglycemia, it is generally adult (60 kg). )), About 0.1 m of the protein per day
g-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, a single dose of the protein may be
For example, when the protein of the present invention is administered to an adult (assuming a body weight of 60 kg) in the form of an injection for the purpose of treating hypoglycemia, the protein may be administered in an amount of about 0.01 to 30 mg per day, although it differs depending on the target disease. Degree, preferably about 0.
About 1 to 20 mg, more preferably about 0.1 to 10 mg
It is convenient to administer the degree by injecting it into the affected area. In the case of other animals, the dose can be administered in terms of 60 kg.

(2) Screening for Binding Inhibitors The protein of the present invention binds to the cytoplasmic domain of IRAP, retains the GLUT4 endoplasmic reticulum in the cells, and suppresses the incorporation of blood sugar into muscle cells and fat cells. . Therefore, a compound that inhibits the binding of the protein of the present invention to IRAP, preferably a compound that inhibits the binding of the protein of the present invention to the cytoplasmic domain of IRAP, is the uptake of blood sugar into muscle cells and fat cells. And can lower blood sugar levels, and is useful as a medicament such as a prophylactic / therapeutic agent for diseases such as hyperglycemia and diabetes. Further, as shown in Example 4 below, the protein of the present invention is GLUT
4 cytoplasmic domains (amino acid numbers 468-510 of GLUT4;
It also binds to SEQ ID NO: 13) and suppresses the uptake of blood sugar into muscle cells and fat cells by retaining the GLUT4 endoplasmic reticulum inside the cells. Therefore, a compound that inhibits the binding of the protein of the present invention to GLUT4, preferably a compound that inhibits the binding of the protein of the present invention to the cytoplasmic domain of GLUT4 is a compound that inhibits the binding of the protein of the present invention to IRAP. Similarly to the above, it is useful as a medicament such as a prophylactic / therapeutic agent for diseases such as hyperglycemia and diabetes. In the screening method of the present invention, the protein of the present invention is used, and IRAP or a peptide corresponding to the cytoplasmic domain of IRAP or GLUT4 or GLUT4
May be used. Further, the screening method of the present invention provides a cell capable of producing the protein of the present invention (preferably,
A transformant (eg, a cell such as a yeast or an animal cell) transformed with the DNA encoding the protein of the present invention may be used. The transformant may be a transformant transformed with DNA encoding the protein of the present invention and DNA encoding IRAP or a peptide corresponding to the cytoplasmic domain of IRAP, or a DNA encoding the protein of the present invention and GLUT4 Alternatively, it may be a transformant transformed with DNA encoding a peptide corresponding to the cytoplasmic domain of GLUT4.

(2-1) Screening by In Vitro Binding Test The protein of the present invention is immobilized on a solid phase (eg, an EIA plate) using an antibody against the protein of the present invention,
Alternatively, the protein of the present invention is replaced with a Tag protein (eg, His
-Tag, GST (glutathione-S-transferase), etc.)
And then immobilized on a solid phase. When the partial peptide of the present invention is used as the protein of the present invention, the partial peptide preferably has a binding activity to IRAP or GLUT4 (amino acids 977 to 1190 of SEQ ID NO: 1;
For example, amino acid numbers 951 to 1164 of SEQ ID NO: 2) are used. When immobilizing proteins on a solid phase, use Hi
Nickel is used for s-Tag, and glutathione is used for GST. In addition, IRAP or a partial peptide corresponding to the cytoplasmic domain of IRAP (amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof, preferably amino acids 55 to 82 of SEQ ID NO: 11) or cytoplasm of GLUT4 or GLUT4 A partial peptide corresponding to the inner domain (SEQ ID NO: 13) labeled with biotin or the like is added and allowed to bind. A test compound is added to this complex, and IRAP or IRAP partial peptide or GLUT4 or GLUT4 partial peptide released as a result of inhibition of the binding of the protein of the present invention to IRAP or GLUT4 is commercially available for detecting a labeled substance such as biotin. Or a known anti-IRAP antibody or a commercially available anti-GLUT4 antibody. A compound that releases IRAP or an IRAP partial peptide or GLUT4 or a GLUT4 partial peptide is selected as a compound that inhibits the binding between the protein of the present invention and IRAP or GLUT4 (hereinafter, may be abbreviated as a binding inhibitor). In addition, IRAP or a partial peptide corresponding to the IRAP cytoplasmic domain (amino acid sequence represented by SEQ ID NO: 11 or a partial sequence thereof, preferably SEQ ID NO: 1)
Amino acid numbers 55 to 82) or GLUT4 or GLU
A partial peptide (SEQ ID NO: 13) corresponding to the cytoplasmic domain of T4 is immobilized on a solid phase, and the protein of the present invention is bound thereto. IRAP or IRAP partial peptide or
When GLUT4 or GLUT4 partial peptide is immobilized on a solid phase, for example, biotin-labeled IRAP or IRAP partial peptide or GLUT4 or GLUT4 partial peptide and avidin-labeled solid phase (eg, plate) are preferably used. . A test compound is added to this complex, and the released protein of the present invention is detected and quantified using an antibody against the protein of the present invention or an antibody against the Tag protein.
In this method, the protein of the present invention may be a protein of the present invention fused with a Tag protein.In this case, the released protein of the present invention may be detected and quantified by using an antibody against the protein of the present invention. Well, also Tag
Detection and quantification may be performed using an antibody against the protein. Compounds that release the protein of the invention are selected as binding inhibitors. Selected compounds are anti-IRAP antibody, anti-GLUT
(4) The inhibitory activity thereof can be confirmed by a method known per se such as immunoprecipitation using an antibody, an antibody against the protein of the present invention or an antibody against Tag. In the immunoprecipitation method, the protein of the present invention or IRAP or GLUT4 released by inhibiting the binding of the protein of the present invention to IRAP or GLUT4 by the selected compound is used.
Is detected by an antibody against the protein of the present invention, an antibody against the Tag protein, an antibody against IRAP or an antibody against GLUT4.

(2-2) Screening by Two-Hybrid method (2-2-1) Yeast Screening by two-hybrid method Yeast (eg, Saccharomyces cerevisiae, more preferably
S. cerevisiae strain Y190), a reporter gene transcriptionally active domain fused with a DNA encoding a partial peptide corresponding to the IRAP cytoplasmic domain or a partial peptide corresponding to the GLUT4 cytoplasmic domain fused with a reporter gene binding domain. When the DNA encoding the protein of the present invention is expressed, the phenotype of the reporter gene β-galactosidase gene and the histidine synthesis system gene HIS3 is expressed by the action of two-hybrid. The yeast strain is cultured in the presence of the test compound for a certain period of time, and a compound that reduces the β-galactosidase activity of the yeast strain or converts the yeast strain to histidine requirement is selected. The yeast strain can be cultured in the same manner as the culture of the above-described transformant whose host is yeast. The activity of β-galactosidase was X-Gal (5-bromo-4-chloro-3-indolyl-
β-D-galactopyranoside), ONPG (o-nitrophenylβ-D-
galactopyranoside) or CPRG (chlorophenyl red-
β-D-galactopyranoside) can be measured as a substrate according to a method known per se. Expression of the HIS3 phenotype can be measured by culturing the yeast in a histidine-deficient minimal medium. Among the selected compounds, cytotoxic compounds and compounds that inhibit the activity of the reporter gene product itself due to interaction with the reporter gene product or the like can be excluded as false positive compounds. (2-2-2) Screening by the animal cell two-hybrid method Animal cell (eg, Chinese hamster ovary (CHO)
As a reporter gene, for example, a chloramphenicol acetyltransferase (CAT) gene or a firefly luciferase gene is introduced into the cell. The transcriptional regulatory region of the reporter gene is, for example, a promoter that functions in animal cells (eg, a minimal promoter derived from adenovirus E1b (TATA box) or the like) and is linked with a transcriptional activation sequence (UAS) of GAL1, for example. , Yeast two-h
The transcriptional regulatory system of GAL4-GAL1 of the ybrid system is introduced into animal cells so that expression of a reporter gene is induced in animal cells. A DNA encoding a partial peptide corresponding to the IRAP cytoplasmic domain or a partial peptide corresponding to the GLUT4 cytoplasmic domain fused to a GAL4-DNA binding domain, and a DNA encoding a VP16 protein derived from herpes simplex virus, for example. Expression of the DNA encoding the fused protein of the present invention yields two-h
An animal cell line in which the reporter gene is expressed by the action of ybrid is obtained. This cell line is incubated in the presence of the test compound.
After culturing for a certain period of time, the activity of the reporter gene product is measured, and a compound that reduces the activity is selected. The animal cell strain can be cultured in the same manner as the culture of the above-mentioned transformant whose host is an animal cell. The activity of reporter gene products such as CAT and luciferase can be measured using a commercially available kit according to a method known per se. Among the selected compounds, cytotoxic compounds and compounds that inhibit the activity of the reporter gene product itself due to interaction with the reporter gene product or the like can be excluded as false positive compounds.

(3) Screening for a compound that promotes or suppresses the expression of the protein of the present invention The transcription regulatory region of the DNA of the present invention is cloned, and a reporter gene (eg, β-galactosidase, firefly luciferase, chloramphenicol, Acetyltransferase (CAT), etc.) and fused with animal cells (eg, C
HO cells). This cell line is cultured for a certain period of time in the presence of the test compound, and a compound that increases or decreases the production of the reporter gene product is selected. The animal cell strain can be cultured in the same manner as the culture of the above-mentioned transformant whose host is an animal cell. The increase or decrease in the production amount of the reporter gene product can be determined, for example, by measuring the activity of the reporter gene product in the culture solution. Among the selected compounds, compounds that increase or decrease the activity of the reporter gene product itself due to interaction with the cytotoxic compound and the reporter gene product can be excluded as false positive compounds. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. Or a known compound. Examples of the compound that suppresses the expression of the protein of the present invention include the compound that suppresses the expression of the protein of the present invention obtained by the above screening, and the antisense DN described above.
A, a compound that inhibits the promoter activity for the DNA of the present invention described later, and the like. Examples of the compound that promotes the expression of the protein of the present invention include a compound that promotes the expression of the protein of the present invention obtained by the above screening, and a compound that promotes the promoter activity of the DNA of the present invention described below. The screening kit of the present invention contains the protein of the present invention, and further comprises
It may contain IRAP or a peptide corresponding to the cytoplasmic domain of IRAP or GLUT4 or a peptide corresponding to the cytoplasmic domain of GLUT4. In addition, the screening kit of the present invention comprises a cell capable of producing the protein of the present invention (preferably, a transformant transformed with the DNA encoding the protein of the present invention (eg, a cell such as a yeast or an animal cell). )). The transformant contains DNA encoding the protein of the present invention and IRAP.
Alternatively, a transformant transformed with a DNA encoding a peptide corresponding to the cytoplasmic domain of IRAP, or a transformant transformed with a DNA encoding the protein of the present invention and a DNA encoding a peptide corresponding to GLUT4 or a cytoplasmic domain of GLUT4. It may be a transformed transformant. Compounds or salts thereof obtained by using the screening method or the screening kit of the present invention are test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts. And compounds selected from animal tissue extracts, plasma, and the like, and include, for example, compounds that inhibit the binding of the protein of the present invention to IRAP or GLUT4, and compounds that promote or suppress the expression of the protein of the present invention. As the salt of the compound, those similar to the aforementioned salts of protein I of the present invention are used.

The protein of the present invention and IRAP or GLUT4
The compound that inhibits the binding to the compound or the compound that suppresses the expression of the protein of the present invention is useful as a medicament such as a prophylactic / therapeutic agent for diseases such as hyperglycemia and diabetes. Compounds that promote the expression of the protein of the present invention,
For example, it is useful as a medicament such as a prophylactic / therapeutic agent for diseases such as hypoglycemia. When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method. For example, in the same manner as the above-mentioned drug containing the protein of the present invention, it is formulated into tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like, and administered orally or parenterally. be able to. Since the preparations obtained in this way are safe and low toxic,
It can be administered to a warm-blooded animal (eg, human, mouse, rat, rabbit, sheep, pig, cow, horse, bird, cat, dog, monkey, chimpanzee, etc.). The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route, and the like.
For the treatment of diabetes, the protein of the present invention and IRAP or
When a compound that inhibits binding to GLUT4 or a compound that suppresses the expression of the protein of the present invention is orally administered, generally, in an adult (with a body weight of 60 kg), about 0.1 to 100 mg of the compound per day is used. Preferably about 1.
0 to 50 mg, more preferably about 1.0 to 20 mg is administered. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., for example, a compound that inhibits the binding of the protein of the present invention to IRAP or GLUT4 for the purpose of treating diabetes Alternatively, when a compound that suppresses the expression of the protein of the present invention is usually administered to an adult (as 60 kg) in the form of an injection, about 0.01 to 30 mg, preferably about 0.1 to 30 mg of the compound per day is administered.
Conveniently, about 20 mg, more preferably about 0.1 to 10 mg is administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. For example, when orally administering a compound that promotes the expression of the protein of the present invention for the purpose of treating hypoglycemia,
Generally, in an adult (assuming a body weight of 60 kg), the compound is used in an amount of about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
Administer. For example, when a compound that promotes the expression of the protein of the present invention for the purpose of treating hypoglycemia is usually administered to an adult (as 60 kg) in the form of an injection, the compound is administered in an amount of about 0.01 to 30 mg per day, Preferably about 0.1 to
It is convenient to administer about 20 mg, more preferably about 0.1 to 10 mg, by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(4) Quantification of the Protein of the Present Invention An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. It can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by a sandwich immunoassay. That is, the present invention
(I) reacting the antibody of the present invention with a test solution and a labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody. And (ii) simultaneously or sequentially reacting the test solution with the antibody of the present invention insolubilized on a carrier and another antibody of the present invention labeled on a carrier. The present invention provides a method for quantifying the protein of the present invention in a test solution, which comprises measuring the activity of a labeling agent on an insolubilized carrier after the reaction. In the quantification method of the above (ii), one of the antibodies binds to the N of the protein of the present invention (preferably, protein I or protein II of the present invention).
It is desirable that the antibody recognize the end and the other antibody react with the C-terminal of the protein of the present invention (preferably, the protein I or the protein II of the present invention). In addition, the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, and F (ab ′) ₂ , Fab ′, or Fab
Fractions may be used. The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, a competitive method, an immunometric method, and a sandwich method are preferably used, and in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably a stable enzyme having a large specific activity, for example, β-galactosidase, β-galactosidase.
Glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent. For the insolubilization of the antigen or the antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent on the carrier, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may. In the method for measuring the protein of the present invention by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different binding site to the protein of the present invention is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is preferably For example, an antibody that recognizes an N-terminal other than the C-terminal is used. The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry, or the like. In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using a polyethylene glycol, a liquid phase method using a second antibody against the antibody, and the like.
An immobilized antibody is used as the antibody, or a solid phase immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used. In the immunometric method,
The antigen in the test solution and the solid-phased antigen are subjected to a competitive reaction with a certain amount of the labeled antibody, and then the solid phase and the liquid phase are separated. After the reaction with the labeled antibody, the immobilized antigen is added and the unreacted labeled antibody is bound to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephelometry utilizing laser scattering is preferably used. In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measurement system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, books, and the like.

For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Radioimmunoassay" edited by Hiroshi Irie (published in Kodansha, 1979), "Enzyme immunoassay" (ed.) Edited by Eiji Ishikawa et al. Shoin, 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), "Me
thods in ENZYMOLOGY ”Vol. 70 (Immunochemical Techn
iques (Part A)), ibid.Vol. 73 (Immunochemical Techn.
iques (Part B)), Ibid.Vol. 74 (Immunochemical Techn)
iques (Part C)), ibid.Vol. 84 (Immunochemical Techn)
iques (Part D: Selected Immunoassays)), ibid.
92 (Immunochemical Techniques (Part E: Monoclonal Ant
ibodiesand General Immunoassay Methods)), ibid.
l. 121 (Immunochemical Techniques (Part I: Hybridoma
Technology and Monoclonal Antibodies)) (above, published by Academic Press).
As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, by quantifying the concentration of the protein of the present invention using the antibody of the present invention, (1) when an increase in the concentration of the protein of the present invention is detected, for example, in diseases such as hyperglycemia or diabetes If it can be diagnosed that there is or is likely to be affected, and (2) a decrease in the concentration of the protein of the present invention is detected, for example,
It can be diagnosed as having a disease, such as hypoglycemia, or is likely to be affected in the future. Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. Also,
Used for preparation of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction at the time of purification, analysis of the behavior of the protein of the present invention in test cells, and the like. be able to.

(5) Gene Diagnostic Agent The DNA of the present invention can be used, for example, as a probe to produce a warm-blooded animal (eg, human, rat, mouse,
Since abnormalities (genetic abnormalities) of DNA or mRNA encoding the protein of the present invention can be detected in guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc., for example, The DNA or mRNA is useful as a gene diagnostic agent for damage, mutation, or decreased expression, and increased or overexpressed DNA or mRNA. The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by Northern hybridization or PCR-SSCP known per se.
Method (Genomics, Vol. 5, 874-87)
9 (1989), The Prossings of the National Academy of Sciences of
USA (Proceedings of the National Academy o
f Sciences of the United States of America), 8th
6, 2766-2770 (1989)). For example, when overexpression is detected by Northern hybridization or PCR
When a DNA mutation is detected by the SSCP method, for example, it is possible to diagnose that there is a high possibility of a disease such as hyperglycemia or diabetes, or hypoglycemia.

(6) Pharmaceuticals Containing Antisense DNA Antisense DNA that can complementarily bind to the DNA of the present invention and suppress the expression of the DNA suppresses the production of the protein of the present invention in vivo. Therefore, it can be used, for example, as a prophylactic / therapeutic agent for hyperglycemia, diabetes, etc., in the same manner as the above-mentioned compound that suppresses the expression of the protein of the present invention. When the antisense DNA is used as the above-mentioned prophylactic / therapeutic agent, it can be carried out in the same manner as the above-mentioned various prophylactic / therapeutic agents containing the DNA of the present invention. For example, when the antisense DNA is used, the method can be carried out according to a conventional method after inserting the antisense DNA alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, and an adenovirus associated virus vector. The antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and administered into the trachea as an inhalant. The dosage of the antisense DNA varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the antisense DNA of the present invention is locally administered into the trachea as an inhalant, generally the adult (body weight) is used. 60 kg), about 0.1 to 100 mg of the antisense DNA is administered per day. Further, the antisense DNA can be used in the tissues and cells of the present invention.
It can also be used as a diagnostic oligonucleotide probe for examining the presence of NA and its expression status.

(7) Pharmaceuticals Containing Antibody of the Present Invention The antibodies of the present invention, which have an activity of neutralizing the activity of the protein of the present invention, are useful as medicaments (prophylactic / therapeutic agents) for diseases such as hyperglycemia and diabetes. Can be used. The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention can be directly used as a liquid preparation or as a pharmaceutical composition in a suitable dosage form as a warm-blooded animal (eg, human, rat, rabbit, sheep, pig, cow, cat) , Dogs, monkeys, etc.) can be administered orally or parenterally. The dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like. For example, when used for the treatment or prevention of diabetes in adults, the antibody of the present invention is usually administered in a single dose, usually 0%. .01-2
0 mg / kg body weight, preferably 0.1-10 mg / kg
Approximately kg body weight, more preferably 0.1 to 5 mg / kg
About 1 to 5 times a day, preferably 1 to 3 times a day
It is convenient to administer by intravenous injection about once. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the above administration contains the antibody of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration. That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly 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 a composition for parenteral administration, for example, injections, suppositories, etc. are used, and the injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections and the like. Is included. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and a suitable solubilizing agent, for example, alcohol (eg,
Ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyeth)
ylene (50mol) adduct of hydrogenated castor oi
l)]. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base. The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg per dosage unit dosage form, especially 5 to 10 mg for injections.
It is preferable that the above-mentioned antibody is contained in 0 mg, and 10 to 250 mg in other dosage forms. Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.

(8) DNA-Transferred Animal The present invention relates to D-encoding an exogenous protein of the present invention.
A non-human mammal having NA (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention) is provided. That is, the present invention provides (1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof, (2) the animal according to (1), wherein the non-human mammal is a rodent, (3) (2) The animal according to (2), wherein the rodent is a mouse or a rat; and (4) a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in a mammal. . Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transferred animal of the present invention) can be used for non-fertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. Preferably, the calcium phosphate method, the electric pulse method, the lipofection method, the microinjection method, at the stage of embryonic development in non-human mammal development (more preferably at the stage of single cells or fertilized egg cells and generally before the 8-cell stage) Target D by the particle gun method, DEAE-dextran method, retrovirus method, etc.
It can be created by transferring NA. Further, by the DNA transfer method, the exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a cell fusion method known per se. As a non-human mammal,
For example, cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Above all, rodents with relatively short ontogeny and biological cycle in terms of the preparation of disease animal model systems and easy reproduction are particularly useful for mice (for example, hybrids such as pure strains such as C57BL / 6 strain and DBA2 strain). B6C3F ₁ system, BDF ₁ system, B6D2F ₁
Strain, BALB / c strain, ICR strain, etc.) or rat (eg, Wistar, SD etc.) is preferred. "Mammals" in a recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals. The exogenous DNA of the present invention is not the DNA of the present invention originally possessed by a non-human mammal, but is the DNA of the present invention once isolated and extracted from a mammal.
NA. As the mutant DNA of the present invention, those having a mutation (eg, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. The resulting DNA or the like is used, and also includes abnormal DNA. The abnormal DNA refers to a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used. The exogenous DNA of the present invention may be derived from a mammal which is the same or different from the animal of interest. D of the present invention
In transferring NA to a target animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when transferring the human DNA of the present invention, DNAs derived from various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology thereto are expressed. Highly expressing the DNA of the present invention by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters that can be made into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA-transferring mammals can be created. Examples of the expression vector of the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus. Are used. Among them, plasmids derived from E. coli,
A plasmid derived from Bacillus subtilis or a plasmid derived from yeast is preferably used. Examples of the promoter that regulates DNA expression include, for example, promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus), various mammals (human , Rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), for example, albumin, insulin II, uroplakin I
I, elastase, erythropoietin, endothelin,
Muscle creatine kinase, glial fibrillary acidic protein, glutathione S-transferase, platelet-derived growth factor β, keratins K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase β I subunit, dystrophin, tartrate resistance Alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chains, metallothionein I and IIA,
Metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TP
O), polypeptide elongation factor 1α (EF-1α), β
Actin, α and β myosin heavy chains, myosin light chains 1 and 2, myelin basic protein, thyroglobulin, T
hy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, promoter such as preproenkephalin A, vasopressin and the like are used. Among them, a cytomegalovirus promoter capable of high expression throughout the body, a promoter of human polypeptide chain elongation factor 1α (EF-1α), and human and chicken β-actin promoters are preferable. The vector preferably has a sequence that terminates transcription of the messenger RNA of interest in a DNA-transferred mammal (generally called a terminator). The simian virus SV40 terminator is preferably used.

In addition, in order to further express the desired foreign DNA, a splicing signal of each DNA, an enhancer region, a part of an intron of eukaryotic DNA, and the like are placed 5 ′ upstream of the promoter region, between the promoter region and the translation region. Alternatively, it can be linked to the 3 'downstream of the translation region depending on the purpose. The normal translation region of the protein of the present invention can be obtained from liver, kidney, thyroid cells, fibroblast-derived DNA derived from various mammals (eg, human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) and commercially available DNA. From the various genomic DNA libraries described above, or as a starting material, a complementary DNA prepared by a known method from RNA derived from liver, kidney, thyroid cells, and fibroblasts. In addition, a translation region obtained by mutating a translation region of a normal protein obtained from the above-described cells or tissues by a point mutagenesis method can be used for the exogenous abnormal DNA. The translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional genetic engineering technique in which it is ligated downstream of the above promoter and, if desired, upstream of the transcription termination site. Transfer of the exogenous 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 mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the DNA transfer means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germ cells and somatic cells. means. The progeny of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells. Foreign normal DNA of the present invention
Is transferred to a non-human mammal by exogenous DN
After confirming that A is stably maintained, the animal having the DNA can be subcultured in a normal breeding environment. Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage
It is ensured that it is present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in germ cells of a produced animal after DNA transfer means that all offspring of the produced animal have an excessive amount of the exogenous DNA of the present invention in all of their germ cells and somatic cells. Means Progeny of such animals that have inherited the exogenous DNA of the present invention have an excess of the exogenous DNA of the present invention in all of their germinal and somatic cells. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and breeding the male and female animals, it is possible to breed and pass all the offspring so as to have the DNA in excess. The non-human mammal having the normal DNA of the present invention, in which the normal DNA of the present invention is highly expressed, promotes the function of the endogenous normal DNA, and eventually causes hyperactivity of the protein of the present invention. It may develop and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. It is possible.

Since the mammal into which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, a screening test for a prophylactic / therapeutic drug against diseases related to the protein of the present invention. Is also available. On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in an ordinary breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. Furthermore, the desired foreign DNA can be incorporated into the above-described plasmid and used as a source material. The DNA construct with the promoter can be prepared by a usual genetic engineering technique. Abnormal D of the present invention at the fertilized egg cell stage
NA transfer is ensured to be present in all germinal and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The offspring of such animals that have inherited the exogenous DNA of the present invention have the abnormal DNA of the present invention in all of their germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all offspring have the DNA. The non-human mammal having the abnormal DNA of the present invention, in which the abnormal DNA of the present invention is highly expressed, inhibits the function of the endogenous normal DNA, and finally, a functionally inactive form of the protein of the present invention. It may become refractory and can be used as a disease model animal. For example, using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease. In addition, as a specific possibility, an animal in which the abnormal DNA of the present invention is highly expressed can be used to inhibit the function of a normal protein by the abnormal protein of the present invention (dominant negat) in the inactive refractory disease of the protein of the present invention.
ive effect). In addition, since the mammal into which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, it can be used for a therapeutic drug screening test for inactive refractory disease of the protein of the present invention. It is. Other possible uses of the above two DNA-transferred animals of the present invention include, for example, use as a cell source for tissue culture, and DNA or R in the tissues of the DNA-transferred animal of the present invention.
Analysis of the relationship with proteins specifically expressed or activated by the protein of the present invention by directly analyzing NA or analyzing protein tissues expressed by DNA; Cultured by standard tissue culture techniques, using these to study the function of cells from tissues that are generally difficult to culture, screening for agents that enhance cell function by using the cells described above, and It is conceivable to isolate and purify the mutant protein and to produce an antibody thereof. Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease related to the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, etc. More detailed pathological findings in each organ of a disease model related to the disease can be obtained, which can contribute to the development of a new treatment method, and further to the research and treatment of a secondary disease caused by the disease.

It is also possible to take out each organ from the DNA-transferred animal of the present invention, cut it into small pieces, and then use a proteolytic enzyme such as trypsin to obtain free DNA-transferred cells, culture them, or systematize the cultured cells. It is possible. Furthermore, it is possible to examine the specificity of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism, and to investigate its abnormalities. It is an effective research material for Further, in order to use the DNA transgenic animal of the present invention to develop a therapeutic agent for a disease related to the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention, the above-described test method and quantification method It is possible to provide an effective and rapid screening method for the therapeutic agent for the disease by using a method or the like. In addition, using the transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for a disease associated with the protein of the present invention.

(9) Knockout Animal The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention. That is, the present invention provides (1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,
(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a drug resistance gene (eg, a neomycin resistance gene) or a reporter gene (eg, a β-galactosidase gene derived from E. coli). (3) The embryonic stem cell according to (1), which is resistant to neomycin, (4) the embryonic stem cell according to (1), wherein the non-human mammal is a rodent, and (5) the mouse is a rodent. (4) an embryonic stem cell according to (4), (6) a non-human mammal deficient in expression of the DNA in which the DNA of the present invention is inactivated, (7) a DNA resistant gene (eg, a neomycin resistance gene) or a reporter. A gene (eg, a β-galactosidase gene derived from Escherichia coli) is inactivated by introduction thereof, and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention. (6) the non-human mammal according to the above,
(8) The non-human mammal according to (6), wherein the non-human mammal is a rodent, (9) the non-human mammal according to (8), wherein the rodent is a mouse, and (10) A) a compound or a salt thereof that promotes or inhibits the promoter activity of the DNA of the present invention, which comprises administering a test compound to the animal according to (7) and detecting the expression of a drug resistance gene or a reporter gene; A screening method is provided. The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated can be obtained by artificially mutating the DNA of the present invention possessed by the non-human mammal.
By suppressing the expression ability of NA or substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA has substantially no expression ability of the protein of the present invention (hereinafter referred to as “ The knockout DNA of the present invention
(Sometimes referred to as "ES cells"). As the non-human mammal, the same one as described above is used. DNA of the present invention
As a method of artificially adding a mutation, for example, deletion of a part or all of the DNA sequence by genetic engineering techniques,
It can be carried out by inserting or substituting another DNA. By these mutations, for example, the knockout DN of the present invention is shifted by shifting the codon reading frame or disrupting the function of the promoter or exon.
A may be prepared. Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and a drug resistance gene represented by a neomycin resistance gene, a hygromycin resistance gene or lacZ
Inserting a reporter gene such as (β-galactosidase gene) or cat (chloramphenicol acetyltransferase gene), etc., destroys the function of exons, or terminates the transcription of genes in the introns between exons. A DNA chain (hereinafter abbreviated as a targeting vector) having a DNA sequence constructed to insert a DNA sequence (for example, a polyA addition signal or the like) so that complete messenger RNA cannot be synthesized, resulting in gene disruption. Is introduced into the chromosome of the animal by, for example, a homologous recombination method, and the obtained ES cells are subjected to Southern hybridization analysis using a DNA sequence at or near the DNA of the present invention as a probe or a DNA sequence on a targeting vector. When Over the DNA sequence of the neighboring region other than the DNA of the present invention used in Getting vector production was analyzed by PCR method using a primer, it can be to select the knockout ES cell of the present invention. As the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like, for example, those already established as described above may be used.
It may be newly established according to the method of Kaufma. For example, in the case of mouse ES cells, currently, generally, 12
Nine ES cells are used, but the immunological background is not clear. For example, C57BL is used for the purpose of obtaining a pure ES cell having an immunologically clear genetic background. BDF which improved the low number of eggs collected for / 6 mice and C57BL / 6 by crossing with DBA / 2
Well as ₁ mice (C57BL / 6 and F ₁ and DBA / 2) which were established by using the usable good. BDF ₁ mice are often egg number, and, in addition to the advantage that the egg is tough, since with C57BL / 6 mice in the background, when the ES cells obtained therefrom, which were generated pathological model mice , C57BL / 6 mice can be advantageously used in that their genetic background can be replaced with C57BL / 6 mice by backcrossing. In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. In addition, a large number of blastocysts can be obtained efficiently by collecting 8-cell stage embryos and culturing them up to blastocysts. Early embryos can be obtained.

Although either male or female ES cells may be used, male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce cumbersome culture labor. Methods for determining the sex of ES cells include, for example, P
Amplify the sex determining region gene on Y chromosome by CR method,
The detection method can be cited as an example.
If this method is used, it is conventionally necessary to perform about one karyotype analysis.
Against 0 G-banding method, requires ^six cell number, since suffices ES cell number of about 1 colony (about 50), be performed the first selection of ES cells at the early stage of culture on sex identification It is possible, and since the selection of male cells is enabled at an early stage, labor in the initial stage of culture can be greatly reduced. Also,
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method.
The number of chromosomes in the obtained ES cells is desirably 100% of the normal number. However, when it is difficult due to physical operations or the like at the time of establishment, after knocking out the gene of the ES cells, the cells can be knocked out of normal cells (for example, chromosome in mice). It is desirable to clone again into cells (number 2n = 40). The embryonic stem cell line obtained in this way usually has very good growth properties, but must be carefully subcultured because it tends to lose its ontogenetic ability. For example, LIF (1-1) on a suitable feeder cell such as STO fibroblast
(0000 U / ml) in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5% carbon dioxide, 90% air) at about 37 ° C. for example. At the time of passage, for example, trypsin / EDTA
Solution (usually 0.001-0.5% trypsin / 0.1-5
mM EDTA, preferably about 0.1% trypsin / 1m
(M EDTA) treatment, and seeding on a newly prepared feeder cell. Such passage is usually performed every 1 to 3 days. At this time, it is desirable to observe the cells and, if morphologically abnormal cells are found, discard the cultured cells. ES cells are differentiated into various types of cells, such as parietal, visceral, and cardiac muscles, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. [MJ Evans and MH
Kaufman, Nature, 292, 154, 1981.
Year: GR Martin, Processings of the National Academy of Sciences of USA (Proc. Natl. Acad. Sci. USA) 78
Vol., 7634, 1981; TCDoetschman et al., Journal of Embryology and Experimental Morphology, Vol. 87, p. 27, 1985], the present invention obtained by differentiating the ES cells of the present invention. DNA expression-deficient cells are useful in in vitro cell biological studies of the protein of the present invention.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level. is there. As the non-human mammal,
The same as described above is used. The non-human mammal deficient in expression of the DNA of the present invention is, for example, a DNA in which the targeting vector prepared as described above is introduced into a mouse embryonic stem cell or a mouse egg cell, and the DNA of the targeting vector of the present invention is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by homologous recombination. Cells in which the DNA of the present invention has been knocked out are those on or near the DNA of the present invention.
A DNA sequence on a Southern hybridization analysis or targeting vector using the sequence as a probe,
The determination can be made by analysis by PCR using a DNA sequence in a nearby region other than the DNA of the present invention derived from the mouse used as the targeting vector as a primer. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention has been inactivated by gene homologous recombination is cloned, and the cell is cultured at an appropriate time, for example, at the 8-cell stage of a non-human mammalian stem cell. The chimeric embryo is injected into an animal embryo or blastocyst, and the produced chimeric embryo is transplanted into the uterus of the pseudopregnant non-human mammal. The produced animal is a chimeric animal comprising both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When a part of the germ cells of the chimeric animal has a mutated DNA locus of the present invention, all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color. The individual obtained in this manner is usually an individual having a hetero-expression defect of the protein of the present invention,
Individuals with heterozygous expression of the protein of the present invention can be obtained from their offspring by mating individuals heterozygously expressing the protein of the present invention. If you use egg cells,
For example, D is injected into the nucleus of an egg by microinjection.
By injecting the NA solution, a transgenic non-human mammal in which the targeting vector has been introduced into the chromosome can be obtained. It can be obtained by selecting those with mutations. In this manner, an individual in which the DNA of the present invention has been knocked out is an animal individual obtained by mating.
After confirming that A has been knocked out, breeding passage can be performed in a normal breeding environment. Furthermore, the acquisition and maintenance of the germ line may be performed according to a conventional method. That is, by crossing male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one homozygote and one normal animal are obtained. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged. The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing a non-human mammal deficient in expressing the DNA of the present invention. In addition, since the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a model of a disease caused by inactivation of the biological activity of the protein of the present invention. It is useful for investigating the cause of these diseases and studying treatment methods.

(10) A method for screening a compound having a therapeutic / preventive effect against diseases caused by DNA deficiency or damage according to the present invention.
It can be used for screening for a compound that has a preventive / therapeutic effect on diseases caused by NA deficiency or damage (eg, hypoglycemia, etc.). That is, the present invention is characterized by administering a test compound to a non-human mammal deficient in expressing the DNA of the present invention, and observing and measuring changes in the animal. Provided is a method for screening a compound or a salt thereof having a therapeutic / prophylactic effect against a disease. Examples of the non-human mammal deficient in expressing DNA of the present invention used in the screening method include the same ones as described above. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound. Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and tested using changes in each organ, tissue, disease symptoms, etc. of the animal as an index. The prophylactic and therapeutic effects of the compounds can be tested. As a method of treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. In addition, the dose of the test compound can be appropriately selected according to the administration method, properties of the test compound, and the like. The compound obtained by using the screening method of the present invention is a compound selected from the test compounds described above, and is used for preventing or preventing diseases caused by decreased expression of the protein of the present invention (eg, hypoglycemia, etc.). Since it has a therapeutic effect, it can be used as a drug such as a safe and low-toxic therapeutic / prophylactic agent for the disease. Further, compounds derived from the compounds obtained by the above screening can be used in the same manner. The compound obtained by the screening method may form a salt, and the salt of the compound may be a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal)
And the like, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, 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) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention. The preparations obtained in this way are safe and of low toxicity, such as, for example, human or warm-blooded animals (for example, rats, mice, guinea pigs,
Rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.). The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the compound is orally administered for the purpose of treating hypoglycemia, it is generally adult (body weight 60%).
kg)).
The dose is 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 of the compound varies depending on the administration subject, target disease, and the like. For example, the compound is usually administered in the form of an injection to an adult (60 kg as an injection) for the treatment of hypoglycemia. )), The compound is administered in an amount of about 0.01 per day.
About 30 mg, preferably about 0.1 to 20 mg,
More preferably, about 0.1 to 10 mg is administered by intravenous injection. For other animals, 60
An amount converted per kg can be administered.

(11) Method for screening for a compound that promotes or inhibits the activity of the promoter for the DNA of the present invention The present invention provides a method for screening a non-human mammal deficient in expressing the DNA of the present invention.
The present invention provides a method for screening a compound or a salt thereof that promotes or inhibits the activity of a promoter for DNA of the present invention, which comprises administering a test compound and detecting the expression of a reporter gene. In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expressing DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene, A reporter gene that can be expressed under the control of a promoter for the DNA of the present invention is used. Examples of the test compound include the same compounds as described above. As the reporter gene, the same one as described above is used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene and the like are preferable. The DN of the present invention in which the DNA of the present invention is replaced with a reporter gene
In a non-human mammal deficient in A expression, since the reporter gene is under the control of the promoter for the DNA of the present invention, the activity of the promoter can be detected by tracing the expression of a substance encoded by the reporter gene. For example, when a part of the DNA region encoding the protein of the present invention is replaced with a β-galactosidase gene (lacZ) derived from Escherichia coli, a tissue that expresses the protein of the present invention originally replaces the protein of the present invention. Β-galactosidase is expressed. Thus, for example, 5-bromo-4-chloro-3-indolyl-β-
By staining with a reagent serving as a substrate for β-galactosidase such as galactopyranoside (X-gal), the expression state of the protein of the present invention in an animal body can be easily observed. More specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, and the buffered saline (PB) is used.
After washing in S), the mixture is reacted with a staining solution containing X-gal at room temperature or about 37 ° C. for about 30 minutes to 1 hour, and then the tissue specimen is washed with a 1 mM EDTA / PBS solution to obtain β-galactosidase. The reaction may be stopped and the color may be observed. Further, mRNA encoding lacZ may be detected according to a conventional method. The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a DNA of the present invention.
A compound that promotes or inhibits promoter activity against The compound obtained by the screening method may form a salt, and the salt of the compound may be a salt with a physiologically acceptable acid (eg, an inorganic acid) or a base (eg, an organic acid). Are used, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, 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) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. A compound or a salt thereof that promotes the promoter activity of the DNA of the present invention can promote the expression of the protein of the present invention and promote the activity of the protein.
It is useful as a drug such as a safe and low toxic prophylactic / therapeutic agent for diseases such as hypoglycemia.

On the other hand, a compound or a salt thereof that inhibits the promoter activity for the DNA of the present invention can inhibit the expression of the protein of the present invention and inhibit the activity of the protein. It is useful as a medicament such as a safe and low toxic prophylactic / therapeutic agent for diseases such as. Further, compounds derived from the compounds obtained by the above screening can be used in the same manner. A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention. The preparation obtained in this way is safe and low toxic, and thus, for example, warm-blooded animals (eg, human, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) Can be administered. The dose of the compound or a salt thereof varies depending on the target disease, the subject of administration, the administration route, and the like.For example, when a compound that inhibits the promoter activity of the DNA of the present invention for the purpose of treating diabetes is orally administered, Adult (weight 6
0 kg), from about 0.1 to 100 mg, preferably from about 1.0 to 50 mg of the compound per day,
More preferably, about 1.0 to 20 mg is administered. When administered parenterally, a single dose of the compound should be administered to the subject,
For example, when a compound that inhibits the promoter activity of the DNA of the present invention for the purpose of treating diabetes is administered to an adult (as 60 kg) in the form of an injection for the purpose of treating diabetes, the compound may be added to the subject every day. 0.01
About 30 mg, preferably about 0.1 to 20 mg,
More preferably, about 0.1 to 10 mg is administered by intravenous injection. For other animals, 60
An amount converted per kg can be administered. On the other hand, for example, when a compound that promotes the promoter activity of the DNA of the present invention is orally administered for the purpose of treating hypoglycemia, generally, in an adult (with a body weight of 60 kg), the compound is used in an amount of about 0.1 per day. ~ 100mg, preferably about 1.0-50mg, more preferably about 1.0 ~
Administer 20 mg. When administered parenterally, the single dose of the compound may vary depending on the administration subject, target disease, and the like. For example, the DNA of the present invention may be used for the treatment of hypoglycemia.
When a compound that promotes the promoter activity of the compound is administered to an adult (as 60 kg) in the form of an injection, the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. Is about 0.1 ~
It is convenient to administer about 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening for a compound or a salt thereof that promotes or inhibits the activity of the promoter of the DNA of the present invention. It can greatly contribute to the investigation of the cause of various diseases caused by the disease or the development of preventive / therapeutic drugs. Further, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene-transferred animal). Once created, it will also be possible to specifically synthesize the protein and study its effects in living organisms. Further, an appropriate reporter gene is linked to the above promoter,
If a cell line that expresses this is established, it can be used as a search system for low-molecular compounds that have the action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body. By analyzing the promoter portion, it is also possible to find a new cis element and a transcription factor binding thereto.

In the present specification and drawings, when bases and amino acids are indicated by abbreviations, IUPAC-IUB
It is based on the abbreviation by the Commission on Biochemical Nomenclature or the abbreviation commonly used in the relevant field. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: thyronine : 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

The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols. Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine -4 (R) - carboxamide group Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ -Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyloxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl 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-hydro Shi-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of human MD36. [SEQ ID NO: 2] This shows the amino acid sequence of human FHOS. [SEQ ID NO: 3] This shows the base sequence of human MD36 gene (cDNA). [SEQ ID NO: 4] This shows the base sequence of human FHOS gene (cDNA). [SEQ ID NO: 5] This shows the amino acid sequence of truncated human FHOS. [SEQ ID NO: 6] This shows the base sequence of truncated human FHOS gene (cDNA). [SEQ ID NO: 7] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 8] This shows the base sequence of the primer used in EXAMPLE 2. [SEQ ID NO: 9] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 10] This shows the base sequence of the primer used in EXAMPLE 3. [SEQ ID NO: 11] This shows the amino acid sequence of N-terminal 109 amino acid residues of IRAP. [SEQ ID NO: 12] This shows the base sequence of DNA encoding the amino acid sequence of N-terminal 109 amino acid residues of IRAP. [SEQ ID NO: 13] This shows the amino acid sequence of amino acid residues 468 to 510 of GLUT4. [SEQ ID NO: 14] This shows the base sequence of DNA encoding the amino acid sequence of amino acid residues 468 to 510 of GLUT4. [SEQ ID NO: 15] Mouse M obtained in Example 6 described later
2 shows the amino acid sequence of D36. [SEQ ID NO: 16] Mouse M obtained in Example 6 described later
1 shows the nucleotide sequence of D36 gene (cDNA). [SEQ ID NO: 17] Mouse F obtained in Example 6 described later
1 shows the amino acid sequence of HOS. [SEQ ID NO: 18] Mouse F obtained in Example 6 described later
Shows the base sequence of the HOS gene (cDNA). [SEQ ID NO: 19] This shows the base sequence of primer M-1 used in EXAMPLE 6, which will be described later. [SEQ ID NO: 20] This shows the base sequence of primer M-5 used in EXAMPLE 6, which will be described later. [SEQ ID NO: 21] This shows the base sequence of primer MMD-3 used in EXAMPLE 6, which will be described later. [SEQ ID NO: 22] This shows the base sequence of primer M-3 used in EXAMPLE 6, which will be described later. [SEQ ID NO: 23] This shows the base sequence of human MD36 gene (cDNA). [SEQ ID NO: 24] This shows the base sequence containing human FHOS gene (cDNA). [SEQ ID NO: 25] Truncate type
1 shows the nucleotide sequence containing the human FHOS gene (cDNA). [SEQ ID NO: 26] Mouse MD36 gene (cDNA)
Shows a base sequence containing [SEQ ID NO: 27] Mouse FHOS gene (cDNA)
Shows a base sequence containing [SEQ ID NO: 28] This shows the amino acid sequence of PFN IIL. [SEQ ID NO: 29] This shows the base sequence encoding the amino acid sequence represented by SEQ ID NO: 28. [SEQ ID NO: 30] This shows the amino acid sequence of PFN II. [SEQ ID NO: 31] This shows the base sequence of the primer used in Reference Example 1. [SEQ ID NO: 32] This shows the base sequence of the primer used in Reference Example 1. [SEQ ID NO: 33] This shows the amino acid sequence of the MD36 used in Example 10, including the region from the N-terminus to the proline-rich domain.

The plasmid pTB20 obtained in Example 2
Escherichai coli DH5α / pTB carrying 77
2077 has been assigned to NIBH from December 16, 1999 by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, under the accession number FE.
RM BP-6969, and November 3, 1999
IFO163 from the fermentation research institute (IFO) from 0th
Deposited as 39. A transformant Escherichai carrying the plasmid pTB2078 obtained in Example 2
coli DH5α / pTB2078 has been available on December 16, 1999 from the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH).
As accession number FERM BP-6970 and 19
From November 30, 1999, Fermentation Research Institute (IFO)
And deposited as IFO 16340.

[0051]

EXAMPLES The present invention will be described more specifically with reference to examples and reference examples, but the present invention is not limited thereto. Genetic manipulation using Escherichia coli is based on molecular cloning.
g) was followed.

Example 1 Cloning of cDNA Encoding IRAP-Binding Protein by Yeast Two-Hybrid Method The cDNA encoding a protein binding to insulin-responsive aminopeptidase (IRAP) was cloned by the yeast two-hybrid method. Yeast two-hybrid method is basically Clonte
This was performed using a MATCHMAKER ^™ two-hybrid system manufactured by Ch. A polypeptide from amino acid residue 55 to 82 of IRAP (K
eller et al, J. Biol. Chem., 270, 23612-23618, 199
5; encodes amino acid number 55 to 82) of SEQ ID NO: 11
A DNA fragment (hereinafter sometimes abbreviated as “IRAP (55-82)”) was chemically synthesized, and this was subjected to GA under the control of the ADH1 promoter.
Plasmid pG expressing L4-DNA binding domain (GAL4-BD)
It was inserted into BT9 (manufactured by CLONTECH) so that the translation frames matched, and this was used as a bait vector pBait-2. As a cDNA library to be screened, a human skeletal muscle-derived cDNA library manufactured by Clontech was used. This library is constructed so that the library cDNA is expressed in yeast in a form fused to the GAL4 transcription activation domain (GAL4-AD) under the control of the ADH1 promoter. Sa for host yeast
ccharomyces cerevisiae Y190 was used. In this yeast strain, GAL1 TATA box and UAS (ups
β-galactosidase gene (LacZ) and histidine synthesis gene (H
IS3) on its chromosome. S. cerevisiae Y1
Introduced pBait-2 (TRP1 marker) and cDNA library plasmid from human skeletal muscle (LEU2 marker) into 90, carrying both plasmids and expressing HIS3, one of the two-hybrid reporter genes Transformed yeast was added with 60 mM 3-aminotriazole as a minimum medium and
Selection was made on SD medium without Trp, Leu and His. Transfer the selected transformant colonies to a nylon membrane by the replica method, disrupt the yeast cell wall by freezing and thawing with liquid nitrogen, and stain with X-Gal (5-bromo-4-chloro-β-D-galactoside) And a strain exhibiting β-galactosidase activity was used as a primary candidate strain. Screened 10 ⁷ or more libraries cDNA in the above manner, it was obtained candidate genes 12 clones. A cell extract was prepared from these yeasts using Zymolyase (manufactured by Seikagaku Corporation), and the DNA fraction was used to prepare leucine-requiring Escherichia coli HB1.
01) was transformed. The transformed Escherichia coli was spread on a leucine-free M9 medium, Escherichia coli strains having a library plasmid (LEU2 marker) were selected, and the plasmid was extracted therefrom. With the extracted library plasmid
Using the IRAP bait vector, pBait-2, S. cer
Evisiae Y190 was transformed, and the obtained transformant was examined for histidine requirement and β-galactosidase activity, and 5 clones showing reproducibility were obtained. Among these, a clone (MD36 strain) having the strongest β-galactosidase activity was selected. The cDNA fragment obtained by the yeast two-hybrid method was 679 bp having the sequence of base numbers 2995 to 3674 in FIGS. 1 to 5 (SEQ ID NO: 3).

Example 2 Full-length cDNA Cloning of Human MD36 In order to know the entire structure of this cDNA, the full-length sequence was cloned by plaque hybridization and polymerase chain reaction (PCR). In plaque hybridization, a cDNA library derived from human skeletal muscle
(Clontech; lTripleEx vector) was screened. For the probe region, a 0.68 kb fragment of human MD36 (base numbers 2995 to 3674 of SEQ ID NO: 3) was used. By screening this phage library, human MD36 cDN
A cDNA clone of about 1.8 kb from the 3 'end of A (SEQ ID NO:
3 base numbers 2015 to 3853). Next, cloning by PCR was attempted. The sequence at the 3 'end of human MD36 cDNA is the sequence of FHOS cDNA derived from human spleen (Westendorf et al.,
Gene, 232, 173-182, 1999; Genbank Accession No. AF
(Blastn; score (bits) = 354)
0, E value = 0.0). Based on the FHOS sequence registered in the Genbank database, two kinds of PCR primers covering almost the full length of this cDNA, (1) 5′-TGAGCCGGCCGCAGAGCCATGG-3 ′ (SEQ ID NO: 7) and (2) 5'-TGCTCCGTGCGTTCAAGGAGCTCAC-3 '(SEQ ID NO:
8) was synthesized, and PCR was performed using these. The PCR template was human skeletal muscle and human spleen cDNA (Clontech, # 7413-
1 and # 7412-1) were used. Reaction is 98 ℃, 20 seconds, 65 ℃,
35 cycles of 40 seconds, 72 ° C. and 3.5 minutes were performed. A fragment of about 3.7 kb obtained from each tissue-derived cDNA by PCR was TA cloned and the nucleotide sequence was determined. In addition, the base sequence substitution site derived from the misreading of Taq polymerase
The R product was identified by comparing the nucleotide sequence of three or more clones.
It was replaced by exchanging the A fragment. In addition, about 1.8 kb from the 3 'end, a cDNA fragment derived from a phage library having no possibility of mutation was used. After determining the base sequence,
All the full-length cDNAs derived from human skeletal muscle had the 78-bp sequence inserted into the reported FHOS cDNA sequence (FIGS. 1 to 5, SEQ ID NO: 3). The sequence corresponding to the inserted part is the base number in FIGS. 1 to 5 (SEQ ID NO: 3).
1339 to 1417. That is, 26 amino acids were inserted between Lys at position 440 and Ala at position 441 of FHOS described in the literature (FIGS. 16 to 17). On the other hand, full-length cDNA from human spleen
Was almost identical to the reported FHOS, but mutations involving some amino acid substitutions were observed (SEQ ID NO: 2, SEQ ID NO: 4, FIGS. 6 to 10 and FIGS. 16 to 17). From the human spleen, a new splicing va containing an additional 24 bp insert
riant was found, which contains a stop codon in the inserted sequence, so that its translated polypeptide was FHOS
After the Lys at position 440, the amino acid is terminated in the form of adding 7 amino acids, and the truncated form (truncate t) containing no C-terminal portion obtained by the yeast two-hybrid method described in Example 1
ype) (SEQ ID NO: 5, SEQ ID NO: 6, FIG. 11).
To 14). The distribution of the presence or absence of the inserted sequence in each human tissue was examined by PCR using human tissue-derived cDNA as a template. CDNA of human tissues is MTC panels (Clontech; # K1420-1, # K1421-1)
Was used. The primer was 5'-CCTACCATCTCTGTGGCACCCTCAGCT-3 '(SEQ ID NO:
9) and 5′-TTGGGGCTTGCTGGTATCAGTGGCTCC-3 ′ (SEQ ID NO: 1)
0) was used. Perform PCR with these primers and convert the product to T
A cloning was performed and their nucleotide sequences were determined. This P
The CR was set to detect a 310 bp band in FHOS. Using the above two kinds of primers (SEQ ID NO: 9 and SEQ ID NO: 10), cDNA derived from each human tissue
As a result of PCR, a 310 bp band derived from FHOS and a 334 bp band containing a 24 bp insertion sequence were clearly detected in all tissues except skeletal muscle. On the other hand, a PCR product (388 bp) derived from human MD36 containing an insert sequence of 78 bp was mainly detected in skeletal muscle, and slightly detected in the heart. That is, the 78 bp insertion sequence is specific to skeletal muscle in the examined organs, and splic without the 78 bp insertion sequence in skeletal muscle.
It was confirmed that there was almost no ing variant (FIG. 15). From these, 6 obtained in Example 1
The 79 bp cDNA fragment is a human MD36 cDNA with a 78 bp insert.
It is considered to be a part of. Known FHOS and 3 obtained above
A comparison of the amino acid sequences encoded by the species cDNAs is shown in FIGS. Obtained in FHOS described in the literature and in Example 2.
In FHOS, a difference of 9 amino acids out of all 1164 amino acids was recognized (Thr of the 249th position of FHOS described in the literature is Ser, 307th Asp is Glu, and 308th Thr of the FHOS obtained in Example 2, respectively). Is Ala, 633rd Asp is Glu, 634th Val is Le
u, 700th Thr is Ser, 751th Gly is Glu, 849th
Asp is Glu, and Leu at position 1061 is Pro). The obtained human FHOS cDNA (SEQ ID NO: 4, FIGS. 6 to 10) was transformed into pBlue
scriptII KS + (Stratagene) SpeI site and XhoI si
The plasmid inserted between te and pTB2077, and similarly, the human MD36 cDNA (SEQ ID NO: 3, FIGS.
The plasmid inserted between the SpeI site and the XhoI site of II KS + was designated as pTB2078. In pTB2078, a base substitution derived from one PCR remains (G at position 1677 is replaced with A), but this does not involve mutation of the amino acid to be translated. FIG. 18 shows a comparison of the protein structures of human FHOS, human MD36 and truncated FHOS obtained above.

Example 3 Confirmation of Binding Activity by Quantitative Measurement of β-Galactosidase Activity CPRG was used to quantitatively confirm the binding of human MD36 to IRAP.
β-galactosidase activity was measured using (chlorophenol red-β-D-galactopyranoside) as a substrate. Yeast holding both bait and prey for which an interaction was to be detected was cultured in liquid, and the cells were recovered. The cell wall was disrupted by freezing and thawing with liquid nitrogen. CPRG was added to the disrupted cell suspension, and the absorbance at 578 nm of those samples was measured as β-galactosidase activity. The unit of β-galactosidase is that one yeast cell per minute converts 1 μmol of CPRG into c.
Enzyme activity hydrolyzing to hlorophenol red and D-galactoside was defined as one unit. IRAP (55-82) for Bait sequence, and
The prey sequence is the yeast two-hybr
A sequence directly isolated by the id method (base number 29 of SEQ ID NO: 3)
679 bp corresponding to 95-3674) was used. Further, as a negative control, a vector pGBT9 expressing a non-fused sequence of bait to GAL4-BD was used. S. cerevis using plasmids with these sequences
iae Y190 was transformed, and the β-galactosidase activity of the reconstructed yeast transformant was measured. The transformant having MD36 cDNA was about 70 uni when baiting IRAP (55-82).
t showed β-galactosidase activity. On the other hand, GAL4-BD
Only a protein having no bait sequence showed almost no binding activity (FIG. 19). The value of β-galactosidase activity in an experiment using a strain that did not have human MD36 cDNA as the prey sequence was below the detection limit.

Example 4 Investigation of Interaction between Human MD36 and Glucose Transporter It is known that GLUT4 endoplasmic reticulum is transferred to the cell surface by a polypeptide at the carboxy-terminal (C-terminal) portion, which is a cytoplasmic domain of GLUT4. (Lee & Jung, J. Biol. Ch
em., 272, 21497-21531, 1997). If the GLUT4 endoplasmic reticulum anchor protein is of one type, the protein that binds to IRAP localized in the GLUT4 endoplasmic reticulum may bind to GLUT4, so this hypothesis was confirmed by the yeast two-hybrid method. . As the Bait sequence, the C-terminal cytoplasmic domain of mouse GLUT4 (amino acids 468 to 510; SEQ ID NOs: 13 and 14) was used. As a control experiment, the same was performed using the C-terminal cytoplasmic domain (amino acids 451 to 492) of mouse GLUT1, another protein of the glucose transporter. These sequences are the respective cDNA
It was isolated by the PCR method using pfu polymerase (manufactured by Stratagene) and fused to the GAL4-BD sequence of pGBT9 (manufactured by Clontech).
It was constructed to be expressed as an ion protein. On the other hand, pre
Human MD36 cDNA fused to GAL4-AD as y sequence
(Base numbers 2995 to 3674 of SEQ ID NO: 3) were used. S. cerevisiae Y190 was transformed with these plasmids, and the binding activity between MD36 and each glucose transporter was measured using β-galactosidase activity as an index. As shown in FIG. 20, it was revealed that human MD36 binds to GLUT4 but hardly binds to GLUT1.

Example 5 Distribution of Expression of Human MD36 mRNA in Human Tissue The distribution of expression of human MD36 mRNA in human tissue was detected by Northern blotting. That is, northern blotting was performed on poly (A) ⁺ RNA (each 2 mg) of each human tissue using human MD36 cDNA (base numbers 2995 to 3674 of SEQ ID NO: 3) as a probe. The nylon membrane to which the mRNA of each human tissue was transferred was manufactured by Clontech. Probe and hybridization conditions are the same as those used in the screening of the lambda phage library described in Example 2. Human M labeled with ³² P
Hybridize and wash the D36 cDNA probe under high stringency conditions and use the image analyzer BAS2
000II (manufactured by Fuji Film Co., Ltd.). Human MD36 mRNA
Was strongly expressed in skeletal muscle.

Example 6 Cloning of Mouse MD36 cDNA The human MD36 cDNA sequence (SEQ ID NO: 3, FIGS. 1 to 5) was published.
When a homology search was performed for the ST sequence, a plurality of mouse ESTs were found as highly homologous sequences mainly on the 3′-side of the cDNA sequence of human MD36. Of these, Genbank
EST; D76497 and Genbank EST; AA109839 were selected and used for PCR primer design. The primers used for cloning of the 3'-side portion have the following sequences. EST; sequence from D76947 (sense) M-1; 5'-GAGTTTGCTGTCAGCAAAGATGGCATTGAG-3 '(SEQ ID NO: 19) EST; sequence from AA109839 (antisense) M-5; 5'-TTGCTTAGTCCCAGTGCCTGCACCAGGTCATCTCC-3'
(SEQ ID NO: 20) Note that M-1 is a partial nucleotide sequence corresponding to positions 231-2343 from the 5 ′ end of SEQ ID NO: 16 (nucleotide sequence of mouse MD36 DNA), and M-5 is SEQ ID NO: 16 (DNA of mouse MD36
Base sequence), the base sequence complementary to the partial base sequence corresponding to positions 3883-3617 from the 5 ′ end of the 5 ′ end. PCR with mouse skeletal muscle cDNA (CLONTECH) as template using primers M-1 and M-5
As a result, about 1.3 kb DNA with homology to human MD36
A fragment was obtained. In the above experiment, the sequence on the 5'-side was still unknown.Therefore, when a fragment having homology with the 5'-terminal portion of human MD36 was searched against the mouse genome sequence database of Celera, CMGD; 90000308
913152 was detected as a homologous sequence. The portion of ATG, which is the initiation codon of human MD36, was also identical in this Celera sequence, and further upstream there was a matching stop codon in frame. Using the portion near the stop codon as a primer, PCR was performed with the nucleotide sequence of the partial cDNA of the mouse obtained above. The primers used for cloning of the 5'-side part have the following sequences. Celera; CMGD; sequence from 90000308913152 (sense) MMD-3; 5'-TGAAGTTGCAGCATTTGCAGGGGACAC-3 '(SEQ ID NO: 21) EST; sequence from D76947 (antisense) M-3; 5'-AGCTGGGCTTCCTCAATCTTCTGCCGCTCT-3' (sequence MMD-3 is a partial nucleotide sequence corresponding to the 1-27th position from the 5 'end of SEQ ID NO: 16 (DNA sequence of mouse MD36), and M-3 is SEQ ID NO: 16 (mouse This is the complementary base sequence of the partial base sequence corresponding to the 2373-2402th position from the 5 'end of the MD36 DNA base sequence). PCR using primers MMD-3 and M-3 and mouse skeletal muscle cDNA (CLONTECH) as template
As a result, about 2.4 kb DN with homology to human MD36
Two types of A fragments were obtained. The two sequences are SEQ ID NO:
16 (base sequence of mouse MD36 DNA) and the 78 bp base sequence from "C" at position 1393 to "A" at position 1470 from the 5 'end of this sequence are missing (Mouse FHOS DNA; SEQ ID NO: 18). In terms of amino acids, mouse MD36 (SEQ ID NO: 15) lacks from the N-terminal from Leu at position 445 to Glu at position 470 in mouse FHOS (mouse FHOS amino acid; SEQ ID NO: 1).
7). This deletion site is the splicing v of human FHOS and human MD36.
The shorter sequence (SEQ ID NO: 17) was considered to be the mouse counterpart corresponding to human FHOS (SEQ ID NO: 2) because it matched the ariant site. MMD-
The two 5'-side fragments obtained by PCR of 3 and M-3
3'-side fragment obtained by PCR of -1 and M-5 and primer M-1
A perfect match was found in the overlapping sequence between and M-3. Therefore, these separately obtained PCR fragments were
Type 6 is also thought to be derived from a common mRNA. In addition, the region from the primer M5 to the stop codon was identical in a plurality of mouse ESTs. These ESTs are Genbank EST
AA109839, AA089340, AW540395 and AW412412.
Therefore, the sequence common to them (5'-AGCTCCTGGTCTAGAG
GTGTGA-3 ′) was considered the consensus sequence from primer M5 to the stop codon. The homology between the amino acid sequences of human and mouse is 86.8% between human FHOS (SEQ ID NO: 2) and mouse FHOS (SEQ ID NO: 17) without the inserted sequence, and 78 bp.
MD36 (SEQ ID NO: 1) and mouse MD
86.7% among 36 (SEQ ID NO: 18).

Example 7 Tissue distribution of mouse MD36 PC obtained using primers M1 and M5 described in Example 6
Northern Blot was performed using the R fragment as a probe. mouse
MD36 was strongly expressed in lung and skeletal muscle.

Example 8 Test for Biochemical Binding of Human MD36 The interaction between IRAP and human MD36 at the protein level was detected by a method different from those in Examples 3 and 4.
A biochemical binding test was performed. The human MD36 protein used was the C-terminal part of SEQ ID NO: 1 (MD36 full length cDNA) (partial peptide encoded by the partial base sequence at position 2995-3674 from the 5 'end of SEQ ID NO: 2). This DNA fragment was inserted into a pGEX expression vector (Amersham Pharmacia), and under the control of the tac promoter, the human MD36 partial protein was GST (Glutathion
eS-transferase) expression plasmid pGEX-MD36N4, which was expressed in a form fused with the protein (GST-MD36N4 protein), was constructed. However, the protease recognition site at these junctions was removed by applying PCR. E. Using pGEX-MD36N4.
E. coli BL21 was transformed, and I
The cells were cultured with expression induction by PTG. After the culture, the collected cells were disrupted by sonication, and the disrupted cell suspension was centrifuged. As a result, most of the GST-MD36N4 protein was present in the insoluble fraction. This insoluble fraction was treated with 8 M urea, 5 mM
The protein was refolded by redissolving it in Phosphate buffered saline (PBS) containing DTT and dialyzing this solution against PBS containing 0.5 M arginine hydrochloride. On the other hand, IRAP is a sequence that is a cytoplasmic domain (SEQ ID NO: 1).
1) was inserted into pET21 (Novagen, Inc.), and His-tag
The plasmid pET21-IRAP (1-109) was constructed to express the ligated product under the control of the T7 promoter, expressed in Escherichia coli according to a conventional method, and the recombinant protein was purified. Hereinafter, this recombinant protein is referred to as IRAP-His. The protein-protein binding test was performed by the following method. Add GST-MD36N4 (10 μg / ml) dissolved in sodium carbonate buffer (25 mM Na ₂ CO ₃ , 25 mM NaHCO ₃ , pH 9.6) to a 96-well EIA plate and leave at 4 ° C overnight In this way to the plate surface. Tris buffer (20 mM Tris-HCl (pH 8.0), 1
After washing the plate three times with 50 mM NaCl (hereinafter abbreviated as TBS) and 0.05% Tween 20 (hereinafter abbreviated as TBS-T), PBS containing 3% bovine serum albumin (BSA) was added. In addition, blocking was performed by allowing the mixture to stand at room temperature for 1 hour. In addition, IRAP was added to the well coated with human MD36.
-His (1.0 μg / ml in TBS) was added and left at room temperature for 2 hours to perform a binding reaction. After washing the wells three times with TBS-T again, the IRAP-His protein remaining in the wells was removed by anti-Penta-
Quantification was performed using a His antibody (Qiagen, Inc.). Detection is HRP-
Anti-mouse IgG goat antibody and ECL system (NEN Life Scienc
e, Inc.). Add IRAP-His (1.0 μg / ml) or anti-Penta-His antibody (2000-fold diluted) and both to wells coated with GST-MD36N4 or uncoated wells, wash with TBS-T The amount of His-Tag remaining in the well was quantified. His-Tag was significantly detected only when both IRAP-His and anti-Penta-His antibody were added to human MD36-coated wells, and the background detected under other conditions was very low (FIG. 21). ). Furthermore, it was confirmed whether these bindings were inhibited by the His-tag-free IRAP peptide. IR for binding inhibition experiments
Amino acid number 55-82 of AP (IRAP (55-82); SEQ ID NO: 1)
A peptide consisting of a partial amino acid sequence at positions 55 to 82 from the N-terminus of No. 1) was chemically synthesized and used. As shown in FIG. 22, the binding of IRAP-His to GST-MD36N4 was inhibited by adding IRAP (55-82) in a concentration-dependent manner. From the above, in this system
The specificity of the binding between IRAP and human MD36 was shown.

Example 9 Screening of a Compound for Inhibition of Binding A compound that inhibits the binding between IRAP and human MD36 was screened according to the method of Example 8 (test for biochemical binding of human MD36). The library compound (dimethylsulfoxide (DMS
O) solution was added to a final concentration of 1 μM to perform a binding test, and a compound that reduced the amount of His-Tag remaining on the plate was selected. As a result, the following two compounds shown in [Formula 1] and [Formula 2] showed binding inhibition. The IC ₅₀ value is the value when the compound is not added (when only DMSO is added) after the background is subtracted from each value as the value when IRAP-His is not added.
It was calculated from the ratio of the measured value at the time of compound addition to the measured value of.

[0061]

Embedded image IC ₅₀ : 0.21 μM

Embedded image IC ₅₀ : 0.24 μM

Reference Example 1 Profilin IIL (SEQ ID NO: 28)
Cloning of a protein having an amino acid represented by the formula: DNA encoding the protein (SEQ ID NO: 29)) Profilin IIL was obtained from Profilin II (Genbank accession #,
678; obtained by polymarase chain reaction (PCR) that amplifies between the start codon and the stop codon of SEQ ID NO: 30). P
Primers used for CR were: (1) 5′-ATGGCCGGTTGGCAGAGCTACGTGGAT-3 ′ (27 mer;
(SEQ ID NO: 31) (2) 5'-TTACACATCAGACCTCCTCAGGTATAAAGC-3 '(30 me
r; SEQ ID NO: 32). The template is cDNA from human skeletal muscle, and the enzyme is Pf
u Using polymerase (STRATAGENE), 95 ℃ 30 seconds, 65 ℃
The reaction was performed for 35 cycles at 60 seconds at 72 ° C. for 45 seconds. As a result of the PCR, in addition to the 423 bp DNA fragment of Profilin II, 745 bp
DNA fragment was found and named Profilin IIL. Pro
The nucleotide sequence of the filin IIL cDNA is shown in SEQ ID NO: 29,
The amino acid sequence of rofilin II is shown in SEQ ID NO: 28. When the tissue distribution of ProfilinIIL was examined by human MTC-panel by PCR using these primers, expression was observed in brain, skeletal muscle, pancreas, placenta and heart.

Example 10 Examination of MD36 (protein having the amino acid sequence represented by SEQ ID NO: 1) and protein-protein interaction of the Profilin family The protein-protein interaction of MD36 and the profilin family was examined using the yeast two-hybrid system. Detected using pr
ofilin I (Evangelista at al., Science, 276, 118-12
2, 1997 Imamura et al, EMBO J., 16, 2745-2755, 199
7 Tanaka, Biochem. Biophys. Res. Commun., 267, 479
-481, 2000), profilin II (Schluter etal., Biochim.
Biophys. Acta, 1359, 97-109, 1997), profilin IIL
Coding region of yeast GAL under the control of yeast ADH1 promoter
Construction of an expression plasmid that is expressed in a form fused to the 4-DNA binding domain protein based on pGBT9 (CLONTECH),
These were pG-PFNI, pG-PFNII, and pG-PFNIIL, respectively (the yeast selection marker was TRP1). On the other hand, the full-length sequence of MD36 which is an IRAP-BP protein (SEQ ID NO: 1)
The coding region from the end to the sequence containing the proline rich domain (SEQ ID NO: 33) is fused to the yeast GAL4-transcriptional activation domain protein under the control of the yeast ADH1 promoter.
pACT2 (CLONTEC
H) and pACT-MD36 and pACT-MD36, respectively.
NT (the yeast selectable marker was LEU2). pG-PFNI, pG-PF
NII, pG-PFNIIL or pACT-pGBT9 as control
Yeast Saccharomyces ce with MD36 or pACT-MD36NT
Yeast strains which were co-transfected into revisiae Y190 and retained both plasmids on SD medium without tryptophan and leucine were selected. S. cerevisiae Y190 is originally a histidine-requiring strain, but when the proteins on the introduced plasmid interact with each other, the reporter gene HIS3 is expressed and can grow on histidine-free plates. The resulting yeast strain was free of tryptophan, leucine and histidine, 40 mM 3-amino-1,2,2.
When applied to SD medium supplemented with 4-triazole, Pro
filin I / MD36, Profilin I / MD36NT, Profilin II / MD36,
Profilin II / MD36NT expression p
As in the control experiment using GBT9, the yeast did not grow and only the combination of expression of Profilin IIL / MD36 and Profilin IIL / MD36NT showed growth of the yeast. These results indicate that MD36 specifically binds to profilin IIL in the profilin family.

[0064]

The protein I of the present invention is strongly expressed in skeletal muscle, and the protein II of the present invention is expressed in spleen and the like. Since the protein of the present invention increases blood glucose level by binding to IRAP, it is useful as a preventive / therapeutic agent for hypoglycemia. Further, the protein of the present invention can be used in a screening method for inhibiting the binding of the protein of the present invention to IRAP or GLUT4. The compound that inhibits the binding between the protein of the present invention and IRAP or GLUT4 is useful as an agent for preventing or treating diseases such as hyperglycemia and diabetes.

[0065]

[Sequence List] [SEQUENCE LISTING] <110> Takeda Chemical Industries, Ltd. <120> Novel Gene and Its Use <130> B00335 <150> JP 11-361679 <151> 1999-12-20 <150> JP 11-365176 <151> 1999-12-22 <160> 33 <210> 1 <211> 1190 <212> PRT <213> Human <400> 1 Met Ala Gly Gly Glu Asp Arg Gly Asp Gly Glu Pro Val Ser Val Val 5 10 15 Thr Val Arg Val Gln Tyr Leu Glu Asp Thr Asp Pro Phe Ala Cys Ala 20 25 30 Asn Phe Pro Glu Pro Arg Arg Ala Pro Thr Cys Ser Leu Asp Gly Ala 35 40 45 Leu Pro Leu Gly Ala Gln Ile Pro Ala Val His Arg Leu Leu Gly Ala 50 55 60 Pro Leu Lys Leu Glu Asp Cys Ala Leu Gln Val Ser Pro Ser Gly Tyr 65 70 75 80 Tyr Leu Asp Thr Glu Leu Ser Leu Glu Glu Gln Arg Glu Met Leu Glu 85 90 95 Gly Phe Tyr Glu Glu Ile Ser Lys Gly Arg Lys Pro Thr Leu Ile Leu 100 105 110 Arg Thr Gln Leu Ser Val Arg Val Asn Ala Ile Leu Glu Lys Leu Tyr 115 120 125 Ser Ser Ser Gly Pro Glu Leu Arg Arg Ser Leu Phe Ser Leu Lys Gln 130 135 140 Ile Phe Gln Glu Asp Lys Asp Leu Val Pro Glu Phe Val His Ser Glu 145 150 155 160 Gly Leu Ser Cys Leu Ile Arg Val Gly Ala Ala Ala Asp His Asn Tyr 165 170 175 Gln Ser Tyr Ile Leu Arg Ala Leu Gly Gln Leu Met Leu Phe Val Asp 180 185 190 Gly Met Leu Gly Val Val Ala His Ser Asp Thr Ile Gln Trp Leu Tyr 195 200 205 Thr Leu Cys Ala Ser Leu Ser Arg Leu Val Val Lys Thr Ala Leu Lys 210 215 220 Leu Leu Leu Val Phe Val Glu Tyr Ser Glu Asn Asn Ala Pro Leu Phe 225 230 235 240 Ile Arg Ala Val Asn Ser Val Ala Ser Thr Thr Gly Ala Pro Pro Trp 245 250 255 Ala Asn Leu Val Ser Ile Leu Glu Glu Lys Asn Gly Ala Asp Pro Glu 260 265 270 Leu Leu Val Tyr Thr Val Thr Leu Ile Asn Lys Thr Leu Ala Ala Leu 275 280 285 Pro Asp Gln Asp Ser Phe Tyr Asp Val Thr Asp Ala Leu Glu Gln Gln 290 295 300 Gly Met Glu Ala Leu Val Gln Arg His Leu Gly Thr Ala Gly Thr Asp 305 310 315 320 Val Asp Leu Arg Thr Gln Leu Val Leu Tyr Glu Asn Ala Leu Lys Leu 325 330 335 Glu Asp Gly Asp Ile Glu Glu Ala Pro Gly Ala Gly Gly Arg Arg Glu 340 345 350 Arg Arg Lys Pro Ser Ser Glu Glu Gly Lys Arg Ser Arg Arg Ser Leu 355 360 365 Glu Gly Gly Gly Cys Pro Ala Arg Ala Pro Glu Pro Gly Pro Thr Gly 370 375 380 Pro Ala Ser Pro Val Gly Pro Thr Ser Ser Thr Gly Pro Ala Leu Leu 385 390 395 400 Thr Gly Pro Ala Ser Ser Pro Val Gly Pro Pro Ser Gly Leu Gln Ala 405 410 415 Ser Val Asn LeuPhe Pro Thr Ile Ser Val Ala Pro Ser Ala Asp Thr 420 425 430 Ser Ser Glu Arg Ser Ile Tyr Lys Leu His Gln Thr Ala Ser Val Trp 435 440 445 Ala Pro Glu Ser Pro Pro Val Pro Gln Ser Pro Pro Gly Gln Ala Arg 450 455 460 Leu Glu Ala Arg Phe Leu Glu Asn Val Ala Ala Ala Glu Thr Glu Lys 465 470 475 480 Gln Val Ala Leu Ala Gln Gly Arg Ala Glu Thr Leu Ala Gly Ala Met 485 490 495 Pro Asn Glu Ala Gly Gly His Pro Asp Ala Arg Gln Leu Trp Asp Ser 500 505 510 Pro Glu Thr Ala Pro Ala Ala Arg Thr Pro Gln Ser Pro Ala Pro Cys 515 520 525 Val Leu Leu Arg Ala Gln Arg Ser Leu Ala Pro Glu Pro Lys Glu Pro 530 535 540 Leu Ile Pro Ala Ser Pro Lys Ala Glu Pro Ile Trp Glu Leu Pro Thr 545 550 555 560 Arg Ala Pro Arg Leu Ser Ile Gly Asp Leu Asp Phe Ser Asp Leu Gly 565 570 575 Glu Asp Glu Asp Gln Asp Met Leu Asn Val Glu Ser Val Glu Ala Gly 580 585 590 Lys Asp Ile Pro Ala Pro Ser Pro Pro Leu Pro Leu Leu Ser Gly Val 595 600 605 Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Ile Lys Gly Pro Phe 610 615 620 620 Pro Pro Pro Pro ProLeu Pro Leu Ala Ala Pro Leu Pro His Ser Val 625 630 635 640 640 Pro Asp Ser Ser Ala Leu Pro Thr Lys Arg Lys Thr Val Lys Leu Phe 645 650 655 Trp Arg Glu Leu Lys Leu Ala Gly Gly His Gly Val Ser Ala Ser Arg 660 665 670 Phe Gly Pro Cys Ala Thr Leu Trp Ala Ser Leu Asp Pro Val Ser Val 675 680 685 Asp Thr Ala Arg Leu Glu His Leu Phe Glu Ser Arg Ala Lys Glu Val 690 695 700 Leu Pro Ser Lys Lys Ala Gly Glu Gly Arg Arg Thr Met Thr Thr Val 705 710 715 720 Leu Asp Pro Lys Arg Ser Asn Ala Ile Asn Ile Gly Leu Thr Thr Leu 725 730 735 Pro Pro Val His Val Ile Lys Ala Ala Leu Leu Asn Phe Asp Glu Phe 740 745 750 Ala Val Ser Lys Asp Gly Ile Glu Lys Leu Leu Thr Met Met Pro Thr 755 760 765 Glu Glu Glu Arg Gln Lys Ile Glu Glu Ala Gln Leu Ala Asn Pro Asp 770 775 780 Ile Pro Leu Gly Pro Ala Glu Asn Phe Leu Met Thr Leu Ala Ser Ile 785 790 795 800 Gly Gly Leu Ala Ala Arg Leu Gln Leu Trp Ala Phe Lys Leu Asp Tyr 805 810 815 Asp Ser Met Glu Arg Glu Ile Ala Glu Pro Leu Phe Asp Leu Lys Val 820 825 830 Gly Met Glu Gln LeuVal Gln Asn Ala Thr Phe Arg Cys Ile Leu Ala 835 840 845 Thr Leu Leu Ala Val Gly Asn Phe Leu Asn Gly Ser Gln Ser Ser Gly 850 855 860 Phe Glu Leu Ser Tyr Leu Glu Lys Val Ser Glu Val Lys Asp Thr Val 865 870 875 880 Arg Arg Gln Ser Leu Leu His His Leu Cys Ser Leu Val Leu Gln Thr 885 890 895 Arg Pro Glu Ser Ser Asp Leu Tyr Ser Glu Ile Pro Ala Leu Thr Arg 900 905 910 Cys Ala Lys Val Asp Phe Glu Gln Leu Thr Glu Asn Leu Gly Gln Leu 915 920 925 Glu Arg Arg Ser Arg Ala Ala Glu Glu Ser Leu Arg Ser Leu Ala Lys 930 935 940 His Glu Leu Ala Pro Ala Leu Arg Ala Arg Leu Thr His Phe Leu Asp 945 950 955 960 Gln Cys Ala Arg Arg Val Ala Met Leu Arg Ile Val His Arg Arg Val 965 970 975 Cys Asn Arg Phe His Ala Phe Leu Leu Tyr Leu Gly Tyr Thr Pro Gln 980 985 990 Ala Ala Arg Glu Val Arg Ile Met Gln Phe Cys His Thr Leu Arg Glu 995 1000 1005 Phe Ala Leu Glu Tyr Arg Thr Cys Arg Glu Arg Val Leu Gln Gln Gln 1010 1015 1020 Gln Lys Gln Ala Thr Tyr Arg Glu Arg Asn Lys Thr Arg Gly Arg Met 1025 1030 1035 1040 Ile Thr Glu Thr Glu Lys Phe Ser Gly Val Ala Gly Glu Ala Pro Ser 1045 1050 1055 Asn Pro Ser Val Pro Val Ala Val Ser Ser Gly Pro Gly Arg Gly Asp 1060 1065 1070 Ala Asp Ser His Ala Ser Met Lys Ser Leu Leu Thr Ser Arg Pro Glu 1075 1080 1085 Asp Thr Thr His Asn Arg Arg Ser Arg Gly Met Val Gln Ser Ser Ser 1090 1095 1100 Pro Ile Met Pro Thr Val Gly Pro Ser Thr Ala Ser Pro Glu Glu Pro 1105 1110 1115 1120 Pro Gly Ser Ser Leu Pro Ser Asp Thr Ser Asp Glu Ile Met Asp Leu 1125 1130 1135 Leu Val Gln Ser Val Thr Lys Ser Ser Pro Arg Ala Leu Ala Ala Arg 1140 1145 1150 Glu Arg Lys Arg Ser Arg Gly Asn Arg Lys Ser Leu Arg Arg Thr Leu 1155 1160 1165 Lys Ser Gly Leu Gly Asp Asp Leu Val Gln Ala Leu Gly Leu Ser Lys 1170 1175 1180 Gly Pro Gly Leu Glu Val 1185 1190 <210> 2 <211> 1164 <212> PRT <213> Human <400> 2 Met Ala Gly Gly Glu Asp Arg Gly Asp Gly Glu Pro Val Ser Val Val 5 10 15 Thr Val Arg Val Gln Tyr Leu Glu Asp Thr Asp Pro Phe Ala Cys Ala 20 25 30 Asn Phe Pro Glu Pro Arg Arg Ala Pro Thr Cys Ser Leu Asp Gly Ala 35 40 45 Leu Pro Leu Gly Ala Gln Ile Pro Ala Val His Arg Leu Leu Gly Ala 50 55 60 Pro Leu Lys Leu Glu Asp Cys Ala Leu Gln Val Ser Pro Ser Gly Tyr 65 70 75 80 Tyr Leu Asp Thr Glu Leu Ser Leu Glu Glu Gln Arg Glu Met Leu Glu 85 90 95 Gly Phe Tyr Glu Glu Ile Ser Lys Gly Arg Lys Pro Thr Leu Ile Leu 100 105 110 Arg Thr Gln Leu Ser Val Arg Val Asn Ala Ile Leu Glu Lys Leu Tyr 115 120 125 Ser Ser Ser Gly Pro Glu Leu Arg Arg Ser Leu Phe Ser Leu Lys Gln 130 135 140 Ile Phe Gln Glu Asp Lys Asp Leu Val Pro Glu Phe Val His Ser Glu 145 150 155 160 Gly Leu Ser Cys Leu Ile Arg Val Gly Ala Ala Ala Asp His Asn Tyr 165 170 175 Gln Ser Tyr Ile Leu Arg Ala Leu Gly Gln Leu Met Leu Phe Val Asp 180 185 190 Gly Met Leu Gly Val Val Ala His Ser Asp Thr Ile Gln Trp Leu Tyr 195 200 205 Thr Leu Cys Ala Ser Leu Ser Arg Leu Val Val Lys Thr Ala Leu Lys 210 215 220 Leu Leu Leu Val Phe Val Glu Tyr Ser Glu Asn Asn Ala Pro Leu Phe 225 230 235 240 Ile Arg Ala Val Asn Ser Val Ala Ser Thr Thr Gly Ala Pro Pro Trp 245 250 255 Ala Asn Leu Val Ser Ile Leu Glu Glu Lys Asn Gly Ala Asp Pro Glu 260 265 270 Leu Leu Val Tyr Thr Val Thr Leu Ile Asn Lys Thr Leu Ala Ala Leu 275 280 285 Pro Asp Gln Asp Ser Phe Tyr Asp Val Thr Asp Ala Leu Glu Gln Gln 290 295 300 Gly Met Glu Ala Leu Val Gln Arg His Leu Gly Thr Ala Gly Thr Asp 305 310 315 320 Val Asp Leu Arg Thr Gln Leu Val Leu Tyr Glu Asn Ala Leu Lys Leu 325 330 335 Glu Asp Gly Asp Ile Glu Glu Ala Pro Gly Ala Gly Gly Arg Arg Glu 340 345 350 Arg Arg Lys Pro Ser Ser Glu Glu Gly Lys Arg Ser Arg Arg Ser Leu 355 360 365 Glu Gly Gly Gly Cys Pro Ala Arg Ala Pro Glu Pro Gly Pro Thr Gly 370 375 380 Pro Ala Ser Pro Val Gly Pro Thr Ser Ser Thr Gly Pro Ala Leu Leu 385 390 395 400 Thr Gly Pro Ala Ser Ser Pro Val Gly Pro Pro Ser Gly Leu Gln Ala 405 410 415 Ser Val Asn Leu Phe Pro Thr Ile Ser Val Ala Pro Ser Ala Asp Thr 420 425 430 Ser Ser Glu Arg Ser Ile Tyr Lys Ala Arg Phe Leu Glu Asn Val Ala 435 440 445 Ala Ala Glu Thr Glu Lys Gln Val Ala Leu Ala Gln Gly Arg Ala Glu 450 455 460 Thr Leu Ala Gly Ala Met Pro Asn Glu Ala Gly Gly His Pro Asp Ala 465 470 475 480 Arg Gln Leu Trp Asp Ser Pro Glu Thr Ala Pro Ala Ala Ala Arg Thr Pro 485 490 495 Gln Ser Pro Ala Pro Cys Val Leu Leu Arg Ala Gln Arg Ser Leu Ala 500 505 510 Pro Glu Pro Lys Glu Pro Leu Ile Pro Ala Ser Pro Lys Ala Glu Pro 515 520 525 Ile Trp Glu Leu Pro Thr Arg Ala Pro Arg Leu Ser Ile Gly Asp Leu 530 535 540 Asp Phe Ser Asp Leu Gly Glu Asp Glu Asp Gln Asp Met Leu Asn Val 545 550 555 560 Glu Ser Val Glu Ala Gly Lys Asp Ile Pro Ala Pro Ser Pro Pro Leu 565 570 575 Pro Leu Leu Ser Gly Val Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro 580 585 590 590 Pro Ile Lys Gly Pro Phe Pro Pro Pro Pro Pro Leu Pro Leu Ala Ala 595 600 605 Pro Leu Pro His Ser Val Pro Asp Ser Ser Ala Leu Pro Thr Lys Arg 610 615 620 620 Lys Thr Val Lys Leu Phe Trp Arg Glu Leu Lys Leu Ala Gly Gly His 625 630 635 640 Gly Val Ser Ala Ser Arg Phe Gly Pro Cys Ala Thr Leu Trp Ala Ser 645 650 655 Leu Asp Pro Val Ser Val Asp Thr Ala Arg Leu Glu His Leu Phe Glu 660 665 670 Ser Arg Ala Lys Glu Val Leu Pro Ser Lys Lys Ala Gly Glu Gly Arg 675 680 685 Arg Thr Met Thr Thr Val Leu Asp Pro Lys Arg Ser Asn Ala Ile Asn 690 695 700 Ile Gly Leu Thr Thr Leu Pro Pro Val His Val Ile Lys Ala Ala Leu 705 710 715 720 720 Leu Asn Phe Asp Glu Phe Ala Val Ser Lys Asp Gly Ile Glu Lys Leu 725 730 735 Leu Thr Met Met Pro Thr Glu Glu Glu Arg Gln Lys Ile Glu Glu Ala 740 745 750 Gln Leu Ala Asn Pro Asp Ile Pro Leu Gly Pro Ala Glu Asn Phe Leu 755 760 765 Met Thr Leu Ala Ser Ile Gly Gly Leu Ala Ala Arg Leu Gln Leu Trp 770 775 780 Ala Phe Lys Leu Asp Tyr Asp Ser Met Glu Arg Glu Ile Ala Glu Pro 785 790 795 800 Leu Phe Asp Leu Lys Val Gly Met Glu Gln Leu Val Gln Asn Ala Thr 805 810 815 Phe Arg Cys Ile Leu Ala Thr Leu Leu Ala Val Gly Asn Phe Leu Asn 820 825 830 Gly Ser Gln Ser Se r Gly Phe Glu Leu Ser Tyr Leu Glu Lys Val Ser 835 840 845 Glu Val Lys Asp Thr Val Arg Arg Gln Ser Leu Leu His His Leu Cys 850 855 860 Ser Leu Val Leu Gln Thr Arg Pro Glu Ser Ser Asp Leu Tyr Ser Glu 865 870 875 880 Ile Pro Ala Leu Thr Arg Cys Ala Lys Val Asp Phe Glu Gln Leu Thr 885 890 895 Glu Asn Leu Gly Gln Leu Glu Arg Arg Ser Arg Ala Ala Glu Glu Ser 900 900 910 910 Leu Arg Ser Leu Ala Lys His Glu Leu Ala Pro Ala Leu Arg Ala Arg 915 920 925 925 Leu Thr His Phe Leu Asp Gln Cys Ala Arg Arg Val Ala Met Leu Arg 930 935 940 Ile Val His Arg Arg Val Cys Asn Arg Phe His Ala Phe Leu Leu Tyr 945 950 955 960 Leu Gly Tyr Thr Pro Gln Ala Ala Arg Glu Val Arg Ile Met Gln Phe 965 970 975 Cys His Thr Leu Arg Glu Phe Ala Leu Glu Tyr Arg Thr Cys Arg Glu 980 985 990 Arg Val Leu Gln Gln Gln Gln Lys Gln Ala Thr Tyr Arg Glu Arg Asn 995 1000 1005 Lys Thr Arg Gly Arg Met Ile Thr Glu Thr Glu Lys Phe Ser Gly Val 1010 1015 1020 Ala Gly Glu Ala Pro Ser Asn Pro Ser Val Pro Val Ala Val Ser Ser 1025 1030 1035 1040 Gly Pro G ly Arg Gly Asp Ala Asp Ser His Ala Ser Met Lys Ser Leu 1045 1050 1055 Leu Thr Ser Arg Pro Glu Asp Thr Thr His Asn Arg Arg Ser Arg Gly 1060 1065 1070 Met Val Gln Ser Ser Ser Pro Ile Met Pro Thr Val Gly Pro Ser Thr 1075 1080 1085 Ala Ser Pro Glu Glu Pro Pro Gly Ser Ser Leu Pro Ser Asp Thr Ser 1090 1095 1100 Asp Glu Ile Met Asp Leu Leu Val Gln Ser Val Thr Lys Ser Ser Pro 1105 1110 1115 1120 Arg Ala Leu Ala Ala Arg Glu Arg Lys Arg Ser Arg Gly Asn Arg Lys 1125 1130 1135 Ser Leu Arg Arg Thr Leu Lys Ser Gly Leu Gly Asp Asp Leu Val Gln 1140 1145 1150 Ala Leu Gly Leu Ser Lys Gly Pro Gly Leu Glu Val 1155 1160 <210> 3 <211> 3570 <212> DNA <213> Human <400> 3 ATGGCGGGCG GGGAAGACCG CGGGGACGGA GAGCCGGTAT CAGTGGTGAC CGTGAGGGTG 60 CAGTACCTGG AAGACACCGA CCCCTTCGCA TGTGCCAACT TTCCGGAGCC GCGCCGGGCC 120 CCCACCTGCA GCCTGGACGG GGCGCTGCCC TTGGGCGCGC AGATACCCGC GGTGCACCGC 180 CTGCTGGGAG CGCCGCTCAA GTTGGAGGAT TGTGCTCTGC AAGTGTCTCC CTCCGGATAC 240 TACCTGGACA CCGAGCTGTC CCTGGAAGAG CAGCGGGAGA TGCTGGAGGG CTTCTATGAA 300 GAGATCAGCA AAGGGCGGAA GCCCACGCTG ATCCTTCGGA CCCAGCTCTC TGTGAGGGTC 360 AACGCTATCT TGGAAAAGCT GTATAGCTCC AGTGGTCCTG AGCTCCGCCG CTCCCTCTTC 420 TCACTGAAGC AGATCTTCCA GGAGGACAAA GACCTGGTGC CTGAATTTGT GCATTCAGAG 480 GGGCTGAGCT GCCTGATCCG TGTGGGTGCT GCTGCCGACC ACAACTACCA GAGCTACATC 540 CTTAGAGCGC TCGGCCAGCT GATGCTCTTT GTGGATGGAA TGCTGGGGGT GGTGGCCCAC 600 AGTGACACTA TTCAGTGGCT GTACACATTG TGTGCCAGCC TGTCCCGCTT GGTGGTGAAG 660 ACAGCCCTGA AGCTGCTGTT GGTGTTTGTA GAATACTCCG AAAACAACGC ACCGCTGTTC 720 ATCCGTGCAG TGAACTCTGT GGCCAGCACC ACCGGTGCTC CTCCCTGGGC CAATCTGGTG 780 TCCATCCTGG AGGAGAAGAA TGGCGCTGAC CCTGAGTTGT TGGTGTACAC GGTCACCCTC 840 ATCAACAAGA CG CTGGCGGC GCTCCCGGAC CAGGACTCCT TCTACGATGT GACGGATGCA 900 CTGGAGCAGC AGGGCATGGA AGCGCTGGTC CAGCGCCACC TGGGCACTGC GGGCACTGAC 960 GTCGACCTGC GCACGCAGCT TGTGCTCTAC GAGAACGCCC TGAAATTGGA GGATGGAGAC 1020 ATCGAAGAAG CCCCAGGCGC TGGTGGGCGG CGGGAACGAC GAAAGCCTTC TTCTGAGGAG 1080 GGCAAGAGGA GCCGCCGTTC TCTGGAAGGC GGGGGCTGCC CCGCGCGTGC CCCGGAACCT 1140 GGCCCCACAG GCCCCGCCTC ACCGGTAGGC CCCACCTCTT CCACCGGCCC CGCCCTGCTG 1200 ACAGGCCCCG CCTCCAGCCC TGTGGGCCCT CCCTCCGGTC TCCAAGCTTC AGTGAACCTT 1260 TTTCCTACCA TCTCTGTGGC ACCCTCAGCT GACACCTCCA GCGAGAGGAG CATCTACAAA 1320 CTTCACCAAA CTGCTTCCGT TTGGGCCCCT GAGAGCCCAC CCGTCCCCCA GTCCCCTCCT 1380 GGGCAGGCCA GGCTGGAAGC CCGGTTCCTG GAGAATGTGG CGGCAGCAGA AACAGAGAAG 1440 CAGGTTGCGC TGGCCCAGGG CCGGGCAGAG ACACTTGCCG GGGCCATGCC CAATGAGGCG 1500 GGTGGACACC CAGATGCCCG GCAACTCTGG GACTCCCCAG AGACAGCCCC TGCAGCCAGA 1560 ACACCCCAGA GCCCTGCCCC CTGTGTCCTG CTCCGGGCCC AGCGAAGCCT TGCACCAGAG 1620 CCCAAGGAGC CACTGATACC AGCAAGCCCC AAGGCTGAGC CCATCTGGGA GCTCCCTACC 1680 CGTGCACCCA GGCTCTCTAT TGGGGACCTG GACTTTTCAG ATCTAGGGGA GGATGAAGAC 1740 CAGGACATGC TGAATGTAGA GTCTGTGGAG GCTGGGAAAG ACATCCCAGC TCCCTCACCC 1800 CCACTGCCCC TGCTCTCGGG AGTACCCCCC CCTCCCCCAC TTCCACCTCC CCCACCCATC 1860 AAAGGCCCCT TCCCACCACC TCCACCTCTA CCTCTGGCTG CCCCTCTTCC CCATTCAGTG 1920 CCTGACAGCT CAGCCCTCCC CACTAAGAGG AAGACAGTAA AACTTTTCTG GCGTGAGCTG 1980 AAGCTGGCTG GGGGCCATGG AGTCTCTGCA AGCCGCTTTG GGCCCTGCGC CACCCTCTGG 2040 GCTTCACTGG ACCCTGTCTC AGTGGACACG GCCCGACTGG AACACCTCTT TGAGTCTCGT 2100 GCCAAAGAGG TGCTGCCCTC CAAGAAAGCT GGAGAGGGCC GCCGGACAAT GACCACAGTG 2160 CTGGACCCCA AGCGCAGCAA CGCCATCAAC ATCGGCCTAA CCACACTGCC ACCTGTGCAT 2220 GTCATTAAGG CTGCTCTGCT CAACTTTGAT GAGTTTGCTG TCAGCAAGGA TGGCATTGAG 2280 AAGCTACTGA CCATGATGCC CACGGAGGAA GAGCGGCAGA AGATTGAGGA AGCCCAGCTG 2340 GCCAACCCTG ACATACCCCT GGGCCCAGCC GAGAACTTCC TGATGACTCT TGCCTCCATT 2400 GGCGGCCTCG CTGCTCGTCT ACAACTCTGG GCCTTCAAGC TGGACTATGA CAGCATGGAG 2460 CGGGAAATTG CTGAGCCACT GTTTGACCTG AAAGTGGGTA TGGAACAGCT GGTACAGAAT 2520 GCCACCTTCC GCTGCATCCT GGCTA CCCTC CTAGCGGTGG GCAACTTCCT CAATGGCTCC 2580 CAGAGCAGCG GCTTTGAGCT GAGCTACCTG GAGAAGGTGT CAGAGGTGAA GGACACGGTG 2640 CGTCGACAGT CACTGCTACA CCATCTCTGC TCCCTAGTGC TCCAGACCCG GCCTGAGTCC 2700 TCTGACCTCT ATTCAGAAAT CCCTGCCCTG ACCCGCTGTG CCAAGGTGGA CTTTGAACAG 2760 CTGACTGAGA ACCTGGGGCA GCTGGAGCGC CGGAGCCGGG CAGCCGAGGA GAGCCTGCGG 2820 AGCTTGGCCA AGCATGAGCT GGCCCCAGCC CTGCGTGCCC GCCTCACCCA CTTCCTGGAC 2880 CAGTGTGCCC GCCGTGTTGC CATGCTAAGG ATAGTGCACC GCCGTGTCTG CAATAGGTTC 2940 CATGCCTTCC TGCTCTACCT GGGCTACACC CCGCAGGCGG CCCGTGAAGT GCGCATCATG 3000 CAGTTCTGCC ACACGCTGCG GGAATTTGCG CTTGAGTATC GGACTTGCCG GGAACGAGTG 3060 CTACAGCAGC AGCAGAAGCA GGCCACATAC CGTGAGCGCA ACAAGACCCG GGGACGCATG 3120 ATCACCGAGA CAGAGAAGTT CTCAGGTGTG GCTGGGGAAG CCCCCAGCAA CCCCTCTGTC 3180 CCAGTAGCAG TGAGCAGCGG GCCAGGCCGG GGAGATGCTG ACAGTCATGC TAGTATGAAG 3240 AGTCTGCTGA CCAGCAGGCC TGAGGACACC ACACACAATC GCCGCAGCAG AGGCATGGTC 3300 CAGAGCAGCT CCCCAATCAT GCCCACAGTG GGGCCCTCCA CTGCATCCCC AGAAGAACCC 3360 CCAGGCTCCA GTTTACCCAG TGATACATCA GATGAGATCA TGGACCTTCT GGTGCAGTCA 3420 GTGACCAAGA GCAGTCCTCG TGCCTTAGCT GCTAGGGAAC GCAAGCGTTC CCGCGGCAAC 3480 CGCAAGTCTT TGAGAAGGAC GTTGAAGAGT GGGCTCGGAG ATGACCTGGT GCAGGCACTG 3540 GGACTAAGCA AGGGTCCTCC CC <210> 4 <211> 3492 <212> DNA <213> Human <400> 4 ATGGCGGGCG GGGAAGACCG CGGGGACGGA GAGCCGGTAT CAGTGGTGAC CGTGAGGGTG 60 CAGTACCTGG AAGACACCGA CCCCTTCGCA TGTGCCAACT TTCCGGAGCC GCGCCGGGCC 120 CCCACCTGCA GCCTGGACGG GGCGCTGCCC TTGGGCGCGC AGATACCCGC GGTGCACCGC 180 CTGCTGGGAG CGCCGCTCAA GTTGGAGGAT TGTGCTCTGC AAGTGTCTCC CTCCGGATAC 240 TACCTGGACA CCGAGCTGTC CCTGGAAGAG CAGCGGGAGA TGCTGGAGGG CTTCTATGAA 300 GAGATCAGCA AAGGGCGGAA GCCCACGCTG ATCCTTCGGA CCCAGCTCTC TGTGAGGGTC 360 AACGCTATCT TGGAAAAGCT GTATAGCTCC AGTGGTCCTG AGCTCCGCCG CTCCCTCTTC 420 TCACTGAAGC AGATCTTCCA GGAGGACAAA GACCTGGTGC CTGAATTTGT GCATTCAGAG 480 GGGCTGAGCT GCCTGATCCG TGTGGGTGCT GCTGCCGACC ACAACTACCA GAGCTACATC 540 CTTAGAGCGC TCGGCCAGCT GATGCTCTTT GTGGATGGAA TGCTGGGGGT GGTGGCCCAC 600 AGTGACACTA TTCAGTGGCT GTACACATTG TGTGCCAGCC TGTCCCGCTT GGTGGTGAAG 660 ACAGCCCTGA AGCTGCTGTT GGTGTTTGTA GAATACTCCG AAAACAACGC ACCGCTGTTC 720 ATCCGTGCAG TGAACTCTGT GGCCAGCACC ACCGGTGCTC CTCCCTGGGC CAATCTGGTG 780 TCCATCCTGG AGGAGAAGAA TGGCGCTGAC CCTGAGTTGT TGGTGTACAC GGTCACCCTC 840 ATCAACAAGA CG CTGGCGGC GCTCCCGGAC CAGGACTCCT TCTACGATGT GACGGATGCA 900 CTGGAGCAGC AGGGCATGGA AGCGCTGGTC CAGCGCCACC TGGGCACTGC GGGCACTGAC 960 GTCGACCTGC GCACGCAGCT TGTGCTCTAC GAGAACGCCC TGAAATTGGA GGATGGAGAC 1020 ATCGAAGAAG CCCCAGGCGC TGGTGGGCGG CGGGAACGAC GAAAGCCTTC TTCTGAGGAG 1080 GGCAAGAGGA GCCGCCGTTC TCTGGAAGGC GGGGGCTGCC CCGCGCGTGC CCCGGAACCT 1140 GGCCCCACAG GCCCCGCCTC ACCGGTAGGC CCCACCTCTT CCACCGGCCC CGCCCTGCTG 1200 ACAGGCCCCG CCTCCAGCCC TGTGGGCCCT CCCTCCGGTC TCCAAGCTTC AGTGAACCTT 1260 TTTCCTACCA TCTCTGTGGC ACCCTCAGCT GACACCTCCA GCGAGAGGAG CATCTACAAA 1320 GCCCGGTTCC TGGAGAATGT GGCGGCAGCA GAAACAGAGA AGCAGGTTGC GCTGGCCCAG 1380 GGCCGGGCAG AGACACTTGC CGGGGCCATG CCCAATGAGG CGGGTGGACA CCCAGATGCC 1440 CGGCAACTCT GGGACTCCCC AGAGACAGCC CCTGCAGCCA GAACACCCCA GAGCCCTGCC 1500 CCCTGTGTCC TGCTCCGGGC CCAGCGAAGC CTTGCACCAG AGCCCAAGGA GCCACTGATA 1560 CCAGCAAGCC CCAAGGCTGA GCCCATCTGG GAGCTCCCTA CCCGTGCACC CAGGCTCTCT 1620 ATTGGGGACC TGGACTTTTC AGATCTAGGG GAGGATGAAG ACCAGGACAT GCTGAATGTA 1680 GAGTCTGTGG AGGCTGGGAA AGACATCCCA GCTCCCTCAC CCCCACTGCC CCTGCTCTCG 1740 GGAGTACCCC CCCCTCCCCC ACTTCCACCT CCCCCACCCA TCAAAGGCCC CTTCCCACCA 1800 CCTCCACCTC TACCTCTGGC TGCCCCTCTT CCCCATTCAG TGCCTGACAG CTCAGCCCTC 1860 CCCACTAAGA GGAAGACAGT AAAACTTTTC TGGCGTGAGC TGAAGCTGGC TGGGGGCCAT 1920 GGAGTCTCTG CAAGCCGCTT TGGGCCCTGC GCCACCCTCT GGGCTTCACT GGACCCTGTC 1980 TCAGTGGACA CGGCCCGACT GGAACACCTC TTTGAGTCTC GTGCCAAAGA GGTGCTGCCC 2040 TCCAAGAAAG CTGGAGAGGG CCGCCGGACA ATGACCACAG TGCTGGACCC CAAGCGCAGC 2100 AACGCCATCA ACATCGGCCT AACCACACTG CCACCTGTGC ATGTCATTAA GGCTGCTCTG 2160 CTCAACTTTG ATGAGTTTGC TGTCAGCAAG GATGGCATTG AGAAGCTACT GACCATGATG 2220 CCCACGGAGG AAGAGCGGCA GAAGATTGAG GAAGCCCAGC TGGCCAACCC TGACATACCC 2280 CTGGGCCCAG CCGAGAACTT CCTGATGACT CTTGCCTCCA TTGGCGGCCT CGCTGCTCGT 2340 CTACAACTCT GGGCCTTCAA GCTGGACTAT GACAGCATGG AGCGGGAAAT TGCTGAGCCA 2400 CTGTTTGACC TGAAAGTGGG TATGGAACAG CTGGTACAGA ATGCCACCTT CCGCTGCATC 2460 CTGGCTACCC TCCTAGCGGT GGGCAACTTC CTCAATGGCT CCCAGAGCAG CGGCTTTGAG 2520 CTGAGCTACC TGGAGAAGGT GTCAG AGGTG AAGGACACGG TGCGTCGACA GTCACTGCTA 2580 CACCATCTCT GCTCCCTAGT GCTCCAGACC CGGCCTGAGT CCTCTGACCT CTATTCAGAA 2640 ATCCCTGCCC TGACCCGCTG TGCCAAGGTG GACTTTGAAC AGCTGACTGA GAACCTGGGG 2700 CAGCTGGAGC GCCGGAGCCG GGCAGCCGAG GAGAGCCTGC GGAGCTTGGC CAAGCATGAG 2760 CTGGCCCCAG CCCTGCGTGC CCGCCTCACC CACTTCCTGG ACCAGTGTGC CCGCCGTGTT 2820 GCCATGCTAA GGATAGTGCA CCGCCGTGTC TGCAATAGGT TCCATGCCTT CCTGCTCTAC 2880 CTGGGCTACA CCCCGCAGGC GGCCCGTGAA GTGCGCATCA TGCAGTTCTG CCACACGCTG 2940 CGGGAATTTG CGCTTGAGTA TCGGACTTGC CGGGAACGAG TGCTACAGCA GCAGCAGAAG 3000 CAGGCCACAT ACCGTGAGCG CAACAAGACC CGGGGACGCA TGATCACCGA GACAGAGAAG 3060 TTCTCAGGTG TGGCTGGGGA AGCCCCCAGC AACCCCTCTG TCCCAGTAGC AGTGAGCAGC 3120 GGGCCAGGCC GGGGAGATGC TGACAGTCAT GCTAGTATGA AGAGTCTGCT GACCAGCAGG 3180 CCTGAGGACA CCACACACAA TCGCCGCAGC AGAGGCATGG TCCAGAGCAG CTCCCCAATC 3240 ATGCCCACAG TGGGGCCCTC CACTGCATCC CCAGAAGAAC CCCCAGGCTC CAGTTTACCC 3300 AGTGATACAT CAGATGAGAT CATGGACCTT CTGGTGCAGT CAGTGACCAA GAGCAGTCCT 3360 CGTGCCTTAG CTGCTAGGGA ACGCAAGCGT TCCCGCGGCA ACCGCAAGTC TTTGAGAAGG 3420 ACGTTGAAGA GTGGGCTCGG AGATGACCTG GTGCAGGCAC TGGGACTAAG CAAGGGTCCT 3480 GGCCTGGAGG TG 3492 <210> 5 <211> 447 <212> PRT <213> Human <400> 5 Met Ala Gly Gly Glu Asp Arg Gly Asp Gly Glu Pro Val Ser Val Val 5 10 15 Thr Val Arg Val Gln Tyr Leu Glu Asp Thr Asp Pro Phe Ala Cys Ala 20 25 30 Asn Phe Pro Glu Pro Arg Arg Ala Pro Thr Cys Ser Leu Asp Gly Ala 35 40 45 Leu Pro Leu Gly Ala Gln Ile Pro Ala Val His Arg Leu Leu Gly Ala 50 55 60 Pro Leu Lys Leu Glu Asp Cys Ala Leu Gln Val Ser Pro Ser Gly Tyr 65 70 75 80 Tyr Leu Asp Thr Glu Leu Ser Leu Glu Glu Gln Arg Glu Met Leu Glu 85 90 95 Gly Phe Tyr Glu Glu Ile Ser Lys Gly Arg Lys Pro Thr Leu Ile Leu 100 105 110 Arg Thr Gln Leu Ser Val Arg Val Asn Ala Ile Leu Glu Lys Leu Tyr 115 120 125 Ser Ser Ser Gly Pro Glu Leu Arg Arg Ser Leu Phe Ser Leu Lys Gln 130 135 140 Ile Phe Gln Glu Asp Lys Asp Leu Val Pro Glu Phe Val His Ser Glu 145 150 155 160 Gly Leu Ser Cys Leu Ile Arg Val Gly Ala Ala Ala Asp His Asn Tyr 165 170 175 Gln Ser Tyr Ile Leu Arg Ala Leu Gly Gln Leu Met Leu Phe Val Asp 180 185 190 Gly Met Leu Gly Val Val Ala His Ser Asp Thr Ile Gln Trp Leu Tyr 195 200 205 Thr Leu Cys Ala Ser Leu Ser Arg Leu Val Val Lys Thr Ala Leu Lys 210 215 220 Leu Leu Leu Val Phe Val Glu Tyr Ser Glu Asn Asn Ala Pro Leu Phe 225 230 235 240 Ile Arg Ala Val Asn Ser Val Ala Ser Thr Thr Gly Ala Pro Pro Trp 245 250 255 Ala Asn Leu Val Ser Ile Leu Glu Glu Lys Asn Gly Ala Asp Pro Glu 260 265 270 Leu Leu Val Tyr Thr Val Thr Leu Ile Asn Lys Thr Leu Ala Ala Leu 275 280 285 Pro Asp Gln Asp Ser Phe Tyr Asp Val Thr Asp Ala Leu Glu Gln Gln 290 295 300 Gly Met Glu Ala Leu Val Gln Arg His Leu Gly Thr Ala Gly Thr Asp 305 310 315 320 Val Asp Leu Arg Thr Gln Leu Val Leu Tyr Glu Asn Ala Leu Lys Leu 325 330 335 Glu Asp Gly Asp Ile Glu Glu Ala Pro Gly Ala Gly Gly Arg Arg Glu 340 345 350 Arg Arg Lys Pro Ser Ser Glu Glu Gly Lys Arg Ser Arg Arg Ser Leu 355 360 365 Glu Gly Gly Gly Cys Pro Ala Arg Ala Pro Glu Pro Gly Pro Thr Gly 370 375 380 Pro Ala Ser Pro Val Gly Pro Thr Ser Ser Thr Gly Pro Ala Leu Leu 385 390 395 400 Thr Gly Pro Ala Ser Ser Pro Val Gly Pro Pro Ser Gly Leu Gln Ala 405 410 415 Ser Val Asn LeuPhe Pro Thr Ile Ser Val Ala Pro Ser Ala Asp Thr 420 425 430 Ser Ser Glu Arg Ser Ile Tyr Lys Leu His Gln Thr Ala Ser Val 435 440 445 <210> 6 <211> 1341 <212> DNA <213> Human <400> 6 ATGGCGGGCG GGGAAGACCG CGGGGACGGA GAGCCGGTAT CAGTGGTGAC CGTGAGGGTG 60 CAGTACCTGG AAGACACCGA CCCCTTCGCA TGTGCCAACT TTCCGGAGCC GCGCCGGGCC 120 CCCACCTGCA GCCTGGACGG GGCGCTGCCC TTGGGCGCGC AGATACCCGC GGTGCACCGC 180 CTGCTGGGAG CGCCGCTCAA GTTGGAGGAT TGTGCTCTGC AAGTGTCTCC CTCCGGATAC 240 TACCTGGACA CCGAGCTGTC CCTGGAAGAG CAGCGGGAGA TGCTGGAGGG CTTCTATGAA 300 GAGATCAGCA AAGGGCGGAA GCCCACGCTG ATCCTTCGGA CCCAGCTCTC TGTGAGGGTC 360 AACGCTATCT TGGAAAAGCT GTATAGCTCC AGTGGTCCTG AGCTCCGCCG CTCCCTCTTC 420 TCACTGAAGC AGATCTTCCA GGAGGACAAA GACCTGGTGC CTGAATTTGT GCATTCAGAG 480 GGGCTGAGCT GCCTGATCCG TGTGGGTGCT GCTGCCGACC ACAACTACCA GAGCTACATC 540 CTTAGAGCGC TCGGCCAGCT GATGCTCTTT GTGGATGGAA TGCTGGGGGT GGTGGCCCAC 600 AGTGACACTA TTCAGTGGCT GTACACATTG TGTGCCAGCC TGTCCCGCTT GGTGGTGAAG 660 ACAGCCCTGA AGCTGCTGTT GGTGTTTGTA GAATACTCCG AAAACAACGC ACCGCTGTTC 720 ATCCGTGCAG TGAACTCTGT GGCCAGCACC ACCGGTGCTC CTCCCTGGGC CAATCTGGTG 780 TCCATCCTGG AGGAGAAGAA TGGCGCTGAC CCTGAGTTGT TGGTGTACAC GGTCACCCTC 840 ATCAACAAGA CG CTGGCGGC GCTCCCGGAC CAGGACTCCT TCTACGATGT GACGGATGCA 900 CTGGAGCAGC AGGGCATGGA AGCGCTGGTC CAGCGCCACC TGGGCACTGC GGGCACTGAC 960 GTCGACCTGC GCACGCAGCT TGTGCTCTAC GAGAACGCCC TGAAATTGGA GGATGGAGAC 1020 ATCGAAGAAG CCCCAGGCGC TGGTGGGCGG CGGGAACGAC GAAAGCCTTC TTCTGAGGAG 1080 GGCAAGAGGA GCCGCCGTTC TCTGGAAGGC GGGGGCTGCC CCGCGCGTGC CCCGGAACCT 1140 GGCCCCACAG GCCCCGCCTC ACCGGTAGGC CCCACCTCTT CCACCGGCCC CGCCCTGCTG 1200 ACAGGCCCCG CCTCCAGCCC TGTGGGCCCT CCCTCCGGTC TCCAAGCTTC AGTGAACCTT 1260 TTTCCTACCA TCTCTGTGGC ACCCTCAGCT GACACCTCCA GCGAGAGGAG CATCTACAAA 1320 CTTCACCAAA CTGCTTCCGT T 1341 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 TGAGCCGGCC GCAGAGCCAT GG 22 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 8 TGCTCCGTGC GTTCAAGGAG CTCAC 25 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 9 CCTACCATCT CTGTGGCACC CTCAGCT 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 TTGGGGCTTG CTGGTATCAG TGGCTCC 27 <210> 11 <211> 109 <212> PRT <213> Human <400> 11 Met Glu Thr Phe Thr Asn Asp Arg Leu Gln Leu Pro Arg Asn Met Ile 5 10 15 Glu Asn Ser Met Phe Glu Glu Glu Pro Asp Val Val Asp Leu Ala Lys 20 25 30 Glu Pro Cys Leu His Pro Leu Glu Pro Asp Glu Val Glu Tyr Glu Pro 35 40 45 Arg Gly Ser Arg Leu Leu Val Arg Gly Leu Gly Glu His Glu Met Asp 50 55 60 Glu Asp Glu Glu Asp Tyr Glu Ser Ser Ala Lys Leu Leu Gly Met Ser 65 70 75 80 Phe Met Asn Arg Ser Ser Gly Leu Arg Asn Ser Ala Thr Gly Tyr Arg 85 90 95 Gln Ser Pro Asp Gly Thr Cys Ser Val Pro Ser Ala Arg 100 105 <210> 12 <211> 327 <212> DNA <213> Human <400> 12 ATGGAGACCT TTACCAATGA TCGACTTCAG CTTCCAAGGA ATATGATTGA AAACAGCATG 60 TTTGAAGAAG AACCAGATGT GGTAGATTTA GCCAAAGAAC CTTGTTTACA TCCTCTGGAA 120 CCTGATGAAG TTGAATATGA GCCCCGAGGT TCGAGGCTTC TGGTGAGAGG TCTTGGTGAG 180 CATGAGATGG ATGAGGATGA AGAGGATTAT GAGTCATCTG CCAAGCTGCT GGGCATGTCC 240 TTCATGAACA GAAGCTCAGG CCTTCGGAAC AGTGCAACAG GCTACAGGCA GAGTCCAGAT 300 GGGACTTGTT CAGTACCCTC TGCCAGG 327 <210> 13 <211> 43 <212> PRT <213> Human <400> 13 Lys Val Pro Glu Thr Arg Gly Arg Thr Phe Asp Gln Ile Ser Ala Ala 5 10 15 Phe Arg Arg Thr Pro Ser Leu Leu Glu Gln Glu Val Lys Pro Ser Thr 20 25 30 Glu Leu Glu Tyr Leu Gly Pro Asp Glu Asn Asp 35 40 <210> 14 <211> 129 <212> DNA <213> Human <400> 14 AAAGTGCCTG AAACCAGAGG CCGGACGTTT GACCAGATCT CAGCTGCCTT CCGACGGACA 60 CCTTCCCTTT TAGAGCAGGA GGTGAAACCC AGTACAGAAC TTGAATACTT AGGGCCAGAT 120 GAGAATGAC 129 <210> 15 <211> 1192 <212> PRT <213> Mouse <400> 15 Met Ala Gly Glu Glu Glu Arg Gly Asp Gly Asp Pro Val Ser Val Val 5 10 15 Thr Val Arg Val Gln Tyr Leu Glu Asp Thr Asp Pro Phe Ala Cys Ala 20 25 30 Asn Phe Pro Glu Pro Arg Arg Ala Pro Thr Cys Ser Leu Asp Gly Ala 35 40 45 Leu Pro Leu Ser Ala Gln Ile Pro Ala Leu His Arg Leu Leu Gly Ala 50 55 60 Pro Leu Lys Leu Glu Asp Cys Ala Leu Gln Val Ser Pro Ser Gly Tyr 65 70 75 80 Tyr Leu Asp Pro Glu Leu Ser Leu Glu Glu Gln Arg Glu Met Leu Glu 85 90 95 Gly Phe Tyr Glu Glu Ile Ser Lys Gly Arg Lys Pro Thr Leu Ile Leu 100 105 110 Arg Thr Gln Leu Ser Val Arg Val Asn Ala Ile Leu Glu Lys Leu Tyr 115 120 125 Gly Ser Ser Gly Pro Glu Leu Arg Arg Ser Leu Phe Ser Leu Lys Gln 130 135 140 Ile Phe Gln Glu Asp Lys Asp Leu Val Pro Glu Phe Val His Ser Glu 145 150 155 160 Gly Leu Ser Cys Leu Ile Arg Val Gly Ala Ala Ala Asp His Asn Tyr 165 170 175 Gln Ser Tyr Ile Leu Arg Ala Leu Gly Gln Leu Met Leu Phe Val Asp 180 185 190 Gly Met Leu Gly Val Val Ala His Ser Glu Thr Val Gln Trp Leu Tyr 195 200 205 Thr Leu Cys Ala Ser Leu Ser Arg Leu Val Val Lys Thr Ala Leu Lys 210 215 220 Leu Leu Leu Val Phe Val Glu Tyr Ser Glu Asn Asn Ala Pro Leu Phe 225 230 235 240 Ile Gln Ala Val Asn Ala Val Ala Ser Ala Thr Gly Thr Leu Pro Trp 245 250 255 Ala Asn Leu Val Ser Ile Leu Glu Glu Lys Asn Gly Ala Asp Ala Glu 260 265 270 Leu Leu Val Tyr Thr Val Thr Leu Ile Asn Lys Thr Leu Ala Ala Leu 275 280 285 285 Pro Asp Gln Asp Ser Phe Tyr Asp Val Thr Asp Ala Leu Glu Gln Gln 290 295 300 Gly Met Glu Ala Leu Val Gln Arg Phe Leu Gly Thr Ala Gly Thr Asp 305 310 315 320 Val Asp Leu Arg Thr Gln Leu Thr Leu Tyr Glu Ser Ala Leu Arg Leu 325 330 335 Glu Asp Gly Asp Met Glu Glu Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly 340 345 350 Arg Arg Glu Arg Arg Lys Pro Ser Ser Glu Glu Gly Lys Arg Ser Arg 355 360 365 Arg Ser Leu Glu Gly Gly Gly Cys Pro Val Arg Ala Pro Glu Pro Gly 370 375 380 Ser Thr Gly Ser Ala Ser Pro Val Gly Ser Thr Pro Ser Thr Gly Ser 385 390 395 400 Ala Pro Pro Thr Asn Pro Ala Phe Ser Ser Thr Gly Pro Ala Ser Gly 405 410 415 Leu Leu Arg Th r Ser Val Asn Leu Phe Pro Thr Ile Ser Val Gly Pro 420 425 430 Ser Val Asp Ser Ser Cys Glu Arg Ser Val Tyr Lys Leu His Gln Thr 435 440 445 Ala Pro Val Cys Ser Pro Glu Ser Pro Pro Val Pro Gln Ser Leu Pro 450 455 460 Gly Gln Ala Arg Leu Glu Ala Arg Phe Leu Glu Asn Val Ala Ala Ala 465 470 475 480 Glu Thr Glu Lys Gln Ala Ala Leu Ala Gln Gly Arg Ala Glu Thr Leu 485 490 495 Ala Gly Ala Thr Val Asp Asp Thr Asp Gly Ser Ser Gly Thr Arg Glu 500 505 510 Leu Trp Asp Ser Pro Glu Pro Ala Ser Ala Pro Arg Thr Pro Gln Ser 515 520 525 Pro Val Ser Arg Ile Leu Leu Arg Thr Gln Arg Ser Leu Glu Pro Glu 530 535 540 Pro Lys Lys Pro Val Ser Pro Pro Ser Pro Lys Ala Glu Pro Ile Gln 545 550 555 560 Glu Pro Pro Thr Cys Val Pro Lys Leu Cys Ile Gly Asp Leu Asp Phe 565 570 575 575 Ser Asp Leu Gly Glu Asp Glu Asp Gln Asp Thr Leu Asn Val Glu Ser 580 585 590 Val Glu Ala Gly Lys Ala Ser Pro Phe Leu Ser Ser Leu Ser Pro Ser 595 600 605 Leu Ser Gly Gly Gly Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Ile Thr 610 615 620 620 Gly Ser Cys Pro Pr o Pro Pro Pro Leu Ala Ala Pro Phe Thr His Ser 625 630 635 640 Ala Leu Asp Gly Pro Arg His Pro Thr Lys Arg Lys Thr Val Lys Leu 645 650 655 Phe Trp Arg Glu Leu Lys Leu Thr Gly Gly Pro Gly Cys Ser Arg Ser 660 665 670 Arg Phe Gly Pro Cys Pro Thr Leu Trp Ala Ser Leu Glu Pro Val Ser 675 680 685 Val Asp Thr Ala Arg Leu Glu His Leu Phe Glu Ser Arg Ala Lys Asp 690 695 700 Val Leu Pro Thr Lys Lys Ala Gly Glu Gly Arg Arg Thr Met Thr Val 705 710 715 715 720 Val Leu Asp Pro Lys Arg Ser Asn Ala Ile Asn Ile Gly Leu Thr Thr 725 730 735 Leu Pro Pro Val His Val Ile Lys Ala Ala Leu Leu Asn Phe Asp Glu 740 745 750 Phe Ala Val Ser Lys Asp Gly Ile Glu Lys Leu Leu Thr Met Met Pro 755 760 765 Thr Glu Glu Glu Arg Gln Lys Ile Glu Glu Ala Gln Leu Ala Asn Pro 770 775 780 Asp Val Pro Leu Gly Pro Ala Glu Asn Phe Leu Met Thr Leu Ala Ser 785 790 795 800 Ile Gly Gly Leu Ala Ala Arg Leu Gln Leu Trp Ala Phe Lys Leu Asp 805 810 815 Tyr Glu Ser Met Glu Arg Glu Ile Ala Glu Pro Leu Phe Asp Leu Lys 820 825 830 Val Gly Met Glu G ln Leu Val His Asn Ala Thr Phe Arg Cys Ile Leu 835 840 845 Ala Thr Leu Leu Ala Val Gly Asn Phe Leu Asn Gly Ser Gln Ser Ser 850 855 860 Gly Phe Glu Leu Ser Tyr Leu Glu Lys Val Ser Glu Val Lys Asp Thr 865 870 875 880 Val Arg Arg Gln Ser Leu Leu Tyr His Leu Cys Ser Leu Val Leu Gln 885 890 895 Thr Arg Pro Asp Ser Ser Asp Leu Tyr Ser Glu Ile Pro Ala Leu Thr 900 905 910 Arg Cys Ala Lys Val Asp Phe Glu Gln Leu Thr Glu Asn Leu Gly Gln 915 920 925 925 Leu Glu Cys Arg Ser Gln Ala Ala Glu Asp Ser Leu Arg Ser Leu Ala 930 935 940 Lys His Glu Leu Ser Pro Ala Leu Arg Ala Arg Leu Thr His Phe Leu 945 950 955 960 Ala Gln Cys Thr Arg Arg Val Ala Met Leu Arg Val Val His Arg Arg 965 970 975 Val Cys Asn Arg Phe His Ala Phe Leu Leu Tyr Leu Gly Tyr Thr Pro 980 985 990 Gln Ala Ala Arg Asp Val Arg Ile Met Gln Phe Cys His Thr Leu Arg 995 1000 1005 Glu Phe Ala Leu Glu Tyr Arg Thr Cys Arg Glu Arg Val Leu Gln Gln 1010 1015 1020 Gln Gln Lys Arg Ala Thr Tyr Arg Glu Arg Asn Lys Thr Arg Gly Arg 1025 1030 1035 1040 Met Ile Thr Glu Thr Glu Lys Phe Ser Gly Val Ala Gly Glu Ala Pro 1045 1050 1055 Asn Asn Leu Ser Val Pro Val Ala Val Gly Ser Gly Pro Gly Gln Gly 1060 1065 1070 Asp Thr Asp Asn His Ala Ser Met Lys Ser Leu Leu Thr Ser Arg Pro 1075 1080 1085 Glu Asp Ala Thr His Ser Arg Arg Ser Arg Gly Met Val Gln Ser Ser 1090 1095 1100 Ser Pro Val Ser His Thr Ala Val Gly Pro Ser Ala Ala Ser Pro Glu 1105 1110 1115 1120 Glu Thr Ala Ala Ser Gly Leu Pro Thr Asp Thr Ser Asp Glu Ile Met 1125 1130 1135 Asp Leu Leu Val Gln Ser Val Thr Lys Ser Gly Pro Arg Ala Leu Ala 1140 1145 1150 Ala Arg Glu Arg Lys Arg Ser Arg Gly Asn Arg Lys Ser Leu Arg Arg 1155 1160 1165 Thr Leu Lys Ser Gly Leu Gly Asp Asp Leu Val Gln Ala Leu Gly Leu 1170 1175 1180 Ser Lys Ala Pro Gly Leu Glu Val 1185 1190 <210> 16 <211> 3576 <212> DNA <213> Mouse <400> 16 ATGGCGGGCG AGGAAGAGCG CGGGGATGGG GACCCAGTAT CCGTGGTAAC TGTGAGAGTG 60 CAGTACCTGG AAGACACCGA CCCTTTCGCT TGTGCCAACT TCCCGGAACC ACGCCGGGCC 120 CCCACCTGCA GCCTGGACGG GGCTCTGCCC CTGAGTGCGC AGATCCCTGC TCTGCACCGA 180 CTCCTGGGGG CGCCTCTTAA GCTGGAGGAC TGTGCATTGC AAGTGTCTCC CTCCGGATAC 240 TACCTGGACC CGGAGCTGTC CCTAGAAGAA CAGCGGGAGA TGCTGGAGGG TTTCTATGAA 300 GAGATCAGCA AAGGGCGGAA GCCCACGTTA ATCCTGCGGA CCCAGCTCTC CGTGAGGGTC 360 AATGCTATCT TGGAAAAGCT GTATGGCTCC AGTGGCCCCG AGCTCCGCCG ATCTCTCTTC 420 TCATTAAAGC AGATATTCCA GGAAGACAAG GACCTGGTGC CCGAATTCGT GCACTCGGAG 480 GGTCTGAGTT GCCTGATCCG CGTGGGTGCT GCTGCAGACC ACAACTACCA GAGCTACATC 540 CTTCGAGCCC TGGGCCAGCT GATGCTGTTT GTGGATGGGA TGCTGGGGGT GGTGGCCCAC 600 AGCGAGACCG TGCAGTGGCT GTATACCCTG TGTGCTAGCC TGTCCCGCTT GGTGGTAAAG 660 ACAGCCCTGA AGCTGCTGCT GGTGTTTGTG GAGTATTCCG AGAACAACGC GCCGCTGTTC 720 ATCCAGGCAG TCAACGCAGT AGCCAGTGCC ACCGGTACTC TTCCCTGGGC CAACTTGGTG 780 TCCATCCTGG AGGAGAAGAA TGGAGCTGAT GCAGAGTTGT TGGTGTACAC TGTCACTCTC 840 ATTAACAAGA C GCTGGCGGC ACTCCCAGAC CAGGACTCCT TCTATGATGT GACAGATGCT 900 CTGGAGCAGC AGGGCATGGA AGCGCTGGTC CAACGTTTCT TGGGCACCGC TGGCACTGAT 960 GTTGACCTGC GAACCCAGCT AACGCTCTAT GAGAGTGCCC TTCGGTTAGA GGATGGAGAT 1020 ATGGAAGAGG CTGCAGCCGC CGCTGCTGCA GGTGGCCGGC GAGAGCGGCG GAAGCCATCC 1080 TCGGAGGAGG GCAAAAGGAG CCGAAGATCA CTAGAAGGTG GAGGCTGCCC TGTGCGTGCC 1140 CCAGAACCTG GCTCTACAGG CTCCGCCTCA CCAGTAGGCT CCACCCCCTC CACTGGCTCC 1200 GCCCCGCCTA CAAATCCAGC CTTCAGCTCT ACTGGCCCAG CCTCTGGCCT TCTTCGAACC 1260 TCAGTGAACC TCTTTCCTAC CATTTCCGTG GGGCCGTCAG TGGACAGTTC CTGTGAGAGA 1320 AGCGTCTACA AACTTCACCA AACTGCTCCC GTTTGCTCCC CTGAGAGTCC ACCTGTCCCC 1380 CAGTCCCTTC CTGGGCAGGC CAGGCTAGAA GCCCGGTTCC TGGAGAATGT GGCGGCAGCA 1440 GAGACGGAGA AGCAGGCTGC TCTGGCCCAA GGCCGAGCGG AGACGCTGGC TGGAGCCACG 1500 GTAGATGACA CTGATGGATC ATCAGGCACA AGGGAACTGT GGGACTCCCC AGAGCCAGCC 1560 TCTGCACCCA GGACACCCCA GAGCCCTGTT TCCCGAATCC TGCTGCGCAC CCAGCGGAGT 1620 CTTGAGCCAG AGCCCAAGAA GCCAGTGTCA CCACCAAGCC CTAAGGCTGA GCCTATCCAG 1680 GAGCCTCCCA CCTGTGTCC C CAAGCTCTGC ATTGGGGACT TGGACTTCTC AGACCTAGGG 1740 GAGGATGAAG ACCAGGACAC ACTGAATGTG GAATCTGTGG AGGCTGGAAA AGCATCTCCC 1800 TTCCTGTCAT CTCTATCGCC CTCACTCTCT GGGGGTCCCC CTCCTCCGCC CCCACCTCCT 1860 CCACCCATCA CAGGCTCCTG CCCACCGCCT CCACCCCTGG CTGCTCCTTT TACCCACTCA 1920 GCACTTGACG GCCCAAGGCA CCCCACCAAA AGGAAGACAG TAAAACTTTT CTGGCGGGAA 1980 CTAAAGCTGA CTGGGGGCCC TGGGTGCTCT AGAAGCCGCT TTGGGCCTTG TCCTACCCTG 2040 TGGGCCTCGC TGGAACCCGT CTCGGTGGAC ACAGCCCGCC TGGAACACCT ATTTGAGTCC 2100 AGAGCCAAGG ATGTGCTACC AACCAAGAAA GCTGGTGAGG GCCGCCGGAC AATGACCGTA 2160 GTGCTGGACC CCAAGCGCAG CAATGCCATC AACATTGGCC TAACCACTCT GCCACCCGTG 2220 CACGTCATCA AGGCTGCCCT GCTCAACTTC GATGAGTTTG CTGTCAGCAA AGATGGCATT 2280 GAGAAACTGC TGACAATGAT GCCCACGGAG GAAGAGCGGC AGAAGATTGA GGAAGCCCAG 2340 CTGGCTAACC CCGATGTACC CCTCGGCCCC GCTGAGAATT TCCTGATGAC GCTTGCTTCC 2400 ATTGGAGGCC TGGCTGCGCG CCTACAGCTC TGGGCTTTCA AGCTGGACTA TGAAAGCATG 2460 GAGCGGGAAA TTGCAGAGCC ACTGTTTGAT CTGAAAGTGG GCATGGAACA GCTGGTACAC 2520 AATGCCACCT TCCGCTGTAT TCTG GCTACC CTTTTGGCTG TGGGCAACTT CCTCAATGGT 2580 TCCCAGAGCA GTGGCTTTGA GCTGAGCTAC CTGGAGAAGG TGTCAGAAGT GAAGGACACA 2640 GTGCGACGGC AGTCATTGCT CTATCATCTC TGCTCCCTGG TGCTCCAGAC CCGACCTGAT 2700 TCCTCTGACC TCTACTCAGA AATTCCTGCC CTCACCCGCT GTGCCAAGGT GGACTTTGAA 2760 CAGCTGACTG AGAACCTAGG GCAGCTGGAG TGCCGGAGCC AGGCTGCCGA GGACAGCCTC 2820 CGGAGCTTGG CTAAGCACGA GCTCTCCCCA GCTCTGCGTG CTCGCCTCAC CCACTTCTTG 2880 GCCCAGTGTA CCCGCCGGGT AGCCATGTTA AGAGTAGTGC ATCGCCGAGT CTGCAATAGG 2940 TTCCATGCCT TCCTGCTCTA CCTGGGCTAC ACCCCACAGG CAGCAAGGGA TGTACGCATC 3000 ATGCAGTTCT GCCACACACT GAGAGAGTTT GCCCTTGAGT ATCGGACTTG TCGGGAACGG 3060 GTACTGCAGC AGCAGCAGAA GCGGGCTACA TACCGTGAGC GCAACAAGAC CCGTGGTCGC 3120 ATGATTACCG AGACAGAGAA GTTCTCAGGT GTGGCTGGGG AGGCCCCCAA TAACCTGTCT 3180 GTCCCAGTGG CTGTGGGCAG CGGGCCAGGT CAGGGTGATA CTGACAATCA TGCCAGCATG 3240 AAGAGCCTGC TTACCAGCAG GCCGGAAGAT GCCACACACA GCCGACGCAG CAGAGGTATG 3300 GTCCAGAGCA GTTCCCCCGT CTCACACACA GCAGTGGGGC CCTCCGCTGC ATCCCCTGAG 3360 GAGACTGCAG CCTCCGGCTT ACCCACCGAC ACGTCAGATG AGATAATGGA CCTGCTGGTG 3420 CAGTCAGTTA CCAAGAGCGG TCCTAGAGCC TTAGCTGCTC GGGAGAGGAA GCGCTCTCGT 3480 GGCAACCGAA AGTCCTTGAG ACGGACACTG AAGAGTGGAC TTGGAGATGA CCTGGTGCAG 3540 GCACTGGGAC TAAGCAAAGCTCGG <210> 17 <211> 1166 <212> PRT <213> Mouse <400> 17 Met Ala Gly Glu Glu Glu Arg Gly Asp Gly Asp Pro Val Ser Val Val 5 10 15 Thr Val Arg Val Gln Tyr Leu Glu Asp Thr Asp Pro Phe Ala Cys Ala 20 25 30 Asn Phe Pro Glu Pro Arg Arg Ala Pro Thr Cys Ser Leu Asp Gly Ala 35 40 45 Leu Pro Leu Ser Ala Gln Ile Pro Ala Leu His Arg Leu Leu Gly Ala 50 55 60 Pro Leu Lys Leu Glu Asp Cys Ala Leu Gln Val Ser Pro Ser Gly Tyr 65 70 75 80 Tyr Leu Asp Pro Glu Leu Ser Leu Glu Glu Gln Arg Glu Met Leu Glu 85 90 95 Gly Phe Tyr Glu Glu Ile Ser Lys Gly Arg Lys Pro Thr Leu Ile Leu 100 105 110 Arg Thr Gln Leu Ser Val Arg Val Asn Ala Ile Leu Glu Lys Leu Tyr 115 120 125 Gly Ser Ser Gly Pro Glu Leu Arg Arg Ser Leu Phe Ser Leu Lys Gln 130 135 140 Ile Phe Gln Glu Asp Lys Asp Leu Val Pro Glu Phe Val His Ser Glu 145 150 155 160 Gly Leu Ser Cys Leu Ile Arg Val Gly Ala Ala Ala Asp His Asn Tyr 165 170 175 Gln Ser Tyr Ile Leu Arg Ala Leu Gly Gln Leu Met Leu Phe Val Asp 180 185 190 Gly Met Leu Gly Val Val Ala His Ser Glu Thr Val Gln Trp Leu Tyr 195 200 205 Thr Leu Cys Al a Ser Leu Ser Arg Leu Val Val Lys Thr Ala Leu Lys 210 215 220 Leu Leu Leu Val Phe Val Glu Tyr Ser Glu Asn Asn Ala Pro Leu Phe 225 230 235 240 Ile Gln Ala Val Asn Ala Val Ala Ser Ala Thr Gly Thr Leu Pro Trp 245 250 255 Ala Asn Leu Val Ser Ile Leu Glu Glu Lys Asn Gly Ala Asp Ala Glu 260 265 270 Leu Leu Val Tyr Thr Val Thr Leu Ile Asn Lys Thr Leu Ala Ala Leu 275 280 280 285 Pro Asp Gln Asp Ser Phe Tyr Asp Val Thr Asp Ala Leu Glu Gln Gln 290 295 300 Gly Met Glu Ala Leu Val Gln Arg Phe Leu Gly Thr Ala Gly Thr Asp 305 310 315 320 Val Asp Leu Arg Thr Gln Leu Thr Leu Tyr Glu Ser Ala Leu Arg Leu 325 330 335 Glu Asp Gly Asp Met Glu Glu Ala Ala Ala Ala Ala Ala Ala Gly Gly 340 345 350 Arg Arg Glu Arg Arg Lys Pro Ser Ser Glu Glu Gly Lys Arg Ser Arg 355 360 365 Arg Ser Leu Glu Gly Gly Gly Cys Pro Val Arg Ala Pro Glu Pro Gly 370 375 380 Ser Thr Gly Ser Ala Ser Pro Val Gly Ser Thr Pro Ser Thr Gly Ser 385 390 395 400 Ala Pro Pro Thr Asn Pro Ala Phe Ser Ser Thr Gly Pro Ala Ser Gly 405 410 415 Leu Leu Arg Th r Ser Val Asn Leu Phe Pro Thr Ile Ser Val Gly Pro 420 425 430 Ser Val Asp Ser Ser Cys Glu Arg Ser Val Tyr Lys Ala Arg Phe Leu 435 440 445 Glu Asn Val Ala Ala Ala Glu Thr Glu Lys Gln Ala Ala Leu Ala Gln 450 455 460 Gly Arg Ala Glu Thr Leu Ala Gly Ala Thr Val Asp Asp Thr Asp Gly 465 470 475 480 Ser Ser Gly Thr Arg Glu Leu Trp Asp Ser Pro Glu Pro Ala Ser Ala 485 490 495 Pro Arg Thr Pro Gln Ser Pro Val Ser Arg Ile Leu Leu Arg Thr Gln 500 505 510 Arg Ser Leu Glu Pro Glu Pro Lys Lys Pro Val Ser Pro Pro Ser Pro 515 520 525 Lys Ala Glu Pro Ile Gln Glu Pro Pro Thr Cys Val Pro Lys Leu Cys 530 535 540 Ile Gly Asp Leu Asp Phe Ser Asp Leu Gly Glu Asp Glu Asp Gln Asp 545 550 555 560 Thr Leu Asn Val Glu Ser Val Glu Ala Gly Lys Ala Ser Pro Phe Leu 565 570 575 Ser Ser Leu Ser Pro Ser Leu Ser Gly Gly Pro Pro Pro Pro Pro Pro 580 585 590 Pro Pro Pro Pro Ile Thr Gly Ser Cys Pro Pro Pro Pro Leu Ala 595 600 605 Ala Pro Phe Thr His Ser Ala Leu Asp Gly Pro Arg His Pro Thr Lys 610 615 620 620 Arg Lys Thr Val L ys Leu Phe Trp Arg Glu Leu Lys Leu Thr Gly Gly 625 630 635 640 Pro Gly Cys Ser Arg Ser Arg Phe Gly Pro Cys Pro Thr Leu Trp Ala 645 650 655 Ser Leu Glu Pro Val Ser Val Asp Thr Ala Arg Leu Glu His Leu Phe 660 665 670 Glu Ser Arg Ala Lys Asp Val Leu Pro Thr Lys Lys Ala Gly Glu Gly 675 680 685 Arg Arg Thr Met Thr Val Val Leu Asp Pro Lys Arg Ser Asn Ala Ile 690 695 700 Asn Ile Gly Leu Thr Thr Leu Pro Pro Val His Val Ile Lys Ala Ala 705 710 715 720 720 Leu Leu Asn Phe Asp Glu Phe Ala Val Ser Lys Asp Gly Ile Glu Lys 725 730 735 Leu Leu Thr Met Met Met Pro Thr Glu Glu Glu Arg Gln Lys Ile Glu Glu 740 745 750 Ala Gln Leu Ala Asn Pro Asp Val Pro Leu Gly Pro Ala Glu Asn Phe 755 760 765 Leu Met Thr Leu Ala Ser Ile Gly Gly Leu Ala Ala Arg Leu Gln Leu 770 775 780 Trp Ala Phe Lys Leu Asp Tyr Glu Ser Met Glu Arg Glu Ile Ala Glu 785 790 795 800 Pro Leu Phe Asp Leu Lys Val Gly Met Glu Gln Leu Val His Asn Ala 805 810 815 Thr Phe Arg Cys Ile Leu Ala Thr Leu Leu Ala Val Gly Asn Phe Leu 820 825 830 Asn Gly Ser Gln S er Ser Gly Phe Glu Leu Ser Tyr Leu Glu Lys Val 835 840 845 Ser Glu Val Lys Asp Thr Val Arg Arg Gln Ser Leu Leu Tyr His Leu 850 855 860 Cys Ser Leu Val Leu Gln Thr Arg Pro Asp Ser Ser Asp Leu Tyr Ser 865 870 875 880 Glu Ile Pro Ala Leu Thr Arg Cys Ala Lys Val Asp Phe Glu Gln Leu 885 890 895 Thr Glu Asn Leu Gly Gln Leu Glu Cys Arg Ser Gln Ala Ala Glu Asp 900 905 910 910 Ser Leu Arg Ser Leu Ala Lys His Glu Leu Ser Pro Ala Leu Arg Ala 915 920 925 925 Arg Leu Thr His Phe Leu Ala Gln Cys Thr Arg Arg Val Ala Met Leu 930 935 940 Arg Val Val His Arg Arg Val Cys Asn Arg Phe His Ala Phe Leu Leu 945 950 955 960 Tyr Leu Gly Tyr Thr Pro Gln Ala Ala Arg Asp Val Arg Ile Met Gln 965 970 975 Phe Cys His Thr Leu Arg Glu Phe Ala Leu Glu Tyr Arg Thr Cys Arg 980 985 990 Glu Arg Val Leu Gln Gln Gln Gln Lys Arg Ala Thr Tyr Arg Glu Arg 995 1000 1005 Asn Lys Thr Arg Gly Arg Met Ile Thr Glu Thr Glu Lys Phe Ser Gly 1010 1015 1020 Val Ala Gly Glu Ala Pro Asn Asn Leu Ser Val Pro Val Ala Val Gly 1025 1030 1035 1040 Ser Gly Pro Gly Gln Gly Asp Thr Asp Asn His Ala Ser Met Lys Ser 1045 1050 1055 Leu Leu Thr Ser Arg Pro Glu Asp Ala Thr His Ser Arg Arg Ser Arg 1060 1065 1070 Gly Met Val Gln Ser Ser Ser Pro Val Ser His Thr Ala Val Gly Pro 1075 1080 1085 Ser Ala Ala Ser Pro Glu Glu Thr Ala Ala Ser Gly Leu Pro Thr Asp 1090 1095 1100 Thr Ser Asp Glu Ile Met Asp Leu Leu Val Gln Ser Val Thr Lys Ser 1105 1110 1115 1120 Gly Pro Arg Ala Leu Ala Ala Arg Glu Arg Lys Arg Ser Arg Gly Asn 1125 1130 1135 Arg Lys Ser Leu Arg Arg Thr Leu Lys Ser Gly Leu Gly Asp Asp Leu 1140 1145 1150 Val Gln Ala Leu Gly Leu Ser Lys Ala Pro Gly Leu Glu Val 1155 1160 1165 <210> 18 <211> 3498 <212> DNA <213> Mouse <400> 18 ATGGCGGGCG AGGAAGAGCG CGGGGATGGG GACCCAGTAT CCGTGGTAAC TGTGAGAGTG 60 CAGTACCTGG AAGACACCGA CCCTTTCGCT TGTGCCAACT TCCCGGAACC ACGCCGGGCC 120 CCCACCTGCA GCCTGGACGG GGCTCTGCCC CTGAGTGCGC AGATCCCTGC TCTGCACCGA 180 CTCCTGGGGG CGCCTCTTAA GCTGGAGGAC TGTGCATTGC AAGTGTCTCC CTCCGGATAC 240 TACCTGGACC CGGAGCTGTC CCTAGAAGAA CAGCGGGAGA TGCTGGAGGG TTTCTATGAA 300 GAGATCAGCA AAGGGCGGAA GCCCACGTTA ATCCTGCGGA CCCAGCTCTC CGTGAGGGTC 360 AATGCTATCT TGGAAAAGCT GTATGGCTCC AGTGGCCCCG AGCTCCGCCG ATCTCTCTTC 420 TCATTAAAGC AGATATTCCA GGAAGACAAG GACCTGGTGC CCGAATTCGT GCACTCGGAG 480 GGTCTGAGTT GCCTGATCCG CGTGGGTGCT GCTGCAGACC ACAACTACCA GAGCTACATC 540 CTTCGAGCCC TGGGCCAGCT GATGCTGTTT GTGGATGGGA TGCTGGGGGT GGTGGCCCAC 600 AGCGAGACCG TGCAGTGGCT GTATACCCTG TGTGCTAGCC TGTCCCGCTT GGTGGTAAAG 660 ACAGCCCTGA AGCTGCTGCT GGTGTTTGTG GAGTATTCCG AGAACAACGC GCCGCTGTTC 720 ATCCAGGCAG TCAACGCAGT AGCCAGTGCC ACCGGTACTC TTCCCTGGGC CAACTTGGTG 780 TCCATCCTGG AGGAGAAGAA TGGAGCTGAT GCAGAGTTGT TGGTGTACAC TGTCACTCTC 840 ATTAACAAGA C GCTGGCGGC ACTCCCAGAC CAGGACTCCT TCTATGATGT GACAGATGCT 900 CTGGAGCAGC AGGGCATGGA AGCGCTGGTC CAACGTTTCT TGGGCACCGC TGGCACTGAT 960 GTTGACCTGC GAACCCAGCT AACGCTCTAT GAGAGTGCCC TTCGGTTAGA GGATGGAGAT 1020 ATGGAAGAGG CTGCAGCCGC CGCTGCTGCA GGTGGCCGGC GAGAGCGGCG GAAGCCATCC 1080 TCGGAGGAGG GCAAAAGGAG CCGAAGATCA CTAGAAGGTG GAGGCTGCCC TGTGCGTGCC 1140 CCAGAACCTG GCTCTACAGG CTCCGCCTCA CCAGTAGGCT CCACCCCCTC CACTGGCTCC 1200 GCCCCGCCTA CAAATCCAGC CTTCAGCTCT ACTGGCCCAG CCTCTGGCCT TCTTCGAACC 1260 TCAGTGAACC TCTTTCCTAC CATTTCCGTG GGGCCGTCAG TGGACAGTTC CTGTGAGAGA 1320 AGCGTCTACA AAGCCCGGTT CCTGGAGAAT GTGGCGGCAG CAGAGACGGA GAAGCAGGCT 1380 GCTCTGGCCC AAGGCCGAGC GGAGACGCTG GCTGGAGCCA CGGTAGATGA CACTGATGGA 1440 TCATCAGGCA CAAGGGAACT GTGGGACTCC CCAGAGCCAG CCTCTGCACC CAGGACACCC 1500 CAGAGCCCTG TTTCCCGAAT CCTGCTGCGC ACCCAGCGGA GTCTTGAGCC AGAGCCCAAG 1560 AAGCCAGTGT CACCACCAAG CCCTAAGGCT GAGCCTATCC AGGAGCCTCC CACCTGTGTC 1620 CCCAAGCTCT GCATTGGGGA CTTGGACTTC TCAGACCTAG GGGAGGATGA AGACCAGGAC 1680 ACACTGAATG TGGAATCTG T GGAGGCTGGA AAAGCATCTC CCTTCCTGTC ATCTCTATCG 1740 CCCTCACTCT CTGGGGGTCC CCCTCCTCCG CCCCCACCTC CTCCACCCAT CACAGGCTCC 1800 TGCCCACCGC CTCCACCCCT GGCTGCTCCT TTTACCCACT CAGCACTTGA CGGCCCAAGG 1860 CACCCCACCA AAAGGAAGAC AGTAAAACTT TTCTGGCGGG AACTAAAGCT GACTGGGGGC 1920 CCTGGGTGCT CTAGAAGCCG CTTTGGGCCT TGTCCTACCC TGTGGGCCTC GCTGGAACCC 1980 GTCTCGGTGG ACACAGCCCG CCTGGAACAC CTATTTGAGT CCAGAGCCAA GGATGTGCTA 2040 CCAACCAAGA AAGCTGGTGA GGGCCGCCGG ACAATGACCG TAGTGCTGGA CCCCAAGCGC 2100 AGCAATGCCA TCAACATTGG CCTAACCACT CTGCCACCCG TGCACGTCAT CAAGGCTGCC 2160 CTGCTCAACT TCGATGAGTT TGCTGTCAGC AAAGATGGCA TTGAGAAACT GCTGACAATG 2220 ATGCCCACGG AGGAAGAGCG GCAGAAGATT GAGGAAGCCC AGCTGGCTAA CCCCGATGTA 2280 CCCCTCGGCC CCGCTGAGAA TTTCCTGATG ACGCTTGCTT CCATTGGAGG CCTGGCTGCG 2340 CGCCTACAGC TCTGGGCTTT CAAGCTGGAC TATGAAAGCA TGGAGCGGGA AATTGCAGAG 2400 CCACTGTTTG ATCTGAAAGT GGGCATGGAA CAGCTGGTAC ACAATGCCAC CTTCCGCTGT 2460 ATTCTGGCTA CCCTTTTGGC TGTGGGCAAC TTCCTCAATG GTTCCCAGAG CAGTGGCTTT 2520 GAGCTGAGCT ACCTGGAGAA GGTG TCAGAA GTGAAGGACA CAGTGCGACG GCAGTCATTG 2580 CTCTATCATC TCTGCTCCCT GGTGCTCCAG ACCCGACCTG ATTCCTCTGA CCTCTACTCA 2640 GAAATTCCTG CCCTCACCCG CTGTGCCAAG GTGGACTTTG AACAGCTGAC TGAGAACCTA 2700 GGGCAGCTGG AGTGCCGGAG CCAGGCTGCC GAGGACAGCC TCCGGAGCTT GGCTAAGCAC 2760 GAGCTCTCCC CAGCTCTGCG TGCTCGCCTC ACCCACTTCT TGGCCCAGTG TACCCGCCGG 2820 GTAGCCATGT TAAGAGTAGT GCATCGCCGA GTCTGCAATA GGTTCCATGC CTTCCTGCTC 2880 TACCTGGGCT ACACCCCACA GGCAGCAAGG GATGTACGCA TCATGCAGTT CTGCCACACA 2940 CTGAGAGAGT TTGCCCTTGA GTATCGGACT TGTCGGGAAC GGGTACTGCA GCAGCAGCAG 3000 AAGCGGGCTA CATACCGTGA GCGCAACAAG ACCCGTGGTC GCATGATTAC CGAGACAGAG 3060 AAGTTCTCAG GTGTGGCTGG GGAGGCCCCC AATAACCTGT CTGTCCCAGT GGCTGTGGGC 3120 AGCGGGCCAG GTCAGGGTGA TACTGACAAT CATGCCAGCA TGAAGAGCCT GCTTACCAGC 3180 AGGCCGGAAG ATGCCACACA CAGCCGACGC AGCAGAGGTA TGGTCCAGAG CAGTTCCCCC 3240 GTCTCACACA CAGCAGTGGG GCCCTCCGCT GCATCCCCTG AGGAGACTGC AGCCTCCGGC 3300 TTACCCACCG ACACGTCAGA TGAGATAATG GACCTGCTGG TGCAGTCAGT TACCAAGAGC 3360 GGTCCTAGAG CCTTAGCTGC TCGGGAGAGG AAGCGCTCTC GTGGCAACCG AAAGTCCTTG 3420 AGACGGACAC TGAAGAGTGG ACTTGGAGAT GACCTGGTGC AGGCACTGGG ACTAAGCAAA 3480 GCTCCTGGTC TAGAGGTG 3498 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 19 GAGTTTGCTG TCAGCAAAGA TGGCATTGAG 30 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 20 TTGCTTAGTC CCAGTGCCTG CACCAGGTCA TCTCC 35 <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 21 TGAAGTTGCA GCATTTGCAG GGGACAC 27 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 22 AGCTGGGCTT CCTCAATCTT CTGCCGCTCT 30 <210> 23 <211> 3853 <212> DNA <213> Human <400> 23 TGAGCCGGCC GCAGAGCCAT GGCGGGCGGG GAAGACCGCG GGGACGGAGA GCCGGTATCA 60 GTGGTGACCG TGAGGGTGCA GTACCTGGAA GACACCGACC CCTTCGCATG TGCCAACTTT 120 CCGGAGCCGC GCCGGGCCCC CACCTGCAGC CTGGACGGGG CGCTGCCCTT GGGCGCGCAG 180 ATACCCGCGG TGCACCGCCT GCTGGGAGCG CCGCTCAAGT TGGAGGATTG TGCTCTGCAA 240 GTGTCTCCCT CCGGATACTA CCTGGACACC GAGCTGTCCC TGGAAGAGCA GCGGGAGATG 300 CTGGAGGGCT TCTATGAAGA GATCAGCAAA GGGCGGAAGC CCACGCTGAT CCTTCGGACC 360 CAGCTCTCTG TGAGGGTCAA CGCTATCTTG GAAAAGCTGT ATAGCTCCAG TGGTCCTGAG 420 CTCCGCCGCT CCCTCTTCTC ACTGAAGCAG ATCTTCCAGG AGGACAAAGA CCTGGTGCCT 480 GAATTTGTGC ATTCAGAGGG GCTGAGCTGC CTGATCCGTG TGGGTGCTGC TGCCGACCAC 540 AACTACCAGA GCTACATCCT TAGAGCGCTC GGCCAGCTGA TGCTCTTTGT GGATGGAATG 600 CTGGGGGTGG TGGCCCACAG TGACACTATT CAGTGGCTGT ACACATTGTG TGCCAGCCTG 660 TCCCGCTTGG TGGTGAAGAC AGCCCTGAAG CTGCTGTTGG TGTTTGTAGA ATACTCCGAA 720 AACAACGCAC CGCTGTTCAT CCGTGCAGTG AACTCTGTGG CCAGCACCAC CGGTGCTCCT 780 CCCTGGGCCA ATCTGGTGTC CATCCTGGAG GAGAAGAATG GCGCTGACCC TGAGTTGTTG 840 GTGTACACGG T CACCCTCAT CAACAAGACG CTGGCGGCGC TCCCGGACCA GGACTCCTTC 900 TACGATGTGA CGGATGCACT GGAGCAGCAG GGCATGGAAG CGCTGGTCCA GCGCCACCTG 960 GGCACTGCGG GCACTGACGT CGACCTGCGC ACGCAGCTTG TGCTCTACGA GAACGCCCTG 1020 AAATTGGAGG ATGGAGACAT CGAAGAAGCC CCAGGCGCTG GTGGGCGGCG GGAACGACGA 1080 AAGCCTTCTT CTGAGGAGGG CAAGAGGAGC CGCCGTTCTC TGGAAGGCGG GGGCTGCCCC 1140 GCGCGTGCCC CGGAACCTGG CCCCACAGGC CCCGCCTCAC CGGTAGGCCC CACCTCTTCC 1200 ACCGGCCCCG CCCTGCTGAC AGGCCCCGCC TCCAGCCCTG TGGGCCCTCC CTCCGGTCTC 1260 CAAGCTTCAG TGAACCTTTT TCCTACCATC TCTGTGGCAC CCTCAGCTGA CACCTCCAGC 1320 GAGAGGAGCA TCTACAAACT TCACCAAACT GCTTCCGTTT GGGCCCCTGA GAGCCCACCC 1380 GTCCCCCAGT CCCCTCCTGG GCAGGCCAGG CTGGAAGCCC GGTTCCTGGA GAATGTGGCG 1440 GCAGCAGAAA CAGAGAAGCA GGTTGCGCTG GCCCAGGGCC GGGCAGAGAC ACTTGCCGGG 1500 GCCATGCCCA ATGAGGCGGG TGGACACCCA GATGCCCGGC AACTCTGGGA CTCCCCAGAG 1560 ACAGCCCCTG CAGCCAGAAC ACCCCAGAGC CCTGCCCCCT GTGTCCTGCT CCGGGCCCAG 1620 CGAAGCCTTG CACCAGAGCC CAAGGAGCCA CTGATACCAG CAAGCCCCAA GGCTGAGCCC 1680 ATCTGGGAGC TCCCTACCC G TGCACCCAGG CTCTCTATTG GGGACCTGGA CTTTTCAGAT 1740 CTAGGGGAGG ATGAAGACCA GGACATGCTG AATGTAGAGT CTGTGGAGGC TGGGAAAGAC 1800 ATCCCAGCTC CCTCACCCCC ACTGCCCCTG CTCTCGGGAG TACCCCCCCC TCCCCCACTT 1860 CCACCTCCCC CACCCATCAA AGGCCCCTTC CCACCACCTC CACCTCTACC TCTGGCTGCC 1920 CCTCTTCCCC ATTCAGTGCC TGACAGCTCA GCCCTCCCCA CTAAGAGGAA GACAGTAAAA 1980 CTTTTCTGGC GTGAGCTGAA GCTGGCTGGG GGCCATGGAG TCTCTGCAAG CCGCTTTGGG 2040 CCCTGCGCCA CCCTCTGGGC TTCACTGGAC CCTGTCTCAG TGGACACGGC CCGACTGGAA 2100 CACCTCTTTG AGTCTCGTGC CAAAGAGGTG CTGCCCTCCA AGAAAGCTGG AGAGGGCCGC 2160 CGGACAATGA CCACAGTGCT GGACCCCAAG CGCAGCAACG CCATCAACAT CGGCCTAACC 2220 ACACTGCCAC CTGTGCATGT CATTAAGGCT GCTCTGCTCA ACTTTGATGA GTTTGCTGTC 2280 AGCAAGGATG GCATTGAGAA GCTACTGACC ATGATGCCCA CGGAGGAAGA GCGGCAGAAG 2340 ATTGAGGAAG CCCAGCTGGC CAACCCTGAC ATACCCCTGG GCCCAGCCGA GAACTTCCTG 2400 ATGACTCTTG CCTCCATTGG CGGCCTCGCT GCTCGTCTAC AACTCTGGGC CTTCAAGCTG 2460 GACTATGACA GCATGGAGCG GGAAATTGCT GAGCCACTGT TTGACCTGAA AGTGGGTATG 2520 GAACAGCTGG TACAGAATGC CACC TTCCGC TGCATCCTGG CTACCCTCCT AGCGGTGGGC 2580 AACTTCCTCA ATGGCTCCCA GAGCAGCGGC TTTGAGCTGA GCTACCTGGA GAAGGTGTCA 2640 GAGGTGAAGG ACACGGTGCG TCGACAGTCA CTGCTACACC ATCTCTGCTC CCTAGTGCTC 2700 CAGACCCGGC CTGAGTCCTC TGACCTCTAT TCAGAAATCC CTGCCCTGAC CCGCTGTGCC 2760 AAGGTGGACT TTGAACAGCT GACTGAGAAC CTGGGGCAGC TGGAGCGCCG GAGCCGGGCA 2820 GCCGAGGAGA GCCTGCGGAG CTTGGCCAAG CATGAGCTGG CCCCAGCCCT GCGTGCCCGC 2880 CTCACCCACT TCCTGGACCA GTGTGCCCGC CGTGTTGCCA TGCTAAGGAT AGTGCACCGC 2940 CGTGTCTGCA ATAGGTTCCA TGCCTTCCTG CTCTACCTGG GCTACACCCC GCAGGCGGCC 3000 CGTGAAGTGC GCATCATGCA GTTCTGCCAC ACGCTGCGGG AATTTGCGCT TGAGTATCGG 3060 ACTTGCCGGG AACGAGTGCT ACAGCAGCAG CAGAAGCAGG CCACATACCG TGAGCGCAAC 3120 AAGACCCGGG GACGCATGAT CACCGAGACA GAGAAGTTCT CAGGTGTGGC TGGGGAAGCC 3180 CCCAGCAACC CCTCTGTCCC AGTAGCAGTG AGCAGCGGGC CAGGCCGGGG AGATGCTGAC 3240 AGTCATGCTA GTATGAAGAG TCTGCTGACC AGCAGGCCTG AGGACACCAC ACACAATCGC 3300 CGCAGCAGAG GCATGGTCCA GAGCAGCTCC CCAATCATGC CCACAGTGGG GCCCTCCACT 3360 GCATCCCCAG AAGAACCCCC AGGCTCCAGT TTACCCAGTG ATACATCAGA TGAGATCATG 3420 GACCTTCTGG TGCAGTCAGT GACCAAGAGC AGTCCTCGTG CCTTAGCTGC TAGGGAACGC 3480 AAGCGTTCCC GCGGCAACCG CAAGTCTTTG AGAAGGACGT TGAAGAGTGG GCTCGGAGAT 3540 GACCTGGTGC AGGCACTGGG ACTAAGCAAG GGTCCTGGCC TGGAGGTGTG AAGGTGCTGT 3600 ATCCCGGAAA TCTATCTGGA CCCTGGACTG CAGTGCAGGA GATGACAGAG TGAGGAGGGC 3660 CCAGAGCAGA ATTCTGGCCC CAGAACTCTG TGCCCAGGAG CCATGCCTTG AGCAGTATTA 3720 GCCGTGTGTG TATGCATGTG AGTGTGTGTG TATGTGTGTG TGTGCATGCA TATGCATGTG 3780 CATGTGTGTG AGCTCCTTGA ACGCACGGAG CAAAATAAAA TTTTCTTAGC TAATCCAAAA 3840 AAAAAAAAAA AAA 3853 <210> 24 <211> 3775 <212> DNA <213> Human <400> 24 TGAGCCGGCC GCAGAGCCAT GGCGGGCGGG GAAGACCGCG GGGACGGAGA GCCGGTATCA 60 GTGGTGACCG TGAGGGTGCA GTACCTGGAA GACACCGACC CCTTCGCATG TGCCAACTTT 120 CCGGAGCCGC GCCGGGCCCC CACCTGCAGC CTGGACGGGG CGCTGCCCTT GGGCGCGCAG 180 ATACCCGCGG TGCACCGCCT GCTGGGAGCG CCGCTCAAGT TGGAGGATTG TGCTCTGCAA 240 GTGTCTCCCT CCGGATACTA CCTGGACACC GAGCTGTCCC TGGAAGAGCA GCGGGAGATG 300 CTGGAGGGCT TCTATGAAGA GATCAGCAAA GGGCGGAAGC CCACGCTGAT CCTTCGGACC 360 CAGCTCTCTG TGAGGGTCAA CGCTATCTTG GAAAAGCTGT ATAGCTCCAG TGGTCCTGAG 420 CTCCGCCGCT CCCTCTTCTC ACTGAAGCAG ATCTTCCAGG AGGACAAAGA CCTGGTGCCT 480 GAATTTGTGC ATTCAGAGGG GCTGAGCTGC CTGATCCGTG TGGGTGCTGC TGCCGACCAC 540 AACTACCAGA GCTACATCCT TAGAGCGCTC GGCCAGCTGA TGCTCTTTGT GGATGGAATG 600 CTGGGGGTGG TGGCCCACAG TGACACTATT CAGTGGCTGT ACACATTGTG TGCCAGCCTG 660 TCCCGCTTGG TGGTGAAGAC AGCCCTGAAG CTGCTGTTGG TGTTTGTAGA ATACTCCGAA 720 AACAACGCAC CGCTGTTCAT CCGTGCAGTG AACTCTGTGG CCAGCACCAC CGGTGCTCCT 780 CCCTGGGCCA ATCTGGTGTC CATCCTGGAG GAGAAGAATG GCGCTGACCC TGAGTTGTTG 840 GTGTACACGG T CACCCTCAT CAACAAGACG CTGGCGGCGC TCCCGGACCA GGACTCCTTC 900 TACGATGTGA CGGATGCACT GGAGCAGCAG GGCATGGAAG CGCTGGTCCA GCGCCACCTG 960 GGCACTGCGG GCACTGACGT CGACCTGCGC ACGCAGCTTG TGCTCTACGA GAACGCCCTG 1020 AAATTGGAGG ATGGAGACAT CGAAGAAGCC CCAGGCGCTG GTGGGCGGCG GGAACGACGA 1080 AAGCCTTCTT CTGAGGAGGG CAAGAGGAGC CGCCGTTCTC TGGAAGGCGG GGGCTGCCCC 1140 GCGCGTGCCC CGGAACCTGG CCCCACAGGC CCCGCCTCAC CGGTAGGCCC CACCTCTTCC 1200 ACCGGCCCCG CCCTGCTGAC AGGCCCCGCC TCCAGCCCTG TGGGCCCTCC CTCCGGTCTC 1260 CAAGCTTCAG TGAACCTTTT TCCTACCATC TCTGTGGCAC CCTCAGCTGA CACCTCCAGC 1320 GAGAGGAGCA TCTACAAAGC CCGGTTCCTG GAGAATGTGG CGGCAGCAGA AACAGAGAAG 1380 CAGGTTGCGC TGGCCCAGGG CCGGGCAGAG ACACTTGCCG GGGCCATGCC CAATGAGGCG 1440 GGTGGACACC CAGATGCCCG GCAACTCTGG GACTCCCCAG AGACAGCCCC TGCAGCCAGA 1500 ACACCCCAGA GCCCTGCCCC CTGTGTCCTG CTCCGGGCCC AGCGAAGCCT TGCACCAGAG 1560 CCCAAGGAGC CACTGATACC AGCAAGCCCC AAGGCTGAGC CCATCTGGGA GCTCCCTACC 1620 CGTGCACCCA GGCTCTCTAT TGGGGACCTG GACTTTTCAG ATCTAGGGGA GGATGAAGAC 1680 CAGGACATGC TGAATGTAG A GTCTGTGGAG GCTGGGAAAG ACATCCCAGC TCCCTCACCC 1740 CCACTGCCCC TGCTCTCGGG AGTACCCCCC CCTCCCCCAC TTCCACCTCC CCCACCCATC 1800 AAAGGCCCCT TCCCACCACC TCCACCTCTA CCTCTGGCTG CCCCTCTTCC CCATTCAGTG 1860 CCTGACAGCT CAGCCCTCCC CACTAAGAGG AAGACAGTAA AACTTTTCTG GCGTGAGCTG 1920 AAGCTGGCTG GGGGCCATGG AGTCTCTGCA AGCCGCTTTG GGCCCTGCGC CACCCTCTGG 1980 GCTTCACTGG ACCCTGTCTC AGTGGACACG GCCCGACTGG AACACCTCTT TGAGTCTCGT 2040 GCCAAAGAGG TGCTGCCCTC CAAGAAAGCT GGAGAGGGCC GCCGGACAAT GACCACAGTG 2100 CTGGACCCCA AGCGCAGCAA CGCCATCAAC ATCGGCCTAA CCACACTGCC ACCTGTGCAT 2160 GTCATTAAGG CTGCTCTGCT CAACTTTGAT GAGTTTGCTG TCAGCAAGGA TGGCATTGAG 2220 AAGCTACTGA CCATGATGCC CACGGAGGAA GAGCGGCAGA AGATTGAGGA AGCCCAGCTG 2280 GCCAACCCTG ACATACCCCT GGGCCCAGCC GAGAACTTCC TGATGACTCT TGCCTCCATT 2340 GGCGGCCTCG CTGCTCGTCT ACAACTCTGG GCCTTCAAGC TGGACTATGA CAGCATGGAG 2400 CGGGAAATTG CTGAGCCACT GTTTGACCTG AAAGTGGGTA TGGAACAGCT GGTACAGAAT 2460 GCCACCTTCC GCTGCATCCT GGCTACCCTC CTAGCGGTGG GCAACTTCCT CAATGGCTCC 2520 CAGAGCAGCG GCTTTGAGCT GAGC TACCTG GAGAAGGTGT CAGAGGTGAA GGACACGGTG 2580 CGTCGACAGT CACTGCTACA CCATCTCTGC TCCCTAGTGC TCCAGACCCG GCCTGAGTCC 2640 TCTGACCTCT ATTCAGAAAT CCCTGCCCTG ACCCGCTGTG CCAAGGTGGA CTTTGAACAG 2700 CTGACTGAGA ACCTGGGGCA GCTGGAGCGC CGGAGCCGGG CAGCCGAGGA GAGCCTGCGG 2760 AGCTTGGCCA AGCATGAGCT GGCCCCAGCC CTGCGTGCCC GCCTCACCCA CTTCCTGGAC 2820 CAGTGTGCCC GCCGTGTTGC CATGCTAAGG ATAGTGCACC GCCGTGTCTG CAATAGGTTC 2880 CATGCCTTCC TGCTCTACCT GGGCTACACC CCGCAGGCGG CCCGTGAAGT GCGCATCATG 2940 CAGTTCTGCC ACACGCTGCG GGAATTTGCG CTTGAGTATC GGACTTGCCG GGAACGAGTG 3000 CTACAGCAGC AGCAGAAGCA GGCCACATAC CGTGAGCGCA ACAAGACCCG GGGACGCATG 3060 ATCACCGAGA CAGAGAAGTT CTCAGGTGTG GCTGGGGAAG CCCCCAGCAA CCCCTCTGTC 3120 CCAGTAGCAG TGAGCAGCGG GCCAGGCCGG GGAGATGCTG ACAGTCATGC TAGTATGAAG 3180 AGTCTGCTGA CCAGCAGGCC TGAGGACACC ACACACAATC GCCGCAGCAG AGGCATGGTC 3240 CAGAGCAGCT CCCCAATCAT GCCCACAGTG GGGCCCTCCA CTGCATCCCC AGAAGAACCC 3300 CCAGGCTCCA GTTTACCCAG TGATACATCA GATGAGATCA TGGACCTTCT GGTGCAGTCA 3360 GTGACCAAGA GCAGTCCTCG TGCCTTAGCT GCTAGGGAAC GCAAGCGTTC CCGCGGCAAC 3420 CGCAAGTCTT TGAGAAGGAC GTTGAAGAGT GGGCTCGGAG ATGACCTGGT GCAGGCACTG 3480 GGACTAAGCA AGGGTCCTGG CCTGGAGGTG TGAAGGTGCT GTATCCCGGA AATCTATCTG 3540 GACCCTGGAC TGCAGTGCAG GAGATGACAG AGTGAGGAGG GCCCAGAGCA GAATTCTGGC 3600 CCCAGAACTC TGTGCCCAGG AGCCATGCCT TGAGCAGTAT TAGCCGTGTG TGTATGCATG 3660 TGAGTGTGTG TGTATGTGTG TGTGTGCATG CATATGCATG TGCATGTGTG TGAGCTCCTT 3720 GAACGCACGG AGCAAAATAA AATTTTCTTA GCTAATCCAA AAAAAAAAAA AAAAA 3775 <210> 25 <211> 3799 <212> DNA <213> Human <400> 25 TGAGCCGGCC GCAGAGCCAT GGCGGGCGGG GAAGACCGCG GGGACGGAGA GCCGGTATCA 60 GTGGTGACCG TGAGGGTGCA GTACCTGGAA GACACCGACC CCTTCGCATG TGCCAACTTT 120 CCGGAGCCGC GCCGGGCCCC CACCTGCAGC CTGGACGGGG CGCTGCCCTT GGGCGCGCAG 180 ATACCCGCGG TGCACCGCCT GCTGGGAGCG CCGCTCAAGT TGGAGGATTG TGCTCTGCAA 240 GTGTCTCCCT CCGGATACTA CCTGGACACC GAGCTGTCCC TGGAAGAGCA GCGGGAGATG 300 CTGGAGGGCT TCTATGAAGA GATCAGCAAA GGGCGGAAGC CCACGCTGAT CCTTCGGACC 360 CAGCTCTCTG TGAGGGTCAA CGCTATCTTG GAAAAGCTGT ATAGCTCCAG TGGTCCTGAG 420 CTCCGCCGCT CCCTCTTCTC ACTGAAGCAG ATCTTCCAGG AGGACAAAGA CCTGGTGCCT 480 GAATTTGTGC ATTCAGAGGG GCTGAGCTGC CTGATCCGTG TGGGTGCTGC TGCCGACCAC 540 AACTACCAGA GCTACATCCT TAGAGCGCTC GGCCAGCTGA TGCTCTTTGT GGATGGAATG 600 CTGGGGGTGG TGGCCCACAG TGACACTATT CAGTGGCTGT ACACATTGTG TGCCAGCCTG 660 TCCCGCTTGG TGGTGAAGAC AGCCCTGAAG CTGCTGTTGG TGTTTGTAGA ATACTCCGAA 720 AACAACGCAC CGCTGTTCAT CCGTGCAGTG AACTCTGTGG CCAGCACCAC CGGTGCTCCT 780 CCCTGGGCCA ATCTGGTGTC CATCCTGGAG GAGAAGAATG GCGCTGACCC TGAGTTGTTG 840 GTGTACACGG T CACCCTCAT CAACAAGACG CTGGCGGCGC TCCCGGACCA GGACTCCTTC 900 TACGATGTGA CGGATGCACT GGAGCAGCAG GGCATGGAAG CGCTGGTCCA GCGCCACCTG 960 GGCACTGCGG GCACTGACGT CGACCTGCGC ACGCAGCTTG TGCTCTACGA GAACGCCCTG 1020 AAATTGGAGG ATGGAGACAT CGAAGAAGCC CCAGGCGCTG GTGGGCGGCG GGAACGACGA 1080 AAGCCTTCTT CTGAGGAGGG CAAGAGGAGC CGCCGTTCTC TGGAAGGCGG GGGCTGCCCC 1140 GCGCGTGCCC CGGAACCTGG CCCCACAGGC CCCGCCTCAC CGGTAGGCCC CACCTCTTCC 1200 ACCGGCCCCG CCCTGCTGAC AGGCCCCGCC TCCAGCCCTG TGGGCCCTCC CTCCGGTCTC 1260 CAAGCTTCAG TGAACCTTTT TCCTACCATC TCTGTGGCAC CCTCAGCTGA CACCTCCAGC 1320 GAGAGGAGCA TCTACAAACT TCACCAAACT GCTTCCGTTT GAGCCCGGTT CCTGGAGAAT 1380 GTGGCGGCAG CAGAAACAGA GAAGCAGGTT GCGCTGGCCC AGGGCCGGGC AGAGACACTT 1440 GCCGGGGCCA TGCCCAATGA GGCGGGTGGA CACCCAGATG CCCGGCAACT CTGGGACTCC 1500 CCAGAGACAG CCCCTGCAGC CAGAACACCC CAGAGCCCTG CCCCCTGTGT CCTGCTCCGG 1560 GCCCAGCGAA GCCTTGCACC AGAGCCCAAG GAGCCACTGA TACCAGCAAG CCCCAAGGCT 1620 GAGCCCATCT GGGAGCTCCC TACCCGTGCA CCCAGGCTCT CTATTGGGGA CCTGGACTTT 1680 TCAGATCTAG GGGAGGATG A AGACCAGGAC ATGCTGAATG TAGAGTCTGT GGAGGCTGGG 1740 AAAGACATCC CAGCTCCCTC ACCCCCACTG CCCCTGCTCT CGGGAGTACC CCCCCCTCCC 1800 CCACTTCCAC CTCCCCCACC CATCAAAGGC CCCTTCCCAC CACCTCCACC TCTACCTCTG 1860 GCTGCCCCTC TTCCCCATTC AGTGCCTGAC AGCTCAGCCC TCCCCACTAA GAGGAAGACA 1920 GTAAAACTTT TCTGGCGTGA GCTGAAGCTG GCTGGGGGCC ATGGAGTCTC TGCAAGCCGC 1980 TTTGGGCCCT GCGCCACCCT CTGGGCTTCA CTGGACCCTG TCTCAGTGGA CACGGCCCGA 2040 CTGGAACACC TCTTTGAGTC TCGTGCCAAA GAGGTGCTGC CCTCCAAGAA AGCTGGAGAG 2100 GGCCGCCGGA CAATGACCAC AGTGCTGGAC CCCAAGCGCA GCAACGCCAT CAACATCGGC 2160 CTAACCACAC TGCCACCTGT GCATGTCATT AAGGCTGCTC TGCTCAACTT TGATGAGTTT 2220 GCTGTCAGCA AGGATGGCAT TGAGAAGCTA CTGACCATGA TGCCCACGGA GGAAGAGCGG 2280 CAGAAGATTG AGGAAGCCCA GCTGGCCAAC CCTGACATAC CCCTGGGCCC AGCCGAGAAC 2340 TTCCTGATGA CTCTTGCCTC CATTGGCGGC CTCGCTGCTC GTCTACAACT CTGGGCCTTC 2400 AAGCTGGACT ATGACAGCAT GGAGCGGGAA ATTGCTGAGC CACTGTTTGA CCTGAAAGTG 2460 GGTATGGAAC AGCTGGTACA GAATGCCACC TTCCGCTGCA TCCTGGCTAC CCTCCTAGCG 2520 GTGGGCAACT TCCTCAATGG CTCC CAGAGC AGCGGCTTTG AGCTGAGCTA CCTGGAGAAG 2580 GTGTCAGAGG TGAAGGACAC GGTGCGTCGA CAGTCACTGC TACACCATCT CTGCTCCCTA 2640 GTGCTCCAGA CCCGGCCTGA GTCCTCTGAC CTCTATTCAG AAATCCCTGC CCTGACCCGC 2700 TGTGCCAAGG TGGACTTTGA ACAGCTGACT GAGAACCTGG GGCAGCTGGA GCGCCGGAGC 2760 CGGGCAGCCG AGGAGAGCCT GCGGAGCTTG GCCAAGCATG AGCTGGCCCC AGCCCTGCGT 2820 GCCCGCCTCA CCCACTTCCT GGACCAGTGT GCCCGCCGTG TTGCCATGCT AAGGATAGTG 2880 CACCGCCGTG TCTGCAATAG GTTCCATGCC TTCCTGCTCT ACCTGGGCTA CACCCCGCAG 2940 GCGGCCCGTG AAGTGCGCAT CATGCAGTTC TGCCACACGC TGCGGGAATT TGCGCTTGAG 3000 TATCGGACTT GCCGGGAACG AGTGCTACAG CAGCAGCAGA AGCAGGCCAC ATACCGTGAG 3060 CGCAACAAGA CCCGGGGACG CATGATCACC GAGACAGAGA AGTTCTCAGG TGTGGCTGGG 3120 GAAGCCCCCA GCAACCCCTC TGTCCCAGTA GCAGTGAGCA GCGGGCCAGG CCGGGGAGAT 3180 GCTGACAGTC ATGCTAGTAT GAAGAGTCTG CTGACCAGCA GGCCTGAGGA CACCACACAC 3240 AATCGCCGCA GCAGAGGCAT GGTCCAGAGC AGCTCCCCAA TCATGCCCAC AGTGGGGCCC 3300 TCCACTGCAT CCCCAGAAGA ACCCCCAGGC TCCAGTTTAC CCAGTGATAC ATCAGATGAG 3360 ATCATGGACC TTCTGGTGCA GTCAGTGACC AAGAGCAGTC CTCGTGCCTT AGCTGCTAGG 3420 GAACGCAAGC GTTCCCGCGG CAACCGCAAG TCTTTGAGAA GGACGTTGAA GAGTGGGCTC 3480 GGAGATGACC TGGTGCAGGC ACTGGGACTA AGCAAGGGTC CTGGCCTGGA GGTGTGAAGG 3540 TGCTGTATCC CGGAAATCTA TCTGGACCCT GGACTGCAGT GCAGGAGATG ACAGAGTGAG 3600 GAGGGCCCAG AGCAGAATTC TGGCCCCAGA ACTCTGTGCC CAGGAGCCAT GCCTTGAGCA 3660 GTATTAGCCG TGTGTGTATG CATGTGAGTG TGTGTGTATG TGTGTGTGTG CATGCATATG 3720 CATGTGCATG TGTGTGAGCT CCTTGAACGC ACGGAGCAAA ATAAAATTTT CTTAGCTAAT 3780 CCAAAAAAAA AAAAAAAAA 3799 <210> 26 <211> 3636 <212> DNA <213> Mouse <400> 26 TGAAGTTGCA GCATTTGCAG GGGACACAAG AGGCGTCCAG CGGTAGCCGG TGGCAGAGCC 60 ATGGCGGGCG AGGAAGAGCG CGGGGATGGG GACCCAGTAT CCGTGGTAAC TGTGAGAGTG 120 CAGTACCTGG AAGACACCGA CCCTTTCGCT TGTGCCAACT TCCCGGAACC ACGCCGGGCC 180 CCCACCTGCA GCCTGGACGG GGCTCTGCCC CTGAGTGCGC AGATCCCTGC TCTGCACCGA 240 CTCCTGGGGG CGCCTCTTAA GCTGGAGGAC TGTGCATTGC AAGTGTCTCC CTCCGGATAC 300 TACCTGGACC CGGAGCTGTC CCTAGAAGAA CAGCGGGAGA TGCTGGAGGG TTTCTATGAA 360 GAGATCAGCA AAGGGCGGAA GCCCACGTTA ATCCTGCGGA CCCAGCTCTC CGTGAGGGTC 420 AATGCTATCT TGGAAAAGCT GTATGGCTCC AGTGGCCCCG AGCTCCGCCG ATCTCTCTTC 480 TCATTAAAGC AGATATTCCA GGAAGACAAG GACCTGGTGC CCGAATTCGT GCACTCGGAG 540 GGTCTGAGTT GCCTGATCCG CGTGGGTGCT GCTGCAGACC ACAACTACCA GAGCTACATC 600 CTTCGAGCCC TGGGCCAGCT GATGCTGTTT GTGGATGGGA TGCTGGGGGT GGTGGCCCAC 660 AGCGAGACCG TGCAGTGGCT GTATACCCTG TGTGCTAGCC TGTCCCGCTT GGTGGTAAAG 720 ACAGCCCTGA AGCTGCTGCT GGTGTTTGTG GAGTATTCCG AGAACAACGC GCCGCTGTTC 780 ATCCAGGCAG TCAACGCAGT AGCCAGTGCC ACCGGTACTC TTCCCTGGGC CAACTTGGTG 840 TCCATCCTGG A GGAGAAGAA TGGAGCTGAT GCAGAGTTGT TGGTGTACAC TGTCACTCTC 900 ATTAACAAGA CGCTGGCGGC ACTCCCAGAC CAGGACTCCT TCTATGATGT GACAGATGCT 960 CTGGAGCAGC AGGGCATGGA AGCGCTGGTC CAACGTTTCT TGGGCACCGC TGGCACTGAT 1020 GTTGACCTGC GAACCCAGCT AACGCTCTAT GAGAGTGCCC TTCGGTTAGA GGATGGAGAT 1080 ATGGAAGAGG CTGCAGCCGC CGCTGCTGCA GGTGGCCGGC GAGAGCGGCG GAAGCCATCC 1140 TCGGAGGAGG GCAAAAGGAG CCGAAGATCA CTAGAAGGTG GAGGCTGCCC TGTGCGTGCC 1200 CCAGAACCTG GCTCTACAGG CTCCGCCTCA CCAGTAGGCT CCACCCCCTC CACTGGCTCC 1260 GCCCCGCCTA CAAATCCAGC CTTCAGCTCT ACTGGCCCAG CCTCTGGCCT TCTTCGAACC 1320 TCAGTGAACC TCTTTCCTAC CATTTCCGTG GGGCCGTCAG TGGACAGTTC CTGTGAGAGA 1380 AGCGTCTACA AACTTCACCA AACTGCTCCC GTTTGCTCCC CTGAGAGTCC ACCTGTCCCC 1440 CAGTCCCTTC CTGGGCAGGC CAGGCTAGAA GCCCGGTTCC TGGAGAATGT GGCGGCAGCA 1500 GAGACGGAGA AGCAGGCTGC TCTGGCCCAA GGCCGAGCGG AGACGCTGGC TGGAGCCACG 1560 GTAGATGACA CTGATGGATC ATCAGGCACA AGGGAACTGT GGGACTCCCC AGAGCCAGCC 1620 TCTGCACCCA GGACACCCCA GAGCCCTGTT TCCCGAATCC TGCTGCGCAC CCAGCGGAGT 1680 CTTGAGCCAG AGCCCAAGA A GCCAGTGTCA CCACCAAGCC CTAAGGCTGA GCCTATCCAG 1740 GAGCCTCCCA CCTGTGTCCC CAAGCTCTGC ATTGGGGACT TGGACTTCTC AGACCTAGGG 1800 GAGGATGAAG ACCAGGACAC ACTGAATGTG GAATCTGTGG AGGCTGGAAA AGCATCTCCC 1860 TTCCTGTCAT CTCTATCGCC CTCACTCTCT GGGGGTCCCC CTCCTCCGCC CCCACCTCCT 1920 CCACCCATCA CAGGCTCCTG CCCACCGCCT CCACCCCTGG CTGCTCCTTT TACCCACTCA 1980 GCACTTGACG GCCCAAGGCA CCCCACCAAA AGGAAGACAG TAAAACTTTT CTGGCGGGAA 2040 CTAAAGCTGA CTGGGGGCCC TGGGTGCTCT AGAAGCCGCT TTGGGCCTTG TCCTACCCTG 2100 TGGGCCTCGC TGGAACCCGT CTCGGTGGAC ACAGCCCGCC TGGAACACCT ATTTGAGTCC 2160 AGAGCCAAGG ATGTGCTACC AACCAAGAAA GCTGGTGAGG GCCGCCGGAC AATGACCGTA 2220 GTGCTGGACC CCAAGCGCAG CAATGCCATC AACATTGGCC TAACCACTCT GCCACCCGTG 2280 CACGTCATCA AGGCTGCCCT GCTCAACTTC GATGAGTTTG CTGTCAGCAA AGATGGCATT 2340 GAGAAACTGC TGACAATGAT GCCCACGGAG GAAGAGCGGC AGAAGATTGA GGAAGCCCAG 2400 CTGGCTAACC CCGATGTACC CCTCGGCCCC GCTGAGAATT TCCTGATGAC GCTTGCTTCC 2460 ATTGGAGGCC TGGCTGCGCG CCTACAGCTC TGGGCTTTCA AGCTGGACTA TGAAAGCATG 2520 GAGCGGGAAA TTGCAGAGCC ACTG TTTGAT CTGAAAGTGG GCATGGAACA GCTGGTACAC 2580 AATGCCACCT TCCGCTGTAT TCTGGCTACC CTTTTGGCTG TGGGCAACTT CCTCAATGGT 2640 TCCCAGAGCA GTGGCTTTGA GCTGAGCTAC CTGGAGAAGG TGTCAGAAGT GAAGGACACA 2700 GTGCGACGGC AGTCATTGCT CTATCATCTC TGCTCCCTGG TGCTCCAGAC CCGACCTGAT 2760 TCCTCTGACC TCTACTCAGA AATTCCTGCC CTCACCCGCT GTGCCAAGGT GGACTTTGAA 2820 CAGCTGACTG AGAACCTAGG GCAGCTGGAG TGCCGGAGCC AGGCTGCCGA GGACAGCCTC 2880 CGGAGCTTGG CTAAGCACGA GCTCTCCCCA GCTCTGCGTG CTCGCCTCAC CCACTTCTTG 2940 GCCCAGTGTA CCCGCCGGGT AGCCATGTTA AGAGTAGTGC ATCGCCGAGT CTGCAATAGG 3000 TTCCATGCCT TCCTGCTCTA CCTGGGCTAC ACCCCACAGG CAGCAAGGGA TGTACGCATC 3060 ATGCAGTTCT GCCACACACT GAGAGAGTTT GCCCTTGAGT ATCGGACTTG TCGGGAACGG 3120 GTACTGCAGC AGCAGCAGAA GCGGGCTACA TACCGTGAGC GCAACAAGAC CCGTGGTCGC 3180 ATGATTACCG AGACAGAGAA GTTCTCAGGT GTGGCTGGGG AGGCCCCCAA TAACCTGTCT 3240 GTCCCAGTGG CTGTGGGCAG CGGGCCAGGT CAGGGTGATA CTGACAATCA TGCCAGCATG 3300 AAGAGCCTGC TTACCAGCAG GCCGGAAGAT GCCACACACA GCCGACGCAG CAGAGGTATG 3360 GTCCAGAGCA GTTCCCCCGT CTCACACACA GCAGTGGGGC CCTCCGCTGC ATCCCCTGAG 3420 GAGACTGCAG CCTCCGGCTT ACCCACCGAC ACGTCAGATG AGATAATGGA CCTGCTGGTG 3480 CAGTCAGTTA CCAAGAGCGG TCCTAGAGCC TTAGCTGCTC GGGAGAGGAA GCGCTCTCGT 3540 GGCAACCGAA AGTCCTAGAGGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGAGCGATCGCTGAGACGAGCGCGATCGAGCGATCGATCGAGCGATCGATCGAGCGATCGATCGAGCGATCGATCGATCGATCGAGCGATCGCTGAGACGAGCGCGATCGATCGATCGAGCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGA) <210> 27 <211> 3558 <212> DNA <213> Mouse <400> 27 TGAAGTTGCA GCATTTGCAG GGGACACAAG AGGCGTCCAG CGGTAGCCGG TGGCAGAGCC 60 ATGGCGGGCG AGGAAGAGCG CGGGGATGGG GACCCAGTAT CCGTGGTAAC TGTGAGAGTG 120 CAGTACCTGG AAGACACCGA CCCTTTCGCT TGTGCCAACT TCCCGGAACC ACGCCGGGCC 180 CCCACCTGCA GCCTGGACGG GGCTCTGCCC CTGAGTGCGC AGATCCCTGC TCTGCACCGA 240 CTCCTGGGGG CGCCTCTTAA GCTGGAGGAC TGTGCATTGC AAGTGTCTCC CTCCGGATAC 300 TACCTGGACC CGGAGCTGTC CCTAGAAGAA CAGCGGGAGA TGCTGGAGGG TTTCTATGAA 360 GAGATCAGCA AAGGGCGGAA GCCCACGTTA ATCCTGCGGA CCCAGCTCTC CGTGAGGGTC 420 AATGCTATCT TGGAAAAGCT GTATGGCTCC AGTGGCCCCG AGCTCCGCCG ATCTCTCTTC 480 TCATTAAAGC AGATATTCCA GGAAGACAAG GACCTGGTGC CCGAATTCGT GCACTCGGAG 540 GGTCTGAGTT GCCTGATCCG CGTGGGTGCT GCTGCAGACC ACAACTACCA GAGCTACATC 600 CTTCGAGCCC TGGGCCAGCT GATGCTGTTT GTGGATGGGA TGCTGGGGGT GGTGGCCCAC 660 AGCGAGACCG TGCAGTGGCT GTATACCCTG TGTGCTAGCC TGTCCCGCTT GGTGGTAAAG 720 ACAGCCCTGA AGCTGCTGCT GGTGTTTGTG GAGTATTCCG AGAACAACGC GCCGCTGTTC 780 ATCCAGGCAG TCAACGCAGT AGCCAGTGCC ACCGGTACTC TTCCCTGGGC CAACTTGGTG 840 TCCATCCTGG A GGAGAAGAA TGGAGCTGAT GCAGAGTTGT TGGTGTACAC TGTCACTCTC 900 ATTAACAAGA CGCTGGCGGC ACTCCCAGAC CAGGACTCCT TCTATGATGT GACAGATGCT 960 CTGGAGCAGC AGGGCATGGA AGCGCTGGTC CAACGTTTCT TGGGCACCGC TGGCACTGAT 1020 GTTGACCTGC GAACCCAGCT AACGCTCTAT GAGAGTGCCC TTCGGTTAGA GGATGGAGAT 1080 ATGGAAGAGG CTGCAGCCGC CGCTGCTGCA GGTGGCCGGC GAGAGCGGCG GAAGCCATCC 1140 TCGGAGGAGG GCAAAAGGAG CCGAAGATCA CTAGAAGGTG GAGGCTGCCC TGTGCGTGCC 1200 CCAGAACCTG GCTCTACAGG CTCCGCCTCA CCAGTAGGCT CCACCCCCTC CACTGGCTCC 1260 GCCCCGCCTA CAAATCCAGC CTTCAGCTCT ACTGGCCCAG CCTCTGGCCT TCTTCGAACC 1320 TCAGTGAACC TCTTTCCTAC CATTTCCGTG GGGCCGTCAG TGGACAGTTC CTGTGAGAGA 1380 AGCGTCTACA AAGCCCGGTT CCTGGAGAAT GTGGCGGCAG CAGAGACGGA GAAGCAGGCT 1440 GCTCTGGCCC AAGGCCGAGC GGAGACGCTG GCTGGAGCCA CGGTAGATGA CACTGATGGA 1500 TCATCAGGCA CAAGGGAACT GTGGGACTCC CCAGAGCCAG CCTCTGCACC CAGGACACCC 1560 CAGAGCCCTG TTTCCCGAAT CCTGCTGCGC ACCCAGCGGA GTCTTGAGCC AGAGCCCAAG 1620 AAGCCAGTGT CACCACCAAG CCCTAAGGCT GAGCCTATCC AGGAGCCTCC CACCTGTGTC 1680 CCCAAGCTCT GCATTGGGG A CTTGGACTTC TCAGACCTAG GGGAGGATGA AGACCAGGAC 1740 ACACTGAATG TGGAATCTGT GGAGGCTGGA AAAGCATCTC CCTTCCTGTC ATCTCTATCG 1800 CCCTCACTCT CTGGGGGTCC CCCTCCTCCG CCCCCACCTC CTCCACCCAT CACAGGCTCC 1860 TGCCCACCGC CTCCACCCCT GGCTGCTCCT TTTACCCACT CAGCACTTGA CGGCCCAAGG 1920 CACCCCACCA AAAGGAAGAC AGTAAAACTT TTCTGGCGGG AACTAAAGCT GACTGGGGGC 1980 CCTGGGTGCT CTAGAAGCCG CTTTGGGCCT TGTCCTACCC TGTGGGCCTC GCTGGAACCC 2040 GTCTCGGTGG ACACAGCCCG CCTGGAACAC CTATTTGAGT CCAGAGCCAA GGATGTGCTA 2100 CCAACCAAGA AAGCTGGTGA GGGCCGCCGG ACAATGACCG TAGTGCTGGA CCCCAAGCGC 2160 AGCAATGCCA TCAACATTGG CCTAACCACT CTGCCACCCG TGCACGTCAT CAAGGCTGCC 2220 CTGCTCAACT TCGATGAGTT TGCTGTCAGC AAAGATGGCA TTGAGAAACT GCTGACAATG 2280 ATGCCCACGG AGGAAGAGCG GCAGAAGATT GAGGAAGCCC AGCTGGCTAA CCCCGATGTA 2340 CCCCTCGGCC CCGCTGAGAA TTTCCTGATG ACGCTTGCTT CCATTGGAGG CCTGGCTGCG 2400 CGCCTACAGC TCTGGGCTTT CAAGCTGGAC TATGAAAGCA TGGAGCGGGA AATTGCAGAG 2460 CCACTGTTTG ATCTGAAAGT GGGCATGGAA CAGCTGGTAC ACAATGCCAC CTTCCGCTGT 2520 ATTCTGGCTA CCCTTTTGGC TGTG GGCAAC TTCCTCAATG GTTCCCAGAG CAGTGGCTTT 2580 GAGCTGAGCT ACCTGGAGAA GGTGTCAGAA GTGAAGGACA CAGTGCGACG GCAGTCATTG 2640 CTCTATCATC TCTGCTCCCT GGTGCTCCAG ACCCGACCTG ATTCCTCTGA CCTCTACTCA 2700 GAAATTCCTG CCCTCACCCG CTGTGCCAAG GTGGACTTTG AACAGCTGAC TGAGAACCTA 2760 GGGCAGCTGG AGTGCCGGAG CCAGGCTGCC GAGGACAGCC TCCGGAGCTT GGCTAAGCAC 2820 GAGCTCTCCC CAGCTCTGCG TGCTCGCCTC ACCCACTTCT TGGCCCAGTG TACCCGCCGG 2880 GTAGCCATGT TAAGAGTAGT GCATCGCCGA GTCTGCAATA GGTTCCATGC CTTCCTGCTC 2940 TACCTGGGCT ACACCCCACA GGCAGCAAGG GATGTACGCA TCATGCAGTT CTGCCACACA 3000 CTGAGAGAGT TTGCCCTTGA GTATCGGACT TGTCGGGAAC GGGTACTGCA GCAGCAGCAG 3060 AAGCGGGCTA CATACCGTGA GCGCAACAAG ACCCGTGGTC GCATGATTAC CGAGACAGAG 3120 AAGTTCTCAG GTGTGGCTGG GGAGGCCCCC AATAACCTGT CTGTCCCAGT GGCTGTGGGC 3180 AGCGGGCCAG GTCAGGGTGA TACTGACAAT CATGCCAGCA TGAAGAGCCT GCTTACCAGC 3240 AGGCCGGAAG ATGCCACACA CAGCCGACGC AGCAGAGGTA TGGTCCAGAG CAGTTCCCCC 3300 GTCTCACACA CAGCAGTGGG GCCCTCCGCT GCATCCCCTG AGGAGACTGC AGCCTCCGGC 3360 TTACCCACCG ACACGTCAGA TGAGATAATG GACCTGCTGG TGCAGTCAGT TACCAAGAGC 3420 GGTCCTAGAG CCTTAGCTGC TCGGGAGAGG AAGCGCTCTC GTGGCAACCG AAAGTCCTTG 3480 AGACGGACAC TGAAGAGTGG ACTTGGAGAT GACCTGGTGC AGGCACTGGG ACTAAGCAAA 3540 GCTCCTGGTC TAGAGGTG 3558 <210> 28 <211> 140 <212> PRT <213> Human <400> 28 Met Ala Gly Trp Gln Ser Tyr Val Asp Asn Leu Met Cys Asp Gly Cys 5 10 15 Cys Gln Glu Ala Ala Ile Val Gly Tyr Cys Asp Ala Lys Tyr Val Trp 20 25 30 Ala Ala Thr Ala Gly Gly Val Phe Gln Ser Ile Thr Pro Ile Glu Ile 35 40 45 Asp Met Ile Val Gly Lys Asp Arg Glu Gly Phe Phe Thr Asn Gly Leu 50 55 60 Thr Leu Gly Ala Lys Lys Cys Ser Val Ile Arg Asp Ser Leu Tyr Val 65 70 75 80 Asp Gly Asp Cys Thr Met Asp Ile Arg Thr Lys Ser Gln Gly Gly Glu 85 90 95 Pro Thr Tyr Asn Val Ala Val Gly Arg Ala Gly Arg Val Leu Val Phe 100 105 110 Val Met Gly Lys Glu Gly Val His Gly Gly Gly Leu Asn Lys Lys Ala 115 120 125 Tyr Ser Met Ala Lys Tyr Leu Arg Asp Ser Gly Phe 130 135 140 <210> 29 <211> 420 <212> DNA <213> Human <400> 29 ATGGCCGGTT GGCAGAGCTA CGTGGATAAC CTGATGTGCG ATGGCTGCTG CCAGGAGGCC 60 GCCATTGTCG GCTACTGCGA CGCCAAATAC GTCTGGGCAG CCACGGCCGG GGGCGTCTTT 120 CAGAGCATTA CGCCAATAGA AATAGATATG ATTGTAGGAA AAGACCGGGA AGGTTTCTTT 180 ACCAACGGTT TGACTCTTGG CGCGAAGAAA TGCTCAGTGA TCAGAGATAG TCTATACGTC 240 GATGGTGACT GCACAATGGA CATCCGGACA AAGAGTCAAG GTGGGGAGCC AACATACAAT 300 GTGGCTGTCG GCAGAGCTGG TAGAGTCTTG GTCTTTGTAA TGGGAAAAGA AGGGGTCCAT 360 GGAGGCGGAT TGAATAAGAA GGCATACTCA ATGGCAAAAT ACTTGAGAGA CTCTGGGTTC 420 <210> 30 <211> 420 <212> DNA <213> Human <400> 30 ATGGCCGGTT GGCAGAGCTA CGTGGATAAC CTGATGTGCG ATGGCTGCTG CCAGGAGGCC 60 GCCATTGTCG GCTACTGCGA CGCCAAATAC GTCTGGGCAG CCACGGCCGG GGGCGTCTTT 120 CAGAGCATTA CGCCAATAGA AATAGATATG ATTGTAGGAA AAGACCGGGA AGGTTTCTTT 180 ACCAACGGTT TGACTCTTGG CGCGAAGAAA TGCTCAGTGA TCAGAGATAG TCTATACGTC 240 GATGGTGACT GCACAATGGA CATCCGGACA AAGAGTCAAG GTGGGGAGCC AACATACAAT 300 GTGGCTGTCG GCAGAGCTGG TAGAGCATTG GTTATAGTCA TGGGAAAGGA AGGTGTCCAC 360 GGAGGCACAC TTAACAAGAA AGCATATGAA CTCGCTTTAT ACCTGAGGAG GTCTGATGTG 420 <210> 31 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> <400> 31 ATGGCCGGTT GGCAGAGCTA CGTGGAT 27 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> <400> 32 TTACACATCA GACCTCCTCA GGTATAAAGC 30 <210> 33 <211> 666 <212> PRT <213> Human <400> 33 Met Ala Gly Gly Glu Asp Arg Gly Asp Gly Glu Pro Val Ser Val Val 5 10 15 Thr Val Arg Val Gln Tyr Leu Glu Asp Thr Asp Pro Phe Ala Cys Ala 20 25 30 Asn Phe Pro Glu Pro Arg Arg Ala Pro Thr Cys Ser Leu Asp Gly Ala 35 40 45 Leu Pro Leu Gly Ala Gln Ile Pro Ala Val His Arg Leu Leu Gly Ala 50 55 60 Pro Leu Lys Leu Glu Asp Cys Ala Leu Gln Val Ser Pro Ser Gly Tyr 65 70 75 80 Tyr Leu Asp Thr Glu Leu Ser Leu Glu Glu Gln Arg Glu Met Leu Glu 85 90 95 Gly Phe Tyr Glu Glu Ile Ser Lys Gly Arg Lys Pro Thr Leu Ile Leu 100 105 110 Arg Thr Gln Leu Ser Val Arg Val Asn Ala Ile Leu Glu Lys Leu Tyr 115 120 125 Ser Ser Ser Gly Pro Glu Leu Arg Arg Ser Leu Phe Ser Leu Lys Gln 130 135 140 Ile Phe Gln Glu Asp Lys Asp Leu Val Pro Glu Phe Val His Ser Glu 145 150 155 160 Gly Leu Ser Cys Leu Ile Arg Val Gly Ala Ala Ala Asp His Asn Tyr 165 170 175 Gln Ser Tyr Ile Leu Arg Ala Leu Gly Gln Leu Met Leu Phe Val Asp 180 185 190 Gly Met Leu Gly Val Val Ala His Ser Asp Thr Ile Gln Trp Leu Tyr 195 200 205 Thr Leu Cys Al a Ser Leu Ser Arg Leu Val Val Lys Thr Ala Leu Lys 210 215 220 Leu Leu Leu Val Phe Val Glu Tyr Ser Glu Asn Asn Ala Pro Leu Phe 225 230 235 240 Ile Arg Ala Val Asn Ser Val Ala Ser Thr Thr Gly Ala Pro Pro Trp 245 250 255 Ala Asn Leu Val Ser Ile Leu Glu Glu Lys Asn Gly Ala Asp Pro Glu 260 265 270 Leu Leu Val Tyr Thr Val Thr Leu Ile Asn Lys Thr Leu Ala Ala Leu 275 280 285 285 Pro Asp Gln Asp Ser Phe Tyr Asp Val Thr Asp Ala Leu Glu Gln Gln 290 295 300 Gly Met Glu Ala Leu Val Gln Arg His Leu Gly Thr Ala Gly Thr Asp 305 310 315 320 Val Asp Leu Arg Thr Gln Leu Val Leu Tyr Glu Asn Ala Leu Lys Leu 325 330 335 Glu Asp Gly Asp Ile Glu Glu Ala Pro Gly Ala Gly Gly Arg Arg Glu 340 345 350 Arg Arg Lys Pro Ser Ser Glu Glu Gly Lys Arg Ser Arg Arg Ser Leu 355 360 365 Glu Gly Gly Gly Cys Pro Ala Arg Ala Pro Glu Pro Gly Pro Thr Gly 370 375 380 Pro Ala Ser Pro Val Gly Pro Thr Ser Ser Thr Gly Pro Ala Leu Leu 385 390 395 400 Thr Gly Pro Ala Ser Ser Pro Val Gly Pro Pro Ser Gly Leu Gln Ala 405 410 415 Ser Val Asn Le u Phe Pro Thr Ile Ser Val Ala Pro Ser Ala Asp Thr 420 425 430 Ser Ser Glu Arg Ser Ile Tyr Lys Leu His Gln Thr Ala Ser Val Trp 435 440 445 Ala Pro Glu Ser Pro Pro Val Pro Gln Ser Pro Pro Gly Gln Ala Arg 450 455 460 Leu Glu Ala Arg Phe Leu Glu Asn Val Ala Ala Ala Glu Thr Glu Lys 465 470 475 480 Gln Val Ala Leu Ala Gln Gly Gly Arg Ala Glu Thr Leu Ala Gly Ala Met 485 490 495 Pro Asn Glu Ala Gly Gly His Pro Asp Ala Arg Gln Leu Trp Asp Ser 500 505 510 Pro Glu Thr Ala Pro Ala Ala Arg Thr Pro Gln Ser Pro Ala Pro Cys 515 520 525 Val Leu Leu Arg Ala Gln Arg Ser Leu Ala Pro Glu Pro Lys Glu Pro 530 535 540 Leu Ile Pro Ala Ser Pro Lys Ala Glu Pro Ile Trp Glu Leu Pro Thr 545 550 555 560 Arg Ala Pro Arg Leu Ser Ile Gly Asp Leu Asp Phe Ser Asp Leu Gly 565 570 575 Glu Asp Glu Asp Gln Asp Met Leu Asn Val Glu Ser Val Glu Ala Gly 580 585 590 Lys Asp Ile Pro Ala Pro Ser Pro Pro Leu Pro Leu Leu Ser Gly Val 595 600 605 Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Ile Lys Gly Pro Phe 610 615 620 620 Pro Pro Pro Pro P ro Leu Pro Leu Ala Ala Pro Leu Pro His Ser Val 625 630 635 640 640 Pro Asp Ser Ser Ala Leu Pro Thr Lys Arg Lys Thr Val Lys Leu Phe 645 650 655 Trp Arg Glu Leu Lys Leu Ala Gly Gly His 660 665 666

[0066]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence and predicted amino acid sequence of the human MD36 gene (cDNA). (Continued to Fig. 2)

FIG. 2 shows the nucleotide sequence and predicted amino acid sequence of the human MD36 gene (cDNA). (Continued to Fig. 3)

FIG. 3 shows the nucleotide sequence and predicted amino acid sequence of the human MD36 gene (cDNA). (Continued to Fig. 4)

FIG. 4 shows the nucleotide sequence and predicted amino acid sequence of the human MD36 gene (cDNA). (Continued to Fig. 5)

FIG. 5 shows the nucleotide sequence and predicted amino acid sequence of the human MD36 gene (cDNA).

FIG. 6 shows the human FHOS gene (cDN) obtained in Example 2.
The base sequence and the predicted amino acid sequence of A) are shown. (Continued to Fig. 7)

FIG. 7 shows the human FHOS gene (cDN) obtained in Example 2.
The base sequence and the predicted amino acid sequence of A) are shown. (Continued to Fig. 8)

FIG. 8 shows the human FHOS gene (cDN) obtained in Example 2.
The base sequence and the predicted amino acid sequence of A) are shown. (Continued to Fig. 9)

FIG. 9 shows the human FHOS gene (cDN) obtained in Example 2.
The base sequence and the predicted amino acid sequence of A) are shown. (Continued to Fig. 10)

FIG. 10 shows the human FHOS gene (cDN) obtained in Example 2.
The base sequence and the predicted amino acid sequence of A) are shown.

FIG. 11. Truncated human FHOS gene (cDN
The base sequence and the predicted amino acid sequence of A) are shown. (Continued to FIG. 12)

FIG. 12 shows a truncated human FHOS gene (cDN
The base sequence and the predicted amino acid sequence of A) are shown. (Continued to FIG. 13)

FIG. 13. Truncated human FHOS gene (cDN
The base sequence and the predicted amino acid sequence of A) are shown. (Continued to FIG. 14)

FIG. 14. Truncated human FHOS gene (cDN
The base sequence and the predicted amino acid sequence of A) are shown.

FIG. 15 shows the distribution of each human tissue of splicing variants. The size (bp) of the PCR product is indicated on the right end.

FIG. 16. Literature (Westendorf et al., Gene, 232, 173-
182, 1999), MD36 derived from skeletal muscle, FHOS derived from spleen obtained in Example 2, and truncated FHOS.
2 shows a comparison of amino acid sequences of OS. In the figure, FHOS is FHOS described in the literature, FHOS0 is FHOS obtained in Example 2, FHOS24.
Indicates a truncated FHOS. (Continued to FIG. 17)

FIG. 17: FHOS described in the literature, MD36 derived from skeletal muscle, FHOS derived from spleen obtained in Example 2, and truncated form
3 shows a comparison of amino acid sequences of FHOS. In the figure, FHOS is FHOS described in the literature, FHOS0 is FHOS obtained in Example 2, FHOS2
4 shows a truncated FHOS.

FIG. 18 shows a comparison of the protein structures of FHOS, MD36 and truncated FHOS obtained in Example 2. In the figure, the portion indicated by Box indicates a typical domain structure portion, an insertion sequence, and the portion obtained in Example 1 (shaded portion).

FIG. 19: IRAP by quantification of β-galactosidase activity
And MD36 interaction. In the figure, Bait (-) and IRAP
(55-82) is the bait sequence used, i.e., each GAL4-B
The D sequence alone and the GAL4-BD fused IRAP (55-82) are shown. The resulting value is β-galactosidase activity units (mean ± standard deviation).

FIG. 20 shows the interaction of MD36 with a glucose transporter by quantification of β-galactosidase activity. In the figure, GLUT1 (451-492) and GLUT4 (468-510)
The mouse glucose transporters used for the bait sequence and their amino acid numbers are shown. The resulting value is β-galactosidase activity units (mean ± standard deviation).

FIG. 21 shows the results of a biochemical binding test of human MD36 performed in Example 8. Specifically, IRAP-His (0.1 μg / ml)
Alternatively, anti-Penta-His antibody (diluted 2000-fold) and both of them are added to wells coated with GST-MD36N4 or uncoated wells, and the amount of His-Tag remaining in the wells after washing with TBS-T. The result of quantifying is shown. In the figure,
+ Indicates an added or coated well, and-indicates a non-added or uncoated well.

FIG. 22 shows the results of a biochemical binding test of human MD36 performed in Example 8. + And-of human MD36 C-terminal region indicate the presence or absence of GST-MD36N4 coating.
+ And-of AP-His represent addition and non-addition of IRAP-His peptide. The numbers of IRAP (55-82) and BSA indicate the added amount (μg / well), respectively.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/08 A61P 43/00 111 3/10 C07K 14/47 43/00 111 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/37 C12P 21/02 G01N 33/15 Z C12Q 1 / 02 33/50 T 1/37 Z G01N 33/15 33/53 D 33/50 C12P 21/08 (C12N 1/19 33/53 C12R 1: 865) // C12P 21/08 (C12P 21/02 (C12N 1/19 C12R 1: 865) C12R 1: 865) (C12Q 1/02 (C12P 21/02 C12R 1:91) C12R 1: 865) (C12Q 1/20 (C12Q 1/02 C12R 1: 865) C12R 1 : 91) C12N 15/00 ZNAA (C12Q 1/02 A61K 37/64 12R 1: 865) C12N 5/00 A

Claims (37)

[Claims] 1. A protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, or a salt thereof. 2. The substantially identical amino acid sequence is represented by SEQ ID NO:
The protein according to claim 1, which has an amino acid sequence represented by 15, or a salt thereof. [3] a D encoding the protein of [1];
DNA containing NA. (4) D encoding the protein of (1)
4. The DN according to claim 3, wherein NA is a DNA containing the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 16.
A. 5. A recombinant vector containing the DNA according to claim 2. [6] A transformant transformed with the recombinant vector according to [5]. 7. A method for producing a protein or a salt thereof according to claim 1, comprising culturing the transformant according to claim 6, producing and accumulating the protein according to claim 1, and collecting the protein. . [8] a pharmaceutical comprising the protein according to [1] or a salt thereof or the DNA according to [2]; 9. The medicament according to claim 8, which is an agent for preventing or treating hypoglycemia. 10. An antibody against the protein according to claim 1 or a salt thereof. (11) A diagnostic agent comprising the antibody according to (10). 12. A protein comprising an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 17 or a salt thereof. (13) A DNA comprising a DNA encoding the protein according to (12). 14. The DNA according to claim 13, wherein the DNA encoding the protein according to claim 12 is a DNA containing the nucleotide sequence represented by SEQ ID NO: 4 or SEQ ID NO: 18. 15. A recombinant vector containing the DNA according to claim 13. [16] A transformant transformed with the recombinant vector according to [15]. (17) culturing the transformant according to (16),
A method for producing the protein or a salt thereof according to claim 12, wherein the protein according to claim 12 is produced, accumulated, and collected. [18] A pharmaceutical comprising the protein or the salt thereof according to [12] or the DNA according to [13]. (19) the agent for preventing or treating hypoglycemia;
The medicament according to claim. 20. An antibody against the protein according to claim 12 or a salt thereof. 21. A diagnostic agent comprising the antibody according to claim 20. 22. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, characterized by using a partial peptide of a protein having the amino acid sequence, or a salt thereof; A partial peptide of a protein having the same or substantially the same amino acid sequence as the represented amino acid sequence, A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2; a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 Or a salt thereof and a compound that inhibits binding between insulin responsive aminopeptidase or glucose transporter 4 or a salt thereof. 23. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2 A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, characterized by using a cell capable of producing a partial peptide of the protein having the amino acid sequence of A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by No. 1 A partial peptide, a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A screening method for a compound or a salt thereof that inhibits the binding between a partial peptide of a protein or a salt thereof and insulin-responsive aminopeptidase or glucose transporter 4. 24. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, comprising a partial peptide of a protein having an amino acid sequence, or a salt thereof; an amino acid represented by SEQ ID NO: 1 Partial peptide of a protein having the same or substantially the same amino acid sequence as the sequence, represented by SEQ ID NO: 2 Amino acid sequence with a protein containing the same or substantially the same amino acid sequence,
A partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or a compound that inhibits the binding of a salt thereof to insulin-responsive aminopeptidase or glucose transporter 4; A kit for screening the salt. 25. A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2 A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, containing cells capable of producing a partial peptide of the protein having the amino acid sequence of SEQ ID NO: 1; A partial peptide of a protein having the same or substantially the same amino acid sequence as the represented amino acid sequence A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a portion of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A kit for screening a compound or a salt thereof that inhibits the binding of the peptide or a salt thereof to insulin responsive aminopeptidase or glucose transporter 4. 26. The screening method according to claim 22, wherein
An amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, obtained by using the screening method according to claim 23, the screening kit according to claim 24, or the screening kit according to claim 25. A protein having a sequence, a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by 2, a partial peptide of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, or A compound or a salt thereof that inhibits binding between the salt and insulin-responsive aminopeptidase or glucose transporter 4. (27) a medicament comprising the compound according to (26) or a salt thereof; (28) a prophylactic / therapeutic agent for hyperglycemia or diabetes comprising the compound according to (26) or a salt thereof; 29. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A medicament comprising a compound that inhibits the binding between a partial peptide of a protein having an amino acid sequence, or a salt thereof, and insulin-responsive aminopeptidase or glucose transporter 4, or a salt thereof. 30. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 A protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 An agent for preventing or treating hyperglycemia or diabetes comprising a partial peptide of a protein having an amino acid sequence, or a salt thereof and a compound that inhibits the binding of insulin responsive aminopeptidase or glucose transporter 4 or a salt thereof. 31. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A pharmaceutical comprising a compound or a salt thereof that promotes or suppresses the expression of a protein containing 32. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A prophylactic / therapeutic agent for hyperglycemia or diabetes comprising a compound or a salt thereof that suppresses the expression of a protein containing the compound. 33. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 A prophylactic / therapeutic agent for hypoglycemia, comprising a compound or a salt thereof that promotes the expression of a protein containing. 34. A method for producing a pharmaceutical comprising the protein or salt thereof according to claim 1, the DNA according to claim 3, the protein or salt thereof according to claim 12, or the DNA according to claim 13. The protein according to item 1, or a salt thereof, the DNA according to item 3, and the DNA according to item 12,
Use of the protein or a salt thereof according to claim 13 or the DNA according to claim 13. (35) a method for administering a protein or a salt thereof according to (1), a DNA according to (3), a protein or a salt thereof according to (12) or a DNA according to (13) to a mammal; How to prevent and treat glycemia. [36] Use of a compound or a salt thereof according to [26] for the manufacture of a medicament comprising the compound or a salt thereof according to [26]; 37. A method for preventing or treating hyperglycemia or diabetes, which comprises administering the compound according to claim 26 or a salt thereof to a mammal.