JP2003235587A - Method for searching protein binding to lk6-a or its derivative, the protein and method for searching immunosuppressant using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for searching a protein binding to LK6-A or a derivative thereof, to provide the protein, and to provide a method for searching autoimmune disease therapeutic agents or immunosuppressants using the protein. <P>SOLUTION: The method for searching the protein comprises contacting a test protein sample with an immobilizing carrier for LK6-A or a derivative thereof to select the protein specifically binding to the LK6-A or the derivative thereof from the sample. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for searching for a protein that binds to LK6-A or a derivative thereof (hereinafter, also referred to as LK6-A binding protein), the protein and an agent for treating autoimmune disease or immunity using the protein. The present invention relates to a method for searching for an inhibitor.

[0002]

2. Description of the Related Art LK6-A and its derivatives (hereinafter, LK6-A and its derivatives are generically referred to as LK6-A derivatives) are used as therapeutic agents for autoimmune diseases having excellent immunosuppressive action, and for organ transplantation. It has been reported to be useful as an immunosuppressant that suppresses rejection [The Journal of Antibiotic
s, 50 , 357 (1997), The Journal of Antibiotics, 50 ,
543 (1997), JP-A-9-151185, WO00 / 02879]. An immunosuppressive drug is a drug used to suppress rejection during organ transplantation. In addition to organ transplantation, immunosuppressants are also used for diseases in which the immune reaction is considered to be involved in the pathological condition such as collagen disease. Major immunosuppressive agents include chemoimmunosuppressive agents, corticosteroids, and T cell function suppressors. Chemical immunosuppressants are classified into alkylating agents (cyclophosphamide etc.), antimetabolites (mizoribine, azathioprine, methotrexate etc.) and the like.

Cyclosporine A is a T cell function inhibitor
(CsA), FK506 and rapamycin [N. Eng. J. Med.,
321 , 1725 (1989); Transplant. Proc., 23 , 2977 (199
1)] etc. The drugs listed above have considerable side effects. For example, azathioprine may cause myelosuppressive effects (leukopenia, thrombocytopenia, anemia, etc.) and liver damage. Mizoribine has the advantages of myelosuppressive action and less liver damage than azathioprine, but it is not so immunosuppressive. Corticosteroids have a broader spectrum of action than other immunosuppressive drugs, but their immunosuppressive action is generally weak, and long-term continuous administration may cause a decrease in efficacy and development of resistance (today). Transplant, Vol. 11, No. 1, p. 37, 1998). CsA, FK5
06 is a drug that has extremely strong immunosuppressive activity and has contributed to improving the results of organ transplantation. However, it has been revealed that these have serious central nervous system toxicity, nephrotoxicity, etc., and their use requires extreme caution (Today's transplantation, Vol. 11, No. 1, p. 37, 1998).

Differentiation antigens CD3, CD4, CD25, CD28, CD
Monoclonal antibodies against 45 and the like are also attracting attention as a new type of immunosuppressant. For example, an anti-CD25 monoclonal antibody has been developed as an immunosuppressant with high specificity and few side effects because it acts only on T lymphocytes expressing the IL-2 receptor. However, immunosuppressive monoclonal antibodies have problems that they cannot be administered orally and that they are expensive. During organ transplantation, either CsA or FK506 and azathioprine or mizoribine [Transplant. Proc., 17 , 1222 (19
85), Clin. Transplant., 4 , 191 (1990)], a treatment using an immunosuppressive drug and a steroid drug in combination is widely used. It is the current situation that it has not yet been demonstrated. In order to search for a new immunosuppressive drug, a model system of organ transplant rejection as described below can be used.

1. The mixed lymphocyte reaction (MLR) was originally developed to assess histocompatibility between donor and recipient during organ transplantation and is one of the best established model systems for in vitro immune response. [J. Immunol.,
105 , 984 (1970)]. MLR using various combinations of mice and rats has been performed. 2. The allogeneic skin transplant model is a system in which major histocompatibility antigens are non-matching combinations of animals of the same species as a donor and a recipient, and skin transplantation is performed to observe the days of graft survival [Transplant. Proc., 28 , 1056 (1996)]. 3. The host-to-host reaction (GVHR) is a system in which lymphocytes are transplanted into a host and a transplantation immune response to the host tissue antigen is observed [Transplantation, 10 , 258 (1970)]. In GVHR, the degree of transplantation immune response can be measured by using the degree of splenomegaly of the host and the increase in the weight of lymph nodes as indicators.

These systems can be used to assess the immunosuppressive activity of compounds. However, since all the systems use animals such as rats and mice, it takes time and cost, and it cannot be said that it is a simple method for searching for immunosuppressive agents. A more convenient way to search for immunosuppressants is to find agonists or antagonists of molecules involved in the immune response. Inhibitor search systems using known immune response-related molecules include cyclophilin [J Immunol., 150 , 2591 (1993)], FK506 binding protein [Biochem Biophys Res Commun., 202 , 437 (19
94)], GYF domain (WO00 / 34309), and other screening systems for compounds are known, but this is not a completely established method. On the other hand, the discovery of new molecules involved in the immune response is also being investigated.

[0007] As one of methods for finding molecules involved in diseases, there are known methods such as DNA array, RT-PCR, Northern blotting and the like, which compare expression of mRNA in cells or tissues and examine expression distribution. [Experimental Medicine,
18 , 1581 (2000)]. For example, when the gene expression levels of antigen-stimulated T cells and unstimulated T cells are compared by a DNA array or the like, the protein encoded by the gene that is significantly increased or decreased in the antigen-stimulated T cells is an immune reaction. It is possible that it is a molecule that is involved in, and it may be a target molecule for immunosuppressant drug search. In addition, a protein encoded by a gene specifically expressed in cells involved in immunity such as T cells is considered to be a molecule involved in immune reaction, and may be a target molecule for immunosuppressive agent search. However, the fluctuation of the mRNA thus selected may be the cause of the immune reaction or the result of the immune reaction. Even if the agonist or antagonist of the protein encoded by the latter fluctuation mRNA is used, it leads to immunosuppression. First, the method of searching for immunosuppressive agents using molecules found from comparison of mRNA expression in cells or tissues and expression distribution is inefficient.

[0008] As another method for finding a molecule involved in a disease, a compound having an activity of treating a disease or a compound which shows activity in the above-mentioned activity evaluation system using animals and cells, but the mechanism of action has not been elucidated Methods for identifying molecules that bind to are also known. In the photoaffinity labeling method, an active compound labeled with a fluorescent dye or the like is introduced with a photoreactive functional group that forms a covalent bond with a molecule in proximity to the active compound, and a cell extract used for activity evaluation. After mixing with, it can be illuminated to identify labeled molecules [B
iochemistry, 31 , 3288 (1992)]. In the affinity purification method, an active compound is immobilized on an insoluble polymer called a carrier, mixed with an extract of cells used for activity evaluation, washed, and then a molecule in which the active compound is bound to the immobilized carrier is used. [Current Protocols in Protein Scienc
e (Chapter 9), John Wiley & Sons (2001)]. Molecules bound to the carrier to which the active compound is immobilized include molecules specifically bound to the active compound and molecules non-specifically bound to the carrier and the like. Therefore, it is necessary to select the former. After mixing the active compound and the cell extract in advance,
After washing with an active compound-immobilized carrier, the molecules bound to the active compound-immobilized carrier are identified, and the molecules that disappear when the active compound is added in advance are specific to the active compound. A method for identifying the molecule bound to the protein is known [Nature Biotech., 18 , 877 (2000)]. In addition, a method is known in which a molecule that specifically elutes when an active compound is added to a washing solution is identified as a molecule that specifically binds to the active compound [Proc Natl Acad Sci US
A., 90 , 11009 (1993)]. However, it is not possible to use these competitive experiments if the active compound is poorly water-soluble and no solution has been reported.

Known carriers include agarose, cellulose, polyacrylamide, and other porous particles formed into beads, latex beads, and the like. Latex beads are known to enable efficient affinity purification because they adsorb nonspecific proteins less than agarose beads [Methods in Enzymology,
254 , 595 (1995)]. It has been confirmed that a high-purity FK506-binding protein can be obtained by a single purification from a cytoplasmic fraction of a cell extract of animal cells using latex beads having FK506 immobilized thereon [Nature Biotech., 18 , 877 ( 200
0)]. Ref-1 has been purified and identified as a binding protein of E3330 having an anti-inflammatory effect using latex beads [JP-A-10-195099, Nature Biotech., 18 , 877].
(2000)].

[0010]

SUMMARY OF THE INVENTION The object of the present invention is to provide LK6-
It is an object of the present invention to provide a method for efficiently searching for a protein that binds to an A derivative, a method for searching for the protein obtained by the method, and a novel therapeutic agent for autoimmune disease or immunosuppressive agent using the protein.

[0011]

Means for Solving the Problems As a result of diligent studies, the inventors have found that LK6-A derivatives are immobilized on latex beads and proteins that bind to the derivatives obtained from various immune-related cells are compared and identified. It was found that the protein that specifically binds to the LK6-A derivative can be efficiently selected. The protein is considered to be a target molecule of LK6-A in vivo, and it became possible to search for a novel therapeutic agent for autoimmune disease or immunosuppressive agent using the target molecule. In the above method for searching for LK6-A binding protein, multiple L
Since K6-A binding protein can be obtained, LK6-A is not preferable among substances that change the binding property between LK6-A derivative and each LK6-A binding protein or the activity of each LK6-A binding protein itself. LK6-A binding protein and LK6 involved in expression of activity
-A remedy for autoimmune diseases with few side effects by selecting a substance that inhibits only the binding with A or a substance that inhibits only the activity of the LK6-A binding protein involved in the expression of the undesirable activity of LK6-A Further, they have found that an immunosuppressive agent can be easily searched for, and completed the present invention.

The present invention relates to the following (1) to (27). (1) The following formula (I)

[0013]

[Chemical 7]

[In the formula, R ¹ is a substituted or unsubstituted lower alkyl, lower alkanoyl, carboxy, lower alkoxycarbonyl, the following formula (II)

[0015]

[Chemical 8]

(Wherein n represents 1 or 2)
Group or COCH = CHR⁹{In the formula, R⁹Is a replacement
Is an unsubstituted lower alkoxy, a substituted or unsubstituted ari
, Substituted or unsubstituted heteroaryl or NR
^TenR¹¹(In the formula, R^TenAnd R ¹¹Are the same or different,
Hydrogen, substituted or unsubstituted lower alkyl, substituted or
Is unsubstituted lower alkenyl, substituted or unsubstituted lower
Alkynyl, substituted or unsubstituted aralkyl, also substituted
Or unsubstituted aryl, substituted or unsubstituted hetero
Aryl, substituted or unsubstituted heteroaryl substituted low
Secondary alkyl, substituted or unsubstituted tetrahydropyrani
Or substituted or unsubstituted tetrahydropyranylme
Represents chill or R^TenAnd R¹¹Together with the adjacent N
To form a substituted or unsubstituted heterocyclic group)},

R ² is hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted Substituted lower alkanoyl, substituted or unsubstituted lower alkanoyloxy, halogen, SR ¹² (in the formula, R ¹² represents substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or Unsubstituted aralkyl, substituted or unsubstituted heteroaryl substituted lower alkyl, substituted or unsubstituted tetrahydropyranyl or substituted or unsubstituted tetrahydropyranylmethyl), NR
¹³ R ¹⁴ (in the formula, R ¹³ and R ¹⁴ are the same as the above R ¹⁰ and R ¹¹ , respectively) or azido,

R ⁴ and R ⁵ are the same or different and are hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aralkyloxycarbonyl or Represents a substituted or unsubstituted heteroaryl-substituted lower alkoxycarbonyl, R ⁶ represents hydrogen or halogen,

R ⁷ and R ⁸ are the same or different and each represents hydrogen, a substituted or unsubstituted lower alkyl or a substituted or unsubstituted lower alkanoyl] (hereinafter abbreviated as LK6-A or a derivative thereof). ) Is immobilized on the carrier and the test protein sample is contacted with the LK6-A or a protein thereof, which is selected from the test protein sample and specifically binds to the compound represented by the above formula (I). A method for searching for a protein that binds to a derivative.

(2) The following formula (I)

[0021]

[Chemical 9]

[Wherein R ¹ is a substituted or unsubstituted lower alkyl, lower alkanoyl, carboxy, lower alkoxycarbonyl, the following formula (II)

[0023]

[Chemical 10]

(Wherein n represents 1 or 2)
Group or COCH = CHR⁹{In the formula, R⁹Is a replacement
Is an unsubstituted lower alkoxy, a substituted or unsubstituted ari
, Substituted or unsubstituted heteroaryl or NR
^TenR¹¹(In the formula, R^TenAnd R ¹¹Are the same or different,
Hydrogen, substituted or unsubstituted lower alkyl, substituted or
Is unsubstituted lower alkenyl, substituted or unsubstituted lower
Alkynyl, substituted or unsubstituted aralkyl, also substituted
Or unsubstituted aryl, substituted or unsubstituted hetero
Aryl, substituted or unsubstituted heteroaryl substituted low
Secondary alkyl, substituted or unsubstituted tetrahydropyrani
Or substituted or unsubstituted tetrahydropyranylme
Represents chill or R^TenAnd R¹¹Together with the adjacent N
To form a substituted or unsubstituted heterocyclic group)},

R ² is hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted Substituted lower alkanoyl, substituted or unsubstituted lower alkanoyloxy, halogen, SR ¹² (in the formula, R ¹² represents substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or Unsubstituted aralkyl, substituted or unsubstituted heteroaryl substituted lower alkyl, substituted or unsubstituted tetrahydropyranyl or substituted or unsubstituted tetrahydropyranylmethyl), NR
¹³ R ¹⁴ (in the formula, R ¹³ and R ¹⁴ are the same as the above R ¹⁰ and R ¹¹ , respectively) or azido,

R ⁴ and R ⁵ are the same or different and are hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aralkyloxycarbonyl or Represents a substituted or unsubstituted heteroaryl-substituted lower alkoxycarbonyl, R ⁶ represents hydrogen or halogen,

R ⁷ and R ⁸ are the same or different and each represents hydrogen, substituted or unsubstituted lower alkyl or substituted or unsubstituted lower alkanoyl] and a carrier immobilized with a compound represented by A protein that binds to the compound represented by the formula (I) when contacted, and the following formula (III)

[0028]

[Chemical 11]

[In the formula, R ¹ is a substituted or unsubstituted lower alkyl, lower alkanoyl, carboxy, lower alkoxycarbonyl, the following formula (II)

[0030]

[Chemical 12]

(Wherein n represents 1 or 2)
Group or COCH = CHR⁹{In the formula, R⁹Is a replacement
Is an unsubstituted lower alkoxy, a substituted or unsubstituted ari
, Substituted or unsubstituted heteroaryl or NR
^TenR¹¹(In the formula, R^TenAnd R ¹¹Are the same or different,
Hydrogen, substituted or unsubstituted lower alkyl, substituted or
Is unsubstituted lower alkenyl, substituted or unsubstituted lower
Alkynyl, substituted or unsubstituted aralkyl, also substituted
Or unsubstituted aryl, substituted or unsubstituted hetero
Aryl, substituted or unsubstituted heteroaryl substituted low
Secondary alkyl, substituted or unsubstituted tetrahydropyrani
Or substituted or unsubstituted tetrahydropyranylme
Represents chill or R^TenAnd R¹¹Together with the adjacent N
To form a substituted or unsubstituted heterocyclic group)},

R ² is hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted Substituted lower alkanoyl, substituted or unsubstituted lower alkanoyloxy, halogen, SR ¹² (in the formula, R ¹² represents substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or Unsubstituted aralkyl, substituted or unsubstituted heteroaryl substituted lower alkyl, substituted or unsubstituted tetrahydropyranyl or substituted or unsubstituted tetrahydropyranylmethyl), NR
¹³ R ¹⁴ (in the formula, R ¹³ and R ¹⁴ are the same as the above R ¹⁰ and R ¹¹ , respectively) or azido,

[0033] R ^{2 'represents} a lower alkanoyloxy hydrogen or a substituted or unsubstituted,, R ³ represents a lower alkanoyl substituted or unsubstituted, R ⁴ and R ⁵ are the same or different, hydrogen, a substituted or unsubstituted A substituted lower alkyl, a substituted or unsubstituted lower alkanoyl, a substituted or unsubstituted lower alkoxycarbonyl, a substituted or unsubstituted aralkyloxycarbonyl or a substituted or unsubstituted heteroaryl substituted lower alkoxycarbonyl, R ⁶ is hydrogen or Represents halogen,

R ⁷ and R ⁸ are the same or different and each represents hydrogen, substituted or unsubstituted lower alkyl or substituted or unsubstituted lower alkanoyl], and a carrier immobilized with a compound represented by The method is characterized by selecting a protein that specifically binds to the compound represented by formula (I) from the test protein sample when compared with a protein that binds to the compound represented by formula (III) when contacted. To
A method for searching a protein that binds to LK6-A or a derivative thereof.

(3) The search method according to (1) or (2) above, wherein the carrier is latex beads. (4) The search method according to any one of (1) to (3) above, wherein the test protein sample is a protein sample derived from a eukaryote. (5) A carrier on which LK6-A or a derivative thereof is immobilized. (6) The carrier according to (5) above, wherein the carrier is latex beads.

(7) Any one protein selected from the following (i) to (v): (I) A protein which binds to LK6-A or a derivative thereof obtained by the method according to any one of (1) to (4) above (ii) any one of SEQ ID NOs: 1 to 5, 43, 45 and 46 A protein (iii) SEQ ID NOS: 1 to 5, 43, which consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added, and which binds to LK6-A or its derivative in the amino acid sequence represented by A protein (iv) consisting of an amino acid sequence having 60-99% homology with the amino acid sequence represented by 45 or 46 and binding to LK6-A or a derivative thereof (iv) the amino acid sequence represented by SEQ ID NO: 2 (V) A protein consisting of the amino acid sequence represented by SEQ ID NO: 49

(8) D encoding the protein of (7) above
NA. (9) A recombinant DNA obtained by ligating the DNA of (8) above to a vector DNA. (10) A transformant containing the recombinant DNA of (9) above. (11) The transformant according to (10) above, which is obtained by using bacteria, yeast, insect cells, plant cells or animal cells as a host. (12) It is characterized in that the transformant of (10) or (11) above is cultured in a medium, the protein of (7) above is produced and accumulated in the culture, and the protein is collected from the culture. The method for producing a protein according to (7) above.

(13) The amount of binding between LK6-A or its derivative and the protein when LK6-A or its derivative is contacted with a protein that binds to LK6-A or its derivative in the presence of a test substance The amount of binding between LK6-A or a derivative thereof and the protein is measured when the LK6-A or a derivative thereof and a protein that binds to the LK6-A or a derivative thereof are contacted with each other in the absence of a test substance. , Comparing the measured value in the presence of the test substance and in the absence of the test substance, the substance that inhibits the binding of LK6-A or its derivative and the protein binding to LK6-A or its derivative from the test substance LK6- characterized by the choice
A method for searching a substance that binds to a protein that binds to A or a derivative thereof.

(14) The activity of the protein when the protein binding to LK6-A or its derivative and the test substance are contacted, and the activity of the protein when the test substance is not contacted are measured, Binding to LK6-A or its derivative characterized by comparing the measured values in the presence and absence of the test substance and selecting a substance that changes the activity of the protein from the test substance A method for searching for a substance that changes the activity of a protein. (15) Cellular response of a transformant that expresses a protein that binds to LK6-A or a derivative thereof and a test substance, and the transformation in the absence of the test substance Characterizing the cell response of the body, comparing the measured values in the presence and absence of the test substance, and selecting a substance that changes the cell response of the transformant from the test substance And a method for searching for a substance that changes the function of a protein that binds to LK6-A or a derivative thereof.

(16) When a transformant expressing a protein that binds to LK6-A or a derivative thereof is brought into contact with a test substance, the expression level of the DNA encoding the protein in the transformant and the test amount The expression level of the DNA encoding the protein of the transformant in the absence of the substance was measured, and the measured values in the presence of the test substance and in the absence of the test substance were compared. A method for searching for a substance that changes the expression level of a DNA encoding a protein that binds to LK6-A or a derivative thereof, which comprises selecting a substance that changes the expression level of a protein-encoding DNA. (17) The expression level of the protein in the transformant when the transformant expressing a protein that binds to LK6-A or a derivative thereof is contacted with the test substance, and the expression level of the protein in the absence of the test substance The expression level of the protein in the transformant is measured, the measured values in the presence and absence of the test substance are compared, and a substance that changes the expression level of the protein is selected from the test substance. A method of searching for a substance which changes the expression level of a protein binding to LK6-A or a derivative thereof, which is characterized in that

(18) In any one of the above (13) to (17), wherein the protein that binds to LK6-A or its derivative is any one protein selected from the following (i) to (v) Search method described. (I) a protein having the amino acid sequence represented by any of SEQ ID NOs: 1 to 5, 43, 45, 46 and 49 (ii) the protein of (7) above (iii) SEQ ID NOs: 1 to 5, 43, 45, 46 and 49
A protein having 60% or more homology with the amino acid sequence represented by any of the above and binding to LK6-A or a derivative thereof (iv) in the amino acid sequence represented by SEQ ID NO: 45, with amino acid numbers 353 to 408 Protein having amino acid sequence represented (v) Protein having amino acid sequence represented by amino acid numbers 64-121 in amino acid sequence represented by SEQ ID NO: 46

(19) In the presence of the test substance, the following (i) to (i)
ii) The activity of any one of the proteins selected from ii) and the activity of the protein in the absence of the test substance are measured, and the measured values in the presence and absence of the test substance are compared. A method for searching for a substance that inhibits the activity of the protein, wherein a substance that inhibits the activity of the protein is selected from test substances. (I) a protein having the amino acid sequence represented by SEQ ID NO: 1 (ii) 1 in the amino acid sequence represented by SEQ ID NO: 1
A protein (iii) SEQ ID NO: 1 which has an amino acid sequence in which the above amino acid residues have been deleted, substituted, inserted or added and which has substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1. A protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence represented by, and having substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1.

(20) Binding of the CD2 protein or its derivative to the protein when the CD2 protein or its derivative is contacted with any one of the following proteins (i) to (iii) in the presence of a test substance: Amount and CD in the absence of analyte
2 The binding amount of the CD2 protein or its derivative and the protein when the protein or its derivative and the protein are contacted is measured, and the measured value in the presence or absence of the test substance is A method for searching for a compound that inhibits the binding between a CD2 protein or a derivative thereof and the protein, comprising comparing and selecting a substance that inhibits the binding from a test substance. (I) a protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1 or 2 And a protein that binds to the CD2 protein (iii) A protein that binds to the CD2 protein and has an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 1 or 2.

(21) A derivative of the CD2 protein has the following (i)
~ The search method according to (20) above, which is any one protein selected from (iii). (I) 1 in the amino acid sequence represented by SEQ ID NO: 6
A protein consisting of an amino acid sequence in which the above amino acids are deleted, substituted, inserted or added, and having an activity of binding to a protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) represented by SEQ ID NO: 6 A protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 or 2 and binding to a protein having the amino acid sequence of SEQ ID NO: 1 or 2 (iii) an amino acid of the amino acid sequence of SEQ ID NO: 6 A protein having the amino acid sequence of Nos. 281 to 310

(22) In the presence of the test substance, the following (i) to (i
ii) Glycogen debranching enzyme activity of any one protein selected from ii) and the activity of the protein in the absence of the test substance were measured, and the activity in the presence or absence of the test substance was measured. A method for searching for a substance that inhibits the activity of the protein, comprising comparing measured values and selecting a substance that inhibits the activity of the protein from the test substance. (I) a protein having the amino acid sequence represented by SEQ ID NO: 3 (ii) in the amino acid sequence represented by SEQ ID NO: 3, 1
A protein having an amino acid sequence in which the above amino acid residues are deleted, substituted, inserted or added, and having glycogen debranching enzyme activity (iii) having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 3. A protein consisting of an amino acid sequence having and having glycogen debranching enzyme activity

(23) In the presence of the test substance, the following (i) to (i)
ii) The peroxidase activity of any one protein selected from ii) and the activity of the protein in the absence of the test substance are measured, and the measured values in the presence and absence of the test substance A method for searching for a substance that inhibits the activity of the protein, comprising comparing and selecting a substance that inhibits the activity of the protein from test substances. (I) a protein having the amino acid sequence represented by SEQ ID NO: 5 (ii) in the amino acid sequence represented by SEQ ID NO: 5, 1
A protein having the above amino acid residue deleted, substituted, inserted or added, and having peroxidase activity (iii) an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 5 And a protein having peroxidase activity

(24) The binding amount of the CD2BP2 protein or its derivative and LK6-A or its derivative when the CD2BP2 protein or its derivative is contacted with LK6-A or its derivative in the presence of the test substance, and the test substance When the CD2 protein or its derivative is contacted with LK6-A or its derivative in the absence, the binding amount of the CD2BP2 protein or its derivative and LK6-A or its derivative is measured. Of the compound that inhibits the binding of the CD2BP2 protein or its derivative and LK6-A or its derivative, which is characterized by comparing the measured values in the absence of a substance and selecting a substance that inhibits the binding from the test substance Search method.

(25) The search method according to the above (24), wherein the derivative of the CD2BP2 protein is any one protein selected from the following (i) to (iii). (I) in the amino acid sequence represented by SEQ ID NO: 43,
A protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added, and having an activity of binding to a protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) represented by SEQ ID NO: 43 (Iii) consisting of an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1 or 2 which binds to a protein having the amino acid sequence shown in SEQ ID NO: 1 or 2 A protein having an amino acid sequence of amino acid numbers 282 to 341

(26) A substance obtained by the search method described in any one of (13) to (25) above, or a pharmaceutically acceptable salt of the substance. (27) An agent for treating an autoimmune disease or an immunosuppressive agent, which comprises the substance of (26) or a pharmaceutically acceptable salt of the substance.

[0050]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The LK6-A derivative used in the search method of the present invention has the following general formula (I):

[0051]

[Chemical 13]

[Wherein R ¹ is a substituted or unsubstituted, substituted or unsubstituted lower alkanoyl, carboxy, lower alkoxycarbonyl, the following formula (II)

[0053]

[Chemical 14]

(Wherein n represents 1 or 2)
Group or COCH = CHR⁹{In the formula, R⁹Is a replacement
Is an unsubstituted lower alkoxy, a substituted or unsubstituted ari
, Substituted or unsubstituted heteroaryl or NR
^TenR¹¹(In the formula, R^TenAnd R ¹¹Are the same or different,
Hydrogen, substituted or unsubstituted lower alkyl, substituted or
Is unsubstituted lower alkenyl, substituted or unsubstituted lower
Alkynyl, substituted or unsubstituted aralkyl, also substituted
Or unsubstituted aryl, substituted or unsubstituted hetero
Aryl, substituted or unsubstituted heteroaryl substituted low
Secondary alkyl, substituted or unsubstituted tetrahydropyrani
Or substituted or unsubstituted tetrahydropyranylme
Represents chill or R^TenAnd R¹¹Together with the adjacent N
To form a substituted or unsubstituted heterocyclic group)},

R ² is hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted Substituted lower alkanoyl, substituted or unsubstituted lower alkanoyloxy, halogen, SR ¹² (in the formula, R ¹² represents substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or Unsubstituted aralkyl, substituted or unsubstituted heteroaryl substituted lower alkyl, substituted or unsubstituted tetrahydropyranyl or substituted or unsubstituted tetrahydropyranylmethyl), NR
¹³ R ¹⁴ (in the formula, R ¹³ and R ¹⁴ are the same as the above R ¹⁰ and R ¹¹ , respectively) or azido,

R ⁴ and R ⁵ are the same or different and are hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aralkyloxycarbonyl or Represents a substituted or unsubstituted heteroaryl-substituted lower alkoxycarbonyl, R ⁶ represents hydrogen or halogen, R ⁷ and R ⁸ are the same or different, and are hydrogen, substituted or unsubstituted lower alkyl or substituted or unsubstituted lower Represents an alkanoyl].

As a more preferred LK6-A derivative used in the present invention, in the above formula (I), R ¹ represents (E) -3-methoxyacryloyl, and R ² , R ⁴ , R ⁵ and R ⁶ Is hydrogen, R ⁷ is hydrogen, and R ⁸ is acetyl.
You can give 6-A. Here, the lower alkyl portion of the lower alkyl, lower alkoxy, lower alkenyl, lower alkynyl, and heteroaryl-substituted lower alkyl preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms. The lower alkanoyl, lower alkanoyloxy, lower alkoxycarbonyl, and heteroaryl-substituted lower alkoxycarbonyl have a lower alkoxycarbonyl moiety preferably having 2 to 7 carbon atoms, and more preferably 2 to 5 carbon atoms.

Substituted lower alkyl, substituted lower alkanoyl, substituted lower alkoxy, substituted aryl, substituted heteroaryl, substituted lower alkenyl, substituted lower alkynyl, substituted aralkyl, substituted aryl, substituted heteroaryl, substituted heteroaryl of substituted lower alkyl, substituted tetrahydro. Pyranyl, substituted tetrahydropyranylmethyl, substituted heterocyclic group, substituted lower alkanoyloxy, substituted lower alkoxycarbonyl, substituted aralkyloxycarbonyl,
And examples of the substituent in the substituted heteroaryl moiety of the substituted heteroaryl-substituted lower alkoxycarbonyl are the same or different and are 1 to the substitutable number, preferably 1 to 4 carbon atoms of lower alkyl, hydroxy, preferably Included are 2 to 5 lower alkoxys, and halogen. Examples of the hetero atom in the heteroaryl include nitrogen atom, oxygen atom, sulfur atom and the like.
As halogen, a fluorine atom, a chlorine atom, a bromine atom,
Iodine atom can be mentioned. Examples of the heterocyclic group formed by R ¹⁰ and R ¹¹ together with the adjacent N include a 5- to 8-membered ring. In addition to N in which R ¹⁰ and R ¹¹ are adjacent to each other, a nitrogen atom and an oxygen atom are also included. , And may further contain a hetero atom such as a sulfur atom.

(1) Method for Preparing Carrier As the carrier used in the present invention, any carrier can be used as long as it can immobilize the LK6-A derivative. A carrier with less non-specific adsorption is preferable, and more preferably SGNEGDE particles having an epoxy group on the surface are preferable. SGNEGDE particles refer to microspheres of styrene-glycidyl methacrylate polymer whose surface is covered with styrene-glycidyl methacrylate. The SGNEGDE
The size of the particles can be appropriately selected, but is usually about 0.05 to 0.5 μm, preferably about 0.1 to 0.3 μm.
m. The particles are described in Methods in Enzymology, 254 , 59.
5 (1995), Japanese Patent No. 3086427, or Colloi
It can be prepared according to ds and Surfaces B, 10 , 41 (1997) and the like. Particles having amino groups on the surface can be produced by reacting SGNEGDE particles with a compound having two or more primary or secondary amines. The solvent and the reaction conditions at this time are a combination of the solvent and the reaction conditions in which the immobilization reaction proceeds, and the compound having two or more SGNEGDE particles, primary or secondary amines is different from the immobilization reaction described above. Any combination of the solvent and the reaction conditions may be used as long as the combination of the solvent and the reaction conditions does not cause the decomposition reaction, the condensation reaction and the like to the extent that the object of the present invention is hindered. More specifically, SGNEGDE particles containing 1 μmol / l to 50 mol / l of a compound having two or more primary or secondary amines, such as 1,2-diaminoethane, 1,3-diaminopropane or piperazine Disperse it in the solution and let it stand still, or while gently stirring with a rotator, mixer, etc.
Particles having amino groups on the surface can be prepared by reacting at 1 to 1000 hours at -100 to 100 ° C. Examples of the solvent include water, DMF, DMSO, THF, dioxane,
Examples include methanol.

After completion of the reaction, the particles are washed to remove unreacted compounds having two or more primary or secondary amines. The cleaning method is stable and efficient recovery of SGNEGDE particles with immobilized compounds having two or more primary or secondary amines, and efficient removal of compounds having two or more primary or secondary amines. Any known method can be used as long as it is a method. For example, after completion of the reaction, centrifugation (15000 rpm, 5 minutes, room temperature) is performed, the supernatant is removed, and the suspension is suspended in a solvent. Further, if necessary, the solvent can be replaced with a solvent suitable for the subsequent reaction by the same method.

LK6-A derivative-immobilized particles can be prepared by reacting the amino group on the particles thus obtained with the LK6-A derivative. Specifically, SGNE in which a compound having two or more primary or secondary amines is immobilized
GDE particles can be dispersed in a solution of the LK6-A derivative and the amino groups on the particle can be reacted with the LK6-A derivative. At this time, the solvent and the reaction condition are a combination of the solvent and the reaction condition in which the immobilization reaction proceeds, and the primary or secondary amine is
SGNEGDE particles having a compound having three or more immobilized, LK6-A derivative is a combination of a solvent and reaction conditions that do not cause decomposition reactions or condensation reactions other than the above-described immobilization reaction to the extent that the object of the present invention is not impaired. Any solvent and reaction conditions may be combined. More specifically, piperazine-immobilized SGNEGDE particles are dispersed in a solution containing 1 μmol / l to 50 mol / l of LK6-A derivative and allowed to stand, or gently with a rotator, mixer, etc. 0 while stirring
Particles having the LK6-A derivative immobilized on the surface can be prepared by reacting at -100 to 100 ° C for 1 second to 1000 hours. Examples of the solvent include water, DMF, DMSO, THF, dioxane, methanol and the like.

After completion of the reaction, the particles having the LK6-A derivative immobilized thereon are washed to remove the unreacted LK6-A derivative.
As a washing method, any known method can be used as long as the particles having the LK6-A derivative immobilized thereon can be stably and efficiently recovered and the LK6-A derivative can be efficiently removed. For example, after completion of the reaction, centrifugation (15000 rpm, 5 minutes, room temperature) is performed, the supernatant is removed, and the suspension is suspended in a solvent.
If necessary, the solvent can be replaced with a solvent suitable for the subsequent storage and mixing with the cell extract by the same method.
When unreacted amino groups remain on the LK6-A derivative-immobilized particles, the unreacted amino groups can be blocked as necessary. Any compound that reacts with an amino group and does not react with the LK6-A derivative immobilized on particles can be used for blocking. For example, succinic anhydride can be used to block unreacted amino groups on LK6-A derivative-immobilized particles.

As another method for producing LK6-A derivative-immobilized particles, a LK6-A derivative is reacted with a compound having two or more primary or secondary amines to form SGNEGDE.
A method of synthesizing a compound into which a primary or secondary amine necessary for binding onto particles is introduced and binding the compound to SGNEGDE particles can be mentioned. Level 1 or 2
The LK6-A derivative into which a primary amine has been introduced can be prepared according to the method described in WO 00/02879. Examples of the compound having two or more primary or secondary amines are 1,2-
Examples thereof include diaminoethane, 1,3-diaminopropane, piperazine and the like.

LK6-A-immobilized particles can be prepared by reacting SGNEGDE particles with the compound prepared using the above method. The solvent and the reaction conditions at this time are a combination of the solvent and the reaction conditions in which the immobilization reaction proceeds, and SGNEGDE particles, the compound is a decomposition reaction or condensation reaction other than the immobilization reaction, the present invention, Any combination of the solvent and the reaction conditions may be used as long as the combination of the solvent and the reaction conditions does not occur to the extent that the object of (1) is not inhibited. More specifically, SGNEGDE particles are dispersed in a solution containing 1 μmol / l to 1 mol / l of a compound represented by the following formula (V) (hereinafter abbreviated as LK6D2), and allowed to stand, or While gently stirring with a rotator, mixer, etc., 0 ℃ -100
LK6-A-immobilized particles can be prepared by reacting at 1 ° C. for 1 second to 1000 hours.

[0065]

[Chemical 15]

Examples of the solvent include water, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMS).
O), tetrahydrofuran (THF), dioxane, methanol and the like. After the reaction is complete, the particles are washed to remove unreacted LK6D2. As a washing method, any known method can be used as long as the LK6-A-immobilized particles are stable and can be efficiently recovered, and LK6D2 can be efficiently removed. For example, after the reaction is complete, centrifuge (15000 rp
m, 5 minutes, room temperature), remove the supernatant, and suspend in a solvent,
Similarly, centrifugation and removal of the supernatant can be repeated for washing. Further, if necessary, the solvent can be replaced with a solvent suitable for the subsequent reaction by the same method.

The LK6-A derivative-immobilized particles thus obtained can be stored after being dispersed in a solvent. The solvent and storage conditions at this time may be any combination of solvent and storage conditions as long as the LK6-A derivative-immobilized particles are stable as long as they do not impair the object of the present invention. More specifically, examples of the solvent include water, DMF, DMSO, THF, dioxane, methanol and the like. It is preferably stored at room temperature or lower, more preferably under shading and under inert gas such as helium gas or argon gas substitution. In the same manner as described above, particles immobilized with LK6-A control compound (hereinafter referred to as LK6-A control compound-immobilized particles) were prepared, and search for a protein that specifically binds to the LK6-A derivative of the present invention was performed. Can be used for the method. LK6-A reference compound is represented by the following formula (III)

[0068]

[Chemical 16]

[Wherein R ¹ is a substituted or unsubstituted lower alkyl, lower alkanoyl, carboxy, lower alkoxycarbonyl, the following formula (II)

[0070]

[Chemical 17]

(Wherein n represents 1 or 2)
Group or COCH = CHR⁹{In the formula, R⁹Is a replacement
Is an unsubstituted lower alkoxy, a substituted or unsubstituted ari
, Substituted or unsubstituted heteroaryl or NR
^TenR¹¹(In the formula, R^TenAnd R ¹¹Are the same or different,
Hydrogen, substituted or unsubstituted lower alkyl, substituted or
Is unsubstituted lower alkenyl, substituted or unsubstituted lower
Alkynyl, substituted or unsubstituted aralkyl, also substituted
Or unsubstituted aryl, substituted or unsubstituted hetero
Aryl, substituted or unsubstituted heteroaryl substituted low
Secondary alkyl, substituted or unsubstituted tetrahydropyrani
Or substituted or unsubstituted tetrahydropyranylme
Represents chill or R^TenAnd R¹¹Together with the adjacent N
To form a substituted or unsubstituted heterocyclic group)},

R ² is hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted Substituted lower alkanoyl, substituted or unsubstituted lower alkanoyloxy, halogen, SR ¹² (in the formula, R ¹² represents substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or Unsubstituted aralkyl, substituted or unsubstituted heteroaryl substituted lower alkyl, substituted or unsubstituted tetrahydropyranyl or substituted or unsubstituted tetrahydropyranylmethyl), NR
¹³ R ¹⁴ (in the formula, R ¹³ and R ¹⁴ are the same as the above R ¹⁰ and R ¹¹ , respectively) or azido,

[0073] R ^{2 'represents} a lower alkanoyloxy hydrogen or a substituted or unsubstituted,, R ³ represents a lower alkanoyl substituted or unsubstituted, R ⁴ and R ⁵ are the same or different, hydrogen, a substituted or unsubstituted Substituted lower alkyl, substituted or unsubstituted lower alkanoyl, substituted or unsubstituted lower alkoxycarbonyl, substituted or unsubstituted aralkyloxycarbonyl or substituted or unsubstituted heteroaryl substituted lower alkoxycarbonyl, R ⁶ is hydrogen or Represents halogen, R ⁷ and R ⁸ are the same or different and each represent hydrogen, substituted or unsubstituted lower alkyl or substituted or unsubstituted lower alkanoyl], and have no immunosuppressive activity, Or its immunosuppressive activity is significantly lower than that of the above LK6-A derivative It is a compound.

More specifically, R ¹ represents (E) -3-methoxyacryloyl, R ² , R ⁵ and R ⁶ represent hydrogen, R ^{2 ′} represents acetoxy, and R ³ and R ⁴ represent LK6D7, which represents acetyl, R ⁷ represents hydrogen, and R ⁸ represents acetyl, may be mentioned. Here, the lower alkyl portion of the lower alkyl, lower alkoxy, lower alkenyl, lower alkynyl, and heteroaryl-substituted lower alkyl preferably has 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms. The lower alkanoyl, lower alkanoyloxy, lower alkoxycarbonyl, and heteroaryl-substituted lower alkoxycarbonyl have a lower alkoxycarbonyl moiety preferably having 2 to 7 carbon atoms, and more preferably 2 to 5 carbon atoms.

Substituted lower alkyl, substituted lower alkanoyl, substituted lower alkoxy, substituted aryl, substituted heteroaryl, substituted lower alkenyl, substituted lower alkynyl, substituted aralkyl, substituted aryl, substituted heteroaryl, substituted heteroaryl of substituted lower alkyl, substituted tetrahydro. Pyranyl, substituted tetrahydropyranylmethyl, substituted heterocyclic group, substituted lower alkanoyloxy, substituted lower alkoxycarbonyl, substituted aralkyloxycarbonyl,
And examples of the substituent in the substituted heteroaryl moiety of the substituted heteroaryl-substituted lower alkoxycarbonyl are the same or different and are 1 to the substitutable number, preferably 1 to 4 carbon atoms of lower alkyl, hydroxy, preferably Included are 2 to 5 lower alkoxys, and halogen.

Examples of the hetero atom in the heteroaryl include nitrogen atom, oxygen atom, sulfur atom and the like.
As halogen, a fluorine atom, a chlorine atom, a bromine atom,
Iodine atom can be mentioned. Examples of the heterocyclic group formed by R ¹⁰ and R ¹¹ together with the adjacent N include a 5- to 8-membered ring. In addition to N in which R ¹⁰ and R ¹¹ are adjacent to each other, a nitrogen atom and an oxygen atom are also included. , And may further contain a hetero atom such as a sulfur atom.

(2) Method for preparing cell extract If the LK6-A binding protein of the present invention is a protein that specifically binds to the LK6-A derivative under conditions capable of binding to the LK6-A derivative, The origin thereof is not limited, but a protein derived from a eukaryote, preferably a protein derived from an animal, more preferably a protein derived from a mammalian organ, organ, tissue or cell can be mentioned. LK of the present invention
The protein sample to be subjected to the 6-A binding protein search method is not particularly limited as long as it is a protein sample, but preferably a eukaryote-derived protein sample, more preferably an animal-derived protein sample, and further preferably a mammalian sample. A protein sample derived from an organ, an organ, a tissue or a cell can be mentioned.

As the organ, organ or tissue, any known tissue may be used, but tissues such as spleen, thymus and lymph nodes are preferred. The cells may be any known cells, but T cell lines, T cell primary culture cells, and the like can be used. Specifically, as a T cell line, human-derived Jurk
Examples of such cells include at cells and mouse-derived CTLL-2 cells.
Examples of T cell primary culture cells include cells obtained by culturing cells collected from tissues such as spleen, thymus, and lymph nodes for a short period of time. These cells can be cultured according to the method described in Introduction to Animal Cell Culture Method, Academic Publishing Center (1993) and the like.

As a method for preparing a protein sample from the above organs, organs, tissues or cells, any known method may be used as long as it is a method capable of recovering a protein, for example,
Basic Experiments for Proteins and Enzymes, Revised 2nd Edition (Nankodo, 1994)
And the like. As a method for crushing an organ, an organ, a tissue or a cell, any known crushing method may be used, such as Waring blender, Polytron, Dounce.
A protein sample is prepared by combining mechanical crushing using a device such as a mold homogenizer or ultrasonic crusher, physical crushing using a hypotonic solution, solubilization using a surfactant, and centrifugation. can do. The protein sample can be stored in a refrigerated state or a frozen state.

(3) Method for identifying LK6-A binding protein Using the LK6-A derivative-immobilized particles prepared by the method (1), LK6-A derivative-immobilized from the protein sample prepared by the method (2) The LK6-A binding protein is bound to the LK6-A control compound-immobilized particles but does not bind to the LK6-A control compound-immobilized particles, or the amount of binding is smaller than that of the LK6-A derivative-immobilized particles. Can be specified. The method for determining the binding protein to both particles may be any known method as long as it can determine the LK6-A binding protein, for example, basic experimental methods for proteins and enzymes, revised second edition (Nankodo, 1994) and the like.

Specifically, LK6- prepared by the method (1)
The A derivative or LK6-A control compound-immobilized particles are dispersed in the cell extract prepared by the method (2), and the mixture is allowed to stand or gently agitated with a rotator or a mixer at 0 ° C.
Incubate at -50 ° C for 1 second to 1000 hours, and then react with LK6-A derivative or L
A protein can be adsorbed on the K6-A control compound-immobilized particles. After this binding reaction, the protein non-specifically adsorbed to the LK6-A derivative or LK6-A control compound-immobilized particles can be removed by washing the LK6-A derivative or LK6-A control compound-immobilized particles. The solution used for binding or washing may be any solution as long as it can be used in this method.
Basic Experiments for Proteins and Enzymes, Revised 2nd Edition (Nankodo, 1994)
The solution described in can be mentioned.

The protein adsorbed on the particles immobilized with the LK6-A derivative or the LK6-A control compound is LK6-A or LK6-A by means of weakening the binding with the LK6-A derivative or the LK6-A control compound.
It can be eluted from the control compound-immobilized particles. LK6-
The means for weakening the binding between the A or LK6-A control compound and the binding protein may be any means as long as it can be used in this method, for example, addition of a denaturant, increase in salt concentration, heating, addition of a surfactant, etc. Can be given. Denaturants include SDS, urea, and guanidine hydrochloride, salts include NaCl, KCl, etc., and surfactants include Nonidet P40, Triton, Twee
n, Digitonin, etc. In addition, heating means raising the temperature to, for example, 30 to 100 ° C. The protein thus purified can be detected by protein analysis techniques such as SDS-PAGE, two-dimensional electrophoresis, mass spectrometry and the like. LK6-
By comparing the binding proteins to A-immobilized particles, LK6-A control compound-immobilized particles, or particles subjected only to blocking, the protein that specifically binds to the LK6-A derivative, that is, the target of the LK6-A derivative A protein can be specified.

(4) Method for identifying LK6-A binding protein The LK6-A binding protein can be excised from the gel after separation by SDS-PAGE and staining with Coomassie Brilliant Blue (CBB). The protein in the gel can be divided into peptide fragments by treatment with endoproteases having specific cleavage points such as lysyl endopeptidase and trypsin. After the obtained peptide fragment is purified by reverse phase HPLC or the like, the partial amino acid sequence of the protein can be examined by using a gas phase protein sequencer or a mass spectrometer. By using the information of these partial amino acid sequences, PIR, SwissProt, and other amino acid sequence databases and Ge
By performing a homology search on a nucleotide sequence database such as nBank, the homology between the protein and a known protein can be known.

The LK6-A binding protein identified by the above method has the amino acid sequence represented by SEQ ID NO: 1.
p150a, p15 having the amino acid sequence represented by SEQ ID NO: 3
0c, p85 having the amino acid sequence represented by SEQ ID NO: 4 and p27 having the amino acid sequence represented by SEQ ID NO: 5. p150a is http // www.kazusa.or.j
KIAA0642 (acc listed in the database of p / huge /
ession No. AB014542) and human cDNA represented by GenBank accession No. AK001739.
It is a protein encoded by DNA and has a GYF domain. In the homology search against public databases, regions other than the GYF domain show no homology with other proteins, and there is no report on the function. The GYF domain was the first domain found in the CD2 binding protein CD2BP2
Binding to the proline-rich region of the intracellular region of CD
Suppressing gene expression of 2BP2 suppresses IL-2 production,
It is known that increased expression increases IL-2 production [Proc. Natl. Acad. Sci. USA., 95 , 14897 (199
8)]. A method for searching for a compound having immunosuppressive activity by searching for a compound that inhibits the binding between the proline-rich region and the GYF domain is known (WO 00 /
34309). The protein having the amino acid sequence represented by SEQ ID NO: 2 is a region containing the GYF domain in p150a, and the DNA encoding the protein is shown in SEQ ID NO: 9.

P150c is two different enzymatic activities involved in glycogen degradation [oligo-1,4 → 1,4-glucantransferase and amylo-1,6-glucosidase. (Amylo-1,6-glucosida
se] activity], which is a protein known as Glycogen debranching enzyme [J. Biol. Chem., 276 , 28824 (20
01)], gene-deficient patients are fatal. However, there are no reports on the relationship between glycogen debranching enzyme and immunosuppression. p85 is the GenBank acces
It is a protein encoded by human cDNA represented by sion No. AK025598, has no homology with other proteins in the homology search against public databases, and has no report on its function. There are no reports on the association between p85 and immunosuppression. p27 is MSP23, an enzyme having an activity of decomposing hydrogen peroxide (peroxidase activity) in the presence of thioredoxin, thioredoxin reductase and NADPH [Antioxidant and Redox Signaling, 1 , 385 (199
9)]. However, there are no reports on the relationship between MSP23 and immunosuppression.

(5) LK6-A binding protein of the present invention As the LK6-A binding protein of the present invention, the LK6-A binding protein obtained by the above-mentioned method excluding known proteins can be mentioned. it can. Specific examples include (i) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, 43, 45 and 46, in which one or more amino acids are deleted, substituted, inserted or added, and LK6-A derivative-binding protein (ii) consisting of an amino acid sequence having 60-99% homology with the amino acid sequence represented by any of SEQ ID NOS: 1-5, 43, 45 and 46, and LK6-A A protein that binds to a derivative (iii) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (iv) a protein consisting of the amino acid sequence represented by SEQ ID NO: 49 can be mentioned.

The amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, 43, 45 and 46 is composed of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added, and is a LK6-A derivative. The binding protein is Mol
ecular Cloning, A Laboratory Manual, Third Editio
n, Cold Spring Harbor Laboratory Press (2001) (hereinafter referred to as Molecular Cloning 3rd Edition), Curr
ent Protocols in Molecular Biology, John Wiley & S
ons (1987-1997) (hereinafter Current Protocols
In Molecular Biology), Nucleic
Acids Research, 10 , 6487 (1982), Proc. Natl. Acad.
Sci. USA, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nu
cleic Acids Research, 13 , 4431 (1985), Proc. Natl.
Acad. Sci. USA, 82 , 488 (1985) and the like, using the site-directed mutagenesis method, for example, SEQ ID NOS: 1 to 5, 43,
It can be obtained by introducing a site-specific mutation into the DNA encoding the protein consisting of the amino acid sequence represented by either 45 or 46.

The number of amino acids to be deleted, substituted, inserted or added is not particularly limited, but it is a number that can be deleted, substituted, inserted or added by well-known methods such as the above site-specific mutation method, 1 to several tens, preferably 1
-20, more preferably 1-10, and even more preferably 1-5. Deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence represented by any of SEQ ID NOs: 1 to 5, 43, 45 and 46 means 1 at any position in the same sequence. Or, it means that there are deletions, substitutions, insertions or additions of a plurality of amino acid residues, and deletions, substitutions, insertions or additions may occur at the same time. And non-natural type. Natural amino acid residues include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-
Histidine, L-isoleucine, L-leucine, L-lysine,
L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine,
Examples include L-valine and L-cysteine.

Examples of amino acid residues that can be mutually substituted are shown below. Amino acid residues included in the same group can be mutually substituted. Group A: Leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid C group: asparagine, glutamine D group: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid E group: proline, 3 -Hydroxyproline, 4-hydroxyproline F group: serine, threonine, homoserine G group: phenylalanine, tyrosine

Further, a protein in which one or more amino acid residues are deleted, substituted, inserted or added in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5, 43, 45 and 46 becomes an LK6-A derivative. In order to be a binding protein, it is preferable that it has 60% or more, usually 80% or more, and particularly 95% or more homology with the amino acid sequence represented by any of SEQ ID NOS: 1 to 5. . The homology of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90 , 5873 (19) by Karlin and Altschul.
93)] or FASTA [Methods Enzymol., 183 , 63 (1990)]. Programs called BLASTN and BLASTX have been developed based on this algorithm BLAST [J. Mol. Biol., 215 , 403 (1990)]. BLAST
When the nucleotide sequence is analyzed by BLASTN based on, the parameters are, for example, Score = 100, wordlength = 12
And When analyzing the amino acid sequence by BLASTX based on BLAST, the parameters are, for example, scor.
Let e = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters for each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

(6) Preparation of LK6-A binding protein used in the present invention (1) The LK6-A binding protein used in the present invention is obtained and identified by the above methods (1) to (4). Examples thereof include the LK6-A binding protein and the LK6-A binding protein of the present invention described in (5) above. The LK6-A binding protein used in the present invention may be prepared by any known method as long as it can be used in the method of the present invention. For example, the basic experiment method for proteins / enzymes, second revised edition (Nankodo, 1994). It can be prepared using the method described in the above.

The LK6-A binding protein can be prepared as an unpurified fraction, a partially purified fraction, or a purified fraction by crushing an eukaryotic organ, organ, tissue or cell. The eukaryote may be any known organism, but mammals are preferred. The organ, organ or tissue may be any known organ, organ or tissue, and examples thereof include spleen, thymus, lymph node and the like. The cells may be any known cells, and examples thereof include Jurkat cells and CTLL-2 cells. Any known crushing method may be used as a method for crushing an organ, an organ, a tissue, or a cell. The protein fraction containing the LK6-A binding protein can be prepared by combining operations such as disruption, physical disruption with a hypotonic solution, solubilization with a surfactant, and centrifugation.

The protein fraction can be stored in a refrigerated state or a frozen state. As a method for purifying LK6-A binding protein from the protein fraction, ion exchange chromatography, hydrophobic chromatography, gel filtration, LK6-A
Examples include a method of fractionating the LK6-A binding protein by a method such as affinity purification using a carrier on which a derivative is immobilized, or a combination thereof. The amount of LK6-A binding protein contained in the fractionated fraction can be measured by an immunoblotting method using an antibody reactive with the LK6-A binding protein. The protein contained in the fractionated fraction is LK6-A
Whether or not the protein binds to the derivative and the degree of its binding activity can be detected using the binding activity to the LK6-A-immobilized particles prepared by the method (1) as an index. In addition, if the LK6-A binding protein is a protein whose biological activity such as other binding activity and enzyme activity is known, it can be evaluated using those binding activity and enzyme activity [CurrentProtoco
ls in Protein Science, John Wiley & Sons (200
1)]. Fractions containing a large amount of LK6-A binding protein and having high LK6-A binding protein activity can be combined to form a partially purified LK6-A binding protein. A highly purified LK6-A binding protein can be prepared by repeating the above operation using about 1 to 6 kinds of different columns.

As an alternative method for purifying the LK6-A binding protein from the protein fraction prepared above, there is a method of purifying by affinity chromatography using an antibody reactive with the LK6-A binding protein. That is, LK6-A
Harlow, E. and Lane, D. Usi after obtaining monoclonal and polyclonal antibodies that react with the binding protein.
ng antibodies: A laboratory manual, Cold Spring Ha
rbor Laboratory Press, Cold Spring Harbor, New Yor
k, USA (1999), etc., and cross-link the antibody with a chromatography carrier to prepare an affinity column. Using this column, a protein fraction or a partially purified fraction of LK6-A binding protein The LK6-A binding protein can be purified from the minutes.

(7) Preparation of LK6-A binding protein used in the present invention (2) The LK6-A binding protein used in the present invention can be prepared using DNA encoding the LK6-A binding protein. .
As the DNA encoding the LK6-A binding protein, (i) the LK obtained or identified by the above method (1) to (4)
DNA encoding the 6-A binding protein (ii) DNA encoding the LK6-A binding protein of the present invention of the above (5), (iii)
DNA having a nucleotide sequence represented by any of SEQ ID NOs: 8 to 12, (iv) hybridizing with the DNA of (ii) or (iii) above under stringent conditions, and LK
A DNA encoding a protein that binds to a 6-A derivative can be mentioned.

A DNA hybridizable under stringent conditions is, for example, a DNA having a nucleotide sequence represented by any of SEQ ID NOs: 8 to 12 as a probe, a colony hybridization method, a plaque
D obtained by using the hybridization method or the Southern blot hybridization method
NA means, specifically, 0.7 using a filter on which DNA derived from colonies or plaques is immobilized.
After hybridization at 65 ° C. in the presence of ˜1.0 mol / l sodium chloride, 0.1 to 2 times concentration of S
SC solution (The composition of 1-fold concentrated SSC solution is 150 mmol
DNAs that can be identified by washing the filter under the conditions of 65 ° C. using 1 / l sodium chloride and 15 mmol / l sodium citrate). Hybridization is described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A
Practical Approach, Second Edition, Oxford Univers
It can be carried out according to the method described in ity (1995) and the like. As the hybridizable DNA, specifically, at least 60% of the nucleotide sequence represented by any of SEQ ID NOs: 8 to 12 when calculated using BLAST, FASTA and the like and the above parameters and the like. The DNA having the above homology, preferably the DNA having 80% or more homology, and more preferably the DNA having 95% or more homology.

As the LK6-A binding protein, the LK6-A binding protein can be prepared by the method described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology, etc. The encoding DNA can be expressed and produced in a host cell. Based on the full-length cDNA, if necessary,
A DNA fragment of an appropriate length containing a portion encoding the protein is prepared. In addition, DNA is prepared by substituting the nucleotide sequence of the portion encoding the LK6-A binding protein so that the codon is optimal for the expression in the host cell, if necessary. The DNA is useful for efficient production of LK6-A binding protein. By inserting the DNA fragment or the full-length cDNA into the downstream of the promoter of an appropriate expression vector,
Make a recombinant vector. The recombinant vector is introduced into a host cell suitable for the expression vector. As the host cell, any cell can be used, such as bacteria, yeast, animal cells, insect cells, and plant cells, as long as it can express the gene of interest.

The expression vector is capable of autonomous replication in the above host cell or can be integrated into the chromosome,
Those containing a promoter at a position where the DNA encoding the polypeptide of the present invention can be transcribed are used.
When using a prokaryote such as a bacterium as a host cell, LK6-
A recombinant vector containing a DNA encoding an A-binding protein is capable of autonomous replication in prokaryotes and
A promoter, a ribosome binding sequence, the DNA of the present invention,
It is preferably a vector composed of a transcription termination sequence. A gene that controls the promoter may be included.

Examples of the expression vector include pBTrp2,
pBTac1, pBTac2 (both are commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invit
rogen), pGEMEX-1 (Promega), pQE-8 (QIAGE
N company), pKYP10 (JP-A-58-110600), pKYP200 [Agri
c. Biol. Chem., 48 , 669 (1984)], pLSA1 [Agric. Bi
ol. Chem., 53 , 277 (1989)], pGEL1 [Proc. Natl. Ac
ad. Sci. USA, 82 , 4306 (1985)], pBluescript II SK
(-) (Manufactured by Stratagene), pTrs30 [ Escherichiacoli JM1
09 / pTrS30 (prepared from FERM BP-5407)], pTrs32 [ Esch
erichia coli JM109 / pTrS32 (FERM BP-5408)], pGHA2 [ Escherichia coli IGHA2 (FERM B-400)
Prepared by JP-A-60-221091], pGKA2 [ Escherichia co
li Prepared from IGKA2 (FERM BP-6798), JP-A-60-22109
1), pTerm2 (US4686191, US4939094, US5160735), pS
upex, pUB110, pTP5, pC194, pEG400 (J. Bacteriol.,
172 , 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen) and the like.

Any promoter can be used, so long as it can function in the host cell. For example, trp
_Examples thereof include promoters (P _trp ), lac promoter, P _L promoter, P _R promoter, T7 promoter and the like derived from Escherichia coli, phage and the like. In addition, a promoter in which two _Ptrp are connected in series (P _trp ×
2), artificially designed and modified promoters such as tac promoter, lacT7 promoter and let I promoter can also be used. It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). In the recombinant vector of the present invention, the transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

Examples of host cells include microorganisms belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, and the like, for example, Escherichia coli XL1-B.
lue, Escherichia coli XL2-Blue, Escherichia coli D
H1, Escherichia coli MC1000, Escherichia coli KY32
76, Escherichia coli W1485, Escherichia coli JM10
9, Escherichia coli HB101, Escherichia coli No.4
9, Escherichia coli W3110, Escherichia coli NY49,
Escherichia coli GI698, Escherichia coli TB1, Serr
atia ficaria , Serratia fonticola , Serratia liquefa
ciens , Serratia marcescens , Bacillus subtilis , Bac
illus amyloliquefacines , Brevibacterium ammoniagen
es , Brevibacterium immariophilum ATCC14068, Brevib
acterium saccharolyticum ATCC14066, Brevibacterium
flavum ATCC14067, Brevibacterium lactofermentum A
TCC13869, Corynebacterium glutamicum ATCC13032, Co
rynebacterium glutamicum ATCC13869, Corynebacteriu
m acetoacidophilum ATCC13870, Microbacterium ammon
iaphilum ATCC15354, Pseudomonas putida , Pseudomon
As sp. D-0110 etc. can be mentioned.

As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into the above-mentioned host cell. For example, a method using calcium ion [Proc. Natl. Acad. Sci. USA, 69 , 2110 (1972)],
Protoplast method (JP-A-63-2483942) or Gene,
17 , 107 (1982) and Molecular & General Genetics, 168 ,
111 (1979) and the like. When yeast is used as a host cell, examples of expression vectors include YEP13 (ATCC37115) and YEp24 (ATCC3705).
1), YCp50 (ATCC37419), pHS19, pHS15 and the like. Any promoter may be used as long as it functions in a yeast strain, and examples thereof include promoters of glycolytic genes such as hexose kinase, PHO5 promoter, PGK promoter, G
Examples thereof include AP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα 1 promoter and CUP 1 promoter.

As host cells, Saccharomyces , Sch
izosaccharomyces , Kluyveromyces , Trichosporon
Genus, Schwanniomyces genus, Pichia genus, Candida genus microorganisms, for example, Saccharomyces cerevisiae , Schizo
saccharomyces pombe , Kluyveromyces lactis , Trichos
poron pullulans , Schwanniomyces alluvius , Candidau
Tilis etc. can be given. As a method of introducing the recombinant vector, any method can be used as long as it is a method of introducing DNA into yeast, for example, electroporation method [Methods Enzymol., 194 , 182 (1990)], spheroplast method [ Proc. Natl. Acad. Sci. USA, 75 , 19
29 (1978)], lithium acetate method [J. Bacteriology, 153 ,
163 (1983)], Proc. Natl. Acad. Sci. USA, 75 , 192.
9 (1978) and the like. When an animal cell is used as a host, as an expression vector, for example, pcDNAI, pCDM8 (manufactured by Invitrogen), pAGE107 [JP-A-3-22979, Cytotechnology, 3 , 133 (1990)], pAS3
-3 (JP-A-2-227075), pcDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Bioche
m., 101 , 1307 (1987)], pAGE210 and the like.

Any promoter can be used, so long as it can function in animal cells.
IE (immediate early) of cytomegalovirus (CMV)
Examples thereof include gene promoters, SV40 early promoters, retrovirus promoters, metallothionein promoters, heat shock promoters, and SRα promoters. In addition, the enhancer of the IE gene of human CMV may be used together with the promoter. As a host cell, a human cell named Namalba (Na
malwa) cells, COS cells that are monkey cells, CHO cells that are Chinese hamster cells, and HBT5637 (JP-A-63)
-299) and so on.

As a method for introducing a recombinant vector into animal cells, any method can be used as long as it is a method for introducing DNA into animal cells. For example, electroporation [Cytotechnology, 3 , 133 (1990)] , Calcium phosphate method (JP-A-2-227075), lipofection method [Pr
oc. Natl. Acad. Sci. USA, 84 , 7413 (1987)], Virol
Ogy, 52 , 456 (1973) can be mentioned. When insect cells are used as a host, for example, Current Protocols in Molecular Biology, Bacu
lovirus Expression Vectors, A Laboratory Manual,
WH Freeman and Company, New York (1992), Bio / Te
chnology, 6 , 47 (1988) etc.
The polypeptide can be expressed.

That is, the recombinant gene transfer vector and the baculovirus are co-introduced into insect cells to obtain recombinant virus in the culture supernatant of insect cells, and then the insect cells are further infected with the recombinant virus to obtain the polypeptide. Can be expressed. Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacII
I (both manufactured by Invitorogen) and the like can be mentioned.
Examples of the baculovirus include Autographa californica nucleea polyhedrosis virus (Autographa virus), which is a virus that infects night-the-moth insects.
californica nuclear polyhedrosis virus) and the like can be used. Insect cells include Spodoptera frugipe
rda ovary cells Sf9, Sf21 [Baculovirus Expressi
on Vectors, A Laboratory Manual, WH Freeman and
Company, New York (1992)], Trichoplusia ni ovary cells such as High 5 (manufactured by Invitrogen) and the like can be used.

Examples of the method for co-introducing the above-mentioned recombinant gene transfer vector and the above-mentioned baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-227075) and the lipofection method [Proc.
Natl. Acad. Sci. USA, 84 , 7413 (1987)] and the like. When a plant cell is used as the host cell, examples of the expression vector include Ti plasmid, tobacco mosaic virus vector and the like. Any promoter may be used as long as it functions in a plant cell, for example, the cauliflower mosaic virus (CaMV) 35S promoter,
The rice actin 1 promoter and the like can be mentioned.
Examples of host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, canola, alfalfa, rice, wheat, barley and the like. As a method of introducing the recombinant vector, any method can be used as long as it is a method of introducing DNA into plant cells. For example, Agrobacterium (Japanese Patent Laid-Open No. 59-
140885, JP-A-60-70080, WO94 / 00977), electroporation method (JP-A-60-251887), a method using a particle gun (gene gun) (JP2606856, JP2517813) and the like. be able to.

The transformant obtained as described above is cultured in a medium, the LK6-A binding protein is produced and accumulated in the culture, and the LK6-A binding protein is collected from the culture to produce the LK6-A binding protein. be able to. The method for culturing the transformant in a medium can be carried out according to a usual method used for culturing a host. When the transformant is a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host, a carbon source that can be assimilated by the transformant as a medium for culturing the transformant A natural medium or a synthetic medium may be used as long as it is a medium containing a nitrogen source, inorganic salts and the like and capable of efficiently culturing the transformant.

Any carbon source may be used as long as it can be assimilated by the transformant, such as glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysate, acetic acid, propionic acid, etc. Organic acids, alcohols such as ethanol and propanol, etc. can be used. As the nitrogen source, ammonia, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other nitrogen-containing compounds, and
Peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used. As the inorganic salt, dibasic potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate,
Sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate,
Calcium carbonate or the like can be used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culturing temperature is preferably 15 to 40 ° C., and the culturing time is usually 16 hours to 7 days. The pH during culture is 3.0 to 9.
It is preferable to keep 0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture. When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with a recombinant vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used, and when culturing a microorganism transformed with a recombinant vector using the trp promoter. You may add indole acrylic acid etc. to a culture medium.

As a medium for culturing a transformant obtained by using animal cells as a host, generally used RPMI16
40 medium (The Journal of the American Medical Associ
ation, 199 , 519 (1967)], Eagle's MEM medium [Science,
122 , 501 (1952)], Dulbecco's modified MEM medium [Virolog
y, 8 , 396 (1959)], 199 medium [Proceeding of the
Society for the Biological Medicine, 73 , 1 (195
0)] or media such as fetal calf serum added to these media can be used. Culturing is usually pH 6-8, 30
It is carried out for 1 to 7 days under the conditions of -40 ° C and the presence of 5% CO ₂ . If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture. As a medium for culturing the transformant obtained using insect cells as a host, TNM-FH medium (Pharmingen), which is generally used, Sf-900 II SFM medium (Life Technologies),
ExCell400, ExCell405 (both manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195 , 788 (196
2)] etc. can be used.

Culturing is usually carried out under conditions of pH 6 to 7, 25 to 30 ° C., etc. for 1 to 5 days. If necessary, an antibiotic such as gentamicin may be added to the medium during the culture. The transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ. The medium for culturing the transformant,
The commonly used Murashige and Scoog (MS)
A medium, a white medium, or a medium in which a plant hormone such as auxin or cytokinin is added to these mediums can be used. Culture is usually pH 5-9,
It is carried out under the condition of 20 to 40 ° C. for 3 to 60 days.

If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culturing. As described above, D encoding the LK6-A binding protein
A microorganism carrying a recombinant vector incorporating NA,
Transformants derived from animal cells, insect cells or plant cells are cultured according to a usual culture method to produce and accumulate LK6-A binding protein, and LK6-A binding protein is collected from the culture to obtain LK6-A binding protein. A-binding protein can be produced. When expressed in yeast, animal cells, insect cells or plant cells, a sugar or a sugar chain-added protein can be obtained. As a mode for producing the LK6-A binding protein, in addition to producing the LK6-A binding protein with the same structure, a marker peptide was linked according to the method described in Molecular Cloning 3rd Edition. It can be produced as a fusion protein or as a secretory protein linked with a signal sequence.

The marker peptide to be fused is m
yc polypeptide, β-galactosidase, protein A, IgG binding region of protein A, chloramphenicol acetyltransferase, poly (Arg),
Poly (Glu), protein G, maltose binding protein, glutathione S-transferase, polyhistidine chain (His-tag), S peptide, DNA binding protein domain, Tac antigen, thioredoxin, green fluorescent protein, FLAG peptide,
And an epitope of any antibody. The signal sequence may be a signal sequence of any secreted protein [Akio Yamakawa, Experimental Medicine, 13 , 469-47.
4 (1995)]. As the method for producing the LK6-A binding protein, there are a method of producing it in the host cell, a method of secreting it outside the host cell, and a method of producing it on the host cell outer membrane,
The method can be selected by changing the host cell used or the structure of the polypeptide to be produced. LK
When the 6-A binding protein is produced in the host cell or on the host extracellular membrane, the method of Paulson et al. [J. Biol. Che.
m., 264 , 17619 (1989)], Law et al. [Proc. Natl.
Acad. Sci. USA, 86 , 8227 (1989), Genes Develop.,
4 , 1288 (1990)], or JP-A-5-336963, WO94 / 2302.
By applying the method described in 1 etc., the LK6-A binding protein can be actively secreted outside the host cell.

That is, using the method of gene recombination,
The polypeptide of the present invention can be positively secreted outside the host cell by expressing it with a signal peptide added in front of the polypeptide containing the active site of the polypeptide of the present invention. Further, according to the method described in JP-A-2-227075, the production amount can be increased by utilizing a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the transgenic animal or plant, transgenic individual (transgenic non-human animal) or plant individual (transgenic plant) is created, and these individuals are used to generate LK6. It is also possible to produce -A binding protein.
When the transformant is an animal individual or a plant individual, it is bred or cultivated according to a usual method, the polypeptide is produced and accumulated, and the protein is collected from the animal individual or the plant individual, thereby binding LK6-A. A protein can be produced.

[0116] As a method for producing an LK6-A binding protein using an individual animal, for example, a known method [American Journ
al of Clinical Nutrition, 63 , 639S (1996), America
n Journal of Clinical Nutrition, 63 , 627S (1996),
Bio / Technology, 9 , 830 (1991)], a method of producing a LK6-A binding protein in an animal constructed by introducing a gene. In the case of an animal individual, for example, a transgenic non-human animal introduced with a DNA encoding the LK6-A binding protein is bred, the polypeptide is produced and accumulated in the animal, and the polypeptide is extracted from the animal. The LK6-A binding protein can be produced by collecting. Examples of the production / accumulation site in the animal include milk of the animal (JP-A-63-309192), eggs, and the like. As the promoter used in this case, any promoter that can be expressed in animals can be used. For example, α which is a mammary cell-specific promoter can be used.
Casein promoter, β casein promoter, β lactoglobulin promoter, whey acidic protein promoter and the like are preferably used.

As a method for producing an LK6-A binding protein using a plant individual, for example, a transgenic plant into which a DNA encoding the LK6-A binding protein has been introduced is known in the art [tissue culture, 20 (1994), tissue Culture, 21 (1995), Tren
ds in Biotechnology, 15 , 45 (1997)], LK6-A binding protein is produced and accumulated in the plant, and LK6-A binding protein is collected from the plant to obtain LK6.
-A-binding protein can be produced. In order to isolate and purify the LK6-A binding protein produced by the above transformant, a general method for isolating and purifying an enzyme can be used. For example, when the LK6-A binding protein is expressed in a lysed state in cells, after the culture is completed, the cells are recovered by centrifugation, suspended in an aqueous buffer solution, ultrasonically disrupted, French press, Mantongaurin. The cells are disrupted with a homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a usual method for isolating and purifying an enzyme, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Ethyl (DEAE) -Sepharose, DIAION HPA-75
(Mitsubishi Chemical Co., Ltd.) anion-exchange chromatography using a resin, S-Sepharose FF (Pharmacia Co.)
Cation exchange chromatography using resins such as, butyl sepharose, phenyl sepharose hydrophobic chromatography using resins such as phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric point A purified sample can be obtained by using techniques such as electrophoresis such as electrophoresis alone or in combination.

When the LK6-A binding protein was expressed by forming an insoluble substance in the cells, the cells were similarly collected, disrupted and centrifuged to give LK6-A as a precipitate fraction.
The insoluble matter of the bound protein is recovered. The recovered insoluble body of LK6-A binding protein is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed, and the concentration of the protein denaturant in the solubilized solution is reduced to restore the LK6-A binding protein to a normal three-dimensional structure. After the operation, a purified preparation of LK6-A binding protein can be obtained by the same isolation and purification method as above. LK6
-When the A-binding protein or a derivative of the LK6-A-binding protein having a sugar chain added thereto is extracellularly secreted, the protein or the derivative of the protein may be recovered in the culture supernatant. it can. That is, a culture supernatant is obtained by treating the culture with a method such as centrifugation similar to the above, and a purified preparation is obtained from the culture supernatant by using the same isolation and purification method as described above. Obtainable. Examples of the LK6-A binding protein thus obtained include a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, 3, 4 or 5.

Further, LK6-A binding protein was analyzed by Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method.
It can also be produced by a chemical synthesis method such as the method (t-butyloxycarbonyl method). Also, Advanced ChemT
ech, Perkin-Elmer, Pharmacia, Protein
Technology Instrument, Synthecell-Vega, PerSep
Chemical synthesis can also be performed using a peptide synthesizer such as tive, Shimadzu. Furthermore, LK6-A binding protein
The DNA encoding the K6-A binding protein can also be produced using an in vitro transcription / translation system. As an in vitro transcription / translation system, a known method [Kigawa T. et al., J. Biomol.
NMR, 6 , 129 (1998), Spirin AS et al., Science, 242 ,
1162 (1988), Kigawa T. & Yokoyama S., J. Biochem.,
110 , 166 (1991)], an in vitro transcription / translation system can be used. That is, a large amount of LK6-A binding protein RNA was synthesized in vitro by connecting DNA encoding the LK6-A binding protein downstream of a promoter such as SP6, T7, T3 and reacting each promoter-specific RNA polymerase. After that, the LK6-A binding protein can be produced using a cell-free translation system, for example, a translation system using a rabbit reticulocyte lysate, a wheat germ extract, an Escherichia coli extract or the like.

(8) Preparation of Antibodies Recognizing LK6-A Binding Protein Antibodies recognizing LK6-A binding protein were prepared by using a purified preparation of LK6-A binding protein or partial fragment polypeptide of the protein, It can be produced as an antibody, a monoclonal antibody, or the like. (8-1) Preparation of Polyclonal Antibody A purified preparation of the LK6-A binding protein or partial fragment polypeptide of the protein is used as an antigen and administered to an animal to prepare a polyclonal antibody. As the animal to be administered, rabbit, goat, rat, mouse, hamster and the like can be used. The dose of the antigen is preferably 50 to 100 μg per animal. When a low-molecular peptide is used, it is desirable to use the peptide covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin as an antigen. The peptide used as an antigen can be synthesized by a peptide synthesizer.

Administration of the antigen is 1 to 2 after the first administration.
Do 3-10 times every week. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the reaction of the serum with the antigen used for immunization is confirmed by enzyme immunoassay [enzyme immunoassay (ELIS
Law A): Published by Medical Shoin (1976), Antibodies-A Lab
oratory Manual, Cold Spring Harbor Laboratory (198
8)] and so on.

A polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum shows a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum. As a method of separation and purification,
Centrifugation, salting out with 40-50% saturated ammonium sulfate, caprylic acid precipitation [Antibodies, A Laboratory manua
l, Cold Spring Harbor Laboratory, (1988)], or DEAE-Sepharose column, anion exchange column,
Examples include a method in which chromatography using a protein A or G-column, a gel filtration column, or the like is used alone or in combination.

(8-2) Preparation of Monoclonal Antibody (a) Preparation of Antibody-Producing Cells Rats whose sera showed a sufficient antibody titer against the partial fragment polypeptide of LK6-A binding protein used for immunization Serve as a source of producer cells. Spleens are extracted 3 to 7 days after the final administration of the antigenic substance to the rats showing the antibody titer. The spleen is cut into small pieces in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded. Spleen cells of the obtained precipitation fraction were treated with Tris-ammonium chloride buffer (pH 7.65).
After removing the erythrocytes by treating the cells for 1-2 minutes, the cells are washed 3 times with MEM medium, and the resulting splenocytes are used as antibody-producing cells.

(B) Preparation of myeloma cells As myeloma cells, cell lines obtained from mouse or rat are used. For example, 8-azaguanine resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 (hereinafter,
Abbreviated as P3-U1) [Curr. Topics. Microbiol. Immuno
l., 81 , 1 (1978), Europ. J. Immunol., 6 , 511 (197
6)), SP2 / 0-Ag14 (SP-2) (Nature, 276 , 269 (197
8)], P3-X63-Ag8653 (653) [J. Immunol., 123 , 1548.
(1979)], P3-X63-Ag8 (X63) (Nature, 256 , 495 (197
5)] etc. can be used. These cell lines are 8-
Azaguanine medium [RPMI-1640 medium with glutamine (1.5 mmol / l), 2-mercaptoethanol (5 x
10 ^-5 mol / l), gentamicin (10 μg / ml) and fetal calf serum (FCS) (CSL, 10%) were added to a medium (hereinafter referred to as normal medium), and 8-azaguanine (15 μg / l) was added. ml) in the medium], but the cells are cultured in a normal medium 3 to 4 days before the cell fusion, and 2 × 10 ⁷ or more of the cells are used for the fusion.

(C) Preparation of hybridoma The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) were mixed with MEM medium or PBS (disodium phosphate 1.83 g, monopotassium phosphate 0.21 g, Salt 7.6
Wash thoroughly with 5 g, distilled water 1 liter, pH 7.2),
The cells are mixed so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, the mixture is centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded. The cell group of the obtained precipitate fraction was thoroughly loosened, and the cell group was stirred at 37 ° C. for 10 ⁸
Polyethylene Glycol-10 per antibody producing cell
00 (PEG-1000) 2 g, MEM 2 ml, and dimethylsulfoxide (DMSO) 0.7 ml mixed solution was added 0.2 to 1 ml, and MEM was added every 1 to 2 minutes.
Add 1-2 ml of medium several times.

After the addition, MEM medium was added to bring the total volume to 50 ml.
To be prepared. The prepared solution is centrifuged at 900 rpm for 5 minutes, and the supernatant is discarded. The cells of the obtained precipitate fraction are loosely loosened and then gently sucked in by a measuring pipette and gently blown out to blow HAT medium [hypoxanthine (10 ⁻⁴ mol / l) and thymidine (1.5 × 10 ^{− 5} m
ol / l) and aminopterin (4 × 10 ⁻⁷ mol / l) -containing medium] 100 ml. The suspension was dispensed on a 96-well culture plate at 100 μl / well, and 5% CO 2 was added.
Incubate at 37 ° C for 7 to 14 days in an incubator.
After culturing, remove a part of the culture supernatant from Antibodies [Antibo
dies, A Laboratory manual, Cold Spring Harbor Labo
ratory, Chapter 14 (1988)] and the like, a hybridoma that specifically reacts with a partial fragment polypeptide of the polypeptide of the present invention is selected. The following methods can be given as specific examples of the enzyme immunoassay method.

During immunization, a partial plate of the polypeptide of the present invention used as an antigen polypeptide is coated on a suitable plate, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below is used as the first antibody. And further reacting with anti-rat or anti-mouse immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like as a second antibody, and then reacting according to the labeling substance, which is specific to the polypeptide of the present invention. Those that react with each other are selected as hybridomas that produce the monoclonal antibody of the present invention. Cloning was repeated twice using the hybridoma by the limiting dilution method [the first time was HT medium (medium obtained by removing aminopterin from HAT medium),
For the second round, use a normal medium], and a stable stable antibody titer is selected as a hybridoma strain producing the monoclonal antibody of the present invention.

(D) Preparation of Monoclonal Antibody 8-10 week-old mice that had been treated with pristane [0.5, 2,10,14-tetramethylpentadecane (Pristane) was intraperitoneally administered and kept for 2 weeks] or In a nude mouse, 5 to 20 hybridoma cells producing the polypeptide monoclonal antibody of the present invention obtained in (c)
× 10 ⁶ cells / mouse are injected intraperitoneally. The hybridoma becomes ascites tumor in 10 to 21 days. Ascites fluid is collected from the ascites tumor-bearing mouse and centrifuged at 3,000 rpm for 5 minutes to remove solids. From the obtained supernatant, a monoclonal antibody can be purified and obtained by a method similar to the method used in polyclonal. The subclass of the antibody is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The amount of polypeptide is determined by the Lowry method or 280n.
It is calculated from the absorbance at m.

(9) Construction of a system for searching for compounds that bind to LK6-A binding protein As a method for searching for compounds that bind to LK6-A binding protein, inhibition of binding between LK6-A binding protein and LK6-A derivative To find compounds that can be used for LK6-A binding protein p150a and CD2 or its CD2BP2
Examples thereof include a method of searching for a compound that suppresses the binding to the binding region, and a method of searching for a compound that inhibits the enzymatic activity of LK6-A binding protein p150c or p27 described later.

Among the compounds that bind to the LK6-A binding protein by combining two or more methods selected from any one of the following, two or more activities preferable as an autoimmune disease therapeutic agent or immunosuppressive agent are obtained. You can also narrow down the compounds you have. On the contrary, it is possible to narrow down compounds having low activity, which are not preferable as immunosuppressants possessed by the LK6-A derivative. Further, if necessary, compounds having more preferable activity as an autoimmune disease therapeutic agent or immunosuppressive agent can be narrowed down by a known method described in the prior art. The compound having a preferable activity as an immunosuppressant refers to a compound having a low activity which is not preferable as an immunosuppressant, for example, a compound having reduced weight loss, bone marrow suppression, liver damage, renal toxicity, neurotoxicity, etc. Can be raised.

The methods 1 to 3 will be described in detail below. In the method of searching for a substance that inhibits the binding between the LK6-A binding protein and the LK6-A derivative, the LK6-A binding protein and the LK6-A derivative are prepared, and the test substance is added to the system for detecting the binding between them. In addition, a compound that inhibits the binding between the LK6-A binding protein and the LK6-A derivative is selected using the inhibition of the binding by the test substance as an index. Here, specifically, in the presence of the test substance, any one protein selected from the following (i) to (vi):
The amount of binding to the LK6-A derivative in the absence of the test substance was measured, and the amount of the protein in the presence of the test substance and the protein in the absence of the test substance
By comparing the binding amount with the LK6-A derivative and selecting a compound that inhibits the binding between the protein and the LK6-A derivative from the test substance, the LK6-A binding protein and the LK6-A derivative are characterized. There is a method of searching for a compound that inhibits binding.

(I) SEQ ID NOS: 1 to 5, 43, 45, 46
(Ii) a protein having an amino acid sequence represented by any one of (1) and (49), wherein one or more amino acid residues are deleted in the amino acid sequence represented by any of SEQ ID NOS: 1 to 5, 43, 45, 46 and 49. (Iii) SEQ ID NOS: 1 to 5, 43, 45, 46 and 49 which consist of a substituted, inserted or added amino acid sequence and bind to the LK6-A derivative
A protein (iv) consisting of an amino acid sequence having 60% or more homology with the amino acid sequence represented by any of the above, and binding to the LK6-A derivative (iv) amino acid numbers 353 to 408 in the amino acid sequence represented by SEQ ID NO: 45. A protein having an amino acid sequence represented by (v) A protein having an amino acid sequence represented by amino acid numbers 64-121 in the amino acid sequence represented by SEQ ID NO: 46 (vi) by the method of (1) to (4) above LK acquired and identified
6-A binding protein Here, an amino acid sequence in which one or more amino acid residues have been deleted, substituted, inserted or added means the same amino acid sequence as defined above. Also, SEQ ID NOS: 45 and 46
The proteins having the amino acid sequence represented by are all proteins having a GYF domain of human origin, and the polypeptide having the amino acid sequence represented by SEQ ID NO: 49 is
It is a polypeptide containing the GYF domain of p150a. A protein having an amino acid sequence represented by amino acid numbers 353 to 408 in the amino acid sequence represented by SEQ ID NO: 45, and a protein having an amino acid sequence represented by amino acid numbers 64 to 121 in the amino acid sequence represented by SEQ ID NO: 46 Are all proteins containing the GYF domain.

In the method of searching for a substance which inhibits the binding between p150a which is the LK6-A binding protein and CD2 or its CD2BP2 binding region, p150a and CD2 or its CD2BP2 binding region polypeptide are prepared, and the binding between them is performed. A test substance is added to the detection system, and inhibition of the binding by the test substance is used as an index to determine p1.
A substance that inhibits the binding between 50a and CD2 or its CD2BP2 binding region is selected. Here, specifically, in the presence of the test substance,
CD2 when the CD2 protein or its derivative is contacted with any one protein selected from the following (i) to (iii)
The amount of binding between the protein or its derivative and the protein and the amount of binding between the CD2 protein or its derivative and the protein when the protein was contacted with the CD2 protein or its derivative in the absence of the test substance Binding of the CD2 protein and the protein characterized by selecting the compound having the activity of inhibiting the binding from the test substance by comparing the measured values of the protein in the presence of the substance and in the absence of the test substance A method of searching for a compound that inhibits

(I) Protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) In the amino acid sequence represented by SEQ ID NO: 1 or 2, one or more amino acids are deleted, substituted, inserted or added A protein that binds to the CD2 protein (iii) a protein that binds to the CD2 protein and has a homology of 60% or more with the amino acid sequence represented by SEQ ID NO: 1 or 2 The amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added means the same amino acid sequence as defined above. As the CD2 protein derivative described in the above search method, any one protein selected from the following (i) to (iii) can be used.

(I) The amino acid sequence represented by SEQ ID NO: 6 comprises an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added, and has the amino acid sequence represented by SEQ ID NO: 1 or 2. A protein having an activity of binding to a protein that it has (ii) a protein having an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having the amino acid sequence represented by SEQ ID NO: 1 or 2. (Iii) A protein having an amino acid sequence of amino acid numbers 281 to 310 of the amino acid sequence represented by SEQ ID NO: 6

Here, an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added means the same amino acid sequence as defined above. The reaction method between LK6-A binding protein and LK6-A derivative, or p150a and CD2 or its CD2BP2 binding region polypeptide, and the amount of binding can be detected by any known method so long as it can detect the binding between the two. good. For example, in the case of the method, either one of the LK6-A binding protein and the LK6-A derivative is used as a multiwell plate, beads such as a chromatographic carrier or latex, a chip for surface plasmon resonance measurement, a target for mass measurement, etc. Physical adsorption, covalent bond, LK6-
Antibodies that react with A-binding proteins, immobilization by binding through a molecule that specifically recognizes a peptide selected from tag peptides, marker peptides, or signal peptides, and blocking that suppresses nonspecific adsorption as necessary After the above treatment, the other molecule is added, and the other unadsorbed molecule is washed away, and the other molecule, LK6-A
The binding of the LK6-A binding protein and the LK6-A derivative by detecting the other molecule bound by the activity of the binding protein or LK6-A derivative, the binding property, the occurrence of surface plasmon resonance, the signal intensity of mass spectrometry, etc. The amount can be detected.

In addition, in the method, the LK6-A binding protein is produced as a fusion protein with another protein, and a substance having an affinity for the fused protein is used to extract a tag peptide, a marker peptide, or a signal peptide. By specifically detecting the selected peptide, LK6-A
It is also possible to measure the binding between the derivative and the LK6-A binding protein. β-galactosidase, chloramphenicol acetyltransferase, glutathione S-transferase, thioredoxin, etc. are used as indicators of green fluorescent protein (GF
P) and the like, using the fluorescence as an index, protein A, an IgG binding region of protein A, poly (Arg), poly (Gl)
u), protein G, maltose-binding polypeptide, polyhistidine chain (His-tag), DNA-binding polypeptide domain, etc., using the binding to the specific binding molecule as an index,
myc polypeptide, S peptide, Tac antigen, FLA
The G peptide, the epitope of an arbitrary antibody, etc. can be detected using the binding to the specific antibody as an index.

Β-galactosidase, chloramphenicol acetyltransferase, glutathione S-
Transferase, thioredoxin, green fluorescent protein (GFP), protein A,
The IgG-binding region of protein A, poly (Arg), poly (Glu), protein G, maltose-binding polypeptide, polyhistidine chain (His-tag), DNA-binding polypeptide domain, etc. also bind to their specific antibodies. It can also be detected as an index. In addition, LK6-A binding protein and LK6
-A derivative is modified with a molecule having specificity for binding biotin or the like, a fluorescent reagent, a RI labeling reagent, etc., and then used in the above reaction system to utilize the binding specificity of biotin, etc. It is also possible to bind to the solid phase, or detect the interaction using the binding specificity of biotin or the like, fluorescence, radioactivity, or the like as an index.

Further, also in the case of, the protein and the polypeptide are labeled with an appropriate fluorescent dye, and by the fluorescence resonance energy transfer (FRET), the LK6-A binding protein is bound to the LK6-A derivative, or p150a is bound to CD2 or That c
Utilizing fluorescence emitted only when it is bound to the D2BP2 binding domain polypeptide, LK6-A binding protein and LK6
The interaction of the -A derivative or p150a with CD2 or its CD2BP2 binding domain polypeptide can be detected.

LK6-A binding protein of p150c or p2
As a method for searching for a compound that inhibits the enzyme activity of the 7 derivative, any method may be used as long as it is a known method capable of measuring these enzyme activities. For example, 6-OaD-glucosyl cyclomaltoheptanose or 6-Oa-maltopentaosyl cyclomaltoheptanose (maltopentaosyl c), which can be substrates for p150c,
The enzymatic activity of p150c can be measured by monitoring the decomposition rate of the substrate by p150c or the production rate of the product by high performance liquid chromatography or the like using a glucose polymer such as yclomaltoheptanose). Also,
The enzymatic activity of p27 can be measured by degrading oxygen peroxide by p27 in the presence of an electron donor such as thioredoxin or dithiothreitol, and measuring the decrease in electron donor or the decrease in oxygen peroxide. A compound having immunosuppressive activity can be found by searching for a compound that inhibits the enzyme activity measured by such a method.

Specifically, in the presence of the test substance, specifically, the glycogen debranching enzyme activity of any one protein selected from the following (i) to (iii) and the presence of the test substance in the absence of the test substance It is characterized in that the activity of the protein is measured, the measured values in the presence and absence of the test substance are compared, and a substance that inhibits the activity of the protein is selected from the test substances. A method of searching for a substance that inhibits the activity of the protein can be mentioned. (I) a protein having the amino acid sequence represented by SEQ ID NO: 3 (ii) in the amino acid sequence represented by SEQ ID NO: 3, 1
A protein having an amino acid sequence in which the above amino acid residues are deleted, substituted, inserted or added, and having glycogen debranching enzyme activity (iii) having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 3. A protein consisting of an amino acid sequence having, and having glycogen debranching enzyme activity, wherein an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added means an amino acid sequence similar to the above definition .

Further, specifically, in the presence of the test substance, the peroxidase activity of any one protein selected from the following (i) to (iii) and the activity of the protein in the absence of the test substance And comparing the measured values in the presence of the test substance and in the absence of the test substance, and selecting a substance that inhibits the activity of the protein from the test substance, the activity of the protein A method of searching for a substance that inhibits the can also be mentioned. (I) a protein having the amino acid sequence represented by SEQ ID NO: 5 (ii) in the amino acid sequence represented by SEQ ID NO: 5, 1
A protein having the above amino acid residue deleted, substituted, inserted or added, and having peroxidase activity (iii) an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 5 And a protein having a peroxidase activity, wherein the amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added means the same amino acid sequence as defined above.

(10) D encoding the LK6-A binding protein
Construction of a search system for a substance that changes NA expression In the presence of a test substance, the expression level of DNA encoding the protein of a transformant expressing the LK6-A binding protein and the presence of the test substance in the absence of the test substance DNA encoding the protein of the transformant
By comparing the measured values in the presence and absence of the test substance, and selecting a substance that changes the expression level of the DNA encoding the protein from the test substance , A substance that changes the expression level of LK6-A binding protein can be obtained. The transformant host may be any as long as it can express the DNA encoding the LK6-A binding protein described above, but is preferably a eukaryotic cell, more preferably an animal cell, and Human cultured cells are preferred.

As a specific method, total RNA or poly (A) ⁺ RN can be prepared from each of the transformed cells expressing the LK6-A binding protein that is in contact with or not in contact with the test substance according to a conventional method.
RT-PCR using A, and primer DNA prepared from DNA encoding LK6-A binding protein
[PCR Protocols, Academic Press (1990)], LK
Measure the expression level of DNA encoding 6-A binding protein,
A method for obtaining a substance that binds to the LK6-A binding protein can be mentioned by comparing the expression levels of the proteins.

(11) Construction of a search system for a substance that changes the expression of LK6-A binding protein The expression level of the protein in a transformant expressing the LK6-A binding protein in the presence of the test substance, and the test substance In the absence of the substance, the expression level of the protein in the transformant was measured, and the measured values in the presence and absence of the test substance were compared to change the expression level of the protein from the test substance. A substance that changes the expression level of the LK6-A binding protein can be obtained by selecting the substance that causes it. As a method for measuring the expression level of the LK6-A binding protein, an immunological quantification method can be mentioned. As an immunological quantification method, for example, sandwich ELIS using two kinds of monoclonal antibodies having different epitopes among antibodies that react with LK6-A binding protein in a liquid phase
Method A, a radioimmunoassay method using an LK6-A binding protein labeled with a radioisotope such as ¹²⁵ I, and an antibody that recognizes the protein can be used.

(12) Construction of a search system for compounds that regulate the function and expression level of LK6-A binding protein The cellular response of transformants expressing the LK6-A binding protein in the presence of the test substance and the test substance In the absence of the substance, the cellular response of the transformant is measured, and the measured values in the presence and absence of the test substance are compared to change the cell response of the transformant strain from the test substance. By selecting compounds, LK6-A
A substance that changes the function or expression level of the binding protein can be obtained. Examples of the method for detecting the cell response of the transformant include known methods for detecting changes in intracellular signal transduction, gene transcription, sugar uptake, proliferation and the like. Examples of the cell response include arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release,
Intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, melanin pigment aggregation or diffusion and the like can be mentioned.

(13) Construction of a search system for substances that inhibit the binding between LK6-A derivative and CD2BP2 It is known that CD2BP2 binds to CD2 via its GYF domain. LK6-A binding protein p150a is also GY
LK6-A is a CD2B protein because it has a F domain.
It is possible that it binds to P2 and exerts its action. Therefore, substances that inhibit the binding between LK6-A and CD2BP2 are
Furthermore, by comparing and examining the action on each LK6-A binding protein, it can be judged whether or not it is a useful substance as an autoimmune disease therapeutic agent or immunosuppressive agent. In the presence of the test substance, the binding amount of the CD2BP2 protein or its derivative and the LK6-A derivative when contacting the LK6-A derivative with the CD2BP2 protein or its derivative, and the CD2 protein or its derivative in the absence of the test substance The binding amount of the CD2BP2 protein or its derivative and the LK6-A derivative when contacted with the LK6-A derivative was measured, and the measured values in the presence and absence of the test substance were compared, A substance that binds to the CD2BP2 protein or its derivative and the LK6-A derivative can be selected from the test substances. As the CD2BP2 protein derivative, the following (i) to
Any one protein selected from (iii) can be used.

(I) The amino acid sequence represented by SEQ ID NO: 43 comprises an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added, and
Or a protein having an activity of binding to a protein having the amino acid sequence represented by 2 (ii) consisting of an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 43, and having SEQ ID NO: 1 or 2 A protein which binds to a protein having the amino acid sequence represented by (iii) a protein having the amino acid sequence of amino acid numbers 282-341 of the amino acid sequence represented by SEQ ID NO: 43, wherein one or more amino acid residues are deleted The substituted, inserted or added amino acid sequence means the same amino acid sequence as defined above.

(14) Evaluation of mixed lymphocyte reaction (MLR) activity Edited by Ivan Lefkovits, Benvennto Peris, translated by Shunsuke Ueda and Atsushi Tanaka; Immunological Experimental Method: Nishimura Shoten p.289-294 (1985), etc. MLR can be performed to measure the immunosuppressive effect of the compound. In the case of MLR using mouse cells, H-2 haplotype or Mls antigen (m
Thymocytes, lymph nodes, or spleen cells are extracted from two types of mice having different inor lymphocyte stimulating antigens and mixed. After 2 to 6 days, the degree of cell proliferation can be detected by a method such as incorporation of thymidine labeled with tritium.

(15) Pharmaceutical containing the substance selected by the method of the present invention For the pharmaceutical containing the substance selected by the method of the present invention as an active ingredient, the active ingredient can be administered alone. However, usually, the active ingredient is mixed with one or more pharmaceutically acceptable carriers to provide a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics. desirable. Preferably, a sterile solution of water or an aqueous carrier such as an aqueous solution of sodium chloride, glycine, glucose or human albumin is used. Further, pharmacologically acceptable additives such as buffering agents and isotonicity agents for bringing the formulation solution closer to physiological conditions, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, citric acid. Sodium or the like can also be added. Alternatively, it may be lyophilized and stored, and may be dissolved in an appropriate solvent before use.

It is desirable to use the most effective route for treatment in the oral administration, or in the oral cavity,
Parenteral administration such as intratracheal, rectal, subcutaneous, intramuscular and intravenous administration can be mentioned. Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like. Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups can be prepared with water, sugars such as sucrose, sorbitol, fructose, polyethylene glycol,
It can be produced by using glycols such as propylene glycol, oils such as sesame oil, olive oil and soybean oil, preservatives such as p-hydroxybenzoic acid esters, flavors such as strawberry flavor and peppermint, etc. as additives. Capsules, tablets, powders, granules, etc., are excipients such as lactose, glucose, sucrose, mannitol, starch,
Disintegrators such as sodium alginate, lubricants such as magnesium stearate and talc, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin are used as additives. Can be manufactured.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like. For example,
It is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid. Also,
The propellant is prepared by using the substance itself or a carrier that does not irritate the oral cavity and respiratory tract mucosa of the recipient and disperses the substance as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin.
Depending on the nature of the substance and the carrier used, formulations such as aerosol and dry powder are possible. Further, also in these parenteral preparations, the components exemplified as the additive in the oral preparation can be added. The dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 μg / kg to 80 mg / day for adults.
It is kg. Hereinafter, the present invention will be described based on Examples and Test Examples, but the present invention is not limited thereto.

[0153]

Example 1 LK6-A-immobilized SGNEGDE particles and LK6D
7 Preparation of immobilized SGNEGDE particles As a control compound for LK6-A, LK6 represented by the following formula (IV)
D7 was selected and used in the following experiments.

[0154]

[Chemical 18]

SGNEGDE particles are Colloids and Surfaces
It was prepared according to the method described in B, 10 , 41 (1997). The amount of epoxy groups on SGNEGDE was determined by opening the epoxy groups with ammonia and then measuring the amount of amine by conductivity titration (J. Appl. Poly. Sci., 26 , 2015 (1
981)). SGNEGDE with approximately 400 nmol / mg particles of epoxy groups
The particles were used in the following experiments. 10 mg SGNEGDE particles were dispersed in the solution of piperazine. The solvent at this time is water, the volume is 1 ml, and the concentration of piperazine is 0.3 mol / l. The mixture was reacted at 37 ° C for 16 hours while gently stirring with a rotator. After completion of the reaction, the particles were centrifuged (15000 rpm, 5 minutes, room temperature) to remove the supernatant. In order to remove unreacted piperazine, 400 μl of water was added to disperse the particles, and the particles were centrifuged (15000 rpm, 5 minutes, room temperature), and the washing operation for removing the supernatant was repeated three times. Hereinafter, the particles thus obtained will be referred to as piperazine particles.

400 μ for pellets of 10 mg piperazine particles
After adding l DMSO to disperse the particles, the particles were centrifuged (15000 rpm, 10 minutes, 25 ° C) to remove the supernatant.
This operation was repeated 3 times. 10 m with solvent exchanged to DMSO
A solution of LK6-A or LK6D7 was added to a pellet of g piperazine particles to disperse the piperazine particles. The solvent at this time is DMSO, the volume is 500 μl, and the concentration of LK6-A or LK6D7 is 2
It is mmol / l. Add a stirrer bar under light-shielded conditions
The mixture was stirred at 37 ° C for 24 hours. After completion of the reaction, the particles were centrifuged (15000 rpm, 10 minutes, 25 ° C) to remove the supernatant.
To remove unreacted LK6-A, 400 μl of DMSO was added to disperse the particles, and then the particles were centrifuged (15000 rpm, 10 minutes, 25 ° C.) to remove the supernatant, which was repeated three times. .
The LK6-A-immobilized particles or LK6D7-immobilized particles thus obtained were dispersed in DMSO at 20 mg particles / ml, and then stored at room temperature under light-shielding conditions.

Example 2 Preparation of cell extract The mouse T cell line CTLL-2 (ATCC TIB-214) was cultured in RPM.
FBS (50 ml) and 7.5% NaHCO in I 1640 medium (500 ml)
₃ (12.5 ml) and 200 mmol / l L-glutamine (15 ml) and 50
mmol / l β-mercaptoethanol (0.5 ml) and Penicill
in-Streptomycin (10000 units / ml and 10 mg / ml respectively;
1.25 ml) and 10 to 100 unit / ml mouse IL-2 were added. Human T cell line Jurkat (ATCC number TIB-15
The culture of 2) was performed by adding FBS (50 ml) to RPMI 1640 medium (500 ml).
ml) and 7.5% NaHCO ₃ (12.5 ml), 200 mmol / l L-glutamine (15 ml) and Penicillin-Streptomycin (each 10
000 units / ml and 10 mg / ml; 1.25 ml) was added to the medium. For CTLL-2 and Jurkat cultures, use a flask containing 5% CO ₂ if the culture volume is 300 ml or less.
The culture was performed by static culture in an incubator at 0 ° C. When the amount of the medium exceeded 500 ml, the culture was performed at 37 ° C. while stirring the medium with a stirrer rotation speed of 80 rpm using a spinner flask.

CTLL-2 or Jurkat was added at a cell concentration of 2 × 10 ⁷ ce.
Buffer A (20 mmol / l HEPES (pH 7.
4), 2 mmol / l EDTA, 150 mmol / l NaCl, 1% NP-40, 5
Suspended in mmol / l DTT, 1 mmol / l phenylmethanesulfonyl fluoride (PMSF), 1 μg / ml aprotinin), 4 ℃
The mixture was gently stirred for 30 minutes on a rotator. Then, the insoluble fraction was precipitated by centrifugation (15000 rpm, 15 minutes, 4 ° C.), and the supernatant was used as a cell extract. By this method, a cell extract with a protein concentration of about 2 mg / ml was obtained. The cell extract was quenched with liquid nitrogen and then stored at -80 ° C.

Example 3 LK6-A immobilized SGNEGDE particles and
Isolation and purification of protein that specifically binds to LK6-A using LK6D7-immobilized SGNEGDE particles Thaw the cryopreserved cell extract of CTLL-2 or Jurkat and centrifuge (15000 rpm, 4 ° C, 10 minutes) The supernatant was taken. Centrifuge the resulting supernatant once again (15000 rpm, 4
The resulting mixture was collected for 10 minutes) and used for subsequent purification. DMSO
LK6-A immobilized SGNEGDE particles, LK6D7 dispersed in 50 μl
To 1 mg of immobilized SGNEGDE particles, 800 μl of buffer solution A was added and suspended, followed by centrifugation (15000 rpm, 3 minutes, 4 ° C.). Remove the supernatant and add 200 μl of Buffer A to immobilize LK6-A.
GNEGDE particles and LK6D7-immobilized SGNEGDE particles were completely dispersed. Unfixed SGNEGDE particles used as a control for purification were prepared as follows. To 1 mg of SGNEGDE particles dispersed in water, 800 μl of buffer solution A was added and suspended, followed by centrifugation (1
5000 rpm, 5 minutes, 4 ° C). Remove the supernatant and
0 μl of buffer A was added to completely disperse the SGNEGDE particles.

Adsorption of protein to LK6-A-immobilized SGNEGDE particles or LK6D7-immobilized SGNEGDE particles or unimmobilized SGNEGDE particles was performed as follows. LK6-A-immobilized SGNEGDE particles or LK6D7-immobilized SGNEGDE dispersed in 200 μl buffer A
1 ml of the cell extract was added to the particles or unfixed SGNEGDE particles, and gently stirred at 4 ° C with a rotator. After 2 hours, centrifugation (15000 rpm, 2 minutes, 4 ° C.) was performed to remove the supernatant. 400 μl of buffer A was added to LK6-A-immobilized SGNEGDE particles, LK6D7-immobilized SGNEGDE particles or unfixed SGNEGDE particles to disperse them. Centrifuge (15000 rpm, 2 minutes, 4 ° C)
The supernatant to remove 400 μl of Buffer B (Buffer A 14 ml
695 mg of NaCl added. NaCl concentration becomes about 1 mol / l) and LK6-A immobilized SGNEGDE particles, LK6D7 immobilized S
GNEGDE particles or unfixed SGNEGDE particles were dispersed. Centrifuge (15000 rpm, 2 minutes, 4 ° C) to remove the supernatant, add 400 μl of buffer B again, and add LK6-A-immobilized SGNEGDE.
Particles, LK6D7 immobilized SGNEGDE particles or unfixed SGNEGDE particles were dispersed. Centrifuge (15000 rpm, 2 minutes, 4 ° C)
Then, the supernatant was removed.

LK6-A immobilized SGNEGDE particles, LK6D7 immobilized SGN
Elution of proteins adsorbed on EGDE particles or unfixed SGN EGDE particles was performed as follows. 30 μl of buffer A was added to completely disperse LK6-A-immobilized SGNEGDE particles, LK6D7-immobilized SGNEGDE particles or unimmobilized SGNEGDE particles. Then 10
μl x 4 sample buffer [0.5 mol / l Tris-HCl (pH 6.
8), 0.08 g / ml SDS, 40% (vol / vol) glycerol, 0.
04 mg / ml bromphenol blue, 4% (vol / vol) 2-mercaptoethanol] was added and mixed. Next, heat at 98 ° C for 4 minutes and centrifuge (15000 rpm, 10 minutes, room temperature)
Then, the supernatant (eluate) was obtained. SDS-PAGE of the eluate was performed using Lady Gel J (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.). Eluate 10 in 1 lane of gel
μl was loaded. The same sample was loaded on gels with a acrylamide concentration of 7.5% (catalog number 161-J311) and 15% gels (catalog number 161-J341). The protein was detected by silver staining.

As shown in FIG. 1, p150c, p27 and p8
5 is purified by LK6-A-immobilized SGNEGDE particles, but LK6D7-immobilized
It is not purified with SGNEGDE particles or unfixed SGNEGDE particles. In addition, p150a is also purified by LK6-A-immobilized SGNEGDE particles. p15
In the case of 0a, bands at the same position were also purified from LK6D7-immobilized SGNEGDE particles, but their intensity was low. Also, p150a is not purified on unfixed SGNEGDE particles. From these results, p150a, p150c, p27 and p85 are LK6-A
It was considered to be a specific binding protein.

Example 4 Identification of p150a CTLL-2 Cell Extract Approximately 100 ml to 100 mg of LK6-A-immobilized S
P150a was purified using GNEGDE particles. The purification method was the same as in Example 3 except for the points described below. Upon elution of the protein described in Example 3, LK6-A-immobilized SGNEGDE
The particles were not in buffer A but buffer C (20 mmol / l HEPES
(pH 7.4), 2 mmol / l EDTA, 150 mmol / l NaCl). Eluate the Centricon-100 (Amicon, Bevery,
MA 01915, USA Cat. No. 4211). The protein obtained was separated by SDS-PAGE at 7.5% and the molecular weight was about 150k.
The Da band was cut out and Lysyl Endopeptidase was added to the gel.
from Achromobacter lyticus M497-1 (EC3.4.21.50) (W
Digested with AKO). The obtained peptide is μRPC C
2 / C18 reverse-phase HPLC column (LC PACKINGS, Catalog No. M
(IC-15-03-MRP) and separated by a linear water / acetonitrile gradient containing 0.065% trifluoroacetic acid. The peptide obtained there was further processed by Applied Biosystems Pr.
The amino acid sequence was determined by the automated Edman degradation method using an ocise 492cLC protein sequencer.

A database was searched using the obtained partial amino acid sequence of p150a (SEQ ID NOs: 15 to 19). Although p150a was purified from the mouse cell line CTLL-2, none of the mouse amino acid sequence databases corresponded to p150a. However, p150a is KIAA0642 (GenBank accession) which is a partial length human cDNA described in the database of Kazusa DNA Research Institute (http // www.kazusa.or.jp / huge /).
No. AB014542) and GenBank accession No. AK00173
It had homology to the protein encoded by human cDNA represented by 9. p150a was considered to be the mouse counterpart of the protein encoded by KIAA0642. GenBan
The amino acid sequence of the protein encoded by the cDNA represented by k accession No. AK001739 is shown in SEQ ID NO: 1.
The nucleotide sequence of is shown in SEQ ID NO: 8.

Example 5 Identification of p150c CTLL-2 Cell Extract Approximately 200 ml to 200 mg of LK6-A-immobilized S
P150c was purified using GNEGDE particles. The purification method was the same as in Example 3 except for the following points. Upon elution of the protein described in Example 3, LK6-A immobilized SGNEGDE
The particles were not in buffer A but buffer C (20 mmol / l HEPES
(pH 7.4), 2 mmol / l EDTA, 150 mmol / l NaCl). Add twice the volume of the eluent to the eluent, mix and place at -30 ° C for 30 minutes, then centrifuge (15000 r
The protein was recovered as a pellet by pm, 10 minutes, 4 ° C. Add approximately 30 μl × 1 sample buffer (10
(A mixture of 0 μl × 4 sample buffer and 300 μl water)
Was added and dissolved.

The obtained protein was separated by 7.5% SDS-PAGE, a band having a molecular weight of about 150 kDa was cut out, and Lysy was analyzed in the gel.
l Endopeptidase from Achromobacter lyticus M497-1
It was digested with (EC3.4.21.50) (WAKO). The obtained peptide was analyzed by μRPC C2 / C18 reverse phase HPLC column (LC PACKINGS
(Catalog No. MIC-15-03-MRP, manufactured by K.K.) and separated by a linear gradient of water / acetonitrile containing 0.065% trifluoroacetic acid. The amino acid sequence of the peptide thus obtained was further determined by an automated Edman degradation method using a Procise 492cLC protein sequencer manufactured by Applied Biosystems. A database was searched using the obtained partial amino acid sequence of p150c (SEQ ID NOs: 20 to 24). p1
50c was purified from the mouse cell line CTLL-2, but none of the mouse amino acid sequence databases corresponded to p150c. However, p150c is a human glycogen debranching enzyme.
(GDE), GenBank accession No. P35573]. p150c was considered to be the mouse counterpart of glycogen debranching enzyme. P35573
The amino acid sequence represented by is shown in SEQ ID NO: 3, and the DNA encoding the protein consisting of the amino acid sequence is shown in SEQ ID NO: 10.

Example 6 Identification of p27 CTLL-2 Cell Extract Approximately 200 ml to 200 mg of LK6-A-immobilized S
P27 was purified using GNEGDE particles. The purification method was the same as in Example 3 except for the following points. Upon elution of the protein described in Example 3, LK6-A-immobilized SGNEGDE particles were not in buffer A but buffer C (20 mmol / l HEPES (p
H 7.4), 2 mmol / l EDTA, 150 mmol / l NaCl). Add twice the volume of the eluate to the eluate, mix and place at -30 ° C for 30 minutes, then centrifuge (15000 rp).
Protein was recovered as a pellet by m, 10 min, 4 ° C). About 30 μl of x1 sample was added to this pellet and dissolved.

The obtained protein was separated by SDS-PAGE at 15%, a band having a molecular weight of about 25 kDa was cut out, and Lysyl was separated in a gel.
Endopeptidase from Achromobacter lyticus M497-1 (E
C3.4.21.50) (manufactured by WAKO). The obtained peptides were separated by a water / acetonitrile linear gradient containing 0.065% trifluoroacetic acid using a μRPC C2 / C18 reverse phase HPLC column (LC PACKINGS, Catalog No. MIC-15-03-MRP). The amino acid sequence of the peptide thus obtained was further determined by an automated Edman degradation method using a Procise 492cLC protein sequencer manufactured by Applied Biosystems. The obtained partial amino acid sequence of p27 (SEQ ID NO: 25
~ 27) was used to search the database. The partial primary sequence of p27 is MSP23 (macrophage 23-kDa stress prot
mouse protein called ein) (GenBank accession No. D
16142). The amino acid sequence of MSP23 is SEQ ID NO: 5.
The nucleotide sequence of the DNA encoding MSP23 is shown in SEQ ID NO: 1.
It was described in 2.

Example 7 Identification of p85 Jurkat Cell Extract Approximately 32 ml to 46 mg LK6-A Immobilized SGN
P85 was purified using EGDE particles. The purification method was performed in the same manner as in Example 3 except for the points described below, and 2.2 mg of L
1.5 ml of cell extract was added per K6-A immobilized SGNEGDE particles. Upon elution of the protein described in Example 3 (6), the LK6-A-immobilized SGNEGDE particles were not in buffer A but buffer C [20 mm
ol / l HEPES (pH 7.4), 2 mmol / l EDTA, 150 mmol / l NaC
l]. Acetone in a volume twice that of the eluate was added to the eluate and mixed, the mixture was kept at -30 ° C for 30 minutes, and then the protein was recovered as a pellet by centrifugation (15000 rpm, 10 minutes, 4 ° C). Add approximately 30 μl x 1 sampl to this pellet
e was added and dissolved, and then subjected to amino acid internal sequence analysis.

The obtained protein was separated by SDS-PAGE at 7.5%, a band having a molecular weight of about 80 kDa was cut out, and Lysyl was separated in a gel.
Endopeptidase from Achromobacter lyticus M497-1 (E
C3.4.21.50) (manufactured by WAKO). The obtained peptides were separated by a water / acetonitrile linear gradient containing 0.065% trifluoroacetic acid using a μRPC C2 / C18 reverse phase HPLC column (LC PACKINGS, Catalog No. MIC-15-03-MRP). The amino acid sequence of the peptide thus obtained was further determined by an automated Edman degradation method using a Procise 492cLC protein sequencer manufactured by Applied Biosystems. The obtained partial amino acid sequence of p85 (SEQ ID NO: 28)
~ 31) was used to search the database. The partial primary sequence of p85 coincided with the cDNA represented by GenBank accession No. AK025598. GenBank accession No. AK02559
The nucleotide sequence represented by 8 is shown in SEQ ID NO: 4, and the amino acid sequence of the protein encoded by the DNA having the nucleotide sequence is shown in SEQ ID NO: 11.

Example 8 Cloning of p150a Gene and Expression of Recombinant Protein As for the p150a gene, two EST clones (IMAGE clone ID number: 27840) sold by Insight Genomics, Inc.
6 and 2188651 (hereinafter referred to as EST1 and EST2, respectively)) and mRNA extracted from Jurkat cells were used as templates to prepare clones of partial fragments of p150a obtained by RT-PCR. First, PCR was performed using EST1 as a template with a primer having a restriction enzyme Xho I cleavage site (SEQ ID NO: 32) and a primer having a restriction enzyme Eco RI cleavage site (SEQ ID NO: 33), and the PCR product was pBluescriptII SK
(-) (Stratagene) was incorporated into the Xho I and Eco RI sites. Then cutting the vector with restriction enzymes Bst EII and Not I, the vector The vector obtained in the restriction enzyme incorporates fragments of p150a obtained by cutting with Bst EII and Not I (such EST2 1). Meanwhile, Jurkat
PCR was performed using the mRNA extracted from the cells as a template and a primer having an Xho I cleavage site (SEQ ID NO: 34) and a primer having an Xba I cleavage site (SEQ ID NO: 35), and the PCR product was converted to Xho of pBluescriptII SK (-). Incorporated into the I and Xba I sites. This vector has restriction enzymes Van 91I and No.
The fragment of p150a obtained by cutting with t I was incorporated into the vector 1 cleaved with restriction enzymes Van 91 I and Not I. In this way, a vector in which p150a was incorporated into pBluescriptII SK (-) was obtained (the vector thus obtained is referred to as vector 2). P150 in vector 2
The sequence of a gene is KIAA0642 (GenBank accessi) which is a partial length human cDNA described in the database of Kazusa DNA Research Institute.
onNo. AB014542) and GenBank accession No. AK0017
The DNA sequencer (Applied Biosystems ABI PRISM310 G
Enetic Analyzer). These series of operations are
Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology, Supple
It can be carried out according to the method described in ment 1-38.

P150a obtained by cutting Vector 2 with Xho I and Not I was used as Sal of pGEX-5x-3 (Cat. No. 27-4586-01) manufactured by Amersham Pharmacia Biotech.
Incorporated into the I and Not I sites. P using this vector
GST fusion protein of 150a (hereinafter referred to as GST-p150a)
Was expressed using Escherichia coli BL21 [Experimental Medicine, 13 , 2051 (1995)] that co-expresses human thioredoxin as a host. Cultivation was carried out at 25 ° C and 0.1 mmol / l IPTG at OD = 0.6.
After the addition, the cells were cultured for 12 hours. 4000 × g, 4 cells
It was recovered by centrifugation at 10 ° C for 10 minutes and stored at -80 ° C. After lysing the stored cells, 2.4 ml of disruption buffer (50 mmol / l
Na ₂ HPO ₄ , 300 mmol / l NaCl, 10 mmol / l imidazole, 1
% NP-40, 1 mmol / l PMSF, 1 μg / ml aprotinin, pH
8.0) was added and ultrasonic disruption was performed. Ultrasonic crushing
Tapered microchip with a tip diameter of 3 mm (TAITEC
It was carried out intermittently for 3 minutes while cooling with ice using classification number MCN-0418). Then, centrifuge (48000 xg, 20 minutes, 4 ° C)
Then, the supernatant was taken and loaded on GSTapFF (Amersham Pharmacia Biotech Code. No. 17-5130-01). PBS containing 1 mmol / l DTT (8.00 g / l NaCl, 0.20 g / l
After washing the column with KCl, 1.15 g / l Na ₂ HPO ₄ , 0.20 g / l KH ₂ PO ₄ , the elution buffer (10 mmol / l reduced glutathione, 1 mmol / l DTT, 50 mmol / l Tris GST-p85 was eluted with -HCl, pH 7.7). Elute GST-p85 with buffer C
(50 mmol / l HEPES-NaOH, 150 mmol / l NaCl, 1 mmol / l E
It was dialyzed against DTA, 1 mmol / l DTT). One-third the volume of the solution of GST-p150a obtained in this way x 4 sam
ple buffer [0.5 M Tris-HCl (pH 6.8), 0.08 g / mL SD
S, 40% (vol / vol) glycerol, 0.04 mg / ml bromphenol blue, 4% (vol / vol) 2-mercaptoethanol] was added to the solution of GST-p150a and heated at 98 ° C for 3 minutes. Samples were prepared for SDS-PAGE. 20μ
loaded l. The results are shown in Fig. 2.

Example 9 Cloning of p150c Gene and Expression of Recombinant Protein The DNA consisting of positions 61 to 4704 in the nucleotide sequence represented by SEQ ID NO: 36 was used as pET14b (Cat. No. 696) manufactured by Novagen.
60-3), which is ligated between the Nde I and Xho I sites, and has SEQ ID NO: 36.
N-terminal His tag fusion p150c (hereinafter, His-p1) having an amino acid sequence encoded by the DNA consisting of the nucleotide sequence represented by
A vector expressing 50c) was constructed. Culture is 5 m
Performed at 25 ° C using 1 l of medium, and 0.02 mmol / l at OD = 0.5
After the addition of the above IPTG, the cells were further cultured for 13 hours. The bacterial cells were collected by centrifugation at 2380 xg at 4 ° C for 30 minutes and stored at -80 ° C. After lysing the stored cells, 1 ml of disruption buffer
(50 mmol / l Na ₂ HPO ₄ , 300 mmol / l NaCl, 10 mmol / l imi
dazole, 1% NP-40, 1 mmol / l PMSF, 1 μg / ml aprotini
n, pH 8.0) was added, and ultrasonication was performed. Ultrasonic disruption uses a tapered microchip with a tip diameter of 3 mm (TAITEC
It was carried out intermittently for 2 minutes while cooling with ice using a product number, classification number MCN-0418). Then, centrifugation (20000 xg, 20 minutes, 4 ° C) was performed to obtain a supernatant. Add 60 μl Ni to the supernatant
-Add NTA agarose (QIAGEN, Cat. No.30210) and add 4 ℃.
Mix gently for 17 hours. The supernatant (permeate) was removed by centrifugation (4000 xg, 1 minute, 4 ° C). Precipitated N
i-NTA agarose with 1.2 ml of wash buffer (50 mmol / l Na ₂ HP
O ₄ , 300 mmol / l NaCl, 20 mmol / l imidazole, 1 mmol
/ l PMSF, 1 μg / ml aprotinin, pH 8.0) and add Ni-
The NTA agarose was washed. In the same manner, after further washing twice with 1.2 ml of the washing buffer, 60 μl of the eluate (50 mmol / l Na ₂ HPO ₄ , 300 mmol / l NaCl, 250 mmol
His-p150c was eluted with / l imidazole, 1 mmol / l PMSF, 1 µg / ml aprotinin, pH 8.0). One-third the volume of the His-p150c solution thus obtained, x4 sampl
e buffer [0.5 M Tris-HCl (pH 6.8), 0.08 g / ml SDS,
40% (vol / vol) glycerol, 0.04 mg / ml bromphenol blue, 4% (vol / vol) 2-mercaptoethanol]
Was added to a solution of His-p150c and heated at 98 ° C for 3 minutes to prepare a sample for SDS-PAGE. 20 μl
Loaded. The results are shown in Fig. 2.

Example 10 Cloning of p85 Gene and Expression of Recombinant Protein The p85 gene having the nucleotide sequence represented by SEQ ID NO: 4 described in Genbank accession No. AKO25598 was used in the development of strategic human cDNA genome application technology. , A clone (HEP Clone No. H
EP04702), which was provided by Associate Professor Sumio Sugano, Genome Structure Analysis Division, Institute of Medical Science, University of Tokyo. A primer having a restriction enzyme Xho I cleavage site (SEQ ID NO: 37) and a primer having an Nco I cleavage site (SEQ ID NO: 3)
8) and PCR using HEP04702 as template
The PCR product was cleaved with Nco I and Xho I (the fragment thus obtained is referred to as fragment 1). The HEP04702 and Nco I
It was cut with Pst I (the fragment of p85 thus obtained is referred to as fragment 1). By cutting pBluescriptII SK (-) with Xho I and Pst I and incorporating fragment 1 and fragment 2, pBlu
A vector in which the full length of p85 was incorporated into escriptII SK (-) was obtained (this vector is referred to as vector 3). A DNA sequencer (ABI PRISM 310, Applied Biosystems) that the sequence of the part amplified by PCR corresponds to the sequence of the corresponding part of the nucleotide sequence represented by SEQ ID NO: 11.
Genetic Analyzer).

[0175] vector 3 Xho I and Not a p150a cut out by cutting in I made of Amersham Pharmacia Biotech pGEX-5x-3 of Sal I and Not I of (Cat.No. 27-4586-01) Incorporated into the site. Using this vector, p150a GST
A fusion protein (hereinafter referred to as GST-p85) was co-expressed with human thioredoxin in E. coli BL21 [Experimental Medicine, 13 , 20
51 (1995)] as a host and expressed. Culture is 25
At 0.1 ° C, and when 0.1 mmol / l IPTG was added when OD = 0.6,
The culture was performed for 12 hours. The cells were collected by centrifugation at 4000 xg at 4 ° C for 10 minutes and stored at -80 ° C. After lysing the stored cells, 2.4 ml of disruption buffer (50 mmol / l Na ₂ HPO ₄ , 300
mmol / l NaCl, 10 mmol / l imidazole, 1% NP-40, 1 m
Mol / l PMSF, 1 μg / ml aprotinin, pH 8.0) was added and sonication was performed. Ultrasonic crushing is performed with a tapered microchip with a tip diameter of 3 mm (TAITEC, classification number MCN-
0418) while intermittently cooling with ice for 3 minutes. Then, centrifugation (48000 × g, 20 minutes, 4 ° C.) was performed, and the supernatant was collected and loaded on GSTapFF (Amersham Pharmacia Biotech Code. No. 17-5130-01). 1 mmol / l
PBS with DTT (8.00 g / l NaCl, 0.20 g / l KCl, 1.15 g /
l Na ₂ HPO ₄ , 0.20 g / l KH ₂ PO ₄ ) After washing the column,
Elution buffer (10 mmol / l reduced glutathione, 1 mmol / l
GST-p85 with DTT, 50 mmol / l Tris-HCl, pH 7.7)
Was eluted. Elute GST-p85 with buffer C (50 mmol / l H
EPES-NaOH, 150 mmol / l NaCl, 1 mmol / l EDTA, 1 mmol /
dialysis against l DTT). GST obtained in this way
-x4 sample buffer [0.
5 M Tris-HCl (pH 6.8), 0.08 g / mL SDS, 40% (vol / vo
l) Glycerol, 0.04 mg / ml bromphenol blue, 4% (vol / vol) 2-mercaptoethanol] with GST-p85
The solution of SDS-
Samples were prepared for PAGE. 20 μl was loaded. The results are shown in Fig. 2.

Example 11 Cloning of p27 Gene and Expression of Recombinant Protein A primer having a restriction enzyme Xho I cleavage site (SEQ ID NO: 3)
RT-PCR was performed using 9) and a primer having a restriction enzyme Bam HI cleavage site (SEQ ID NO: 40) as a template with mRNA extracted from RAW (J774) cells, which is a mouse macrophage-derived cell line. The PCR product was labeled with pET14b (Novagen
Cat. No. 69660-3) was incorporated into the Xho I and Bam HI sites (the vector thus obtained is referred to as vector 4). The fact that the sequence of the MSP23 gene in Vector 4 matches the nucleotide sequence represented by SEQ ID NO: 12 means that the DNA sequencer (Applied Biosystems ABI PRISM 31
0 Genetic Analyzer). These series of operations are described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology, Supp.
It can be carried out according to the method described in lements 1 to 38 and the like.

E. coli BL21 (DE transformed with vector 4
Using 3) as a host, MSP23 was expressed as an N-terminal His-tag fusion protein (hereinafter referred to as His-MSP23).
The culture was carried out at 30 ° C. using 250 ml of LB medium, and when OD = 0.5, 1 mmol / l of IPTG was added, and then the culture was further carried out for 2 hours. The cells were collected by centrifugation at 4000 xg at 4 ° C for 10 minutes and stored at -80 ° C. 2.5 ml after lysing the stored cells
Crushing buffer (50 mmol / l Na ₂ HPO ₄ , 300 mmol / l NaCl,
10 mmol / l imidazole, 1% NP-40, 1 mmol / l PMSF, 1
μg / ml aprotinin, pH 8.0) was added and sonication was performed. The ultrasonic disruption was intermittently performed for 2 minutes while cooling with ice using a tapered microchip (TAITEC, classification number MCN-0418) having a tip diameter of 3 mm. Then centrifuge
(48000 × g, 20 minutes, 4 ° C.) and the supernatant was collected. 1.5 ml of Ni-NTA agarose (QIAGEN, Cat.No. 30) was added to the supernatant.
210) was added and mixed gently at 4 ° C. for 1 hour and loaded onto an empty column. 16 ml wash buffer (50 mmol / l Na ₂ HP
O ₄ , 300 mmol / l NaCl, 20 mmol / l imidazole, 1 mmol
After washing the column with / l PMSF, 1 μg / ml aprotinin, pH 8.0), 1 ml eluate (50 mmol / l Na ₂ HPO ₄ , 300 mmol /
l NaCl, 250 mmol / l imidazole, 1 mmol / l PMSF, 1
His-MSP23 was eluted with μg / ml aprotinin, pH 8.0). Using HiTrap Desalting (Amersham Pharmacia Biotech, Code No.17-1408-01), the buffer solution of the eluate was 50 mmol / l HEPES, 150 mmol / l NaCl (pH 7.
I replaced it with 0). One-third the volume of the thus-obtained His-MSP23 solution, x4 sample buffer [0.5 M Tris-
HCl (pH 6.8), 0.08 g / mL SDS, 40% (vol / vol) glycerol, 0.04 mg / ml bromphenol blue, 4% (vol
/ vol) 2-mercaptoethanol] was added to the solution of His-MSP23 and heated at 98 ° C for 3 minutes to prepare a sample for SDS-PAGE. 5 μl was loaded. The results are shown in Fig. 2.

Example 12 Cloning of gene for CD2BP2 binding region polypeptide of CD2 and expression of recombinant protein Primer having restriction site Xho I cleavage site (SEQ ID NO: 4)
RT-PCR was performed using 1) and a primer (SEQ ID NO: 42) having a restriction enzyme Bam HI cleavage site as a template with mRNA extracted from Jurkat cells. PCR product is pET14b
(Novegen Cat. No. 69660-3) was integrated into the Xho I and Bam HI sites (the vector thus obtained is referred to as vector 5). The sequence of the gene in Vector 5 is GenBan
k accession No. M16445 sequence matches the DNA
Sequencer (ABI PR, Applied Biosystems)
ISM 310 Genetic Analyzer). These series of operations can be carried out according to the method described in Molecular Cloning, Third Edition, Current Protocols in Molecular Biology, Supplements 1 to 38, and the like. The nucleotide sequence of the CD2BP2 binding region of CD2 in vector 5 is shown in SEQ ID NO: 14. The amino acid sequence encoded by the DNA having the nucleotide sequence represented by SEQ ID NO: 14 is shown in SEQ ID NO: 7.

E. coli BL21 (DE3) transformed with vector 5
Was used as a host to express the CD2BP2-binding domain polypeptide of CD2 as an N-terminal His-tag fusion protein (hereinafter referred to as His-CD2). Culture was performed using 250 ml of LB medium for 30
It was carried out at 0 ° C., 1 mmol / l of IPTG was added at the time of OD = 0.5, and further culturing was carried out for 3 hours. The cells were collected by centrifugation at 4000 xg at 4 ° C for 10 minutes and stored at -80 ° C. After lysing the stored cells, 2.5 ml of disruption buffer (50 mmol / l Na ₂ HPO ₄ , 3
00 mmol / l NaCl, 10 mmol / l imidazole, 1% NP-40,
1 mmol / l PMSF, 1 μg / ml aprotinin, pH 8.0) was added and sonication was performed. Ultrasonic crushing, tip diameter 3 mm
Taper type microchip (manufactured by TAITEC, classification number MC
N-0418) while cooling with ice for 2 minutes intermittently.
Then, centrifugation (48000 × g, 20 minutes, 4 ° C.) was performed and the supernatant was collected. 1.5 ml of Ni-NTA agarose (QIAG
EN company, Cat. No. 30210) was added and mixed gently at 4 ° C. for 1 hour and loaded on an empty column. 16 ml of wash buffer (5
0 mmol / l Na ₂ HPO ₄ , 300 mmol / l NaCl, 20 mmol / l imidazole, 1 mmol / l PMSF, 1 μg / ml aprotinin, pH
After washing the column with 8.0), 1 ml of eluate (50 mmol / l Na ₂
HPO ₄ , 300 mmol / l NaCl, 250 mmol / l imidazole, 1
His-CD with mmol / l PMSF, 1 μg / ml aprotinin, pH 8.0)
2 was eluted. The buffer solution of the eluate was exchanged with 50 mmol / l HEPES-NaOH (pH 6.5) by a usual dialysis operation. One-third the volume of × 4 sample buffer [0.5 M Tris-HCl (pH 6.8), 0.08 g of the thus obtained His-CD2 solution
/ mL SDS, 40% (vol / vol) glycerol, 0.04 mg / ml bromphenol blue, 4% (vol / vol) 2-mercaptoethanol] to the His-CD2 solution and heat at 98 ℃ for 3 minutes. Prepared a sample for SDS-PAGE. 10 μl was loaded. The results are shown in Fig. 2.

Example 13 Search for autoimmune disease therapeutic agent or immunosuppressive agent using cell extract and LK6-A-immobilized particles Jurkat or CTLL-2 cell extract 1 prepared in Example 2
Add 100 μmol / l test substance to ml and stir gently at 4 ° C for 1 hour. Jurk without added test substance as a control
1 ml of cell extract of at or CTLL-2 is gently stirred at 4 ° C. for 1 hour. The proteins in the cell extract treated with the test substance and in the cell extract not treated with the test substance were treated with LK6-A-immobilized S by the same method as described in Example 3.
Adsorb to GNEGDE particles. After that, the LK6-A-immobilized SGNEGDE particles are washed, the protein is eluted, and SDS-PAGE is carried out in the same manner as in Example 3. Purification with LK6-A-immobilized SGNEGDE particles from cell extract containing test substance gave p150
By examining whether or not a, p150c, p85 or p27 can be found, it is possible to detect the inhibition of LK6-A binding to p150a, p150c, p85 or p27 by the test substance. It is possible to determine whether the substance selected by the above-mentioned search method is a substance useful as an autoimmune disease therapeutic agent or immunosuppressant by further comparing and examining the action on each LK6-A binding protein. it can.

Example 14 Search for autoimmune disease therapeutic agent or immunosuppressive agent using LK6-A binding protein (1) His-p27 which is LK6-A binding protein prepared in Example 8, 9, 10 or 11 , His-p150c, GST-p85 or GST-p15
100 μl of PBS containing 10 μg of 0a protein was added to each well of a 96-well ELISA plate and allowed to stand overnight at 4 ° C for adsorption, and 1% BSA / P was added.
Add 300 μl of BS to each well, leave at 4 ° C for 1 hour to block, and in the presence or absence of 100 μmol / l analyte, 5 μmol / l fluorescein isothiocyanate (FI
TC) Labeled LK6-A solution is brought into contact with 100 μl, the supernatant is removed, washed with PBS containing 0.05% Tween20, and the remaining FITC-labeled LK6-A is measured by a fluorescence plate reader to bind LK6-A. The amount of LK6-A bound to the protein and the inhibition of LK6-A binding by the test substance can be detected. It is possible to determine whether the substance selected by the above-mentioned search method is a substance useful as an autoimmune disease therapeutic agent or immunosuppressant by further comparing and examining the action on each LK6-A binding protein. it can.

Example 15 Search for autoimmune disease therapeutic agent or immunosuppressive agent using LK6-A binding protein (2) His-p27, which is the LK6-A binding protein prepared in Example 8, 9, 10 or 11 , His-p150c, GST-p85 or GST-p15
The supernatant of the disrupted liquid of recombinant Escherichia coli expressing 100 μg of 0a protein is mixed with 10 μl of Zn ²⁺ ion chelating sepharose or glutathione sepharose, the protein is bound, and washed with PBS to obtain affinity beads. The affinity beads in the presence or absence of 500 μmol / l of the test substance,
After mixing with 1 ml of a solution containing 1 μmol / l FITC-labeled LK6-A and removing the supernatant and washing,
Extraction with 100 μl of a% SDS aqueous solution, and measuring the fluorescence intensity of the extract with a spectrofluorometric system showed that LK6 to LK6-A binding protein
-A binding amount and LK6-A binding inhibition by the test substance can be detected. The substances selected by the above-mentioned search method are
By comparing and examining the action on 6-A binding protein,
It can be judged whether or not the substance is useful as a therapeutic agent for autoimmune diseases or an immunosuppressive agent.

Example 16 Search for immunosuppressant using LK6-A binding protein (3) 10 μmol / l LK6D2 solution [20 mmol / l HEPES · NaOH, 200 mmol / l
l NaCl (pH 7.2)] was applied onto a sensor chip CA for a BIACORE device (manufactured by BIOCORE) to immobilize the same, and then, Examples 8, 9, and 1 were performed.
LK6-A binding protein His-p2 prepared with 0 or 11
7, 200-n His-p150c, GST-p85, or GST-p150a protein
20 mmol / l HEPES ・ NaOH containing 200 mol / l, 200 mmol / l NaCl (pH 7.
2) By flowing the solution over the sensor chip and observing the sensorgram, the binding between the LK6-A binding protein and LK6D2 immobilized on the sensor chip can be detected. Immobilize the LK6-A binding protein by the test substance and the sensor chip by observing the sensorgram by mixing 500 μmol / l of the test substance in advance to the solution containing the LK6-A binding protein flowing on the sensor chip. It is possible to detect the inhibition of the binding with LK6D2. It is possible to determine whether the substance selected by the above-mentioned search method is a substance useful as an autoimmune disease therapeutic agent or immunosuppressant by further comparing and examining the action on each LK6-A binding protein. it can. The LK6D2 used above is the following formula (V)

[0184]

[Chemical 19]

It is a compound represented by: and can be prepared using the method described in WO00 / 02879.

Example 17 Search for autoimmune disease therapeutic agent or immunosuppressant using LK6-A binding protein (4) Solution of 10 μmol / l CD2BP2 binding domain polypeptide His-CD2 of CD2 prepared in Example 12 [20mmol / l HEPES ・ NaOH, 200
mmol / l NaCl (pH 7.2)] BIACORE device (manufactured by BIOCORE)
GST-p150a protein, which is the LK6-A binding protein prepared in Example 9, after being immobilized on the sensor chip CA for use by immobilizing it in 200 n
20 mmol / l HEPES ・ NaOH containing 200 mol / l, 200 mmol / l NaCl (pH 7.
2) By flowing the solution over the sensor chip and observing the sensorgram, it is possible to detect the binding between the GST-p150a protein and the CD2BP2 binding domain polypeptide of CD2 immobilized on the sensor chip. LK6-A flowing on the sensor chip
The solution containing the binding protein was mixed with 500 μmol / l of the test substance in advance, and the sensorgram was observed. Inhibition of peptide binding can be detected. It is possible to determine whether the substance selected by the above-mentioned search method is a substance useful as an autoimmune disease therapeutic agent or immunosuppressant by further comparing and examining the action on each LK6-A binding protein. it can.

Example 18 Search for Autoimmune Disease Therapeutic Agent or Immunosuppressant Using LK6-A Binding Protein (5) The His-p27 protein, which is the LK6-A binding protein prepared in Example 11, was added to 500 nmol / l. , NADPH 0.2 mmol / l, thioredoxin reductase 50 nmol / l, thioredoxin 5 μmol / l
20 mmol / l HEPES ・ NaOH, 200 mmol / l NaCl (pH 7.2)
0.5 mmol / l of hydrogen peroxide is added to the solution consisting of and the mixture is incubated at 37 ° C., and the decrease of NADPH accompanying the decomposition of hydrogen peroxide is detected by measuring the absorbance at 340 nm. By adding a test substance to this system, inhibition of the peroxidase activity of His-p27 by the test substance can be detected. It is possible to determine whether the substance selected by the above-mentioned search method is a substance useful as an autoimmune disease therapeutic agent or immunosuppressant by further comparing and examining the action on each LK6-A binding protein. it can.

Example 19 Search for Drugs Using LK6-A Binding Protein (6) 50 mmol / l phosphate buffer containing 2 μmol / l of LK6-A binding protein His-p150c protein prepared in Example 9 (pH 6.5)
6-OaD-glucosylcyclomalt, a substrate of p150c, was added to the solution.
oheptaose or 6-Oa-maltopentaosyl cyclomalto hep
Add taose and incubate at 37 ° C to detect the degradation of the substrate by p150c by HPLC [J. Biol. Chem., 276 , 2
8824 (2001)]. By adding a test substance to this system, inhibition of the enzymatic activity of His-p150c by the test substance can be detected. It is possible to determine whether the substance selected by the above-mentioned search method is a substance useful as an autoimmune disease therapeutic agent or immunosuppressant by further comparing and examining the action on each LK6-A binding protein. it can.

Example 20 Drug Search Using CD2BP2 Protein or Derivatives Thereof The CD2BP2 protein is described in Nature Structure Biology, 6 , 656.
According to the method described in (1999), a protein consisting of the amino acid sequence of amino acid numbers 282 to 341 of the amino acid sequence represented by SEQ ID NO: 43 can be expressed as a protein having a His tag attached to the N-terminus. After purifying the protein by a known method, 100 μl of PBS containing 10 μg of the protein was subjected to 96-well ELISA.
Add to each well of the plate, let stand 4 ° C overnight to adsorb, add 1% BSA / PBS 300μl to each well, let sit at 4 ° C for 1 hour, block, 100μmol / l test substance In the presence or absence of fluorescein isothiocyanate (FITC) -labeled LK6-A solution in the presence or absence of 5 μmol / l, remove the supernatant, and wash with PBS containing 0.05% Tween20 to remove residual F
The amount of LK6-A bound to the CD2BP2 protein derivative and the inhibition of LK6-A binding by the test substance can be detected by measuring ITC-labeled LK6-A with a fluorescence plate reader. It is possible to determine whether the substance selected by the above-mentioned search method is a substance useful as an autoimmune disease therapeutic agent or immunosuppressant by further comparing and examining the action on each LK6-A binding protein. it can.

[0190] The region containing the GYF domain expression P150a cloning and recombinant proteins of the region containing the GYF-domains of Example 21 P150a, the vector 2 prepared in Example 8 as a template, a primer DNA with Bam HI cleavage site ( DNA consisting of the nucleotide sequence represented by SEQ ID NO: 47) and a primer DNA having Not I cleavage site
PCR is performed using (DNA consisting of the nucleotide sequence represented by SEQ ID NO: 48), and the amplified DNA fragment is pGEX-6p-1 (Amersham Pharmacia Biotech, Cat. No. 27).
-4597-01) was incorporated into the Bam HI and Not I sites to prepare a recombinant DNA. Using the recombinant DNA, a fusion protein of a polypeptide having the amino acid sequence represented by SEQ ID NO: 49 (region containing the GYF domain of p150a) and GST (hereinafter referred to as GST-p150a-GYF) was transformed into E. coli. BL21 (R
P) (manufactured by STRATAGENE, Cat. No. 230255) was expressed as a host. Cultivation is performed at 25 ℃, 0.1 mmol / L when OD = 0.6
After adding IPTG, the cells were cultured for 6 hours. 8000 × after culturing
Collect the cells by centrifuging at g, 4 ℃ for 10 minutes,
Stored at ° C. After lysing the stored cells, 10 mL of disruption buffer was added and ultrasonic disruption was performed. The ultrasonic disruption was intermittently performed for 2 minutes while cooling with ice using a taper type microchip with a tip diameter of 3 mm (TAITEC, classification number MCN-0418). Then, centrifugation (48000 xg, 20 minutes, 4 ° C) was performed to collect the supernatant, and Glutathione Sepharose 4B (Amersham Pharmacia Biotech, Code. No. 17-0
756-01). After washing the column with the disruption buffer, GST-p150a-GYF was eluted with an elution buffer (PBS solution containing 10 mmol / L reduced glutathione).
The eluted GST-p150a-GYF was dialyzed against PBS and GST-p150
Obtained a-GYF.

Example 22 Synthesis of biotinylated LK6-A 122.2 mg (0.5 mmol) biotin was dissolved in 2 mL DMSO to give 2
27.6 mg (0.6 mmol) of HBTU [2- (1H-benzotriazole-
1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate], 91.9 mg (0.6 mmol) HOBt · H ₂ O
[N-hydroxybenzotriazole monohydrate] and 1
31.9 μL (1.2 mmol) of N-methylmorpholine was added, and the mixture was stirred at room temperature for 1 hour. 4 mL of this solution in advance
To 430.7 mg (5 mmol) of piperazine dissolved in DMSO was slowly added over 20 minutes, and after the addition was completed, the mixture was stirred at room temperature for another 30 minutes. After adding 100 mL of water and 100 mL of ether to the reaction solution and shaking, the aqueous phase was recovered and freeze-dried. To the residue was added 100 mL of 0.1 mol / l sodium hydrogen carbonate aqueous solution, and reverse phase cartridge Dispo SPE (1 g resin filling, Y
I went to MC). After washing the cartridge with 5 mL of water, the fraction eluted with water containing 20% methanol was collected. Lyophilization yielded 30.9 mg of biotinylated piperazine. 9.2 mg (29.6 μmol) LK6-A was dissolved in 9.2 mL DMSO, 13.9 mg (44.4 μmol) of biotinylated piperazine obtained above was added, and the mixture was stirred at room temperature for 23 hours. Invertsil ODS-3 (20 mm ID x 250 mm, reverse phase column)
HPLC system equipped with GL Sciences (LC)
-8A, manufactured by Shimadzu Corp.) was used for purification with a mixed solvent of water and methanol, and an eluate containing the desired product was recovered. The eluate was freeze-dried to obtain 11.3 mg of biotinylated LK6-A.

Example 23 Search for autoimmune disease therapeutic agent or immunosuppressive agent using GST-p150a-GYF 96-well ELISA plate (PIERCE, Cat. No. 23-0151-40) precoated with glutathione was used. 0.1% BSA in each hole
(bovine serum albumin) [Bovine serum albumin (F-
V); manufactured by Nacalai Tesque, Inc.] and 200 μl of a blocking buffer containing 0.05% Tween-20 were added, and the mixture was allowed to stand at room temperature for 1 hour for blocking. The biotinylated LK6-A and the test substance prepared in Example 22 were treated with dimethyl sulfoxide (D
MSO) to prepare a DMSO solution having a concentration of 10 mmol / L. DMSO of 5 μg of GST-p150a-GYF prepared in Example 21 and 2 μL of biotinylated LK6-A in 2 mL of blocking buffer.
The solution was added, and 100 μL of this solution was dispensed into each tube. DMSO solution of the substance to be tested is previously diluted with DMSO to 1/100, 1/1
After diluting to 0 and 1/1, 1 μL was added to each so that the final concentration of the test substance was 1, 10 and 100 μmol / L. Test substances include LK6-A, LK6D2, LK6D7 and the following formula (VI)
Compounds 1 to 3 represented by (VIII) were used.

[0193]

[Chemical 20]

[0194]

[Chemical 21]

[0195]

[Chemical formula 22]

The solution containing this GST-p150a-GYF, biotinylated LK6-A and the test substance was blocked as described above.
It was added to each well of the well plate and left overnight at 4 ° C. After completion of the reaction, the supernatant was removed and the well was washed with PBS containing 0.05% Tween-20. Add 1 μL HRP (Horse
radish peroxidase) binding streptavidin solution (Nakarai, Cat. No. 02517-61) was added, 50 μL of each was added to each well of the plate, and the mixture was allowed to stand at room temperature for 1 hour. After removing the supernatant, the wells were washed with PBS containing 0.05% Tween20, TMB (3, 3 ',
5, 5'-tetramethyl-benzidine; manufactured by SIGMA, Code.No.
T-0440) solution was added in an amount of 50 μL and reacted at room temperature for 1 hour. Then, 50 μl of a 0.1 mol / l HCl solution was added to stop the reaction. GST-p150 immobilized on the plate was measured by measuring the absorbance at a wavelength of 450 nm using a 96-well plate reader.
Binding of biotinylated LK6-A to a-GYF and binding inhibition of biotinylated LK6-A by the test substance were detected. GST-p150a-GYF / biotinylated LK6-A binding by each concentration of the test substance, with the absorbance without addition of compound as the binding inhibition rate of 0% and the absorbance at 100 μmol / L LK6-A addition as the binding inhibition rate of 100%. The inhibition rate was calculated.

As a result, as shown in Table 1, LK6-A and LK6
It was confirmed that D2 and Compound 2 inhibited the binding between GST-p150a-GYF and biotinylated LK6-A in a concentration-dependent manner.

[0198]

[Table 1]

Test Example 1 Evaluation of MLR activity Lymph nodes were taken from B10BR / SgSn mice (Japanese SLC, female, 4-6 weeks old) and spleens were taken from AKR / N mice (Japanese SLC, female, 4-6 weeks old). After homogenizing the tissue with a homogenizer, nylon plankton net (Mesh Opening
= 250 μm) for filtration to prepare cells. Spleen cells from AKR / N mice were treated with 0.5 mg / ml mitomycin C. B10BR / SgSn mouse lymph node cells 1.5 x 1
0 ⁵ cells and 5 × 10 ⁵ cells of AKR / N mouse spleen are mixed to obtain 200
The mixture was dispensed into a 96-well plate so that the concentration became μl / well. Culture was performed in 1640 medium (500 ml) with FBS (50 ml) and 7.5% NaH.
CO ₃ (12.5 ml) and 200 mmol / l L-glutamine (5 ml)
Penicillin-Streptomycin (10000 units / ml and 1 respectively
0 mg / ml; 2.5 ml) and NCTC109 (GIBCO, Code 21340-039; 2
5 ml) and 1 mol / l HEPES buffer (5 ml) and 50 mmol / l β-
It was performed using a medium to which mercaptoethanol (0.5 ml) was added. When a test substance is added, the substance dissolved in DMSO is mixed with two types of cells, and then the final concentration of DMSO is 0.1%.
It was added to the 96-well plate so that it became%. The cells were cultured for 72 hours at 37 ° C in the presence of 5% carbon dioxide, thymidine labeled with tritium was added, and the culture was continued for another 16 hours. Thereafter, the cells are adsorbed on a glass filter, the amount of tritium taken into the cells is measured by a scintillation counter, and the obtained tritium count number can be used as an index of cell proliferation.

Test Example 2 Evaluation of MLR activity According to Test Example 1 and the method described in JP 2000-802879 A, LK6 was used.
The MLR activity of the -A derivative was investigated. The results are shown in Table 2.

[0201]

[Table 2]

[0202]

INDUSTRIAL APPLICABILITY The present invention provides a method for efficiently searching for LK6-A binding protein. LK6-useful in the search for a novel therapeutic agent for autoimmune disease or immunosuppressive agent by the search method
The A-binding protein is obtained, and a method for searching for a novel therapeutic agent for autoimmune disease or immunosuppressive agent using the LK6-A binding protein is provided.

[0203]

[Sequence free text]

SEQ ID NO: 32-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 33-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 34-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 35-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 36-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 37-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 38-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 39-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 40-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 41-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 42-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 47-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 48-Description of Artificial Sequence: Synthetic DNA

[0204]

[Sequence list] <110> KYOWA HAKKO KOGYO, CO., LTD.       TOKYO INSTITUTE OF TECHNOLOGY <120> Method for screening of a LK6-A-derivative binding protein, a LK6- A-derivative binding protein and method for screening of an immunosuppre ssive drug. <130> H13-2191A4 <140> <141> <160> 49 <170> PatentIn Ver. 2.1 <210> 1 <211> 1299 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ala Glu Thr Gln Thr Leu Asn Phe Gly Pro Glu Trp Leu Arg   1 5 10 15 Ala Leu Ser Ser Gly Gly Ser Ile Thr Ser Pro Pro Leu Ser Pro Ala              20 25 30 Leu Pro Lys Tyr Lys Leu Ala Asp Tyr Arg Tyr Gly Arg Glu Glu Met          35 40 45 Leu Ala Leu Phe Leu Lys Asp Asn Lys Ile Pro Ser Asp Leu Leu Asp      50 55 60 Lys Glu Phe Leu Pro Ile Leu Gln Glu Glu Pro Leu Pro Pro Leu Ala  65 70 75 80 Leu Val Pro Phe Thr Glu Glu Glu Gln Arg Asn Phe Ser Met Ser Val                  85 90 95 Asn Ser Ala Ala Val Leu Arg Leu Thr Gly Arg Gly Gly Gly Gly Thr             100 105 110 Val Val Gly Ala Pro Arg Gly Arg Ser Ser Ser Arg Gly Arg Gly Arg         115 120 125 Gly Arg Gly Glu Cys Gly Phe Tyr Gln Arg Ser Phe Asp Glu Val Glu     130 135 140 Gly Val Phe Gly Arg Gly Gly Gly Arg Glu Met His Arg Ser Gln Ser 145 150 155 160 Trp Glu Glu Arg Gly Asp Arg Arg Phe Glu Lys Pro Gly Arg Lys Asp                 165 170 175 Val Gly Arg Pro Asn Phe Glu Glu Gly Gly Pro Thr Ser Val Gly Arg             180 185 190 Lys His Glu Phe Ile Arg Ser Glu Ser Glu Asn Trp Arg Ile Phe Arg         195 200 205 Glu Glu Gln Asn Gly Glu Asp Glu Asp Gly Gly Trp Arg Leu Ala Gly     210 215 220 Ser Arg Arg Asp Gly Glu Arg Trp Arg Pro His Ser Pro Asp Gly Pro 225 230 235 240 Arg Ser Ala Gly Trp Arg Glu His Met Glu Arg Arg Arg Arg Phe Glu                 245 250 255 Phe Asp Phe Arg Asp Arg Asp Asp Glu Arg Gly Tyr Arg Arg Val Arg             260 265 270 Ser Gly Ser Gly Ser Ile Asp Asp Asp Arg Asp Ser Leu Pro Glu Trp         275 280 285 Cys Leu Glu Asp Ala Glu Glu Glu Met Gly Thr Phe Asp Ser Ser Gly     290 295 300 Ala Phe Leu Ser Leu Lys Lys Val Gln Lys Glu Pro Ile Pro Glu Glu 305 310 315 320 Gln Glu Met Asp Phe Arg Pro Val Asp Glu Gly Glu Glu Cys Ser Asp                 325 330 335 Ser Glu Gly Ser His Asn Glu Glu Ala Lys Glu Pro Asp Lys Thr Asn             340 345 350 Lys Lys Glu Gly Glu Lys Thr Asp Arg Val Gly Val Glu Ala Ser Glu         355 360 365 Glu Thr Pro Gln Thr Ser Ser Ser Ser Ala Arg Pro Gly Thr Pro Ser     370 375 380 Asp His Gln Ser Gln Glu Ala Ser Gln Phe Glu Arg Lys Asp Glu Pro 385 390 395 400 Lys Thr Glu Gln Thr Glu Lys Ala Glu Glu Glu Thr Arg Met Glu Asn                 405 410 415 Ser Leu Pro Ala Lys Val Pro Ser Arg Gly Asp Glu Met Val Ala Asp             420 425 430 Val Gln Gln Pro Leu Ser Gln Ile Pro Ser Asp Thr Ala Ser Pro Leu         435 440 445 Leu Ile Leu Pro Pro Pro Val Pro Asn Pro Ser Pro Thr Leu Arg Pro     450 455 460 Val Glu Thr Pro Val Val Gly Ala Pro Gly Met Gly Ser Val Ser Thr 465 470 475 480 Glu Pro Asp Asp Glu Glu Gly Leu Lys His Leu Glu Gln Gln Ala Glu                 485 490 495 Lys Met Val Ala Tyr Leu Gln Asp Ser Ala Leu Asp Asp Glu Arg Leu             500 505 510 Ala Ser Lys Leu Gln Glu His Arg Ala Lys Gly Val Ser Ile Pro Leu         515 520 525 Met His Glu Ala Met Gln Lys Trp Tyr Tyr Lys Asp Pro Gln Gly Glu     530 535 540 Ile Gln Gly Pro Phe Asn Asn Gln Glu Met Ala Glu Trp Phe Gln Ala 545 550 555 560 Gly Tyr Phe Thr Met Ser Leu Leu Val Lys Arg Ala Cys Asp Glu Ser                 565 570 575 Phe Gln Pro Leu Gly Asp Ile Met Lys Met Trp Gly Arg Val Pro Phe             580 585 590 Ser Pro Gly Pro Ala Pro Pro Pro His Met Gly Glu Leu Asp Gln Glu         595 600 605 Arg Leu Thr Arg Gln Gln Glu Leu Thr Ala Leu Tyr Gln Met Gln His     610 615 620 Leu Gln Tyr Gln Gln Phe Leu Ile Gln Gln Gln Tyr Ala Gln Val Leu 625 630 635 640 Ala Gln Gln Gln Lys Ala Ala Leu Ser Ser Gln Gln Gln Gln Gln Leu                 645 650 655 Ala Leu Leu Leu Gln Gln Phe Gln Thr Leu Lys Met Arg Ile Ser Asp             660 665 670 Gln Asn Ile Ile Pro Ser Val Thr Arg Ser Val Ser Val Pro Asp Thr         675 680 685 Gly Ser Ile Trp Glu Leu Gln Pro Thr Ala Ser Gln Pro Thr Val Trp     690 695 700 Glu Gly Gly Ser Val Trp Asp Leu Pro Leu Asp Thr Thr Thr Pro Gly 705 710 715 720 Pro Ala Leu Glu Gln Leu Gln Gln Leu Glu Lys Ala Lys Ala Ala Lys                 725 730 735 Leu Glu Gln Glu Arg Arg Glu Ala Glu Met Arg Ala Lys Arg Glu Glu             740 745 750 Glu Glu Arg Lys Arg Gln Glu Glu Leu Arg Arg Gln Gln Glu Glu Ile         755 760 765 Leu Arg Arg Gln Gln Glu Glu Glu Arg Lys Arg Arg Glu Glu Glu Glu     770 775 780 Leu Ala Arg Arg Lys Gln Glu Glu Ala Leu Arg Arg Gln Arg Glu Gln 785 790 795 800 Glu Ile Ala Leu Arg Arg Gln Arg Glu Glu Glu Glu Arg Gln Gln Gln                 805 810 815 Glu Glu Ala Leu Arg Arg Leu Glu Glu Arg Arg Arg Glu Glu Glu Glu             820 825 830 Arg Arg Lys Gln Glu Glu Leu Leu Arg Lys Gln Glu Glu Glu Ala Ala         835 840 845 Lys Trp Ala Arg Glu Glu Glu Glu Ala Gln Arg Arg Leu Glu Glu Asn     850 855 860 Arg Leu Arg Met Glu Glu Glu Ala Ala Arg Leu Arg His Glu Glu Glu 865 870 875 880 Glu Arg Lys Arg Lys Glu Leu Glu Val Gln Arg Gln Lys Glu Leu Met                 885 890 895 Arg Gln Arg Gln Gln Gln Gln Glu Ala Leu Arg Arg Leu Gln Gln Gln             900 905 910 Gln Gln Gln Gln Gln Leu Ala Gln Met Lys Leu Pro Ser Ser Ser Thr         915 920 925 Trp Gly Gln Gln Ser Asn Thr Thr Ala Cys Gln Ser Gln Ala Thr Leu     930 935 940 Ser Leu Ala Glu Ile Gln Lys Leu Glu Glu Glu Arg Glu Arg Gln Leu 945 950 955 960 Arg Glu Glu Gln Arg Arg Gln Gln Arg Glu Leu Met Lys Ala Leu Gln                 965 970 975 Gln Gln Gln Gln Gln Gln Gln Gln Lys Leu Ser Gly Trp Gly Asn Val             980 985 990 Ser Lys Pro Ser Gly Thr Thr Lys Ser Leu Leu Glu Ile Gln Gln Glu         995 1000 1005 Glu Ala Arg Gln Met Gln Lys Gln Gln Gln Gln Gln Gln Gln His Gln    1010 1015 1020 Gln Pro Asn Arg Ala Arg Asn Asn Thr His Ser Asn Leu His Thr Ser 1025 1030 1035 1040 Ile Gly Asn Ser Val Trp Gly Ser Ile Asn Thr Gly Pro Pro Asn Gln                1045 1050 1055 Trp Ala Ser Asp Leu Val Ser Ser Ile Trp Ser Asn Ala Asp Thr Lys            1060 1065 1070 Asn Ser Asn Met Gly Phe Trp Asp Asp Ala Val Lys Glu Val Gly Pro        1075 1080 1085 Arg Asn Ser Thr Asn Lys Asn Lys Asn Asn Ala Ser Leu Ser Lys Ser    1090 1095 1100 Val Gly Val Ser Asn Arg Gln Asn Lys Lys Val Glu Glu Glu Glu Lys 1105 1110 1115 1120 Leu Leu Lys Leu Phe Gln Gly Val Asn Lys Ala Gln Asp Gly Phe Thr                1125 1130 1135 Gln Trp Cys Glu Gln Met Leu His Ala Leu Asn Thr Ala Asn Asn Leu            1140 1145 1150 Asp Val Pro Thr Phe Val Ser Phe Leu Lys Glu Val Glu Ser Pro Tyr        1155 1160 1165 Glu Val His Asp Tyr Ile Arg Ala Tyr Leu Gly Asp Thr Ser Glu Ala    1170 1175 1180 Lys Glu Phe Ala Lys Gln Phe Leu Glu Arg Arg Ala Lys Gln Lys Ala 1185 1190 1195 1200 Asn Gln Gln Arg Gln Gln Gln Gln Leu Pro Gln Gln Gln Gln Gln Gln                1205 1210 1215 Pro Pro Gln Gln Pro Pro Gln Gln Pro Gln Gln Gln Asp Ser Val Trp            1220 1225 1230 Gly Met Asn His Ser Thr Leu His Ser Val Phe Gln Thr Asn Gln Ser        1235 1240 1245 Asn Asn Gln Gln Ser Asn Phe Glu Ala Val Gln Ser Gly Lys Lys Lys    1250 1255 1260 Lys Lys Gln Lys Met Val Arg Ala Asp Pro Ser Leu Leu Gly Phe Ser 1265 1270 1275 1280 Val Asn Ala Ser Ser Glu Arg Leu Asn Met Gly Glu Ile Glu Thr Leu                1285 1290 1295 Asp Asp Tyr <210> 2 <211> 60 <212> PRT <213> Homo sapiens <400> 2 Glu Ala Met Gln Lys Trp Tyr Tyr Lys Asp Pro Gln Gly Glu Ile Gln   1 5 10 15 Gly Pro Phe Asn Asn Gln Glu Met Ala Glu Trp Phe Gln Ala Gly Tyr              20 25 30 Phe Thr Met Ser Leu Leu Val Lys Arg Ala Cys Asp Glu Ser Phe Gln          35 40 45 Pro Leu Gly Asp Ile Met Lys Met Trp Gly Arg Val      50 55 <210> 3 <211> 1532 <212> PRT <213> Homo sapiens <400> 3 Met Gly His Ser Lys Gln Ile Arg Ile Leu Leu Leu Asn Glu Met Glu   1 5 10 15 Lys Leu Glu Lys Thr Leu Phe Arg Leu Glu Gln Gly Tyr Glu Leu Gln              20 25 30 Phe Arg Leu Gly Pro Thr Leu Gln Gly Lys Ala Val Thr Val Tyr Thr          35 40 45 Asn Tyr Pro Phe Pro Gly Glu Thr Phe Asn Arg Glu Lys Phe Arg Ser      50 55 60 Leu Asp Trp Glu Asn Pro Thr Glu Arg Glu Asp Asp Ser Asp Lys Tyr  65 70 75 80 Cys Lys Leu Asn Leu Gln Gln Ser Gly Ser Phe Gln Tyr Tyr Phe Leu                  85 90 95 Gln Gly Asn Glu Lys Ser Gly Gly Gly Tyr Ile Val Val Asp Pro Ile             100 105 110 Leu Arg Val Gly Ala Asp Asn His Val Leu Pro Leu Asp Cys Val Thr         115 120 125 Leu Gln Thr Phe Leu Ala Lys Cys Leu Gly Pro Phe Asp Glu Trp Glu     130 135 140 Ser Arg Leu Arg Val Ala Lys Glu Ser Gly Tyr Asn Met Ile His Phe 145 150 155 160 Thr Pro Leu Gln Thr Leu Gly Leu Ser Arg Ser Cys Tyr Ser Leu Ala                 165 170 175 Asn Gln Leu Glu Leu Asn Pro Asp Phe Ser Arg Pro Asn Arg Lys Tyr             180 185 190 Thr Trp Asn Asp Val Gly Gln Leu Val Glu Lys Leu Lys Lys Glu Trp         195 200 205 Asn Val Ile Cys Ile Thr Asp Val Val Tyr Asn His Thr Ala Ala Asn     210 215 220 Ser Lys Trp Ile Gln Glu His Pro Glu Cys Ala Tyr Asn Leu Val Asn 225 230 235 240 Ser Pro His Leu Lys Pro Ala Trp Val Leu Asp Arg Ala Leu Trp Arg                 245 250 255 Phe Ser Cys Asp Val Ala Glu Gly Lys Tyr Lys Glu Lys Gly Ile Pro             260 265 270 Ala Leu Ile Glu Asn Asp His His Met Asn Ser Ile Arg Lys Ile Ile         275 280 285 Trp Glu Asp Ile Phe Pro Lys Leu Lys Leu Trp Glu Phe Phe Gln Val     290 295 300 Asp Val Asn Lys Ala Val Glu Gln Phe Arg Arg Leu Leu Thr Gln Glu 305 310 315 320 Asn Arg Arg Val Thr Lys Ser Asp Pro Asn Gln His Leu Thr Ile Ile                 325 330 335 Gln Asp Pro Glu Tyr Arg Arg Phe Gly Cys Thr Val Asp Met Asn Ile             340 345 350 Ala Leu Thr Thr Phe Ile Pro His Asp Lys Gly Pro Ala Ala Ile Glu         355 360 365 Glu Cys Cys Asn Trp Phe His Lys Arg Met Glu Glu Leu Asn Ser Glu     370 375 380 Lys His Arg Leu Ile Asn Tyr His Gln Glu Gln Ala Val Asn Cys Leu 385 390 395 400 Leu Gly Asn Val Phe Tyr Glu Arg Leu Ala Gly His Gly Pro Lys Leu                 405 410 415 Gly Pro Val Thr Arg Lys His Pro Leu Val Thr Arg Tyr Phe Thr Phe             420 425 430 Pro Phe Glu Glu Ile Asp Phe Ser Met Glu Glu Ser Met Ile His Leu         435 440 445 Pro Asn Lys Ala Cys Phe Leu Met Ala His Asn Gly Trp Val Met Gly     450 455 460 Asp Asp Pro Leu Arg Asn Phe Ala Glu Pro Gly Ser Glu Val Tyr Leu 465 470 475 480 Arg Arg Glu Leu Ile Cys Trp Gly Asp Ser Val Lys Leu Arg Tyr Gly                 485 490 495 Asn Lys Pro Glu Asp Cys Pro Tyr Leu Trp Ala His Met Lys Lys Tyr             500 505 510 Thr Glu Ile Thr Ala Thr Tyr Phe Gln Gly Val Arg Leu Asp Asn Cys         515 520 525 His Ser Thr Pro Leu His Val Ala Glu Tyr Met Leu Asp Ala Ala Arg     530 535 540 Asn Leu Gln Pro Asn Leu Tyr Val Val Ala Glu Leu Phe Thr Gly Ser 545 550 555 560 Glu Asp Leu Asp Asn Val Phe Val Thr Arg Leu Gly Ile Ser Ser Leu                 565 570 575 Ile Arg Glu Ala Met Ser Ala Tyr Asn Ser His Glu Glu Gly Arg Leu             580 585 590 Val Tyr Arg Tyr Gly Gly Glu Pro Val Gly Ser Phe Val Gln Pro Cys         595 600 605 Leu Arg Pro Leu Met Pro Ala Ile Ala His Ala Leu Phe Met Asp Ile     610 615 620 Thr His Asp Asn Glu Cys Pro Ile Val His Arg Ser Ala Tyr Asp Ala 625 630 635 640 Leu Pro Ser Thr Thr Ile Val Ser Met Ala Cys Cys Ala Ser Gly Ser                 645 650 655 Thr Arg Gly Tyr Asp Glu Leu Val Pro His Gln Ile Ser Val Val Ser             660 665 670 Glu Glu Arg Phe Tyr Thr Lys Trp Asn Pro Glu Ala Leu Pro Ser Asn         675 680 685 Thr Gly Glu Val Asn Phe Gln Ser Gly Ile Ile Ala Ala Arg Cys Ala     690 695 700 Ile Ser Lys Leu His Gln Glu Leu Gly Ala Lys Gly Phe Ile Gln Val 705 710 715 720 Tyr Val Asp Gln Val Asp Glu Asp Ile Val Ala Val Thr Arg His Ser                 725 730 735 Pro Ser Ile His Gln Ser Val Val Ala Val Ser Arg Thr Ala Phe Arg             740 745 750 Asn Pro Lys Thr Ser Phe Tyr Ser Lys Glu Val Pro Gln Met Cys Ile         755 760 765 Pro Gly Lys Ile Glu Glu Val Val Leu Glu Ala Arg Thr Ile Glu Arg     770 775 780 Asn Thr Lys Pro Tyr Arg Lys Asp Glu Asn Ser Ile Asn Gly Thr Pro 785 790 795 800 Asp Ile Thr Val Glu Ile Arg Glu His Ile Gln Leu Asn Glu Ser Lys                 805 810 815 Ile Val Lys Gln Ala Gly Val Ala Thr Lys Gly Pro Asn Glu Tyr Ile             820 825 830 Gln Glu Ile Glu Phe Glu Asn Leu Ser Pro Gly Ser Val Ile Ile Phe         835 840 845 Arg Val Ser Leu Asp Pro His Ala Gln Val Ala Val Gly Ile Leu Arg     850 855 860 Asn His Leu Thr Gln Phe Ser Pro His Phe Lys Ser Gly Ser Leu Ala 865 870 875 880 Val Asp Asn Ala Asp Pro Ile Leu Lys Ile Pro Phe Ala Ser Leu Ala                 885 890 895 Ser Arg Leu Thr Leu Ala Glu Leu Asn Gln Ile Leu Tyr Arg Cys Glu             900 905 910 Ser Glu Glu Lys Glu Asp Gly Gly Gly Cys Tyr Asp Ile Pro Asn Trp         915 920 925 Ser Ala Leu Lys Tyr Ala Gly Leu Gln Gly Leu Met Ser Val Leu Ala     930 935 940 Glu Ile Arg Pro Lys Asn Asp Leu Gly His Pro Phe Cys Asn Asn Leu 945 950 955 960 Arg Ser Gly Asp Trp Met Ile Asp Tyr Val Ser Asn Arg Leu Ile Ser                 965 970 975 Arg Ser Gly Thr Ile Ala Glu Val Gly Lys Trp Leu Gln Ala Met Phe             980 985 990 Phe Tyr Leu Lys Gln Ile Pro Arg Tyr Leu Ile Pro Cys Tyr Phe Asp         995 1000 1005 Ala Ile Leu Ile Gly Ala Tyr Thr Thr Leu Leu Asp Thr Ala Trp Lys    1010 1015 1020 Gln Met Ser Ser Phe Val Gln Asn Gly Ser Thr Phe Val Lys His Leu 1025 1030 1035 1040 Ser Leu Gly Ser Val Gln Leu Cys Gly Val Gly Lys Phe Pro Ser Leu                1045 1050 1055 Pro Ile Leu Ser Pro Ala Leu Met Asp Val Pro Tyr Arg Leu Asn Glu            1060 1065 1070 Ile Thr Lys Glu Lys Glu Gln Cys Cys Val Ser Leu Ala Ala Gly Leu        1075 1080 1085 Pro His Phe Ser Ser Gly Ile Phe Arg Cys Trp Gly Arg Asp Thr Phe    1090 1095 1100 Ile Ala Leu Arg Gly Ile Leu Leu Ile Thr Arg Arg Tyr Val Glu Ala 1105 1110 1115 1120 Arg Asn Ile Ile Leu Ala Phe Ala Gly Thr Leu Arg His Gly Leu Ile                1125 1130 1135 Pro Asn Leu Leu Gly Glu Gly Ile Tyr Ala Arg Tyr Asn Cys Arg Asp            1140 1145 1150 Ala Val Trp Trp Trp Leu Gln Cys Ile Gln Asp Tyr Cys Lys Met Val        1155 1160 1165 Pro Asn Gly Leu Asp Ile Leu Lys Cys Pro Val Ser Arg Met Tyr Pro    1170 1175 1180 Thr Asp Asp Ser Ala Pro Leu Pro Ala Gly Thr Leu Asp Gln Pro Leu 1185 1190 1195 1200 Phe Glu Val Ile Gln Glu Ala Met Gln Lys His Met Gln Gly Ile Gln                1205 1210 1215 Phe Arg Glu Arg Asn Ala Gly Pro Gln Ile Asp Arg Asn Met Lys Asp            1220 1225 1230 Glu Gly Phe Asn Ile Thr Ala Gly Val Asp Glu Glu Thr Gly Phe Val        1235 1240 1245 Tyr Gly Gly Asn Arg Phe Asn Cys Gly Thr Trp Met Asp Lys Met Gly    1250 1255 1260 Glu Ser Asp Arg Ala Arg Asn Arg Gly Ile Pro Ala Thr Pro Arg Asp 1265 1270 1275 1280 Gly Ser Ala Val Glu Ile Val Gly Leu Ser Lys Ser Ala Val Arg Trp                1285 1290 1295 Leu Leu Glu Leu Ser Lys Lys Asn Ile Phe Pro Tyr His Glu Val Pro            1300 1305 1310 Val Lys Arg His Gly Lys Ala Ile Lys Val Ser Tyr Asp Glu Trp Thr        1315 1320 1325 Arg Lys Ile Gln Asp Asn Phe Glu Lys Leu Phe His Val Ser Glu Asp    1330 1335 1340 Pro Ser Asp Leu Asn Glu Lys His Pro Asn Leu Val His Lys Arg Gly 1345 1350 1355 1360 Ile Tyr Lys Asp Ser Tyr Gly Ala Ser Ser Pro Trp Cys Asp Tyr Gln                1365 1370 1375 Leu Arg Pro Asn Phe Thr Ile Ala Met Val Val Ala Pro Glu Leu Phe            1380 1385 1390 Thr Thr Glu Lys Ala Trp Lys Ala Leu Glu Ile Ala Glu Lys Lys Leu        1395 1400 1405 Leu Gly Pro Leu Gly Met Lys Thr Leu Asp Pro Asp Asp Met Val Tyr    1410 1415 1420 Cys Gly Ile Tyr Asp Asn Ala Leu Asp Asn Asp Asn Tyr Asn Leu Ala 1425 1430 1435 1440 Lys Gly Phe Asn Tyr His Gln Gly Pro Glu Trp Leu Trp Pro Ile Gly                1445 1450 1455 Tyr Phe Leu Arg Ala Lys Leu Tyr Phe Ser Arg Leu Met Gly Pro Glu            1460 1465 1470 Thr Thr Ala Lys Thr Ile Val Leu Val Lys Asn Val Leu Ser Arg His        1475 1480 1485 Tyr Val His Leu Glu Arg Ser Pro Trp Lys Gly Leu Pro Glu Leu Thr    1490 1495 1500 Asn Glu Asn Ala Gln Tyr Cys Pro Phe Ser Cys Glu Thr Gln Ala Trp 1505 1510 1515 1520 Ser Ile Ala Thr Ile Leu Glu Thr Leu Tyr Asp Leu                1525 1530 <210> 4 <211> 721 <212> PRT <213> Homo sapiens <400> 4 Met Glu Leu Gly Lys Gly Lys Leu Leu Arg Thr Gly Leu Asn Ala Leu   1 5 10 15 His Gln Ala Val His Pro Ile His Gly Leu Ala Trp Thr Asp Gly Asn              20 25 30 Gln Val Val Leu Thr Asp Leu Arg Leu His Ser Gly Glu Val Lys Phe          35 40 45 Gly Asp Ser Lys Val Ile Gly Gln Phe Glu Cys Val Cys Gly Leu Ser      50 55 60 Trp Ala Pro Pro Val Ala Asp Asp Thr Pro Val Leu Leu Ala Val Gln  65 70 75 80 His Glu Lys His Val Thr Val Trp Gln Leu Cys Pro Ser Pro Met Glu                  85 90 95 Ser Ser Lys Trp Leu Thr Ser Gln Thr Cys Glu Ile Arg Gly Ser Leu             100 105 110 Pro Ile Leu Pro Gln Gly Cys Val Trp His Pro Lys Cys Ala Ile Leu         115 120 125 Thr Val Leu Thr Ala Gln Asp Val Ser Ile Phe Pro Asn Val His Ser     130 135 140 Asp Asp Ser Gln Val Lys Ala Asp Ile Asn Thr Gln Gly Arg Ile His 145 150 155 160 Cys Ala Cys Trp Thr Gln Asp Gly Leu Arg Leu Val Val Ala Val Gly                 165 170 175 Ser Ser Leu His Ser Tyr Ile Trp Asp Ser Ala Gln Lys Thr Leu His             180 185 190 Arg Cys Ser Ser Cys Leu Val Phe Asp Val Asp Ser His Val Cys Ser         195 200 205 Ile Thr Ala Thr Val Asp Ser Gln Val Ala Ile Ala Thr Glu Leu Pro     210 215 220 Leu Asp Lys Ile Cys Gly Leu Asn Ala Ser Glu Thr Phe Asn Ile Pro 225 230 235 240 Pro Asn Ser Lys Asp Met Thr Pro Tyr Ala Leu Pro Val Ile Gly Glu                 245 250 255 Val Arg Ser Met Asp Lys Glu Ala Thr Asp Ser Glu Thr Asn Ser Glu             260 265 270 Val Ser Val Ser Ser Ser Tyr Leu Glu Pro Leu Asp Leu Thr His Ile         275 280 285 His Phe Asn Gln His Lys Ser Glu Gly Asn Ser Leu Ile Cys Leu Arg     290 295 300 Lys Lys Asp Tyr Leu Thr Gly Thr Gly Gln Asp Ser Ser His Leu Val 305 310 315 320 Leu Val Thr Phe Lys Lys Ala Val Thr Met Thr Arg Lys Val Thr Ile                 325 330 335 Pro Gly Ile Leu Val Pro Asp Leu Ile Ala Phe Asn Leu Lys Ala His             340 345 350 Val Val Ala Val Ala Ser Asn Thr Cys Asn Ile Ile Leu Ile Tyr Ser         355 360 365 Val Ile Pro Ser Ser Val Pro Asn Ile Gln Gln Ile Arg Leu Glu Asn     370 375 380 Thr Glu Arg Pro Lys Gly Ile Cys Phe Leu Thr Asp Gln Leu Leu Leu 385 390 395 400 Ile Leu Val Gly Lys Gln Lys Leu Thr Asp Thr Thr Phe Leu Pro Ser                 405 410 415 Ser Lys Ser Asp Gln Tyr Ala Ile Ser Leu Ile Val Arg Glu Ile Met             420 425 430 Leu Glu Glu Glu Pro Ser Ile Thr Ser Gly Glu Ser Gln Thr Thr Tyr         435 440 445 Ser Thr Phe Ser Ala Pro Leu Asn Lys Ala Asn Arg Lys Lys Leu Ile     450 455 460 Glu Ser Leu Ser Pro Asp Phe Cys His Gln Asn Lys Gly Leu Leu Leu 465 470 475 480 Thr Val Asn Thr Ser Ser Gln Asn Gly Arg Pro Gly Arg Thr Leu Ile                 485 490 495 Lys Glu Ile Gln Ser Pro Leu Ser Ser Ile Cys Asp Gly Ser Ile Ala             500 505 510 Leu Asp Ala Glu Pro Val Thr Gln Pro Ala Ser Leu Pro Arg His Ser         515 520 525 Ser Thr Pro Asp His Thr Ser Thr Leu Glu Pro Pro Arg Leu Pro Gln     530 535 540 Arg Lys Asn Leu Gln Ser Glu Lys Glu Thr Tyr Gln Leu Ser Lys Glu 545 550 555 560 Val Glu Ile Leu Ser Arg Asn Leu Val Glu Met Gln Arg Cys Leu Ser                 565 570 575 Glu Leu Thr Asn Arg Leu His Asn Gly Lys Lys Ser Ser Ser Val Tyr             580 585 590 Pro Leu Ser Gln Asp Leu Pro Tyr Val His Ile Ile Tyr Gln Lys Pro         595 600 605 Tyr Tyr Leu Gly Pro Val Val Glu Lys Arg Ala Val Leu Leu Cys Asp     610 615 620 Gly Lys Leu Arg Leu Ser Thr Val Gln Gln Thr Phe Gly Leu Ser Leu 625 630 635 640 Ile Glu Met Leu His Asp Ser His Trp Ile Leu Leu Ser Ala Asp Ser                 645 650 655 Glu Gly Phe Ile Pro Leu Thr Phe Thr Ala Thr Gln Glu Ile Ile Ile             660 665 670 Arg Asp Gly Ser Leu Ser Arg Ser Asp Val Phe Arg Asp Ser Phe Ser         675 680 685 His Ser Pro Gly Ala Val Ser Ser Leu Lys Val Phe Thr Gly Leu Ala     690 695 700 Ala Pro Ser Leu Asp Thr Thr Gly Cys Cys Asn His Val Asp Gly Met 705 710 715 720 Ala <210> 5 <211> 199 <212> PRT <213> Mus musculus <400> 5 Met Ser Ser Gly Asn Ala Lys Ile Gly Tyr Pro Ala Pro Asn Phe Lys   1 5 10 15 Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser Leu Ser              20 25 30 Glu Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu Asp Phe          35 40 45 Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala Asp      50 55 60 Glu Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp Ser  65 70 75 80 His Phe Cys His Leu Ala Trp Ile Asn Thr Pro Lys Lys Gln Gly Gly                  85 90 95 Leu Gly Pro Met Asn Ile Pro Leu Ile Ser Asp Pro Lys Arg Thr Ile             100 105 110 Ala Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly Ile Ser Phe Arg         115 120 125 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg Gln Ile Thr Ile     130 135 140 Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ile Ile Arg Leu Val 145 150 155 160 Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly                 165 170 175 Trp Lys Pro Gly Ser Asp Thr Ile Lys Pro Asp Val Asn Lys Ser Lys             180 185 190 Glu Tyr Phe Ser Lys Gln Lys         195 <210> 6 <211> 351 <212> PRT <213> Homo sapiens <400> 6 Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn   1 5 10 15 Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu              20 25 30 Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe          35 40 45 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp      50 55 60 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu  65 70 75 80 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His                  85 90 95 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr             100 105 110 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu         115 120 125 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu     130 135 140 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln 145 150 155 160 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp                 165 170 175 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val             180 185 190 Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu         195 200 205 Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met     210 215 220 Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Lys Arg Lys Lys Gln 225 230 235 240 Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val                 245 250 255 Ala Thr Glu Glu Arg Gly Arg Lys Pro Gln Gln Ile Pro Ala Ser Thr             260 265 270 Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro Pro Pro Pro Gly His         275 280 285 Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro Pro Gly His Arg Val     290 295 300 Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val 305 310 315 320 His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys                 325 330 335 Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn             340 345 350 <210> 7 <211> 107 <212> PRT <213> Homo sapiens <400> 7 Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val Ala Thr Glu Glu   1 5 10 15 Arg Gly Arg Lys Pro Gln Gln Ile Pro Ala Ser Thr Pro Gln Asn Pro              20 25 30 Ala Thr Ser Gln His Pro Pro Pro Pro Gly His Arg Ser Gln Ala          35 40 45 Pro Ser His Arg Pro Pro Pro Pro Gly His Arg Val Gln His Gln Pro      50 55 60 Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val His Gln Gln Lys  65 70 75 80 Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys Pro Pro His Gly                  85 90 95 Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn             100 105 <210> 8 <211> 3897 <212> DNA <213> Homo sapiens <400> 8 atg gca gcg gaa acg cag aca ctg aac ttt ggg cct gaa tgg ctc cga 48 Met Ala Ala Glu Thr Gln Thr Leu Asn Phe Gly Pro Glu Trp Leu Arg   1 5 10 15 gct ctg tcc agt ggt ggg agt att aca tcc cct cct ctt tct cca gca 96 Ala Leu Ser Ser Gly Gly Ser Ile Thr Ser Pro Pro Leu Ser Pro Ala              20 25 30 ttg ccg aag tat aaa tta gca gat tat cgt tac ggc aga gaa gaa atg 144 Leu Pro Lys Tyr Lys Leu Ala Asp Tyr Arg Tyr Gly Arg Glu Glu Met          35 40 45 tta gca ctt ttc ctt aaa gac aac aag ata cct tca gac ctt ctg gat 192 Leu Ala Leu Phe Leu Lys Asp Asn Lys Ile Pro Ser Asp Leu Leu Asp      50 55 60 aaa gaa ttt ctg cct atc ctc cag gag gaa ccc ctt cca cca ttg gct 240 Lys Glu Phe Leu Pro Ile Leu Gln Glu Glu Pro Leu Pro Pro Leu Ala  65 70 75 80 ctg gta ccc ttt aca gaa gaa gaa cag aga aac ttt tcc atg tct gta 288 Leu Val Pro Phe Thr Glu Glu Glu Gln Arg Asn Phe Ser Met Ser Val                  85 90 95 aat agt gct gct gtc ctg cga ttg aca gga cga gga gga gga gga aca 336 Asn Ser Ala Ala Val Leu Arg Leu Thr Gly Arg Gly Gly Gly Gly Thr             100 105 110 gtg gtg ggg gct cct aga ggt cga agt tct tca aga ggg cga ggc aga 384 Val Val Gly Ala Pro Arg Gly Arg Ser Ser Ser Arg Gly Arg Gly Arg         115 120 125 ggc aga ggt gaa tgt ggt ttc tac caa aga agt ttt gat gaa gta gag 432 Gly Arg Gly Glu Cys Gly Phe Tyr Gln Arg Ser Phe Asp Glu Val Glu     130 135 140 ggt gtt ttt ggt cga gga ggt ggc aga gaa atg cat aga tcg cag agc 480 Gly Val Phe Gly Arg Gly Gly Gly Arg Glu Met His Arg Ser Gln Ser 145 150 155 160 tgg gag gaa agg ggt gac aga cgt ttt gaa aaa cca gga cga aaa gat 528 Trp Glu Glu Arg Gly Asp Arg Arg Phe Glu Lys Pro Gly Arg Lys Asp                 165 170 175 gta ggg aga cca aat ttt gag gaa ggt gga cca aca tca gta ggg aga 576 Val Gly Arg Pro Asn Phe Glu Glu Gly Gly Pro Thr Ser Val Gly Arg             180 185 190 aag cat gaa ttt ata cgc tca gaa agt gaa aat tgg cgc atc ttt aga 624 Lys His Glu Phe Ile Arg Ser Glu Ser Glu Asn Trp Arg Ile Phe Arg         195 200 205 gag gaa caa aat gga gaa gat gaa gat gga ggt tgg cga cta gct gga 672 Glu Glu Gln Asn Gly Glu Asp Glu Asp Gly Gly Trp Arg Leu Ala Gly     210 215 220 tca agg agg gat gga gag agg tgg cga cct cac agt cct gat ggc cct 720 Ser Arg Arg Asp Gly Glu Arg Trp Arg Pro His Ser Pro Asp Gly Pro 225 230 235 240 cgt tct gca ggc tgg cgg gaa cac atg gaa cga cgt cgg agg ttt gag 768 Arg Ser Ala Gly Trp Arg Glu His Met Glu Arg Arg Arg Arg Phe Glu                 245 250 255 ttt gat ttt cga gat aga gat gat gaa cgg ggt tac cga agg gtt cgc 816 Phe Asp Phe Arg Asp Arg Asp Asp Glu Arg Gly Tyr Arg Arg Val Arg             260 265 270 tct ggc agt ggg agc ata gat gat gac agg gat agc ttg ccc gaa tgg 864 Ser Gly Ser Gly Ser Ile Asp Asp Asp Arg Asp Ser Leu Pro Glu Trp         275 280 285 tgc tta gag gat gct gaa gaa gaa atg ggt aca ttt gac tca tct gga 912 Cys Leu Glu Asp Ala Glu Glu Glu Met Gly Thr Phe Asp Ser Ser Gly     290 295 300 gca ttc ctt tct cta aaa aaa gta cag aaa gag cct att cca gaa gag 960 Ala Phe Leu Ser Leu Lys Lys Val Gln Lys Glu Pro Ile Pro Glu Glu 305 310 315 320 cag gag atg gac ttc cgg cct gtg gac gaa ggg gag gag tgc tct gac 1008 Gln Glu Met Asp Phe Arg Pro Val Asp Glu Gly Glu Glu Cys Ser Asp                 325 330 335 tct gag ggt agc cat aat gaa gag gcc aaa gaa ccc gat aag aca aat 1056 Ser Glu Gly Ser His Asn Glu Glu Ala Lys Glu Pro Asp Lys Thr Asn             340 345 350 aag aaa gaa gga gag aaa aca gat aga gta gga gtt gaa gct agt gag 1104 Lys Lys Glu Gly Glu Lys Thr Asp Arg Val Gly Val Glu Ala Ser Glu         355 360 365 gaa act ccc cag acc tca tca tca tct gct aga cca ggt act cct tca 1152 Glu Thr Pro Gln Thr Ser Ser Ser Ser Ala Arg Pro Gly Thr Pro Ser     370 375 380 gac cat cag tct cag gaa gca tca cag ttt gag agg aaa gat gaa cca 1200 Asp His Gln Ser Gln Glu Ala Ser Gln Phe Glu Arg Lys Asp Glu Pro 385 390 395 400 aaa act gag caa acg gaa aaa gct gaa gag gag act cgg atg gaa aat 1248 Lys Thr Glu Gln Thr Glu Lys Ala Glu Glu Glu Thr Arg Met Glu Asn                 405 410 415 agt cta cca gcc aaa gtg ccc agc aga ggg gat gaa atg gtt gct gat 1296 Ser Leu Pro Ala Lys Val Pro Ser Arg Gly Asp Glu Met Val Ala Asp             420 425 430 gtc cag cag ccc ctg tcg cag att cct tca gat aca gcc tct cct ctt 1344 Val Gln Gln Pro Leu Ser Gln Ile Pro Ser Asp Thr Ala Ser Pro Leu         435 440 445 ctc ata ctt cca cct cct gtt ccc aat cct agt cct act ctc cgg cca 1392 Leu Ile Leu Pro Pro Pro Val Pro Asn Pro Ser Pro Thr Leu Arg Pro     450 455 460 gtt gaa aca cca gtt gta ggt gct cct ggt atg ggc agt gtt tcc aca 1440 Val Glu Thr Pro Val Val Gly Ala Pro Gly Met Gly Ser Val Ser Thr 465 470 475 480 gaa cct gat gat gaa gaa ggt ctc aaa cat ttg gag cag caa gct gag 1488 Glu Pro Asp Asp Glu Glu Gly Leu Lys His Leu Glu Gln Gln Ala Glu                 485 490 495 aaa atg gtg gct tat ctc caa gac agt gca cta gat gat gaa aga ttg 1536 Lys Met Val Ala Tyr Leu Gln Asp Ser Ala Leu Asp Asp Glu Arg Leu             500 505 510 gca tca aaa ctg caa gag cac aga gct aaa gga gtg tcg att cca ttg 1584 Ala Ser Lys Leu Gln Glu His Arg Ala Lys Gly Val Ser Ile Pro Leu         515 520 525 atg cat gaa gca atg cag aag tgg tat tac aaa gat cct cag gga gaa 1632 Met His Glu Ala Met Gln Lys Trp Tyr Tyr Lys Asp Pro Gln Gly Glu     530 535 540 att caa ggt ccc ttc aat aat cag gag atg gca gaa tgg ttt cag gcg 1680 Ile Gln Gly Pro Phe Asn Asn Gln Glu Met Ala Glu Trp Phe Gln Ala 545 550 555 560 ggc tat ttt act atg tct tta ttg gtg aag aga gcg tgt gat gaa agc 1728 Gly Tyr Phe Thr Met Ser Leu Leu Val Lys Arg Ala Cys Asp Glu Ser                 565 570 575 ttc caa cct ctt ggc gat atc atg aaa atg tgg gga agg gtt ccc ttt 1776 Phe Gln Pro Leu Gly Asp Ile Met Lys Met Trp Gly Arg Val Pro Phe             580 585 590 tct cca ggt cca gct ccc cct cct cat atg gga gag ctg gac cag gaa 1824 Ser Pro Gly Pro Ala Pro Pro Pro His Met Gly Glu Leu Asp Gln Glu         595 600 605 cga ctg acc agg cag caa gaa ctc aca gcc tta tac cag atg cag cac 1872 Arg Leu Thr Arg Gln Gln Glu Leu Thr Ala Leu Tyr Gln Met Gln His     610 615 620 ctg cag tac cag cag ttt tta ata caa caa caa tat gca cag gtt ttg 1920 Leu Gln Tyr Gln Gln Phe Leu Ile Gln Gln Gln Tyr Ala Gln Val Leu 625 630 635 640 gcc caa cag cag aaa gca gca ctg tct tcc cag cag cag cag cag ttg 1968 Ala Gln Gln Gln Lys Ala Ala Leu Ser Ser Gln Gln Gln Gln Gln Leu                 645 650 655 gca ctt ctt ctt caa cag ttt cag acc ttg aag atg aga ata tct gat 2016 Ala Leu Leu Leu Gln Gln Phe Gln Thr Leu Lys Met Arg Ile Ser Asp             660 665 670 cag aac atc att ccc tca gta act agg tct gtg tcc gtg cca gat act 2064 Gln Asn Ile Ile Pro Ser Val Thr Arg Ser Val Ser Val Pro Asp Thr         675 680 685 ggc tct atc tgg gag ctt cag cca aca gct tca cag cct aca gtt tgg 2112 Gly Ser Ile Trp Glu Leu Gln Pro Thr Ala Ser Gln Pro Thr Val Trp     690 695 700 gaa ggt ggt agt gta tgg gat ctt cct ctg gac acc acg aca cca ggc 2160 Glu Gly Gly Ser Val Trp Asp Leu Pro Leu Asp Thr Thr Thr Pro Gly 705 710 715 720 cct gcc ctg gaa cag ctt cag cag cta gag aag gcc aaa gct gca aag 2208 Pro Ala Leu Glu Gln Leu Gln Gln Leu Glu Lys Ala Lys Ala Ala Lys                 725 730 735 cta gag caa gag aga aga gag gca gaa atg agg gca aaa cgg gaa gag 2256 Leu Glu Gln Glu Arg Arg Glu Ala Glu Met Arg Ala Lys Arg Glu Glu             740 745 750 gaa gag cga aag agg cag gaa gaa ctc cga aga caa cag gag gaa att 2304 Glu Glu Arg Lys Arg Gln Glu Glu Leu Arg Arg Gln Gln Glu Glu Ile         755 760 765 ctt cgg cga cag cag gaa gaa gaa agg aaa agg cga gag gaa gaa gaa 2352 Leu Arg Arg Gln Gln Glu Glu Glu Arg Lys Arg Arg Glu Glu Glu Glu     770 775 780 ctt gcc cga agg aaa cag gaa gag gct ctg cgt cgc cag cgg gag caa 2400 Leu Ala Arg Arg Lys Gln Glu Glu Ala Leu Arg Arg Gln Arg Glu Gln 785 790 795 800 gaa att gca tta agg cga cag cga gaa gag gaa gaa aga cag cag caa 2448 Glu Ile Ala Leu Arg Arg Gln Arg Glu Glu Glu Glu Arg Gln Gln Gln                 805 810 815 gaa gaa gct ctt aga aga ctg gaa gag agg aga aga gaa gag gaa gaa 2496 Glu Glu Ala Leu Arg Arg Leu Glu Glu Arg Arg Arg Glu Glu Glu Glu             820 825 830 agg cgg aag cag gaa gaa ttg tta cgc aaa cag gaa gag gag gct gca 2544 Arg Arg Lys Gln Glu Glu Leu Leu Arg Lys Gln Glu Glu Glu Ala Ala         835 840 845 aaa tgg gcc cgg gaa gaa gaa gaa gcc cag cgt cga tta gag gag aac 2592 Lys Trp Ala Arg Glu Glu Glu Glu Ala Gln Arg Arg Leu Glu Glu Asn     850 855 860 cgg ctg cgg atg gaa gag gag gca gcc aga ctc cgg cat gag gaa gaa 2640 Arg Leu Arg Met Glu Glu Glu Ala Ala Arg Leu Arg His Glu Glu Glu 865 870 875 880 gaa cgg aag aga aag gag ctg gag gtc cag cgg cag aag gag tta atg 2688 Glu Arg Lys Arg Lys Glu Leu Glu Val Gln Arg Gln Lys Glu Leu Met                 885 890 895 cgc cag agg cag cag cag caa gag gct ctc cgg agg ttg cag cag cag 2736 Arg Gln Arg Gln Gln Gln Gln Glu Ala Leu Arg Arg Leu Gln Gln Gln             900 905 910 cag cag caa caa cag ctg gcg cag atg aag ctt cct tct tct tca acg 2784 Gln Gln Gln Gln Gln Leu Ala Gln Met Lys Leu Pro Ser Ser Ser Thr         915 920 925 tgg ggc cag cag tcc aat aca aca gca tgt cag tcc cag gcc acg ctg 2832 Trp Gly Gln Gln Ser Asn Thr Thr Ala Cys Gln Ser Gln Ala Thr Leu     930 935 940 tcg ttg gct gaa atc caa aaa cta gag gaa gaa cga gaa cgg cag ctt 2880 Ser Leu Ala Glu Ile Gln Lys Leu Glu Glu Glu Arg Glu Arg Gln Leu 945 950 955 960 cga gaa gag caa agg cgc cag cag agg gag ttg atg aaa gct ctt cag 2928 Arg Glu Glu Gln Arg Arg Gln Gln Arg Glu Leu Met Lys Ala Leu Gln                 965 970 975 cag cag cag caa cag caa cag cag aaa ctc tca ggt tgg ggg aat gtc 2976 Gln Gln Gln Gln Gln Gln Gln Gln Lys Leu Ser Gly Trp Gly Asn Val             980 985 990 agc aaa cct tca ggt acc acg aaa tct ctt ctg gag atc cag cag gaa 3024 Ser Lys Pro Ser Gly Thr Thr Lys Ser Leu Leu Glu Ile Gln Gln Glu         995 1000 1005 gag gcc agg caa atg caa aag cag cag cag cag cag cag caa cac cag 3072 Glu Ala Arg Gln Met Gln Lys Gln Gln Gln Gln Gln Gln Gln His Gln     1010 1015 1020 caa cca aac aga gct cgt aac aat acg cat tcc aac ctg cac acc agc 3120 Gln Pro Asn Arg Ala Arg Asn Asn Thr His Ser Asn Leu His Thr Ser 1025 1030 1035 1040 att ggg aat tct gtt tgg ggc tct ata aat act ggt cct cct aac cag 3168 Ile Gly Asn Ser Val Trp Gly Ser Ile Asn Thr Gly Pro Pro Asn Gln                 1045 1050 1055 tgg gca tct gac cta gtc agt agt att tgg agt aat gct gac act aaa 3216 Trp Ala Ser Asp Leu Val Ser Ser Ile Trp Ser Asn Ala Asp Thr Lys             1060 1065 1070 aac tcc aac atg gga ttc tgg gat gat gca gtg aaa gag gtg gga cct 3264 Asn Ser Asn Met Gly Phe Trp Asp Asp Ala Val Lys Glu Val Gly Pro         1075 1080 1085 agg aat tca aca aat aaa aat aaa aac aac gcc agt ctc agt aaa tct 3312 Arg Asn Ser Thr Asn Lys Asn Lys Asn Asn Ala Ser Leu Ser Lys Ser     1090 1095 1100 gta ggt gtg tct aac cgg cag aat aag aaa gta gaa gaa gaa gaa aag 3360 Val Gly Val Ser Asn Arg Gln Asn Lys Lys Val Glu Glu Glu Glu Lys 1105 1110 1115 1120 ttg ctg aag ctc ttt cag gga gta aat aaa gcc caa gat gga ttt acg 3408 Leu Leu Lys Leu Phe Gln Gly Val Asn Lys Ala Gln Asp Gly Phe Thr                 1125 1130 1135 cag tgg tgt gaa cag atg ctt cat gcc ctt aat acg gca aat aac ttg 3456 Gln Trp Cys Glu Gln Met Leu His Ala Leu Asn Thr Ala Asn Asn Leu             1140 1145 1150 gat gtt ccc aca ttt gtt tct ttc ctg aaa gaa gta gaa tct cct tat 3504 Asp Val Pro Thr Phe Val Ser Phe Leu Lys Glu Val Glu Ser Pro Tyr         1155 1160 1165 gag gtc cat gat tat atc agg gcc tat tta gga gat act tct gag gcc 3552 Glu Val His Asp Tyr Ile Arg Ala Tyr Leu Gly Asp Thr Ser Glu Ala     1170 1175 1180 aag gag ttt gcc aag cag ttc ctt gag cgc cgt gcc aaa cag aaa gcc 3600 Lys Glu Phe Ala Lys Gln Phe Leu Glu Arg Arg Ala Lys Gln Lys Ala 1185 1190 1195 1200 aac cag cag cgt cag cag cag cag ctg cca cag cag cag cag cag cag 3648 Asn Gln Gln Arg Gln Gln Gln Gln Leu Pro Gln Gln Gln Gln Gln Gln                 1205 1210 1215 ccg cca cag cag ccg cca cag cag cca caa cag cag gac tct gtg tgg 3696 Pro Pro Gln Gln Pro Pro Gln Gln Pro Gln Gln Gln Asp Ser Val Trp             1220 1225 1230 ggg atg aac cac agt aca ctc cat tca gta ttt cag acc aat caa agc 3744 Gly Met Asn His Ser Thr Leu His Ser Val Phe Gln Thr Asn Gln Ser         1235 1240 1245 aac aac caa caa tcc aat ttt gag gct gtg cag agt ggc aag aag aag 3792 Asn Asn Gln Gln Ser Asn Phe Glu Ala Val Gln Ser Gly Lys Lys Lys     1250 1255 1260 aaa aag cag aag atg gtc cga gca gat ccc agt tta tta gga ttt tca 3840 Lys Lys Gln Lys Met Val Arg Ala Asp Pro Ser Leu Leu Gly Phe Ser 1265 1270 1275 1280 gtc aat gca tca tcg gag cga ctc aac atg ggt gaa atc gag acg ttg 3888 Val Asn Ala Ser Ser Glu Arg Leu Asn Met Gly Glu Ile Glu Thr Leu                 1285 1290 1295 gat gac tac 3897 Asp Asp Tyr <210> 9 <211> 180 <212> DNA <213> Homo sapiens <400> 9 gaa gca atg cag aag tgg tat tac aaa gat cct cag gga gaa att caa 48 Glu Ala Met Gln Lys Trp Tyr Tyr Lys Asp Pro Gln Gly Glu Ile Gln   1 5 10 15 ggt ccc ttc aat aat cag gag atg gca gaa tgg ttt cag gcg ggc tat 96 Gly Pro Phe Asn Asn Gln Glu Met Ala Glu Trp Phe Gln Ala Gly Tyr              20 25 30 ttt act atg tct tta ttg gtg aag aga gcg tgt gat gaa agc ttc caa 144 Phe Thr Met Ser Leu Leu Val Lys Arg Ala Cys Asp Glu Ser Phe Gln          35 40 45 cct ctt ggc gat atc atg aaa atg tgg gga agg gtt 180 Pro Leu Gly Asp Ile Met Lys Met Trp Gly Arg Val      50 55 60 <210> 10 <211> 4596 <212> DNA <213> Homo sapiens <400> 10 atg gga cac agt aaa cag att cga att tta ctt ctg aac gaa atg gag 48 Met Gly His Ser Lys Gln Ile Arg Ile Leu Leu Leu Asn Glu Met Glu   1 5 10 15 aaa ctg gaa aag acc ctc ttc aga ctt gaa caa ggg tat gag cta cag 96 Lys Leu Glu Lys Thr Leu Phe Arg Leu Glu Gln Gly Tyr Glu Leu Gln              20 25 30 ttc cga tta ggc cca act tta cag gga aaa gca gtt acc gtg tat aca 144 Phe Arg Leu Gly Pro Thr Leu Gln Gly Lys Ala Val Thr Val Tyr Thr          35 40 45 aat tac cca ttt cct gga gaa aca ttt aat aga gaa aaa ttc cgt tct 192 Asn Tyr Pro Phe Pro Gly Glu Thr Phe Asn Arg Glu Lys Phe Arg Ser      50 55 60 ctg gat tgg gaa aat cca aca gaa aga gaa gat gat tct gat aaa tac 240 Leu Asp Trp Glu Asn Pro Thr Glu Arg Glu Asp Asp Ser Asp Lys Tyr  65 70 75 80 tgt aaa ctt aat ctg caa caa tct ggt tca ttt cag tat tat ttc ctt 288 Cys Lys Leu Asn Leu Gln Gln Ser Gly Ser Phe Gln Tyr Tyr Phe Leu                  85 90 95 caa gga aat gag aaa agt ggt gga ggt tac ata gtt gtg gac ccc att 336 Gln Gly Asn Glu Lys Ser Gly Gly Gly Tyr Ile Val Val Asp Pro Ile             100 105 110 tta cgt gtt ggt gct gat aat cat gtg cta ccc ttg gac tgt gtt act 384 Leu Arg Val Gly Ala Asp Asn His Val Leu Pro Leu Asp Cys Val Thr         115 120 125 ctt cag aca ttt tta gct aag tgt ttg gga cct ttt gat gaa tgg gaa 432 Leu Gln Thr Phe Leu Ala Lys Cys Leu Gly Pro Phe Asp Glu Trp Glu     130 135 140 agc aga ctt agg gtt gca aaa gaa tca ggc tac aac atg att cat ttt 480 Ser Arg Leu Arg Val Ala Lys Glu Ser Gly Tyr Asn Met Ile His Phe 145 150 155 160 acc cca ttg cag act ctt gga cta tct agg tca tgc tac tcc ctt gcc 528 Thr Pro Leu Gln Thr Leu Gly Leu Ser Arg Ser Cys Tyr Ser Leu Ala                 165 170 175 aat cag tta gaa tta aat cct gac ttt tca aga cct aat aga aag tat 576 Asn Gln Leu Glu Leu Asn Pro Asp Phe Ser Arg Pro Asn Arg Lys Tyr             180 185 190 acc tgg aat gat gtt gga cag cta gtg gaa aaa tta aaa aag gaa tgg 624 Thr Trp Asn Asp Val Gly Gln Leu Val Glu Lys Leu Lys Lys Glu Trp         195 200 205 aat gtt att tgt att act gat gtt gtc tac aat cat act gct gct aat 672 Asn Val Ile Cys Ile Thr Asp Val Val Tyr Asn His Thr Ala Ala Asn     210 215 220 agt aaa tgg atc cag gaa cat cca gaa tgt gcc tat aat ctt gtg aat 720 Ser Lys Trp Ile Gln Glu His Pro Glu Cys Ala Tyr Asn Leu Val Asn 225 230 235 240 tct cca cac tta aaa cct gcc tgg gtc tta gac aga gca ctt tgg cgt 768 Ser Pro His Leu Lys Pro Ala Trp Val Leu Asp Arg Ala Leu Trp Arg                 245 250 255 ttc tcc tgt gat gtt gca gaa ggg aaa tac aaa gaa aag gga ata cct 816 Phe Ser Cys Asp Val Ala Glu Gly Lys Tyr Lys Glu Lys Gly Ile Pro             260 265 270 gct ttg att gaa aat gat cac cat atg aat tcc atc cga aaa ata att 864 Ala Leu Ile Glu Asn Asp His His Met Asn Ser Ile Arg Lys Ile Ile         275 280 285 tgg gag gat att ttt cca aag ctt aaa ctc tgg gaa ttt ttc caa gta 912 Trp Glu Asp Ile Phe Pro Lys Leu Lys Leu Trp Glu Phe Phe Gln Val     290 295 300 gat gtc aac aaa gcg gtt gag caa ttt aga aga ctt ctt aca caa gaa 960 Asp Val Asn Lys Ala Val Glu Gln Phe Arg Arg Leu Leu Thr Gln Glu 305 310 315 320 aat agg cga gta acc aag tct gat cca aac caa cac ctt acg att att 1008 Asn Arg Arg Val Thr Lys Ser Asp Pro Asn Gln His Leu Thr Ile Ile                 325 330 335 caa gat cct gaa tac aga cgg ttt ggc tgt act gta gat atg aac att 1056 Gln Asp Pro Glu Tyr Arg Arg Phe Gly Cys Thr Val Asp Met Asn Ile             340 345 350 gca cta acg act ttc ata cca cat gac aag ggg cca gca gca att gaa 1104 Ala Leu Thr Thr Phe Ile Pro His Asp Lys Gly Pro Ala Ala Ile Glu         355 360 365 gaa tgc tgt aat tgg ttt cat aaa aga atg gag gaa tta aat tca gag 1152 Glu Cys Cys Asn Trp Phe His Lys Arg Met Glu Glu Leu Asn Ser Glu     370 375 380 aag cat cga ctc att aac tat cat cag gaa cag gca gtt aat tgc ctt 1200 Lys His Arg Leu Ile Asn Tyr His Gln Glu Gln Ala Val Asn Cys Leu 385 390 395 400 ttg gga aat gtg ttt tat gaa cga ctg gct ggc cat ggt cca aaa cta 1248 Leu Gly Asn Val Phe Tyr Glu Arg Leu Ala Gly His Gly Pro Lys Leu                 405 410 415 gga cct gtc act aga aag cat cct tta gtt acc agg tat ttt act ttc 1296 Gly Pro Val Thr Arg Lys His Pro Leu Val Thr Arg Tyr Phe Thr Phe             420 425 430 cca ttt gaa gag ata gac ttc tcc atg gaa gaa tct atg att cat ctg 1344 Pro Phe Glu Glu Ile Asp Phe Ser Met Glu Glu Ser Met Ile His Leu         435 440 445 cca aat aaa gct tgt ttt ctg atg gca cac aat gga tgg gta atg gga 1392 Pro Asn Lys Ala Cys Phe Leu Met Ala His Asn Gly Trp Val Met Gly     450 455 460 gat gat cct ctt cga aac ttt gct gaa ccg ggt tca gaa gtt tac cta 1440 Asp Asp Pro Leu Arg Asn Phe Ala Glu Pro Gly Ser Glu Val Tyr Leu 465 470 475 480 agg aga gaa ctt att tgc tgg gga gac agt gtt aaa tta cgc tat ggg 1488 Arg Arg Glu Leu Ile Cys Trp Gly Asp Ser Val Lys Leu Arg Tyr Gly                 485 490 495 aat aaa cca gag gac tgt cct tat ctc tgg gca cac atg aaa aaa tac 1536 Asn Lys Pro Glu Asp Cys Pro Tyr Leu Trp Ala His Met Lys Lys Tyr             500 505 510 act gaa ata act gca act tat ttc cag gga gta cgt ctt gat aac tgc 1584 Thr Glu Ile Thr Ala Thr Tyr Phe Gln Gly Val Arg Leu Asp Asn Cys         515 520 525 cac tca aca cct ctt cac gta gct gag tac atg ttg gat gct gct agg 1632 His Ser Thr Pro Leu His Val Ala Glu Tyr Met Leu Asp Ala Ala Arg     530 535 540 aat ttg caa ccc aat tta tat gta gta gct gaa ctg ttc aca gga agt 1680 Asn Leu Gln Pro Asn Leu Tyr Val Val Ala Glu Leu Phe Thr Gly Ser 545 550 555 560 gaa gat ctg gac aat gtc ttt gtt act aga ctg ggc att agt tcc tta 1728 Glu Asp Leu Asp Asn Val Phe Val Thr Arg Leu Gly Ile Ser Ser Leu                 565 570 575 ata aga gag gca atg agt gca tat aat agt cat gaa gag ggc aga tta 1776 Ile Arg Glu Ala Met Ser Ala Tyr Asn Ser His Glu Glu Gly Arg Leu             580 585 590 gtt tac cga tat gga gga gaa cct gtt gga tcc ttt gtt cag ccc tgt 1824 Val Tyr Arg Tyr Gly Gly Glu Pro Val Gly Ser Phe Val Gln Pro Cys         595 600 605 ttg agg cct tta atg cca gct att gca cat gcc ctg ttt atg gat att 1872 Leu Arg Pro Leu Met Pro Ala Ile Ala His Ala Leu Phe Met Asp Ile     610 615 620 acg cat gat aat gag tgt cct att gtg cat aga tca gcg tat gat gct 1920 Thr His Asp Asn Glu Cys Pro Ile Val His Arg Ser Ala Tyr Asp Ala 625 630 635 640 ctt cca agt act aca att gtt tct atg gca tgt tgt gct agt gga agt 1968 Leu Pro Ser Thr Thr Ile Val Ser Met Ala Cys Cys Ala Ser Gly Ser                 645 650 655 aca aga ggc tat gat gaa tta gtg cct cat cag att tca gtg gtt tct 2016 Thr Arg Gly Tyr Asp Glu Leu Val Pro His Gln Ile Ser Val Val Ser             660 665 670 gaa gaa cgg ttt tac act aag tgg aat cct gaa gca ttg cct tca aac 2064 Glu Glu Arg Phe Tyr Thr Lys Trp Asn Pro Glu Ala Leu Pro Ser Asn         675 680 685 aca ggt gaa gtt aat ttc caa agc ggc att att gca gcc agg tgt gct 2112 Thr Gly Glu Val Asn Phe Gln Ser Gly Ile Ile Ala Ala Arg Cys Ala     690 695 700 atc agt aaa ctt cat cag gag ctt gga gcc aag ggt ttt att cag gtg 2160 Ile Ser Lys Leu His Gln Glu Leu Gly Ala Lys Gly Phe Ile Gln Val 705 710 715 720 tat gtg gat caa gtt gat gaa gac ata gtg gca gta aca aga cac tca 2208 Tyr Val Asp Gln Val Asp Glu Asp Ile Val Ala Val Thr Arg His Ser                 725 730 735 cct agc atc cat cag tct gtt gtg gct gta tct aga act gct ttc agg 2256 Pro Ser Ile His Gln Ser Val Val Ala Val Ser Arg Thr Ala Phe Arg             740 745 750 aat ccc aag act tca ttt tac agc aag gaa gtg cct caa atg tgc atc 2304 Asn Pro Lys Thr Ser Phe Tyr Ser Lys Glu Val Pro Gln Met Cys Ile         755 760 765 cct ggc aaa att gaa gaa gta gtt ctt gaa gct aga act att gag aga 2352 Pro Gly Lys Ile Glu Glu Val Val Leu Glu Ala Arg Thr Ile Glu Arg     770 775 780 aac acg aaa cct tat agg aag gat gag aat tca atc aat gga aca cca 2400 Asn Thr Lys Pro Tyr Arg Lys Asp Glu Asn Ser Ile Asn Gly Thr Pro 785 790 795 800 gat atc aca gta gaa att aga gaa cat att cag ctt aat gaa agt aaa 2448 Asp Ile Thr Val Glu Ile Arg Glu His Ile Gln Leu Asn Glu Ser Lys                 805 810 815 att gtt aaa caa gct gga gtt gcc aca aaa ggg ccc aat gaa tat att 2496 Ile Val Lys Gln Ala Gly Val Ala Thr Lys Gly Pro Asn Glu Tyr Ile             820 825 830 caa gaa ata gaa ttt gaa aac ttg tct cca gga agt gtt att ata ttc 2544 Gln Glu Ile Glu Phe Glu Asn Leu Ser Pro Gly Ser Val Ile Ile Phe         835 840 845 aga gtt agt ctt gat cca cat gca caa gtc gct gtt gga att ctt cga 2592 Arg Val Ser Leu Asp Pro His Ala Gln Val Ala Val Gly Ile Leu Arg     850 855 860 aat cat ctg aca caa ttc agt cct cac ttt aaa tct ggc agc cta gct 2640 Asn His Leu Thr Gln Phe Ser Pro His Phe Lys Ser Gly Ser Leu Ala 865 870 875 880 gtt gac aat gca gat cct ata tta aaa att cct ttt gct tct ctt gcc 2688 Val Asp Asn Ala Asp Pro Ile Leu Lys Ile Pro Phe Ala Ser Leu Ala                 885 890 895 tcc aga tta act ttg gct gag cta aat cag atc ctt tac cga tgt gaa 2736 Ser Arg Leu Thr Leu Ala Glu Leu Asn Gln Ile Leu Tyr Arg Cys Glu             900 905 910 tca gaa gaa aag gaa gat ggt gga ggg tgc tat gac ata cca aac tgg 2784 Ser Glu Glu Lys Glu Asp Gly Gly Gly Cys Tyr Asp Ile Pro Asn Trp         915 920 925 tca gcc ctt aaa tat gca ggt ctt caa ggt tta atg tct gta ttg gca 2832 Ser Ala Leu Lys Tyr Ala Gly Leu Gln Gly Leu Met Ser Val Leu Ala     930 935 940 gaa ata aga cca aag aat gac ttg ggg cat cct ttt tgt aat aat ttg 2880 Glu Ile Arg Pro Lys Asn Asp Leu Gly His Pro Phe Cys Asn Asn Leu 945 950 955 960 aga tct gga gat tgg atg att gac tat gtc agt aac cgg ctt att tca 2928 Arg Ser Gly Asp Trp Met Ile Asp Tyr Val Ser Asn Arg Leu Ile Ser                 965 970 975 cga tca gga act att gct gaa gtt ggt aaa tgg ttg cag gct atg ttc 2976 Arg Ser Gly Thr Ile Ala Glu Val Gly Lys Trp Leu Gln Ala Met Phe             980 985 990 ttc tac ctg aag cag atc cca cgt tac ctt atc cca tgt tac ttt gat 3024 Phe Tyr Leu Lys Gln Ile Pro Arg Tyr Leu Ile Pro Cys Tyr Phe Asp         995 1000 1005 gct ata tta att ggt gca tat acc act ctt ctg gat aca gca tgg aag 3072 Ala Ile Leu Ile Gly Ala Tyr Thr Thr Leu Leu Asp Thr Ala Trp Lys     1010 1015 1020 cag atg tca agc ttt gtt cag aat ggt tca acc ttt gtg aaa cac ctt 3120 Gln Met Ser Ser Phe Val Gln Asn Gly Ser Thr Phe Val Lys His Leu 1025 1030 1035 1040 tca ttg ggt tca gtt caa ctg tgt gga gta gga aaa ttc cct tcc ctg 3168 Ser Leu Gly Ser Val Gln Leu Cys Gly Val Gly Lys Phe Pro Ser Leu                 1045 1050 1055 cca att ctt tca cct gcc cta atg gat gta cct tat agg tta aat gag 3216 Pro Ile Leu Ser Pro Ala Leu Met Asp Val Pro Tyr Arg Leu Asn Glu             1060 1065 1070 atc aca aaa gaa aag gag caa tgt tgt gtt tct cta gct gca ggc tta 3264 Ile Thr Lys Glu Lys Glu Gln Cys Cys Val Ser Leu Ala Ala Gly Leu         1075 1080 1085 cct cat ttt tct tct ggt att ttc cgc tgc tgg gga agg gat act ttt 3312 Pro His Phe Ser Ser Gly Ile Phe Arg Cys Trp Gly Arg Asp Thr Phe     1090 1095 1100 att gca ctt aga ggt ata ctg ctg att act aga cgc tat gta gaa gcc 3360 Ile Ala Leu Arg Gly Ile Leu Leu Ile Thr Arg Arg Tyr Val Glu Ala 1105 1110 1115 1120 agg aat att att tta gca ttt gcg ggt acc ctg agg cat ggt ctc att 3408 Arg Asn Ile Ile Leu Ala Phe Ala Gly Thr Leu Arg His Gly Leu Ile                 1125 1130 1135 cct aat cta ctg ggt gaa gga att tat gcc aga tac aat tgt cgg gat 3456 Pro Asn Leu Leu Gly Glu Gly Ile Tyr Ala Arg Tyr Asn Cys Arg Asp             1140 1145 1150 gct gtg tgg tgg tgg ctg cag tgt atc cag gat tac tgt aaa atg gtt 3504 Ala Val Trp Trp Trp Leu Gln Cys Ile Gln Asp Tyr Cys Lys Met Val         1155 1160 1165 cca aat ggt cta gac att ctc aag tgc cca gtt tcc aga atg tat cct 3552 Pro Asn Gly Leu Asp Ile Leu Lys Cys Pro Val Ser Arg Met Tyr Pro     1170 1175 1180 aca gat gat tct gct cct ttg cct gct ggc aca ctg gat cag cca ttg 3600 Thr Asp Asp Ser Ala Pro Leu Pro Ala Gly Thr Leu Asp Gln Pro Leu 1185 1190 1195 1200 ttt gaa gtc ata cag gaa gca atg caa aaa cac atg cag ggc ata cag 3648 Phe Glu Val Ile Gln Glu Ala Met Gln Lys His Met Gln Gly Ile Gln                 1205 1210 1215 ttc cga gaa agg aat gct ggt ccc cag ata gat cga aac atg aag gac 3696 Phe Arg Glu Arg Asn Ala Gly Pro Gln Ile Asp Arg Asn Met Lys Asp             1220 1225 1230 gaa ggt ttt aat ata act gca gga gtt gat gaa gaa aca gga ttt gtt 3744 Glu Gly Phe Asn Ile Thr Ala Gly Val Asp Glu Glu Thr Gly Phe Val         1235 1240 1245 tat gga gga aat cgt ttc aat tgt ggc aca tgg atg gat aaa atg gga 3792 Tyr Gly Gly Asn Arg Phe Asn Cys Gly Thr Trp Met Asp Lys Met Gly     1250 1255 1260 gaa agt gac aga gct aga aac aga gga atc cca gcc aca cca aga gat 3840 Glu Ser Asp Arg Ala Arg Asn Arg Gly Ile Pro Ala Thr Pro Arg Asp 1265 1270 1275 1280 ggg tct gct gtg gaa att gtg ggc ctg agt aaa tct gct gtt cgc tgg 3888 Gly Ser Ala Val Glu Ile Val Gly Leu Ser Lys Ser Ala Val Arg Trp                 1285 1290 1295 ttg ctg gaa tta tcc aaa aaa aat att ttc cct tat cat gaa gtc cca 3936 Leu Leu Glu Leu Ser Lys Lys Asn Ile Phe Pro Tyr His Glu Val Pro             1300 1305 1310 gta aaa aga cat gga aag gct ata aag gtc tca tat gat gag tgg acc 3984 Val Lys Arg His Gly Lys Ala Ile Lys Val Ser Tyr Asp Glu Trp Thr         1315 1320 1325 aga aaa ata caa gac aac ttt gaa aag cta ttt cat gtt tcc gaa gac 4032 Arg Lys Ile Gln Asp Asn Phe Glu Lys Leu Phe His Val Ser Glu Asp     1330 1335 1340 cct tca gat tta aat gaa aag cat cca aat ctg gtt cac aaa cgt ggc 4080 Pro Ser Asp Leu Asn Glu Lys His Pro Asn Leu Val His Lys Arg Gly 1345 1350 1355 1360 ata tac aaa gat agt tat gga gct tca agt cct tgg tgt gac tat cag 4128 Ile Tyr Lys Asp Ser Tyr Gly Ala Ser Ser Pro Trp Cys Asp Tyr Gln                 1365 1370 1375 ctc agg cct aat ttt acc ata gca atg gtt gtg gcc cct gag ctc ttt 4176 Leu Arg Pro Asn Phe Thr Ile Ala Met Val Val Ala Pro Glu Leu Phe             1380 1385 1390 act aca gaa aaa gca tgg aaa gct ttg gag att gca gaa aaa aaa ttg 4224 Thr Thr Glu Lys Ala Trp Lys Ala Leu Glu Ile Ala Glu Lys Lys Leu         1395 1400 1405 ctt ggt ccc ctt ggc atg aaa act tta gat cca gat gat atg gtt tac 4272 Leu Gly Pro Leu Gly Met Lys Thr Leu Asp Pro Asp Asp Met Val Tyr     1410 1415 1420 tgt gga att tat gac aat gca tta gac aat gac aac tac aat ctt gct 4320 Cys Gly Ile Tyr Asp Asn Ala Leu Asp Asn Asp Asn Tyr Asn Leu Ala 1425 1430 1435 1440 aaa ggt ttc aat tat cac caa gga cct gag tgg ctg tgg cct att ggg 4368 Lys Gly Phe Asn Tyr His Gln Gly Pro Glu Trp Leu Trp Pro Ile Gly                 1445 1450 1455 tat ttt ctt cgt gca aaa tta tat ttt tcc aga ttg atg ggc ccg gag 4416 Tyr Phe Leu Arg Ala Lys Leu Tyr Phe Ser Arg Leu Met Gly Pro Glu             1460 1465 1470 act act gca aag act ata gtt ttg gtt aaa aat gtt ctt tcc cga cat 4464 Thr Thr Ala Lys Thr Ile Val Leu Val Lys Asn Val Leu Ser Arg His         1475 1480 1485 tat gtt cat ctt gag aga tcc cct tgg aaa gga ctt cca gaa ctg acc 4512 Tyr Val His Leu Glu Arg Ser Pro Trp Lys Gly Leu Pro Glu Leu Thr     1490 1495 1500 aat gag aat gcc cag tac tgt cct ttc agc tgt gaa aca caa gcc tgg 4560 Asn Glu Asn Ala Gln Tyr Cys Pro Phe Ser Cys Glu Thr Gln Ala Trp 1505 1510 1515 1520 tca att gct act att ctt gag aca ctt tat gat tta 4596 Ser Ile Ala Thr Ile Leu Glu Thr Leu Tyr Asp Leu                 1525 1530 <210> 11 <211> 2163 <212> DNA <213> Homo sapiens <400> 11 atg gag ttg gga aaa gga aaa cta ctc agg act gga ctg aat gcg ttg 48 Met Glu Leu Gly Lys Gly Lys Leu Leu Arg Thr Gly Leu Asn Ala Leu   1 5 10 15 cat caa gca gtg cat ccg atc cat ggc ctt gcc tgg acc gat ggg aat 96 His Gln Ala Val His Pro Ile His Gly Leu Ala Trp Thr Asp Gly Asn              20 25 30 caa gtt gtc cta act gat ttg cgg ctt cac agt gga gag gtc aag ttt 144 Gln Val Val Leu Thr Asp Leu Arg Leu His Ser Gly Glu Val Lys Phe          35 40 45 ggg gac tcc aaa gtc att gga cag ttt gaa tgt gtc tgt ggg ttg tcc 192 Gly Asp Ser Lys Val Ile Gly Gln Phe Glu Cys Val Cys Gly Leu Ser      50 55 60 tgg gcc cca cct gtt gca gat gat aca cct gtt cta ctc gct gtc cag 240 Trp Ala Pro Pro Val Ala Asp Asp Thr Pro Val Leu Leu Ala Val Gln  65 70 75 80 cat gag aag cat gtc act gtg tgg cag ctg tgt ccc agc cct atg gag 288 His Glu Lys His Val Thr Val Trp Gln Leu Cys Pro Ser Pro Met Glu                  85 90 95 tca agc aaa tgg ctg acg tct cag act tgt gag att aga gga tca cta 336 Ser Ser Lys Trp Leu Thr Ser Gln Thr Cys Glu Ile Arg Gly Ser Leu             100 105 110 cct atc ctt ccc cag ggc tgt gtg tgg cac cca aaa tgt gct att ctg 384 Pro Ile Leu Pro Gln Gly Cys Val Trp His Pro Lys Cys Ala Ile Leu         115 120 125 act gtg ttg act gct cag gat gtc tcc att ttc cct aat gtt cac tct 432 Thr Val Leu Thr Ala Gln Asp Val Ser Ile Phe Pro Asn Val His Ser     130 135 140 gat gat tcc cag gta aag gca gac atc aac acc cag ggc cgc att cac 480 Asp Asp Ser Gln Val Lys Ala Asp Ile Asn Thr Gln Gly Arg Ile His 145 150 155 160 tgt gca tgt tgg acc cag gat ggc ctg agg ctg gtg gtg gca gta ggc 528 Cys Ala Cys Trp Thr Gln Asp Gly Leu Arg Leu Val Val Ala Val Gly                 165 170 175 agc agc ctg cat tct tat att tgg gac agc gct cag aag act ctt cac 576 Ser Ser Leu His Ser Tyr Ile Trp Asp Ser Ala Gln Lys Thr Leu His             180 185 190 agg tgc tcc tcc tgc ctg gtg ttt gat gtg gac agc cac gtc tgc tcc 624 Arg Cys Ser Ser Cys Leu Val Phe Asp Val Asp Ser His Val Cys Ser         195 200 205 atc aca gca act gtg gac tca cag gtt gct ata gct act gag ctt cca 672 Ile Thr Ala Thr Val Asp Ser Gln Val Ala Ile Ala Thr Glu Leu Pro     210 215 220 ttg gat aag atc tgt ggc tta aat gca tct gaa acc ttt aat atc cca 720 Leu Asp Lys Ile Cys Gly Leu Asn Ala Ser Glu Thr Phe Asn Ile Pro 225 230 235 240 cct aac agt aaa gac atg act ccg tat gct tta cca gtt att ggt gaa 768 Pro Asn Ser Lys Asp Met Thr Pro Tyr Ala Leu Pro Val Ile Gly Glu                 245 250 255 gta cgc tct atg gat aaa gag gca act gat tct gaa aca aat tct gaa 816 Val Arg Ser Met Asp Lys Glu Ala Thr Asp Ser Glu Thr Asn Ser Glu             260 265 270 gta tca gtt tct tct tcc tat tta gaa cct ctg gat cta act cac ata 864 Val Ser Val Ser Ser Ser Tyr Leu Glu Pro Leu Asp Leu Thr His Ile         275 280 285 cat ttc aat caa cat aag tct gag ggt aat tct ctt att tgt cta aga 912 His Phe Asn Gln His Lys Ser Glu Gly Asn Ser Leu Ile Cys Leu Arg     290 295 300 aaa aag gac tac ttg aca gga act ggc caa gat tct tca cat ttg gtc 960 Lys Lys Asp Tyr Leu Thr Gly Thr Gly Gln Asp Ser Ser His Leu Val 305 310 315 320 ctt gtg acc ttt aag aag gca gtt acc atg acg aga aaa gtc act att 1008 Leu Val Thr Phe Lys Lys Ala Val Thr Met Thr Arg Lys Val Thr Ile                 325 330 335 cca ggc att ctg gtt cct gat ctg ata gca ttt aat ctt aaa gcc cac 1056 Pro Gly Ile Leu Val Pro Asp Leu Ile Ala Phe Asn Leu Lys Ala His             340 345 350 gta gtg gca gtg gct tcc aac act tgt aat ata att ttg atc tac tct 1104 Val Val Ala Val Ala Ser Asn Thr Cys Asn Ile Ile Leu Ile Tyr Ser         355 360 365 gtc att cca tct tca gtc cca aac atc cag caa att cga tta gag aac 1152 Val Ile Pro Ser Ser Val Pro Asn Ile Gln Gln Ile Arg Leu Glu Asn     370 375 380 act gaa aga cca aaa ggg ata tgt ttc ttg aca gac caa cta tta cta 1200 Thr Glu Arg Pro Lys Gly Ile Cys Phe Leu Thr Asp Gln Leu Leu Leu 385 390 395 400 att ttg gta gga aaa caa aaa ctc act gat aca aca ttt ctt cct tct 1248 Ile Leu Val Gly Lys Gln Lys Leu Thr Asp Thr Thr Phe Leu Pro Ser                 405 410 415 tca aag tct gat cag tat gcc att agc ttg att gtt aga gaa ata atg 1296 Ser Lys Ser Asp Gln Tyr Ala Ile Ser Leu Ile Val Arg Glu Ile Met             420 425 430 ttg gaa gaa gaa cct tca ata aca tca ggt gaa agc cag act acc tac 1344 Leu Glu Glu Glu Pro Ser Ile Thr Ser Gly Glu Ser Gln Thr Thr Tyr         435 440 445 tct act ttc agt gct ccg tta aat aaa gca aat aga aaa aag tta att 1392 Ser Thr Phe Ser Ala Pro Leu Asn Lys Ala Asn Arg Lys Lys Leu Ile     450 455 460 gaa agt ctt tcc cca gat ttt tgt cac caa aac aaa ggg ctg ttg ctg 1440 Glu Ser Leu Ser Pro Asp Phe Cys His Gln Asn Lys Gly Leu Leu Leu 465 470 475 480 aca gtt aat acc agt agt cag aat gga agg cct gga aga aca ctt att 1488 Thr Val Asn Thr Ser Ser Gln Asn Gly Arg Pro Gly Arg Thr Leu Ile                 485 490 495 aaa gaa atc cag agt cct ctg tct agt atc tgt gat ggc tcc ata gct 1536 Lys Glu Ile Gln Ser Pro Leu Ser Ser Ile Cys Asp Gly Ser Ile Ala             500 505 510 cta gat gct gag cct gtt acc cag cca gca tcg ctg ccc aga cac agc 1584 Leu Asp Ala Glu Pro Val Thr Gln Pro Ala Ser Leu Pro Arg His Ser         515 520 525 agc aca cca gac cac acc agc aca ctg gag cct cct cgt ttg cct caa 1632 Ser Thr Pro Asp His Thr Ser Thr Leu Glu Pro Pro Arg Leu Pro Gln     530 535 540 aga aag aac tta caa agt gaa aag gaa act tat cag ctg tct aag gag 1680 Arg Lys Asn Leu Gln Ser Glu Lys Glu Thr Tyr Gln Leu Ser Lys Glu 545 550 555 560 gtg gaa att tta tct agg aac ctg gtt gaa atg caa cgg tgt ctt tct 1728 Val Glu Ile Leu Ser Arg Asn Leu Val Glu Met Gln Arg Cys Leu Ser                 565 570 575 gaa ctt aca aac cgt ctg cat aat ggg aag aaa tcc tct tca gtg tat 1776 Glu Leu Thr Asn Arg Leu His Asn Gly Lys Lys Ser Ser Ser Val Tyr             580 585 590 cca ctc tct caa gat ctt cct tat gtt cac atc att tac cag aaa cct 1824 Pro Leu Ser Gln Asp Leu Pro Tyr Val His Ile Ile Tyr Gln Lys Pro         595 600 605 tat tat cta ggt cct gtt gtt gaa aaa aga gcg gtg ctt ctc tgt gat 1872 Tyr Tyr Leu Gly Pro Val Val Glu Lys Arg Ala Val Leu Leu Cys Asp     610 615 620 ggt aaa cta agg ctc agt aca gtt cag cag act ttt ggc ctt tct ctc 1920 Gly Lys Leu Arg Leu Ser Thr Val Gln Gln Thr Phe Gly Leu Ser Leu 625 630 635 640 att gaa atg cta cat gat tcc cac tgg att ctt ctc tct gct gac agt 1968 Ile Glu Met Leu His Asp Ser His Trp Ile Leu Leu Ser Ala Asp Ser                 645 650 655 gag ggc ttt atc ccg tta acc ttc aca gcc aca cag gaa ata atc ata 2016 Glu Gly Phe Ile Pro Leu Thr Phe Thr Ala Thr Gln Glu Ile Ile Ile             660 665 670 aga gat ggc agc ctg tcc agg tca gat gtc ttc aga gac tct ttt tct 2064 Arg Asp Gly Ser Leu Ser Arg Ser Asp Val Phe Arg Asp Ser Phe Ser         675 680 685 cac agt cca ggt gct gtt tct tct ctt aaa gtc ttt aca ggc ctt gct 2112 His Ser Pro Gly Ala Val Ser Ser Leu Lys Val Phe Thr Gly Leu Ala     690 695 700 gcc ccc agt tta gat acc act ggc tgt tgt aac cat gta gat ggc atg 2160 Ala Pro Ser Leu Asp Thr Thr Gly Cys Cys Asn His Val Asp Gly Met 705 710 715 720 gct 2163 Ala <210> 12 <211> 597 <212> DNA <213> Mus musculus <400> 12 atg tct tca gga aat gca aaa att ggg tat cct gct ccc aac ttc aaa 48 Met Ser Ser Gly Asn Ala Lys Ile Gly Tyr Pro Ala Pro Asn Phe Lys   1 5 10 15 gct aca gct gtt atg cca gat gga caa ttc aaa gat atc agc cta agt 96 Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser Leu Ser              20 25 30 gaa tac aaa gga aaa tat gtt gtg ttc ttc ttt tac cct ctt gac ttt 144 Glu Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu Asp Phe          35 40 45 act ttt gtg tgt ccc acg gag atc att gct ttc agt gat aga gcc gat 192 Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala Asp      50 55 60 gaa ttt aag aaa ctc aac tgc caa gtg att ggc gct tct gtg gat tct 240 Glu Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp Ser  65 70 75 80 cac ttc tgt cat ctg gca tgg att aac aca ccc aag aaa caa gga gga 288 His Phe Cys His Leu Ala Trp Ile Asn Thr Pro Lys Lys Gln Gly Gly                  85 90 95 ttg gga ccc atg aac att ccc tta ata tca gat ccc aag cgc acc att 336 Leu Gly Pro Met Asn Ile Pro Leu Ile Ser Asp Pro Lys Arg Thr Ile             100 105 110 gct cag gat tat gga gtc tta aag gct gat gaa ggt atc tct ttc agg 384 Ala Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly Ile Ser Phe Arg         115 120 125 ggc ctt ttt att att gat gat aaa ggt atc ctt cga cag ata aca ata 432 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg Gln Ile Thr Ile     130 135 140 aac gat ctt ccc gtt ggc cgc tct gtg gat gag att ata cga cta gtc 480 Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ile Ile Arg Leu Val 145 150 155 160 cag gcc ttc cag ttc act gac aaa cat ggt gaa gtg tgt cca gct ggc 528 Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu Val Cys Pro Ala Gly                 165 170 175 tgg aaa cct ggc agt gat acc atc aag cct gat gtc aat aag agc aaa 576 Trp Lys Pro Gly Ser Asp Thr Ile Lys Pro Asp Val Asn Lys Ser Lys             180 185 190 gag tat ttc tct aag cag aag 597 Glu Tyr Phe Ser Lys Gln Lys         195 <210> 13 <211> 1053 <212> DNA <213> Homo sapiens <400> 13 atg agc ttt cca tgt aaa ttt gta gcc agc ttc ctt ctg att ttc aat 48 Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn   1 5 10 15 gtt tct tcc aaa ggt gca gtc tcc aaa gag att acg aat gcc ttg gaa 96 Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu              20 25 30 acc tgg ggt gcc ttg ggt cag gac atc aac ttg gac att cct agt ttt 144 Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe          35 40 45 caa atg agt gat gat att gac gat ata aaa tgg gaa aaa act tca gac 192 Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp      50 55 60 aag aaa aag att gca caa ttc aga aaa gag aaa gag act ttc aag gaa 240 Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu  65 70 75 80 aaa gat aca tat aag cta ttt aaa aat gga act ctg aaa att aag cat 288 Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His                  85 90 95 ctg aag acc gat gat cag gat atc tac aag gta tca ata tat gat aca 336 Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr             100 105 110 aaa gga aaa aat gtg ttg gaa aaa ata ttt gat ttg aag att caa gag 384 Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu         115 120 125 agg gtc tca aaa cca aag atc tcc tgg act tgt atc aac aca acc ctg 432 Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu     130 135 140 acc tgt gag gta atg aat gga act gac ccc gaa tta aac ctg tat caa 480 Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln 145 150 155 160 gat ggg aaa cat cta aaa ctt tct cag agg gtc atc aca cac aag tgg 528 Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp                 165 170 175 acc acc agc ctg agt gca aaa ttc aag tgc aca gca ggg aac aaa gtc 576 Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val             180 185 190 agc aag gaa tcc agt gtc gag cct gtc agc tgt cca gag aaa ggt ctg 624 Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu         195 200 205 gac atc tat ctc atc att ggc ata tgt gga gga ggc agc ctc ttg atg 672 Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met     210 215 220 gtc ttt gtg gca ctg ctc gtt ttc tat atc acc aaa agg aaa aaa cag 720 Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Lys Arg Lys Lys Gln 225 230 235 240 agg agt cgg aga aat gat gag gag ctg gag aca aga gcc cac aga gta 768 Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val                 245 250 255 gct act gaa gaa agg ggc cgg aag ccc caa caa att cca gct tca acc 816 Ala Thr Glu Glu Arg Gly Arg Lys Pro Gln Gln Ile Pro Ala Ser Thr             260 265 270 cct cag aat cca gca act tcc caa cat cct cct cca cca cct ggt cat 864 Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro Pro Pro Pro Gly His         275 280 285 cgt tcc cag gca cct agt cat cgt ccc ccg cct cct gga cac cgt gtt 912 Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro Pro Gly His Arg Val     290 295 300 cag cac cag cct cag aag agg cct cct gct ccg tcg ggc aca caa gtt 960 Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val 305 310 315 320 cac cag cag aaa ggc ccg ccc ctc ccc aga cct cga gtt cag cca aaa 1008 His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys                 325 330 335 cct ccc cat ggg gca gca gaa aac tca ttg tcc cct tcc tct aat 1053 Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn             340 345 350 <210> 14 <211> 321 <212> DNA <213> Homo sapiens <400> 14 aat gat gag gag ctg gag aca aga gcc cac aga gta gct act gaa gaa 48 Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val Ala Thr Glu Glu   1 5 10 15 agg ggc cgg aag ccc caa caa att cca gct tca acc cct cag aat cca 96 Arg Gly Arg Lys Pro Gln Gln Ile Pro Ala Ser Thr Pro Gln Asn Pro              20 25 30 gca act tcc caa cat cct cct cca cca cct ggt cat cgt tcc cag gca 144 Ala Thr Ser Gln His Pro Pro Pro Pro Gly His Arg Ser Gln Ala          35 40 45 cct agt cat cgt ccc ccg cct cct gga cac cgt gtt cag cac cag cct 192 Pro Ser His Arg Pro Pro Pro Pro Gly His Arg Val Gln His Gln Pro      50 55 60 cag aag agg cct cct gct ccg tcg ggc aca caa gtt cac cag cag aaa 240 Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val His Gln Gln Lys  65 70 75 80 ggc ccg ccc ctc ccc aga cct cga gtt cag cca aaa cct ccc cat ggg 288 Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys Pro Pro His Gly                  85 90 95 gca gca gaa aac tca ttg tcc cct tcc tct aat 321 Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn             100 105 <210> 15 <211> 18 <212> PRT <213> Mus musculus <220> <221> unsure <222> (1) <223> Xaa is unsure <220> <221> unsure <222> (2) <223> Xaa is unsure <400> 15 Xaa Xaa Glu Glu Leu Arg Arg Gln Gln Glu Glu Ile Leu Arg Arg Gln   1 5 10 15 Gln Glu <210> 16 <211> 13 <212> PRT <213> Mus musculus <220> <221> unsure <222> (1) <223> Xaa is unsure <220> <221> unsure <222> (3) <223> Xaa is unsure <220> <221> unsure <222> (10) <223> Xaa is unsure <220> <221> unsure <222> (11) <223> Xaa is unsure <220> <221> unsure <222> (12) <223> Xaa is unsure <400> 16 Xaa Val Xaa Arg Pro Asn Phe Glu Glu Xaa Xaa Xaa Thr   1 5 10 <210> 17 <211> 15 <212> PRT <213> Mus musculus <220> <221> unsure <222> (1) <223> Xaa is unsure <220> <221> unsure <222> (8) <223> Xaa is unsure <220> <221> unsure <222> (9) <223> Xaa is unsure <220> <221> unsure <222> (14) <223> Xaa is unsure <400> 17 Xaa Leu Leu Glu Ile Gln Gln Xaa Xaa Ala Arg Gln Met Xaa Lys   1 5 10 15 <210> 18 <211> 10 <212> PRT <213> Mus musculus <220> <221> unsure <222> (1) <223> Xaa is unsure <220> <221> unsure <222> (8) <223> Xaa is unsure <400> 18 Xaa Val Ala Tyr Leu Gln Asp Xaa Ala Leu   1 5 10 <210> 19 <211> 8 <212> PRT <213> Mus musculus <220> <221> unsure <222> (1) <223> Xaa is unsure <400> 19 Xaa Pro Ser Asp Leu Leu Glu Lys   1 5 <210> 20 <211> 7 <212> PRT <213> Mus musculus <400> 20 Ile Glu Asn Asn Phe Glu Lys   1 5 <210> 21 <211> 18 <212> PRT <213> Mus musculus <400> 21 Ala Val Glu Gln Phe Arg Arg Leu Leu Thr Gln Glu Asn Arg Arg Val   1 5 10 15 Thr Lys <210> 22 <211> 9 <212> PRT <213> Mus musculus <220> <221> unsure <222> (6) <223> Xaa is unsure <400> 22 Ile Pro Phe Ala Ser Xaa Ala Ser Arg   1 5 <210> 23 <211> 9 <212> PRT <213> Mus musculus <220> <221> unsure <222> (5) <223> Xaa is unsure <220> <221> unsure <222> (7) <223> Xaa is unsure <400> 23 Phe Pro Ser Leu Xaa Ile Xaa Ser Pro   1 5 <210> 24 <211> 19 <212> PRT <213> Mus musculus <400> 24 Ala Val Thr Val Tyr Thr Asp Tyr Pro Leu Pro Gly Glu Ala Phe Asp   1 5 10 15 Arg Glu Lys <210> 25 <211> 10 <212> PRT <213> Mus musculus <220> <221> unsure <222> (4) <223> Xaa is unsure <220> <221> unsure <222> (9) <223> Xaa is unsure <400> 25 His Gly Glu Val Xaa Pro Ala Gly Xaa Lys   1 5 10 <210> 26 <211> 7 <212> PRT <213> Mus musculus <400> 26 Asp Ile Ser Leu Ser Gle Tyr   1 5 <210> 27 <211> 16 <212> PRT <213> Mus musculus <220> <221> unsure <222> (13) <223> Xaa is unsure <400> 27 Glu Gly Gly Leu Gly Pro Met Asn Ieu Pro Leu Ile Xaa Asp Pro Lys   1 5 10 15 <210> 28 <211> 7 <212> PRT <213> Homo sapiens <400> 28 Glu Thr Tyr Gln Lys Ser Lys   1 5 <210> 29 <211> 10 <212> PRT <213> Homo sapiens <400> 29 Pro Tyr Tyr Leu Gly Pro Val Val Glu Lys   1 5 10 <210> 30 <211> 6 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (4) <223> Xaa is unsure <400> 30 Leu Ile Glu Xaa Leu Ser   1 5 <210> 31 <211> 13 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (4) <223> Xaa is unsure <220> <221> unsure <222> (12) <223> Xaa is unsure <400> 31 Glu Ala Thr Xaa Ser Glu Thr Asn Ser Glu Val Xaa Val   1 5 10 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <400> 32 gatcctcgag atggcagcgg aaacgcagac 30 <210> 33 <211> 31 <212> DNA <213> Artificial Sequence <400> 33 gtgatcgaat tcccttcggt aaccccgttc a 31 <210> 34 <211> 34 <212> DNA <213> Artificial Sequence <400> 34 gatcctcgag ggcacttctt cttcaacagt ttca 34 <210> 35 <211> 33 <212> DNA <213> Artificial Sequence <400> 35 gatctctaga tcagtagtca tccaacgtct cga 33 <210> 36 <211> 4704 <212> DNA <213> Artificial Sequence <400> 36 atg ggc agc agc cat cat cat cat cat cac agc agc ggc ctg gtg ccg 48 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro   1 5 10 15 cgc ggc agc cat agc ccc acc atg gac tac aag gac gac gat gac aag 96 Arg Gly Ser His Ser Pro Thr Met Asp Tyr Lys Asp Asp Asp Asp Lys              20 25 30 cat agg ccg caa atg gga cac agt aaa cag att cga att tta ctt ctg 144 His Arg Pro Gln Met Gly His Ser Lys Gln Ile Arg Ile Leu Leu Leu          35 40 45 aac gaa atg gag aaa ctg gaa aag acc ctc ttc aga ctt gaa caa ggg 192 Asn Glu Met Glu Lys Leu Glu Lys Thr Leu Phe Arg Leu Glu Gln Gly      50 55 60 tat gag cta cag ttc cga tta ggc cca act tta cag gga aaa gca gtt 240 Tyr Glu Leu Gln Phe Arg Leu Gly Pro Thr Leu Gln Gly Lys Ala Val  65 70 75 80 acc gtg tat aca aat tac cca ttt cct gga gaa aca ttt aat aga gaa 288 Thr Val Tyr Thr Asn Tyr Pro Phe Pro Gly Glu Thr Phe Asn Arg Glu                  85 90 95 aaa ttc cgt tct ctg gat tgg gaa aat cca aca gaa aga gaa gat gat 336 Lys Phe Arg Ser Leu Asp Trp Glu Asn Pro Thr Glu Arg Glu Asp Asp             100 105 110 tct gat aaa tac tgt aaa ctt aat ctg caa caa tct ggt tca ttt cag 384 Ser Asp Lys Tyr Cys Lys Leu Asn Leu Gln Gln Ser Gly Ser Phe Gln         115 120 125 tat tat ttc ctt caa gga aat gag aaa agt ggt gga ggt tac ata gtt 432 Tyr Tyr Phe Leu Gln Gly Asn Glu Lys Ser Gly Gly Gly Tyr Ile Val     130 135 140 gtg gac ccc att tta cgt gtt ggt gct gat aat cat gtg cta ccc ttg 480 Val Asp Pro Ile Leu Arg Val Gly Ala Asp Asn His Val Leu Pro Leu 145 150 155 160 gac tgt gtt act ctt cag aca ttt tta gct aag tgt ttg gga cct ttt 528 Asp Cys Val Thr Leu Gln Thr Phe Leu Ala Lys Cys Leu Gly Pro Phe                 165 170 175 gat gaa tgg gaa agc aga ctt agg gtt gca aaa gaa tca ggc tac aac 576 Asp Glu Trp Glu Ser Arg Leu Arg Val Ala Lys Glu Ser Gly Tyr Asn             180 185 190 atg att cat ttt acc cca ttg cag act ctt gga cta tct agg tca tgc 624 Met Ile His Phe Thr Pro Leu Gln Thr Leu Gly Leu Ser Arg Ser Cys         195 200 205 tac tcc ctt gcc aat cag tta gaa tta aat cct gac ttt tca aga cct 672 Tyr Ser Leu Ala Asn Gln Leu Glu Leu Asn Pro Asp Phe Ser Arg Pro     210 215 220 aat aga aag tat acc tgg aat gat gtt gga cag cta gtg gaa aaa tta 720 Asn Arg Lys Tyr Thr Trp Asn Asp Val Gly Gln Leu Val Glu Lys Leu 225 230 235 240 aaa aag gaa tgg aat gtt att tgt att act gat gtt gtc tac aat cat 768 Lys Lys Glu Trp Asn Val Ile Cys Ile Thr Asp Val Val Tyr Asn His                 245 250 255 act gct gct aat agt aaa tgg atc cag gaa cat cca gaa tgt gcc tat 816 Thr Ala Ala Asn Ser Lys Trp Ile Gln Glu His Pro Glu Cys Ala Tyr             260 265 270 aat ctt gtg aat tct cca cac tta aaa cct gcc tgg gtc tta gac aga 864 Asn Leu Val Asn Ser Pro His Leu Lys Pro Ala Trp Val Leu Asp Arg         275 280 285 gca ctt tgg cgt ttc tcc tgt gat gtt gca gaa ggg aaa tac aaa gaa 912 Ala Leu Trp Arg Phe Ser Cys Asp Val Ala Glu Gly Lys Tyr Lys Glu     290 295 300 aag gga ata cct gct ttg att gaa aat gat cac cat atg aat tcc atc 960 Lys Gly Ile Pro Ala Leu Ile Glu Asn Asp His His Met Asn Ser Ile 305 310 315 320 cga aaa ata att tgg gag gat att ttt cca aag ctt aaa ctc tgg gaa 1008 Arg Lys Ile Ile Trp Glu Asp Ile Phe Pro Lys Leu Lys Leu Trp Glu                 325 330 335 ttt ttc caa gta gat gtc aac aaa gcg gtt gag caa ttt aga aga ctt 1056 Phe Phe Gln Val Asp Val Asn Lys Ala Val Glu Gln Phe Arg Arg Leu             340 345 350 ctt aca caa gaa aat agg cga gta acc aag tct gat cca aac caa cac 1104 Leu Thr Gln Glu Asn Arg Arg Val Thr Lys Ser Asp Pro Asn Gln His         355 360 365 ctt acg att att caa gat cct gaa tac aga cgg ttt ggc tgt act gta 1152 Leu Thr Ile Ile Gln Asp Pro Glu Tyr Arg Arg Phe Gly Cys Thr Val     370 375 380 gat atg aac att gca cta acg act ttc ata cca cat gac aag ggg cca 1200 Asp Met Asn Ile Ala Leu Thr Thr Phe Ile Pro His Asp Lys Gly Pro 385 390 395 400 gca gca att gaa gaa tgc tgt aat tgg ttt cat aaa aga atg gag gaa 1248 Ala Ala Ile Glu Glu Cys Cys Asn Trp Phe His Lys Arg Met Glu Glu                 405 410 415 tta aat tca gag aag cat cga ctc att aac tat cat cag gaa cag gca 1296 Leu Asn Ser Glu Lys His Arg Leu Ile Asn Tyr His Gln Glu Gln Ala             420 425 430 gtt aat tgc ctt ttg gga aat gtg ttt tat gaa cga ctg gct ggc cat 1344 Val Asn Cys Leu Leu Gly Asn Val Phe Tyr Glu Arg Leu Ala Gly His         435 440 445 ggt cca aaa cta gga cct gtc act aga aag cat cct tta gtt acc agg 1392 Gly Pro Lys Leu Gly Pro Val Thr Arg Lys His Pro Leu Val Thr Arg     450 455 460 tat ttt act ttc cca ttt gaa gag ata gac ttc tcc atg gaa gaa tct 1440 Tyr Phe Thr Phe Pro Phe Glu Glu Ile Asp Phe Ser Met Glu Glu Ser 465 470 475 480 atg att cat ctg cca aat aaa gct tgt ttt ctg atg gca cac aat gga 1488 Met Ile His Leu Pro Asn Lys Ala Cys Phe Leu Met Ala His Asn Gly                 485 490 495 tgg gta atg gga gat gat cct ctt cga aac ttt gct gaa ccg ggt tca 1536 Trp Val Met Gly Asp Asp Pro Leu Arg Asn Phe Ala Glu Pro Gly Ser             500 505 510 gaa gtt tac cta agg aga gaa ctt att tgc tgg gga gac agt gtt aaa 1584 Glu Val Tyr Leu Arg Arg Glu Leu Ile Cys Trp Gly Asp Ser Val Lys         515 520 525 tta cgc tat ggg aat aaa cca gag gac tgt cct tat ctc tgg gca cac 1632 Leu Arg Tyr Gly Asn Lys Pro Glu Asp Cys Pro Tyr Leu Trp Ala His     530 535 540 atg aaa aaa tac act gaa ata act gca act tat ttc cag gga gta cgt 1680 Met Lys Lys Tyr Thr Glu Ile Thr Ala Thr Tyr Phe Gln Gly Val Arg 545 550 555 560 ctt gat aac tgc cac tca aca cct ctt cac gta gct gag tac atg ttg 1728 Leu Asp Asn Cys His Ser Thr Pro Leu His Val Ala Glu Tyr Met Leu                 565 570 575 gat gct gct agg aat ttg caa ccc aat tta tat gta gta gct gaa ctg 1776 Asp Ala Ala Arg Asn Leu Gln Pro Asn Leu Tyr Val Val Ala Glu Leu             580 585 590 ttc aca gga agt gaa gat ctg gac aat gtc ttt gtt act aga ctg ggc 1824 Phe Thr Gly Ser Glu Asp Leu Asp Asn Val Phe Val Thr Arg Leu Gly         595 600 605 att agt tcc tta ata aga gag gca atg agt gca tat aat agt cat gaa 1872 Ile Ser Ser Leu Ile Arg Glu Ala Met Ser Ala Tyr Asn Ser His Glu     610 615 620 gag ggc aga tta gtt tac cga tat gga gga gaa cct gtt gga tcc ttt 1920 Glu Gly Arg Leu Val Tyr Arg Tyr Gly Gly Glu Pro Val Gly Ser Phe 625 630 635 640 gtt cag ccc tgt ttg agg cct tta atg cca gct att gca cat gcc ctg 1968 Val Gln Pro Cys Leu Arg Pro Leu Met Pro Ala Ile Ala His Ala Leu                 645 650 655 ttt atg gat att acg cat gat aat gag tgt cct att gtg cat aga tca 2016 Phe Met Asp Ile Thr His Asp Asn Glu Cys Pro Ile Val His Arg Ser             660 665 670 gcg tat gat gct ctt cca agt act aca att gtt tct atg gca tgt tgt 2064 Ala Tyr Asp Ala Leu Pro Ser Thr Thr Ile Val Ser Met Ala Cys Cys         675 680 685 gct agt gga agt aca aga ggc tat gat gaa tta gtg cct cat cag att 2112 Ala Ser Gly Ser Thr Arg Gly Tyr Asp Glu Leu Val Pro His Gln Ile     690 695 700 tca gtg gtt tct gaa gaa cgg ttt tac act aag tgg aat cct gaa gca 2160 Ser Val Val Ser Glu Glu Arg Phe Tyr Thr Lys Trp Asn Pro Glu Ala 705 710 715 720 ttg cct tca aac aca ggt gaa gtt aat ttc caa agc ggc att att gca 2208 Leu Pro Ser Asn Thr Gly Glu Val Asn Phe Gln Ser Gly Ile Ile Ala                 725 730 735 gcc agg tgt gct atc agt aaa ctt cat cag gag ctt gga gcc aag ggt 2256 Ala Arg Cys Ala Ile Ser Lys Leu His Gln Glu Leu Gly Ala Lys Gly             740 745 750 ttt att cag gtg tat gtg gat caa gtt gat gaa gac ata gtg gca gta 2304 Phe Ile Gln Val Tyr Val Asp Gln Val Asp Glu Asp Ile Val Ala Val         755 760 765 aca aga cac tca cct agc atc cat cag tct gtt gtg gct gta tct aga 2352 Thr Arg His Ser Pro Ser Ile His Gln Ser Val Val Ala Val Ser Arg     770 775 780 act gct ttc agg aat ccc aag act tca ttt tac agc aag gaa gtg cct 2400 Thr Ala Phe Arg Asn Pro Lys Thr Ser Phe Tyr Ser Lys Glu Val Pro 785 790 795 800 caa atg tgc atc cct ggc aaa att gaa gaa gta gtt ctt gaa gct aga 2448 Gln Met Cys Ile Pro Gly Lys Ile Glu Glu Val Val Leu Glu Ala Arg                 805 810 815 act att gag aga aac acg aaa cct tat agg aag gat gag aat tca atc 2496 Thr Ile Glu Arg Asn Thr Lys Pro Tyr Arg Lys Asp Glu Asn Ser Ile             820 825 830 aat gga aca cca gat atc aca gta gaa att aga gaa cat att cag ctt 2544 Asn Gly Thr Pro Asp Ile Thr Val Glu Ile Arg Glu His Ile Gln Leu         835 840 845 aat gaa agt aaa att gtt aaa caa gct gga gtt gcc aca aaa ggg ccc 2592 Asn Glu Ser Lys Ile Val Lys Gln Ala Gly Val Ala Thr Lys Gly Pro     850 855 860 aat gaa tat att caa gaa ata gaa ttt gaa aac ttg tct cca gga agt 2640 Asn Glu Tyr Ile Gln Glu Ile Glu Phe Glu Asn Leu Ser Pro Gly Ser 865 870 875 880 gtt att ata ttc aga gtt agt ctt gat cca cat gca caa gtc gct gtt 2688 Val Ile Ile Phe Arg Val Ser Leu Asp Pro His Ala Gln Val Ala Val                 885 890 895 gga att ctt cga aat cat ctg aca caa ttc agt cct cac ttt aaa tct 2736 Gly Ile Leu Arg Asn His Leu Thr Gln Phe Ser Pro His Phe Lys Ser             900 905 910 ggc agc cta gct gtt gac aat gca gat cct ata tta aaa att cct ttt 2784 Gly Ser Leu Ala Val Asp Asn Ala Asp Pro Ile Leu Lys Ile Pro Phe         915 920 925 gct tct ctt gcc tcc aga tta act ttg gct gag cta aat cag atc ctt 2832 Ala Ser Leu Ala Ser Arg Leu Thr Leu Ala Glu Leu Asn Gln Ile Leu     930 935 940 tac cga tgt gaa tca gaa gaa aag gaa gat ggt gga ggg tgc tat gac 2880 Tyr Arg Cys Glu Ser Glu Glu Lys Glu Asp Gly Gly Gly Cys Tyr Asp 945 950 955 960 ata cca aac tgg tca gcc ctt aaa tat gca ggt ctt caa ggt tta atg 2928 Ile Pro Asn Trp Ser Ala Leu Lys Tyr Ala Gly Leu Gln Gly Leu Met                 965 970 975 tct gta ttg gca gaa ata aga cca aag aat gac ttg ggg cat cct ttt 2976 Ser Val Leu Ala Glu Ile Arg Pro Lys Asn Asp Leu Gly His Pro Phe             980 985 990 tgt aat aat ttg aga tct gga gat tgg atg att gac tat gtc agt aac 3024 Cys Asn Asn Leu Arg Ser Gly Asp Trp Met Ile Asp Tyr Val Ser Asn         995 1000 1005 cgg ctt att tca cga tca gga act att gct gaa gtt ggt aaa tgg ttg 3072 Arg Leu Ile Ser Arg Ser Gly Thr Ile Ala Glu Val Gly Lys Trp Leu     1010 1015 1020 cag gct atg ttc ttc tac ctg aag cag atc cca cgt tac ctt atc cca 3120 Gln Ala Met Phe Phe Tyr Leu Lys Gln Ile Pro Arg Tyr Leu Ile Pro 1025 1030 1035 1040 tgt tac ttt gat gct ata tta att ggt gca tat acc act ctt ctg gat 3168 Cys Tyr Phe Asp Ala Ile Leu Ile Gly Ala Tyr Thr Thr Leu Leu Asp                 1045 1050 1055 aca gca tgg aag cag atg tca agc ttt gtt cag aat ggt tca acc ttt 3216 Thr Ala Trp Lys Gln Met Ser Ser Phe Val Gln Asn Gly Ser Thr Phe             1060 1065 1070 gtg aaa cac ctt tca ttg ggt tca gtt caa ctg tgt gga gta gga aaa 3264 Val Lys His Leu Ser Leu Gly Ser Val Gln Leu Cys Gly Val Gly Lys         1075 1080 1085 ttc cct tcc ctg cca att ctt tca cct gcc cta atg gat gta cct tat 3312 Phe Pro Ser Leu Pro Ile Leu Ser Pro Ala Leu Met Asp Val Pro Tyr     1090 1095 1100 agg tta aat gag atc aca aaa gaa aag gag caa tgt tgt gtt tct cta 3360 Arg Leu Asn Glu Ile Thr Lys Glu Lys Glu Gln Cys Cys Val Ser Leu 1105 1110 1115 1120 gct gca ggc tta cct cat ttt tct tct ggt att ttc cgc tgc tgg gga 3408 Ala Ala Gly Leu Pro His Phe Ser Ser Gly Ile Phe Arg Cys Trp Gly                 1125 1130 1135 agg gat act ttt att gca ctt aga ggt ata ctg ctg att act aga cgc 3456 Arg Asp Thr Phe Ile Ala Leu Arg Gly Ile Leu Leu Ile Thr Arg Arg             1140 1145 1150 tat gta gaa gcc agg aat att att tta gca ttt gcg ggt acc ctg agg 3504 Tyr Val Glu Ala Arg Asn Ile Ile Leu Ala Phe Ala Gly Thr Leu Arg         1155 1160 1165 cat ggt ctc att cct aat cta ctg ggt gaa gga att tat gcc aga tac 3552 His Gly Leu Ile Pro Asn Leu Leu Gly Glu Gly Ile Tyr Ala Arg Tyr     1170 1175 1180 aat tgt cgg gat gct gtg tgg tgg tgg ctg cag tgt atc cag gat tac 3600 Asn Cys Arg Asp Ala Val Trp Trp Trp Leu Gln Cys Ile Gln Asp Tyr 1185 1190 1195 1200 tgt aaa atg gtt cca aat ggt cta gac att ctc aag tgc cca gtt tcc 3648 Cys Lys Met Val Pro Asn Gly Leu Asp Ile Leu Lys Cys Pro Val Ser                 1205 1210 1215 aga atg tat cct aca gat gat tct gct cct ttg cct gct ggc aca ctg 3696 Arg Met Tyr Pro Thr Asp Asp Ser Ala Pro Leu Pro Ala Gly Thr Leu             1220 1225 1230 gat cag cca ttg ttt gaa gtc ata cag gaa gca atg caa aaa cac atg 3744 Asp Gln Pro Leu Phe Glu Val Ile Gln Glu Ala Met Gln Lys His Met         1235 1240 1245 cag ggc ata cag ttc cga gaa agg aat gct ggt ccc cag ata gat cga 3792 Gln Gly Ile Gln Phe Arg Glu Arg Asn Ala Gly Pro Gln Ile Asp Arg     1250 1255 1260 aac atg aag gac gaa ggt ttt aat ata act gca gga gtt gat gaa gaa 3840 Asn Met Lys Asp Glu Gly Phe Asn Ile Thr Ala Gly Val Asp Glu Glu 1265 1270 1275 1280 aca gga ttt gtt tat gga gga aat cgt ttc aat tgt ggc aca tgg atg 3888 Thr Gly Phe Val Tyr Gly Gly Asn Arg Phe Asn Cys Gly Thr Trp Met                 1285 1290 1295 gat aaa atg gga gaa agt gac aga gct aga aac aga gga atc cca gcc 3936 Asp Lys Met Gly Glu Ser Asp Arg Ala Arg Asn Arg Gly Ile Pro Ala             1300 1305 1310 aca cca aga gat ggg tct gct gtg gaa att gtg ggc ctg agt aaa tct 3984 Thr Pro Arg Asp Gly Ser Ala Val Glu Ile Val Gly Leu Ser Lys Ser         1315 1320 1325 gct gtt cgc tgg ttg ctg gaa tta tcc aaa aaa aat att ttc cct tat 4032 Ala Val Arg Trp Leu Leu Glu Leu Ser Lys Lys Asn Ile Phe Pro Tyr     1330 1335 1340 cat gaa gtc cca gta aaa aga cat gga aag gct ata aag gtc tca tat 4080 His Glu Val Pro Val Lys Arg His Gly Lys Ala Ile Lys Val Ser Tyr 1345 1350 1355 1360 gat gag tgg acc aga aaa ata caa gac aac ttt gaa aag cta ttt cat 4128 Asp Glu Trp Thr Arg Lys Ile Gln Asp Asn Phe Glu Lys Leu Phe His                 1365 1370 1375 gtt tcc gaa gac cct tca gat tta aat gaa aag cat cca aat ctg gtt 4176 Val Ser Glu Asp Pro Ser Asp Leu Asn Glu Lys His Pro Asn Leu Val             1380 1385 1390 cac aaa cgt ggc ata tac aaa gat agt tat gga gct tca agt cct tgg 4224 His Lys Arg Gly Ile Tyr Lys Asp Ser Tyr Gly Ala Ser Ser Pro Trp         1395 1400 1405 tgt gac tat cag ctc agg cct aat ttt acc ata gca atg gtt gtg gcc 4272 Cys Asp Tyr Gln Leu Arg Pro Asn Phe Thr Ile Ala Met Val Val Ala     1410 1415 1420 cct gag ctc ttt act aca gaa aaa gca tgg aaa gct ttg gag att gca 4320 Pro Glu Leu Phe Thr Thr Glu Lys Ala Trp Lys Ala Leu Glu Ile Ala 1425 1430 1435 1440 gaa aaa aaa ttg ctt ggt ccc ctt ggc atg aaa act tta gat cca gat 4368 Glu Lys Lys Leu Leu Gly Pro Leu Gly Met Lys Thr Leu Asp Pro Asp                 1445 1450 1455 gat atg gtt tac tgt gga att tat gac aat gca tta gac aat gac aac 4416 Asp Met Val Tyr Cys Gly Ile Tyr Asp Asn Ala Leu Asp Asn Asp Asn             1460 1465 1470 tac aat ctt gct aaa ggt ttc aat tat cac caa gga cct gag tgg ctg 4464 Tyr Asn Leu Ala Lys Gly Phe Asn Tyr His Gln Gly Pro Glu Trp Leu         1475 1480 1485 tgg cct att ggg tat ttt ctt cgt gca aaa tta tat ttt tcc aga ttg 4512 Trp Pro Ile Gly Tyr Phe Leu Arg Ala Lys Leu Tyr Phe Ser Arg Leu     1490 1495 1500 atg ggc ccg gag act act gca aag act ata gtt ttg gtt aaa aat gtt 4560 Met Gly Pro Glu Thr Thr Ala Lys Thr Ile Val Leu Val Lys Asn Val 1505 1510 1515 1520 ctt tcc cga cat tat gtt cat ctt gag aga tcc cct tgg aaa gga ctt 4608 Leu Ser Arg His Tyr Val His Leu Glu Arg Ser Pro Trp Lys Gly Leu                 1525 1530 1535 cca gaa ctg acc aat gag aat gcc cag tac tgt cct ttc agc tgt gaa 4656 Pro Glu Leu Thr Asn Glu Asn Ala Gln Tyr Cys Pro Phe Ser Cys Glu             1540 1545 1550 aca caa gcc tgg tca att gct act att ctt gag aca ctt tat gat tta 4704 Thr Gln Ala Trp Ser Ile Ala Thr Ile Leu Glu Thr Leu Tyr Asp Leu         1555 1560 1565 <210> 37 <211> 32 <212> DNA <213> Artificial Sequence <400> 37 gatcctcgag atggagttgg gaaaaggaaa ac 32 <210> 38 <211> 31 <212> DNA <213> Artificial Sequence <400> 38 gatcgaattc tcaaacacca ggcaggagga g 31 <210> 39 <211> 34 <212> DNA <213> Artificial Sequence <400> 39 gactctcgag atgtcttcag gaaatgcaaa aatt 34 <210> 40 <211> 32 <212> DNA <213> Artificial Sequence <400> 40 gatcggatcc tggtccagtg ctcacttctg ct 32 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <400> 41 gatcctcgag aatgatgagg agctggagac aag 33 <210> 42 <211> 35 <212> DNA <213> Artificial Sequence <400> 42 gatcggatcc ttaattagag gaaggggaca ataag 35 <210> 43 <211> 341 <212> PRT <213> Homo sapiens <400> 43 Met Pro Lys Arg Lys Val Thr Phe Gln Gly Val Gly Asp Glu Glu Asp   1 5 10 15 Glu Asp Glu Ile Ile Val Pro Lys Lys Lys Leu Val Asp Pro Val Ala              20 25 30 Gly Ser Gly Gly Pro Gly Ser Arg Phe Lys Gly Lys His Ser Leu Asp          35 40 45 Ser Asp Glu Glu Glu Asp Asp Asp Asp Gly Gly Ser Ser Lys Tyr Asp      50 55 60 Ile Leu Ala Ser Glu Asp Val Glu Gly Gln Glu Ala Ala Thr Leu Pro  65 70 75 80 Ser Glu Gly Gly Val Arg Ile Thr Pro Phe Asn Leu Gln Glu Glu Met                  85 90 95 Glu Glu Gly His Phe Asp Ala Asp Gly Asn Tyr Phe Leu Asn Arg Asp             100 105 110 Ala Gln Ile Arg Asp Ser Trp Leu Asp Asn Ile Asp Trp Val Lys Ile         115 120 125 Arg Glu Arg Pro Pro Gly Gln Arg Gln Ala Ser Asp Ser Glu Glu Glu     130 135 140 Asp Ser Leu Gly Gln Thr Ser Met Ser Ala Gln Ala Leu Leu Glu Gly 145 150 155 160 Leu Leu Glu Leu Leu Leu Pro Arg Glu Thr Val Ala Gly Ala Leu Arg                 165 170 175 Arg Leu Gly Ala Arg Gly Gly Gly Lys Gly Arg Lys Gly Pro Gly Gln             180 185 190 Pro Ser Ser Pro Gln Arg Leu Asp Arg Leu Ser Gly Leu Ala Asp Gln         195 200 205 Met Val Ala Arg Gly Asn Leu Gly Val Tyr Gln Glu Thr Arg Glu Arg     210 215 220 Leu Ala Met Arg Leu Lys Gly Leu Gly Cys Gln Thr Leu Gly Pro His 225 230 235 240 Asn Pro Thr Pro Pro Pro Ser Leu Asp Met Phe Ala Glu Glu Leu Ala                 245 250 255 Glu Glu Glu Leu Glu Thr Pro Thr Pro Thr Gln Arg Gly Glu Ala Glu             260 265 270 Ser Arg Gly Asp Gly Leu Val Asp Val Met Trp Glu Tyr Lys Trp Glu         275 280 285 Asn Thr Gly Asp Ala Glu Leu Tyr Gly Pro Phe Thr Ser Ala Gln Met     290 295 300 Gln Thr Trp Val Ser Glu Gly Tyr Phe Pro Asp Gly Val Tyr Cys Arg 305 310 315 320 Lys Leu Asp Pro Pro Gly Gly Gln Phe Tyr Asn Ser Lys Arg Ile Asp                 325 330 335 Phe Asp Leu Tyr Thr             340 <210> 44 <211> 1023 <212> DNA <213> Homo sapiens <400> 44 atg cca aag agg aaa gtg acc ttc caa ggc gtg gga gat gag gag gat 48 Met Pro Lys Arg Lys Val Thr Phe Gln Gly Val Gly Asp Glu Glu Asp   1 5 10 15 gag gat gaa atc att gtc ccc aag aag aag ctg gtg gac cct gtg gct 96 Glu Asp Glu Ile Ile Val Pro Lys Lys Lys Leu Val Asp Pro Val Ala              20 25 30 ggg tca ggg ggt cct ggg agc cgc ttt aaa ggc aaa cac tct ttg gat 144 Gly Ser Gly Gly Pro Gly Ser Arg Phe Lys Gly Lys His Ser Leu Asp          35 40 45 agc gat gag gag gag gat gat gat gat ggg ggg tcc agc aaa tat gac 192 Ser Asp Glu Glu Glu Asp Asp Asp Asp Gly Gly Ser Ser Lys Tyr Asp      50 55 60 atc ttg gcc tca gag gat gta gaa ggt cag gag gca gcc aca ctc ccc 240 Ile Leu Ala Ser Glu Asp Val Glu Gly Gln Glu Ala Ala Thr Leu Pro  65 70 75 80 agc gag ggg ggt gtt cgg atc aca ccc ttt aac ctg cag gag gag atg 288 Ser Glu Gly Gly Val Arg Ile Thr Pro Phe Asn Leu Gln Glu Glu Met                  85 90 95 gag gaa ggc cac ttt gat gcc gat ggc aac tac ttc ctg aac cgg gat 336 Glu Glu Gly His Phe Asp Ala Asp Gly Asn Tyr Phe Leu Asn Arg Asp             100 105 110 gct cag atc cga gac agc tgg ctg gac aac att gac tgg gtg aag atc 384 Ala Gln Ile Arg Asp Ser Trp Leu Asp Asn Ile Asp Trp Val Lys Ile         115 120 125 cgg gag cgg cca cct ggc cag cgc cag gcc tca gac tcg gag gag gag 432 Arg Glu Arg Pro Pro Gly Gln Arg Gln Ala Ser Asp Ser Glu Glu Glu     130 135 140 gac agc ttg ggc cag acc tca atg agt gcc caa gcc ctc ttg gag gga 480 Asp Ser Leu Gly Gln Thr Ser Met Ser Ala Gln Ala Leu Leu Glu Gly 145 150 155 160 ctt ttg gag ctc cta ttg cct aga gag aca gtg gct ggg gca ctg agg 528 Leu Leu Glu Leu Leu Leu Pro Arg Glu Thr Val Ala Gly Ala Leu Arg                 165 170 175 cgt ctg ggg gcc cga gga gga ggc aaa ggg aga aag ggg cct ggg caa 576 Arg Leu Gly Ala Arg Gly Gly Gly Lys Gly Arg Lys Gly Pro Gly Gln             180 185 190 ccc agt tcc cct cag cgc ctg gac cgg ctc tcc ggg ttg gcc gac cag 624 Pro Ser Ser Pro Gln Arg Leu Asp Arg Leu Ser Gly Leu Ala Asp Gln         195 200 205 atg gtg gcc cgg ggc aac ctt ggt gtg tac cag gaa aca agg gaa cgg 672 Met Val Ala Arg Gly Asn Leu Gly Val Tyr Gln Glu Thr Arg Glu Arg     210 215 220 ttg gct atg cgt ctg aag ggt ttg ggg tgt cag acc cta gga ccc cac 720 Leu Ala Met Arg Leu Lys Gly Leu Gly Cys Gln Thr Leu Gly Pro His 225 230 235 240 aat ccc aca ccc cca ccc tcc ctg gac atg ttc gct gag gag ttg gcg 768 Asn Pro Thr Pro Pro Pro Ser Leu Asp Met Phe Ala Glu Glu Leu Ala                 245 250 255 gag gag gaa ctg gag acc cca acc cct acc cag aga gga gaa gca gag 816 Glu Glu Glu Leu Glu Thr Pro Thr Pro Thr Gln Arg Gly Glu Ala Glu             260 265 270 tcg cgg gga gat ggt ctg gtg gat gtg atg tgg gaa tat aag tgg gag 864 Ser Arg Gly Asp Gly Leu Val Asp Val Met Trp Glu Tyr Lys Trp Glu         275 280 285 aac acg ggg gat gcc gag ctg tat ggg ccc ttc acc agc gcc cag atg 912 Asn Thr Gly Asp Ala Glu Leu Tyr Gly Pro Phe Thr Ser Ala Gln Met     290 295 300 cag acc tgg gtg agt gaa ggc tac ttc ccg gac ggt gtt tat tgc cgg 960 Gln Thr Trp Val Ser Glu Gly Tyr Phe Pro Asp Gly Val Tyr Cys Arg 305 310 315 320 aag ctg gac ccc cct ggt ggt cag ttc tac aac tcc aaa cgc att gac 1008 Lys Leu Asp Pro Pro Gly Gly Gln Phe Tyr Asn Ser Lys Arg Ile Asp                 325 330 335 ttt gac ctc tac acc 1023 Phe Asp Leu Tyr Thr             340 <210> 45 <211> 817 <212> PRT <213> Homo sapience <400> 45 Met Gly Glu Arg Lys Cys Trp Leu Ser Thr Ser Arg Arg Thr Arg Trp   1 5 10 15 Val Pro Glu Glu Leu Gln Asp Lys Glu Phe Ala Ala Val Leu Gln Asp              20 25 30 Glu Pro Leu Gln Pro Leu Ala Leu Glu Pro Leu Thr Glu Glu Glu Gln          35 40 45 Ala Ala Ala Val Val Thr Ala Ala Phe Thr Lys Glu Ala Ser Lys Lys      50 55 60 Ala Met Gly Pro Leu Asp Glu Ala Pro Gly Lys Ser Ser Ala Ala Arg  65 70 75 80 Ala Gly Met Thr Ala Arg Cys Gly Phe Glu Glu Gly Gly Ala Gly Pro                  85 90 95 Arg Lys Glu His Ala Arg Ser Asp Ser Glu Asn Trp Arg Ser Leu Arg             100 105 110 Glu Glu Gln Glu Glu Glu Glu Glu Gly Ser Trp Arg Leu Gly Ala Gly         115 120 125 Pro Arg Arg Asp Gly Asp Arg Trp Arg Ser Ala Ser Pro Asp Gly Gly     130 135 140 Pro Arg Ser Ala Gly Trp Arg Glu His Gly Glu Arg Arg Arg Lys Phe 145 150 155 160 Glu Phe Asp Leu Arg Gly Asp Arg Gly Gly Cys Gly Glu Glu Glu Gly                 165 170 175 Arg Gly Gly Gly Gly Ser Ser His Leu Arg Arg Cys Arg Ala Pro Glu             180 185 190 Gly Phe Glu Glu Asp Lys Asp Gly Leu Pro Glu Trp Cys Leu Asp Asp         195 200 205 Glu Asp Glu Glu Met Gly Thr Phe Asp Ala Ser Gly Ala Phe Leu Pro     210 215 220 Leu Lys Leu Phe Ser Ala Pro His Pro Gly Pro Met Gln Lys Gly Pro 225 230 235 240 Lys Glu Pro Ile Pro Glu Glu Gln Glu Leu Asp Phe Gln Gly Leu Glu                 245 250 255 Glu Glu Glu Glu Pro Ser Glu Gly Leu Glu Glu Glu Gly Pro Glu Ala             260 265 270 Asp Asp Ile Arg Gly Ile Gln Leu Ser Pro Gly Val Gly Ser Ser Ala         275 280 285 Gly Pro Pro Gly Asp Leu Glu Asp Asp Glu Gly Leu Lys His Leu Gln     290 295 300 Gln Gly Ile Gln Glu Ala Glu Lys Leu Val Ala Ser Leu Gln Asp Ser 305 310 315 320 Ser Leu Glu Glu Glu Gln Phe Thr Ala Ala Met Gln Thr Gln Gly Leu                 325 330 335 Arg His Ser Ala Ala Ala Thr Ala Leu Pro Leu Ser His Gly Ala Ala             340 345 350 Arg Lys Trp Phe Tyr Lys Asp Pro Gln Gly Glu Ile Gln Gly Pro Phe         355 360 365 Thr Thr Gln Glu Met Ala Glu Trp Phe Gln Ala Gly Tyr Phe Ser Met     370 375 380 Ser Leu Leu Val Lys Arg Gly Cys Asp Glu Gly Phe Gln Pro Leu Gly 385 390 395 400 Glu Val Ile Lys Met Trp Gly Arg Val Pro Phe Ala Pro Gly Pro Ser                 405 410 415 Pro Pro Pro Leu Leu Gly Asn Met Asp Gln Glu Arg Leu Lys Lys Gln             420 425 430 Gln Glu Leu Ala Ala Ala Ala Leu Tyr Gln Gln Leu Gln His Gln Gln         435 440 445 Phe Leu Gln Leu Val Ser Ser Arg Gln Leu Pro Gln Cys Ala Leu Arg     450 455 460 Glu Lys Ala Ala Leu Gly Asp Leu Thr Pro Pro Pro Pro Pro Pro Pro 465 470 475 480 Gln Gln Gln Gln Gln Gln Leu Thr Ala Phe Leu Gln Gln Leu Gln Ala                 485 490 495 Leu Lys Pro Pro Ser Ser Arg Ser Ala Glu Lys Trp Ser Ser Gly Arg             500 505 510 Ser Gly Arg Lys Arg Asn Ala Ser Val Glu Arg Arg Ser Ala Ala Ser         515 520 525 Ser Ser Arg Arg Ser Arg Ser Gly Gly Arg Arg Arg Lys Ser Cys Phe     530 535 540 Gly Ala Ser Thr Trp Gly Leu Ser Met Lys Thr Leu Leu Glu Leu Gln 545 550 555 560 Leu Glu Gly Glu Arg Gln Leu His Lys Gln Pro Pro Pro Arg Glu Pro                 565 570 575 Ala Arg Ala Gln Ala Pro Asn His Arg Val Gln Leu Gly Gly Leu Gly             580 585 590 Thr Ala Pro Leu Asn Gln Trp Val Ser Glu Ala Gly Pro Leu Trp Gly         595 600 605 Gly Pro Asp Lys Ser Gly Gly Gly Ser Ser Gly Leu Gly Leu Trp Glu     610 615 620 Asp Thr Pro Lys Ser Gly Gly Ser Leu Val Arg Gly Leu Gly Leu Lys 625 630 635 640 Asn Ser Arg Ser Ser Pro Ser Leu Ser Asp Ser Tyr Ser His Leu Ser                 645 650 655 Gly Arg Pro Ile Arg Lys Lys Thr Glu Glu Glu Glu Lys Leu Leu Lys             660 665 670 Leu Leu Gln Gly Ile Pro Arg Pro Gln Asp Gly Phe Thr Gln Trp Cys         675 680 685 Glu Gln Met Leu His Thr Leu Ser Ala Thr Gly Ser Leu Asp Val Pro     690 695 700 Met Ala Val Ala Ile Leu Lys Glu Val Glu Ser Pro Tyr Asp Val His 705 710 715 720 Asp Tyr Ile Arg Ser Cys Leu Gly Asp Thr Leu Glu Ala Lys Glu Phe                 725 730 735 Ala Lys Gln Phe Leu Glu Arg Arg Ala Lys Gln Lys Ala Ser Gln Gln             740 745 750 Arg Gln Gln Gln Gln Glu Ala Trp Leu Ser Ser Ala Ser Leu Gln Thr         755 760 765 Ala Phe Gln Ala Asn His Ser Thr Lys Leu Gly Pro Gly Glu Gly Ser     770 775 780 Lys Ala Lys Arg Arg Ala Leu Met Leu His Ser Asp Pro Ser Ile Leu 785 790 795 800 Gly Tyr Ser Leu His Gly Ser Ser Gly Glu Ile Glu Ser Val Asp Asp                 805 810 815 Tyr <210> 46 <211> 124 <212> PRT <213> Homo sapience <400> 46 Tyr Gln Glu Thr Arg Glu Arg Leu Ala Met Arg Leu Lys Gly Leu Gly   1 5 10 15 Cys Gln Thr Leu Gly Pro His Asn Pro Thr Pro Pro Pro Ser Leu Asp              20 25 30 Met Phe Ala Glu Glu Leu Ala Glu Glu Glu Leu Glu Thr Pro Thr Pro          35 40 45 Thr Gln Arg Gly Glu Ala Glu Ser Arg Gly Asp Gly Leu Val Asp Val      50 55 60 Met Trp Glu Tyr Lys Trp Glu Asn Thr Gly Asp Ala Glu Leu Tyr Gly  65 70 75 80 Pro Phe Thr Ser Ala Gln Met Gln Thr Trp Val Ser Glu Gly Tyr Phe                  85 90 95 Pro Asp Gly Val Tyr Cys Arg Lys Leu Asp Pro Pro Gly Gly Gln Phe             100 105 110 Tyr Asn Ser Lys Arg Ile Asp Phe Asp Leu Tyr Thr         115 120 <210> 47 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 47 gatcggatcc atggtggctt atctccaaga ca 32 <210> 48 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 48 gatcgcggcc gctcaggaag acagtgctgc tttctg 36 <210> 49 <211> 153 <212> PRT <213> Homo sapience <400> 49 Met Val Ala Tyr Leu Gln Asp Ser Ala Leu Asp Asp Glu Arg Leu Ala   1 5 10 15 Ser Lys Leu Gln Glu His Arg Ala Lys Gly Val Ser Ile Pro Leu Met              20 25 30 His Glu Ala Met Gln Lys Trp Tyr Tyr Lys Asp Pro Gln Gly Glu Ile          35 40 45 Gln Gly Pro Phe Asn Asn Gln Glu Met Ala Glu Trp Phe Gln Ala Gly      50 55 60 Tyr Phe Thr Met Ser Leu Leu Val Lys Arg Ala Cys Asp Glu Ser Phe  65 70 75 80 Gln Pro Leu Gly Asp Ile Met Lys Met Trp Gly Arg Val Pro Phe Ser                  85 90 95 Pro Gly Pro Ala Pro Pro Pro His Met Gly Glu Leu Asp Gln Glu Arg             100 105 110 Leu Thr Arg Gln Gln Glu Leu Thr Ala Leu Tyr Gln Met Gln His Leu         115 120 125 Gln Tyr Gln Gln Phe Leu Ile Gln Gln Gln Tyr Ala Gln Val Leu Ala     130 135 140 Gln Gln Gln Lys Ala Ala Leu Ser Ser 145 150

[Brief description of drawings]

FIG. 1 is a diagram showing purification of proteins from cell extracts of CTLL-2 and Jurkat. (a) shows SDS-PAGE by 7.5% acrylamide gel, (b) shows SDS-PAGE by 15% acrylamide gel. The arrow above lane 2 in (a) is p150a,
The arrow below lane2 in (a) is p150c, and the arrow in lane5 in (a) is p8.
The lane 2 arrow in (5) and (b) indicates p27.

[Fig. 2] Recombinant GST-p150a, recombinant His-p150c, recombinant GST-
It is a figure which shows the result of SDS-PAGE of p85, recombinant His-p27, and recombinant His-CD2.

[Explanation of symbols]

1 Protein purified from CTLL-2 cell extract with SGNEGDE particles 2 Protein purified from CTLL-2 cell extract with LK6-A immobilized particles 3 Protein purified from CTLL-2 cell extract with LK6D7 immobilized particles Protein 4 Protein purified from Jurkat cell extract with SGNEGDE particles Protein 5 purified from Jurkat cell extract with LK6-A immobilized particles 6 Protein M purified from Jurkat cell extract with LK6D7 immobilized particles M molecular weight marker

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12P 21/02 C 4C084 5/10 C12Q 1/68 A 4C086 C12P 21/02 G01N 33/15 Z 4H045 C12Q 1/68 33/50 Z G01N 33/15 33/53 D 33/50 33/566 33/53 A61K 31/4745 33/566 C12N 15/00 ZNAA // A61K 31/4745 5/00 B C (72) Inventor Akira Okazaki 3-6-6 Asahimachi, Machida-shi, Tokyo Kyowa Fermentation Industry Co., Ltd. Tokyo Laboratory (72) Inventor Seiji Sugimoto 1-1-6 Otemachi, Chiyoda-ku, Tokyo Kyowa In-house of Fermentation Industry Co., Ltd. (72) Inventor Chitose Koneyama 3-6-6 Asahimachi, Machida, Tokyo Kyowa Fermentation Co., Ltd. Tokyo Research Laboratory F-term (reference) 2G045 AA34 AA35 BB01 BB0 7 BB10 BB20 BB24 BB50 BB51 DA13 DA36 FB02 4B024 AA11 BA80 CA04 DA06 EA04 GA11 HA12 4B063 QA18 QQ08 QQ13 QQ42 QR59 QR77 QR80 QS12 QS24 QS28 QS28 QS28 QS28 4860 A4 CA0 A4 CA0 A4 CA0 CA24 CC13 CC24 CA13 MA01 MA04 NA14 ZB07 4H045 AA10 AA20 AA30 BA10 CA40 DA76 DA86 EA50 FA74

Claims (27)

[Claims] 1. The following formula (I) [Chemical 1] [In the formula, R¹Is a substituted or unsubstituted lower alkyl, low
Grade alkanoyl, carboxy, lower alkoxy carbonyl
The following formula (II) [Chemical 2] (In the formula, n represents 1 or 2) or a group represented by C
OCH = CHR⁹{In the formula, R⁹Is a substituted or unsubstituted low
Primary alkoxy, substituted or unsubstituted aryl, substituted also
Or unsubstituted heteroaryl or NR^TenR¹¹(formula
Medium, R^TenAnd R ¹¹Are the same or different and are hydrogen or substituted
Or unsubstituted lower alkyl, substituted or unsubstituted
Lower alkenyl, substituted or unsubstituted lower alkynyl
, Substituted or unsubstituted aralkyl, substituted or non-substituted
Substituted aryl, substituted or unsubstituted heteroaryl
Group, substituted or unsubstituted heteroaryl-substituted lower alkyl
Killed, substituted or unsubstituted tetrahydropyranyl or
Is substituted or unsubstituted tetrahydropyranylmethyl
Represents or R^TenAnd R¹¹Can be replaced together with the adjacent N
To form an unsubstituted heterocyclic group)}, R²Is
Hydrogen, substituted or unsubstituted lower alkyl, substituted or
Is unsubstituted lower alkenyl, substituted or unsubstituted lower
Alkynyl, substituted or unsubstituted aryl, substituted if
Or unsubstituted heteroaryl, substituted or unsubstituted low
Alkanoyl, substituted or unsubstituted lower alkanoyl
Roxy, halogen, SR¹²(In the formula, R¹²Is a replacement
Is unsubstituted lower alkyl, substituted or unsubstituted aryl
Group, substituted or unsubstituted heteroaryl, substituted or
Is an unsubstituted aralkyl, a substituted or unsubstituted heteroaralkyl
Reel-substituted lower alkyl, substituted or unsubstituted tetra
Hydropyranyl or substituted or unsubstituted tetrahydr
Represents lopyranylmethyl), NR¹³R¹⁴(In the formula, R¹³Oh
And R¹⁴Is the above R^TenAnd R¹¹Synonymous with
R) or azide, and R^FourAnd R^FiveAre the same or different
Becomes hydrogen, substituted or unsubstituted lower alkyl,
Substituted or unsubstituted lower alkanoyl, substituted or unsubstituted
Substituted lower alkoxycarbonyl, substituted or unsubstituted
Aralkyloxycarbonyl or substituted or non-substituted
Alternative heteroaryl substituted lower alkoxycarbonyl
And R⁶Represents hydrogen or halogen, R⁷And R⁸Is
Identical or different, hydrogen, substituted or unsubstituted lower
Alkyl or substituted or unsubstituted lower alkanoyl
Represents the compound] (hereinafter referred to as LK6-A or its derivative).
Contact the carrier to which the conductor (abbreviated) is immobilized with the test protein sample
The compound represented by the above formula (I) from the test protein sample.
Characterized by selecting a protein that specifically binds to an object
To find proteins that bind to LK6-A or its derivatives
Law. 2. The following formula (I) [Chemical 3] [In the formula, R¹Is a substituted or unsubstituted lower alkyl, low
Grade alkanoyl, carboxy, lower alkoxy carbonyl
The following formula (II) [Chemical 4] (In the formula, n represents 1 or 2) or a group represented by C
OCH = CHR⁹{In the formula, R⁹Is a substituted or unsubstituted low
Primary alkoxy, substituted or unsubstituted aryl, substituted also
Or unsubstituted heteroaryl or NR^TenR¹¹(formula
Medium, R^TenAnd R ¹¹Are the same or different and are hydrogen or substituted
Or unsubstituted lower alkyl, substituted or unsubstituted
Lower alkenyl, substituted or unsubstituted lower alkynyl
, Substituted or unsubstituted aralkyl, substituted or non-substituted
Substituted aryl, substituted or unsubstituted heteroaryl
Group, substituted or unsubstituted heteroaryl-substituted lower alkyl
Killed, substituted or unsubstituted tetrahydropyranyl or
Is substituted or unsubstituted tetrahydropyranylmethyl
Represents or R^TenAnd R¹¹Can be replaced together with the adjacent N
To form an unsubstituted heterocyclic group)}, R²Is
Hydrogen, substituted or unsubstituted lower alkyl, substituted or
Is unsubstituted lower alkenyl, substituted or unsubstituted lower
Alkynyl, substituted or unsubstituted aryl, substituted if
Or unsubstituted heteroaryl, substituted or unsubstituted low
Alkanoyl, substituted or unsubstituted lower alkanoyl
Roxy, halogen, SR¹²(In the formula, R¹²Is a replacement
Is unsubstituted lower alkyl, substituted or unsubstituted aryl
Group, substituted or unsubstituted heteroaryl, substituted or
Is an unsubstituted aralkyl, a substituted or unsubstituted heteroaralkyl
Reel-substituted lower alkyl, substituted or unsubstituted tetra
Hydropyranyl or substituted or unsubstituted tetrahydr
Represents lopyranylmethyl), NR¹³R¹⁴(In the formula, R¹³Oh
And R¹⁴Is the above R^TenAnd R¹¹Synonymous with
R) or azide, and R^FourAnd R^FiveAre the same or different
Becomes hydrogen, substituted or unsubstituted lower alkyl,
Substituted or unsubstituted lower alkanoyl, substituted or unsubstituted
Substituted lower alkoxycarbonyl, substituted or unsubstituted
Aralkyloxycarbonyl or substituted or non-substituted
Alternative heteroaryl substituted lower alkoxycarbonyl
And R⁶Represents hydrogen or halogen, R⁷And R⁸Is
Identical or different, hydrogen, substituted or unsubstituted lower
Alkyl or substituted or unsubstituted lower alkanoyl
And a carrier to which a compound represented by
When a quality sample is brought into contact with the compound of formula (I),
And the following formula (III) [Chemical 5] [In the formula, R¹Is a substituted or unsubstituted lower alkyl, low
Grade alkanoyl, carboxy, lower alkoxy carbonyl
The following formula (II) [Chemical 6] (In the formula, n represents 1 or 2) or a group represented by C
OCH = CHR⁹{In the formula, R⁹Is a substituted or unsubstituted low
Primary alkoxy, substituted or unsubstituted aryl, substituted also
Or unsubstituted heteroaryl or NR^TenR¹¹(formula
Medium, R^TenAnd R ¹¹Are the same or different and are hydrogen or substituted
Or unsubstituted lower alkyl, substituted or unsubstituted
Lower alkenyl, substituted or unsubstituted lower alkynyl
, Substituted or unsubstituted aralkyl, substituted or non-substituted
Substituted aryl, substituted or unsubstituted heteroaryl
Group, substituted or unsubstituted heteroaryl-substituted lower alkyl
Killed, substituted or unsubstituted tetrahydropyranyl or
Is substituted or unsubstituted tetrahydropyranylmethyl
Represents or R^TenAnd R¹¹Can be replaced together with the adjacent N
To form an unsubstituted heterocyclic group)}, R²Is
Hydrogen, substituted or unsubstituted lower alkyl, substituted or
Is unsubstituted lower alkenyl, substituted or unsubstituted lower
Alkynyl, substituted or unsubstituted aryl, substituted if
Or unsubstituted heteroaryl, substituted or unsubstituted low
Alkanoyl, substituted or unsubstituted lower alkanoyl
Roxy, halogen, SR¹²(In the formula, R¹²Is a replacement
Is unsubstituted lower alkyl, substituted or unsubstituted aryl
Group, substituted or unsubstituted heteroaryl, substituted or
Is an unsubstituted aralkyl, a substituted or unsubstituted heteroaralkyl
Reel-substituted lower alkyl, substituted or unsubstituted tetra
Hydropyranyl or substituted or unsubstituted tetrahydr
Represents lopyranylmethyl), NR¹³R¹⁴(In the formula, R¹³Oh
And R¹⁴Is the above R^TenAnd R¹¹Synonymous with
R) or azide, and R^{2 '}Is hydrogen or if substituted
Represents an unsubstituted lower alkanoyloxy, R³Is
Represents a substituted or unsubstituted lower alkanoyl, R^FourAnd
And R^FiveAre the same or different and are hydrogen, substituted or non-substituted
Substituted lower alkyl, substituted or unsubstituted lower alkano
Yl, substituted or unsubstituted lower alkoxycarbonyl
Or substituted or unsubstituted aralkyloxycarbonyl
Or a substituted or unsubstituted heteroaryl-substituted lower aryl
Rucoxycarbonyl, R⁶Is hydrogen or halogen
Represents R⁷And R⁸Are the same or different, hydrogen,
Substituted or unsubstituted lower alkyl or substituted or unsubstituted
Represents a substituted lower alkanoyl]
When contacting the standardized carrier with the test protein sample,
Compare with the protein that binds to the compound represented by (III)
The test protein sample was used to identify the compound represented by the formula (I).
LK6-characterized by selecting proteins that bind to different proteins
A method for searching for a protein that binds to A or a derivative thereof. 3. The carrier is latex beads.
Alternatively, the search method described in 2. 4. The search method according to claim 1, wherein the test protein sample is a protein sample derived from a eukaryote. 5. A carrier having LK6-A or a derivative thereof immobilized thereon. 6. The carrier is latex beads.
The carrier described. 7. A protein selected from the following (i) to (v): (I) a protein that binds to LK6-A or a derivative thereof obtained by the method according to any one of claims 1 to 4 (ii) represented by any one of SEQ ID NOs: 1 to 5, 43, 45 and 46 Protein (iii) consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added, and binding to LK6-A or its derivative (SEQ ID NOS: 1 to 5, 43, 45 and 46) A protein consisting of an amino acid sequence having 60-99% homology with the amino acid sequence represented by any of the above, and binding to LK6-A or a derivative thereof (iv) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. (V) a protein consisting of the amino acid sequence represented by SEQ ID NO: 49 8. A DN encoding the protein according to claim 7.
A. 9. A recombinant DNA obtained by ligating the DNA according to claim 8 to a vector DNA. 10. A transformant containing the recombinant DNA according to claim 9. 11. The transformant is a bacterium, yeast, insect cell,
A plant cell or an animal cell which can be obtained as a host.
The transformant described in 0. 12. The transformant according to claim 10 or 11 is cultivated in a medium, the protein according to claim 7 is produced and accumulated in the culture, and the protein is collected from the culture. The method for producing the protein according to claim 7, wherein 13. A method for measuring the amount of binding between LK6-A or its derivative and said protein when LK6-A or its derivative is brought into contact with a protein that binds to LK6-A or its derivative in the presence of a test substance. Then, in the absence of the test substance, the binding amount of LK6-A or a derivative thereof and the protein when the LK6-A or a derivative thereof is contacted with a protein that binds to LK6-A or a derivative thereof is measured, Compare the measured values in the presence and absence of the test substance, and select the substance that inhibits the binding of LK6-A or its derivative and the protein binding to LK6-A or its derivative from the test substance. A method of searching for a substance that binds to a protein that binds to LK6-A or a derivative thereof, comprising: 14. An activity of a protein that binds to LK6-A or a derivative thereof, when the protein is brought into contact with a test substance,
A substance that measures the activity of the protein in the absence of contact with a test substance, compares the measured values in the presence of the test substance and in the absence of the test substance, and changes the activity of the protein from the test substance A method for searching for a substance that alters the activity of a protein that binds to LK6-A or a derivative thereof, characterized in that 15. A cellular response of a transformant expressing a protein that binds to LK6-A or a derivative thereof and a test substance, and the cell response of the transformant in the absence of the test substance. Measuring the cellular response of transformants, comparing the measured values in the presence and absence of the test substance, and selecting a substance that changes the cell response of the transformant from the test substance A method for searching for a substance that changes the function of a protein that binds to LK6-A or a derivative thereof, which is characterized by: 16. An expression amount of DNA encoding the protein of a transformant, which is obtained when a transformant expressing a protein that binds to LK6-A or a derivative thereof is contacted with the test substance, and the test substance. The expression level of the DNA encoding the protein of the transformant in the absence of the protein was measured, and the measured value in the presence of the test substance was compared with that in the absence of the test substance. A method for searching for a substance that changes the expression level of DNA encoding a protein that binds to LK6-A or a derivative thereof, which comprises selecting a substance that changes the expression level of DNA that encodes LK6-A. 17. An expression level of the protein in the transformant when a transformant expressing a protein that binds to LK6-A or a derivative thereof is contacted with the test substance, and in the absence of the test substance. Measuring the expression level of the protein of the transformant, comparing the measured value in the presence of the test substance and the absence of the test substance, a substance that changes the expression level of the protein from the test substance A method for searching for a substance that changes the expression level of a protein that binds to LK6-A or a derivative thereof, which is characterized in that it is selected. 18. The search method according to claim 13, wherein the protein that binds to LK6-A or a derivative thereof is any one protein selected from the following (i) to (v). . (I) a protein having the amino acid sequence represented by any of SEQ ID NOS: 1 to 5, 43, 45, 46 and 49 (ii) the protein according to claim 7 (iii) SEQ ID NOS: 1 to 5, 43, 45, 46 and 49
A protein having 60% or more homology with the amino acid sequence represented by any of the above and binding to LK6-A or a derivative thereof (iv) in the amino acid sequence represented by SEQ ID NO: 45, with amino acid numbers 353 to 408 Protein having amino acid sequence represented (v) Protein having amino acid sequence represented by amino acid numbers 64-121 in amino acid sequence represented by SEQ ID NO: 46 19. The test substance is measured in the presence of the test substance to measure the activity of any one of the following proteins (i) to (iii) and the activity of the protein in the absence of the test substance. A method for searching for a substance that inhibits the activity of the protein, comprising comparing the measured values in the presence and absence of the test substance and selecting a substance that inhibits the activity of the protein from the test substance. (I) a protein having the amino acid sequence represented by SEQ ID NO: 1 (ii) 1 in the amino acid sequence represented by SEQ ID NO: 1
A protein (iii) SEQ ID NO: 1 which has an amino acid sequence in which the above amino acid residues have been deleted, substituted, inserted or added and which has substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1. A protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence represented by, and having substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1. 20. A binding amount of a CD2 protein or a derivative thereof and the protein when the CD2 protein or a derivative thereof is contacted with any one protein selected from the following (i) to (iii) in the presence of a test substance: And when the CD2 protein or its derivative and the protein are contacted in the absence of the test substance.
The binding amount of the CD2 protein or its derivative and the protein is measured, and the measured values in the presence and absence of the test substance are compared, and a substance that inhibits the binding is selected from the test substances. A method of searching for a compound that inhibits the binding between a CD2 protein or a derivative thereof and the protein, the method comprising: (I) a protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 1 or 2 And a protein that binds to the CD2 protein (iii) A protein that binds to the CD2 protein and has an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 1 or 2. 21. A derivative of CD2 protein is represented by the following (i) to (ii)
It is any one protein selected from i).
The search method described in 0. (I) 1 in the amino acid sequence represented by SEQ ID NO: 6
A protein consisting of an amino acid sequence in which the above amino acids are deleted, substituted, inserted or added, and having an activity of binding to a protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) represented by SEQ ID NO: 6 A protein consisting of an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 or 2 and binding to a protein having the amino acid sequence of SEQ ID NO: 1 or 2 (iii) an amino acid of the amino acid sequence of SEQ ID NO: 6 A protein having the amino acid sequence of Nos. 281 to 310 22. The glycogen debranching enzyme activity of any one protein selected from the following (i) to (iii) in the presence of a test substance, and the activity of the protein in the absence of the test substance are measured. And comparing the measured values in the presence of the test substance and in the absence of the test substance, and selecting a substance that inhibits the activity of the protein from the test substance, which inhibits the activity of the protein Search method for substances to be used. (I) a protein having the amino acid sequence represented by SEQ ID NO: 3 (ii) in the amino acid sequence represented by SEQ ID NO: 3, 1
A protein having an amino acid sequence in which the above amino acid residues are deleted, substituted, inserted or added, and having glycogen debranching enzyme activity (iii) having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 3. A protein consisting of an amino acid sequence having and having glycogen debranching enzyme activity 23. The peroxidase activity of any one protein selected from the following (i) to (iii) in the presence of a test substance and the activity of the protein in the absence of the test substance are measured, Comparing the measured values in the presence of the test substance and in the absence of the test substance, and selecting a substance that inhibits the activity of the protein from the test substance, a substance that inhibits the activity of the protein, Search method. (I) a protein having the amino acid sequence represented by SEQ ID NO: 5 (ii) in the amino acid sequence represented by SEQ ID NO: 5, 1
A protein having the above amino acid residue deleted, substituted, inserted or added, and having peroxidase activity (iii) an amino acid sequence having 60% or more homology with the amino acid sequence represented by SEQ ID NO: 5 And a protein having peroxidase activity 24. C when a CD2BP2 protein or its derivative is contacted with LK6-A or its derivative in the presence of a test substance
The amount of binding between D2BP2 protein or its derivative and LK6-A or its derivative, and C when CD2 protein or its derivative and LK6-A or its derivative were contacted in the absence of the test substance.
A substance that inhibits the binding of the D2BP2 protein or its derivative and the LK6-A or its derivative by measuring the binding amount and comparing the measured values in the presence and absence of the test substance A method for searching for a compound that inhibits the binding between the CD2BP2 protein or its derivative and LK6-A or its derivative, characterized in that 25. A CD2BP2 protein derivative according to the following (i) to
25. The search method according to claim 24, which is any one protein selected from (iii). (I) in the amino acid sequence represented by SEQ ID NO: 43,
A protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added, and having an activity of binding to a protein having the amino acid sequence represented by SEQ ID NO: 1 or 2 (ii) represented by SEQ ID NO: 43 (Iii) consisting of an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1 or 2 which binds to a protein having the amino acid sequence shown in SEQ ID NO: 1 or 2 A protein having an amino acid sequence of amino acid numbers 282 to 341 26. A substance obtained by the searching method according to any one of claims 13 to 25, or a pharmaceutically acceptable salt of the substance. 27. An agent for treating an autoimmune disease or an immunosuppressive agent, which comprises the substance according to claim 26 or a pharmaceutically acceptable salt of the substance.