JP2001231578A - Protein belonging to il-1 family - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein having an activity as a factor belonging to an IL-family useful for the search and development of a treating agent of diseases associated with the IL-1, a DNA encoding the above protein, an antibody recognizing the above protein and a method for utilizing these. SOLUTION: This protein is a protein belonging to the IL family containing a specific human origin amino acid sequence and includes a protein having an amino acid sequence in which >=1 amino acids are lost, substituted and/or added in the above protein and having a higher binding affinity to a receptor of a protein belonging to the IL-1 family than that of Tango-77; a protein containing an amino acid sequence having >=60% homology with the amino acid sequence of the above protein and having a higher binding affinity to the receptor of a protein belonging to the IL-1 family than that of Tango-77; and a protein recognized by a monoclonal antibody produced by a hybridoma FERM BP-7368 and not recognized by a monoclonal antibody produced by a hybridoma FERM BP-7369.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel protein, a DNA encoding the protein, an antibody recognizing the protein, and methods of using the same.

[0002]

BACKGROUND OF THE INVENTION Interleukin-1 (IL-1) refers to two types of proteins (IL-1α and IL-1β) that play a key role in the early stage of the inflammatory response (for review,
Blood, 87 , 2095, 1996; Bio Science term library,
See cytokines and growth factors, revised edition, p14-17, 1998; and references in their reviews). IL-1 has been known for its existence since the 1940's and has been referred to by names such as endogenous pyrogens or lymphocyte activating factors. By 1985, the cDNA for IL-1 had been isolated,
Two molecules derived from different genes, namely IL-1α
And the existence of IL-1β (Nature, 315 , 641,
1985). IL-1α and IL-1β have different isoelectric points,
271 and 269, both without signal peptide
After being produced as a precursor of 31 kD consisting of amino acids,
It is cleaved into a 17 kD mature molecule consisting of 159 and 153 amino acids, respectively. IL-1α is produced by calpain
It is known that IL-1β is converted from a precursor to a mature molecule by IL-1β converting enzyme (Nature, 356 , 768, 19
92; and Blood, 87 , 2095, 1996). Although the amino acid sequence homology between IL-1α and IL-1β is 27%, it is known that they have similar higher-order structures. The mature IL-1β molecule does not contain an α-helix, the β-sheet is 59%, and the rest is a globular protein of random coil and has a tetrahedral-like structure consisting of 12 β-sheets (EMBO J., 7 , 339). , 1988).
In addition, it has been revealed that both IL-1α and IL-1β genes are located at 2q13 on the human chromosome (Nu
cleic Acids Res., 14 , 3167, 1986; and Nucleic Acid
s Res., 14 , 7897, 1986).

[0003] IL-1α and IL-1β exhibit biological activity by binding to the same receptor. IL-1 receptors include 80 kD type I (IL-1RI: CD121a) and 68 kD type II (IL-1RI
I: CD121wb), which were cloned in 1988 and 1991, respectively (Science, 241 , 585,
1988; and EMBO J., 10 , 2821, 1991). The type I gene is located on human chromosome 2q13-21, and the type II gene is located on human chromosome 2q13-22. It has also been shown that the type II extracellular domain exists as a soluble receptor (sIL-1R) (FEBS Lett., 260 , 213, 19).
90). Type I and type II receptors are both members of the immunoglobulin superfamily and are composed of an extracellular region containing three immunoglobulin-like domains, a transmembrane region and an intracellular region. The homology of the amino acid composition of the extracellular regions of type I and type II is 2
Although it is 8%, a large difference is observed in the structure of the intracellular region. That is, the intracellular region of type I is composed of 215 amino acids, and a sequence similar to the protein kinase C phosphorylation structure exists therein, whereas the intracellular region of type II is as short as 29 amino acids, No sequence thought to be involved in signal transduction has been found. Differences were also observed in the expression distribution. Type I was T cells, fibroblasts, epidermal cells, endothelial cells, synovial cells, chondrocytes, hepatocytes, and type II was B cells, monocytes, neutrophils. Expressed in bone marrow cells. These two receptors have large structural differences in the intracellular region and differ in expression distribution. Therefore, besides the binding affinity with IL-1, they also regulate the intracellular signal transduction system and expression control. It is thought that they have different biological functions due to differences.

In 1990, an IL-1 receptor antagonist (IL-1ra), which binds to the IL-1 receptor and inhibits the binding between IL-1 and the IL-1 receptor, was cloned (Natu).
re, 343 , 341, 1990). IL-1ra is a 17 kD protein consisting of 152 amino acids, and its amino acid composition is IL-1α and 19
%, And 26% homology with IL-1β, and can bind to both type I and type II receptors. IL
It is known that -1ra has at least three types of splice forms, one encoding a secreted protein and the other two encoding an intracellular protein (Proc. Natl. Acad. Sci. USA, 88 , 3681, 1991; and Blood, 87 , 2095, 1996). Secreted IL-1ra is produced by monocytes, macrophages, neutrophils, fibroblasts, and the like, and non-secreted IL-1ra is produced by keratinocytes, epithelial cells, and macrophages. The IL-1ra gene is located at 2q13-14.1 on the human chromosome.

In 1998, an IL-1 receptor antagonist β (IL-1raβ) was identified as a gene having significant homology to IL-1ra through a search of an EST database (Japanese Patent Application Laid-Open No. 10-304888). Tango-77 in 1999
Was identified as a gene having significant homology to IL-1ra through analysis of genomic sequence data (International Publication WO99
/ 06426). Since the obtained IL-1raβ cDNA does not have a signal sequence, it has been pointed out that another splice form including the signal sequence may exist, but it has not been identified yet. In Tango-77, using a computer program Procrustes, it is estimated that there may be other splice forms other than the obtained cDNA, and the sequence of the putative splice form T77-procrustes has been clarified. However, with the current informatics technology, it is impossible to accurately predict all splice forms, and it is possible that there are still other splice forms that play an unknown biological role.

Thus, IL-1α, IL-1β, IL-1ra, I
L-1raβ and Tango-77 have been shown to form a family of highly homologous families, suggesting the existence of additional members. In addition, it has been revealed that factors belonging to the IL-1 family have various biological activities, and are considered to be physiologically important factors including their association with disease states (for review, see Blood, 87 , 2095, 199
6; BioScience Term Library, Cytokines and Growth Factors, Revised Edition, p14-17, 1998; and references in their review). It has also been reported that partial peptides of factors belonging to the IL-1 family have various activities (Blood, 177 , 1627, 1991). That is, IL-1β 2
The partial peptides corresponding to the 08th to 240th positions lack the ability to activate T cells but have a hypnotic effect or a pyrogen effect (Am. J. Physiol., 259 , R439, 199).
0), a partial peptide corresponding to positions 237 to 269 of IL-1β inhibits the activation of T cells by IL-1 (Biochem. Biophys. Res. Comm., 147 , 204, 198).
7), a partial peptide corresponding to positions 163 to 171 of IL-1β promotes T cell activation and glycosaminoglycan synthesis, but lacks pyrogen action (Adv.
Exp. Med. Biol., 251 , 153, 1989, Eur. Cytokine Ne.
t., 1 , 21, 1990), it has been reported that a partial peptide corresponding to positions 165 to 186 of IL-1β has a fibroblast growth promoting activity (Scand. J. Immunol.,
30 , 549, 1989).

Factors belonging to the IL-1 family include microbial infection, HIV infection, chronic B
Hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, traumatic brain injury, inflammatory bowel disease, or other diseases accompanied by inflammation, abnormal smoothness such as arteriosclerosis, restenosis, etc. Diseases involving differentiation and proliferation of muscle cells, abnormal fibroblasts such as rheumatoid arthritis or diseases associated with activation of synovial tissue, diseases associated with disorders of pancreatic β-cells such as diabetes, abnormal osteoclasts such as osteoporosis Diseases, allergies,
It has been shown to be involved in diseases involving abnormal cell proliferation, such as atopy, asthma, hay fever, airway hyperreactivity, and autoimmune diseases, and abnormal cell proliferation such as acute myeloid leukemia and malignant tumors. I have. Further, an IL-1 antagonist capable of inhibiting the IL-1 activity of a factor belonging to the IL-1 family, an antibody,
Inhibitors such as antisense DNA are useful for microbial infection, HIV
Infections, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, traumatic brain injury, inflammatory bowel disease, and other diseases involving infection or inflammation, arteriosclerosis, restenosis, etc. Diseases involving abnormal proliferation and proliferation of smooth muscle cells, diseases involving abnormal activation of fibroblasts and synovial tissue such as rheumatoid arthritis, diseases involving disorders of pancreatic β-cells such as diabetes, and abnormalities such as osteoporosis Diseases associated with the activation of various osteoclasts, allergies, atopy, asthma, hay fever, airway irritability,
Diseases associated with abnormal activation of immune cells, such as autoimmune diseases,
The activity has been shown to treat diseases associated with abnormal cell proliferation, such as acute myeloid leukemia and malignant tumors, and diseases based on neuronal disorders such as ischemic dysfunction. Furthermore, a factor having IL-1 activity belonging to the IL-1 family is a disease associated with abnormal cell proliferation such as acute myeloid leukemia, malignant tumor, Alzheimer's disease, It has been shown to be effective for diseases based on neuronal disorders such as diseases and ischemic diseases.
Therefore, factors belonging to the IL-1 family have received much attention as targets for the development of useful new drugs. Also, IL-1
It is recalled that there may be new factors belonging to the family.

[0008]

SUMMARY OF THE INVENTION The present invention relates to microbial infection, HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes,
Diseases involving infection and inflammation such as traumatic brain injury, inflammatory bowel disease, diseases involving abnormal proliferation and proliferation of smooth muscle cells such as arteriosclerosis and restenosis, abnormal fibroblasts and synovium such as rheumatoid arthritis Diseases associated with tissue activation, diseases associated with pancreatic β-cell damage such as diabetes, diseases associated with abnormal activation of osteoclasts such as osteoporosis, allergies, atopy, asthma, hay fever, airway hypersensitivity, autoimmune diseases Diseases associated with abnormal cell proliferation, such as abnormal myelogenous leukemia, malignant tumors, etc., diseases caused by neuronal disorders such as Alzheimer's disease, Parkinson's disease, ischemic disease, etc. , A protein having an activity as a factor belonging to the IL-1 family useful for the search and development of therapeutic agents, and a DN encoding the protein
A, an antibody recognizing the protein, and a method of using the same.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, obtained a factor belonging to the IL-1 family containing a novel amino acid sequence and a DNA encoding the factor. And succeeded in completing the present invention. That is, the present invention provides the following (1) to
(36) is provided. (1) a protein selected from the following (a) to (f): (a) a protein having the amino acid sequence represented by SEQ ID NO: 2; (b) a protein having the amino acid sequence represented by SEQ ID NO: 2;
A protein having an amino acid sequence of positions 6 to 208; (c) an IL-containing amino acid sequence represented by SEQ ID NO: 1
A protein belonging to one family; (d) a protein having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the protein of (a) or (b), and belonging to the human IL-1 family; A protein having a binding affinity to a receptor higher than Tango-77; (e) an amino acid sequence having 60% or more homology with the amino acid sequence of the protein of (a) or (b),
A protein belonging to the human IL-1 family and having a binding affinity to a receptor higher than that of Tango-77; and The protein according to the above (d) or (e), which is not recognized by the monoclonal antibody produced by (1).

(2) a DNA selected from the following (a) to (d): (a) a DNA encoding the protein of (1); (b) a base sequence represented by SEQ ID NO: 4;
(C) a DNA having a nucleotide sequence represented by SEQ ID NO: 4;
(D) a DNA having a nucleotide sequence of the 99th base sequence; and (d) a DNA which hybridizes with any of the DNAs of (a) to (c) under stringent conditions and which is a protein receptor belonging to the human IL-1 family. Tang binding affinity
DNA encoding a protein higher than o-77.

(3) a protein selected from the following (a) to (c): (a) a protein having the amino acid sequence represented by SEQ ID NO: 1; (b) a protein having the amino acid sequence represented by SEQ ID NO: 1 A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, and / or added to the protein, and having an activity of binding to a receptor of a protein belonging to the human IL-1 family; and (c) SEQ ID NO: 1 Has an amino acid sequence having 60% or more homology with the amino acid sequence represented by
A protein having a binding activity of a protein belonging to one family to a receptor. (4) DNA selected from the following (a) to (c): (a) DNA encoding the protein of (3); (b) DNA having a base sequence represented by SEQ ID NO: 3;
And (c) a DNA that hybridizes with the DNA of (a) or (b) under stringent conditions and encodes a protein having a binding affinity for a receptor of a protein belonging to the human IL-1 family.

(5) DN according to (2) or (4)
A recombinant DNA obtained by incorporating A into a vector. (6) A transformant having the recombinant DNA according to (5). (7) The accession number is FERM BP-6943.
The transformant according to (6). (8) The transformant according to (6) or (7) is cultured in a medium, the protein according to (1) or (3) is produced and accumulated in the culture, and the protein is collected from the culture. The method for producing a protein according to (1) or (3), wherein

(9) An antibody that recognizes the protein according to (1) or (3). (10) The antibody has an activity of inhibiting the activity of the protein according to (1) or (3),
The antibody according to (9). (11) The antibody according to (10), wherein the antibody is an antibody that recognizes a protein having the amino acid sequence represented by SEQ ID NO: 1. (12) The antibody according to (10) or (1), wherein the antibody recognizes an amino acid sequence at positions 1 to 20 in the amino acid sequence represented by SEQ ID NO: 1 as an antigenic epitope.
The antibody according to 1). (13) A monoclonal antibody produced by a hybridoma having a deposit number of FERM BP-7368. (14) A monoclonal antibody produced by a hybridoma having a deposit number of FERM BP-7369.

(15) Consisting of 5 to 60 consecutive nucleotides in the nucleotide sequence of the DNA encoding the region of the amino acid sequence of SEQ ID NO: 1 or the region corresponding to the amino acid sequence of SEQ ID NO: 1 in the protein according to (1). An oligonucleotide having a sequence or an oligonucleotide having a sequence complementary to the oligonucleotide. (16) performing hybridization using the DNA according to (2) or (4) or the oligonucleotide according to (15) as a probe,
A method for detecting the expression of a gene encoding the protein according to (1) or (3). (17) A method for detecting the expression of a gene encoding the protein according to (1) or (3), comprising performing polymerase chain reaction using the oligonucleotide according to (15) as a primer. (18) Using the DNA according to (2) or (4) or the oligonucleotide according to (15), a mutation in the gene encoding the protein according to (1) or (3) is detected by a hybridization method. how to. (19) A method for detecting a mutation in a gene encoding the protein according to (1) or (3), comprising performing polymerase chain reaction using the oligonucleotide according to (15).

(20) Diseases associated with infection and inflammation, diseases associated with abnormal differentiation and proliferation of smooth muscle cells, diseases associated with abnormal fibroblast activation, diseases associated with abnormal synovial tissue activation, pancreas diseases with β-cell disorders, diseases with abnormal activation of osteoclasts, diseases with abnormal activation of immune cells,
Alternatively, the method according to any one of (16) to (19), which is used for detecting a disease associated with abnormal cell proliferation. (21) Diseases accompanied by infection or inflammation are caused by microbial infection, HIV
Infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, traumatic injury or inflammatory bowel disease, with abnormal smooth muscle cell differentiation and proliferation. A disease with arteriosclerosis or restenosis, which involves abnormal fibroblast activation or abnormal synovial tissue activation, is rheumatoid arthritis; and a disease, which involves pancreatic β-cell damage, is diabetes. The disease associated with abnormal activation of osteoclasts is osteoporosis, and the disease associated with abnormal activation of immune cells is allergy, atopy, asthma, hay fever, airway hyperreactivity or autoimmune disease, and abnormal cell proliferation. The method according to (20), wherein the disease associated with is myeloid leukemia or malignant tumor. (22) Transcription or mRNA of a gene encoding the protein according to (1) or (3), wherein the DNA according to (2) or (4) or the oligonucleotide according to (15) is used. How to suppress translations. (23) The DNA according to (2) or (4) or the oligonucleotide according to (15), wherein the promoter region of the gene encoding the protein according to (1) or (3) is used. How to get.

(24) A medicament comprising the protein according to (1) or (3). (25) The DNA according to (2) or (4),
Medicine. (26) A medicament comprising the antibody according to any one of (9) to (14). (27) A medicament comprising the oligonucleotide according to (15). (28) Diseases associated with infection or inflammation, diseases associated with abnormal differentiation and proliferation of smooth muscle cells, diseases associated with abnormal fibroblast activation, diseases associated with abnormal synovial tissue activation, pancreatic β-cell Treatment and / or prevention of disorders associated with disorders, disorders involving abnormal activation of osteoclasts, disorders involving abnormal activation of immune cells, disorders involving abnormal cell proliferation or disorders based on neuronal cell disorders. (24) ~
The medicament according to any one of (27). (29) Diseases associated with infection or inflammation, diseases associated with abnormal differentiation and proliferation of smooth muscle cells, diseases associated with abnormal fibroblast activation, diseases associated with abnormal synovial tissue activation, pancreatic β-cell (24) a medicament for the diagnosis of a disease associated with a disorder, a disease associated with abnormal activation of osteoclasts, a disease associated with abnormal activation of immune cells, or a disease associated with abnormal cell proliferation.
-The medicament according to any of (27).

(30) Microbial infection, HIV infection, chronic hepatitis B, rheumatoid arthritis,
Sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, traumatic injury or inflammatory bowel disease, a disease with abnormal smooth muscle cell differentiation and proliferation is arteriosclerosis or restenosis, abnormal fiber A disease with blast cell activation or abnormal synovial tissue activation is rheumatoid arthritis, a disease with pancreatic β-cell damage is diabetes, with abnormal osteoclast activation The disease is osteoporosis, the disease associated with abnormal activation of immune cells is allergy, atopy, asthma, hay fever, airway hyperreactivity or autoimmune disease, and the disease associated with abnormal cell proliferation is acute myeloid leukemia or malignancy. (28) or (29), wherein the disease based on a nerve cell disorder is Alzheimer's disease or ischemic brain disease.

(31) A method for screening a receptor that specifically interacts with the protein according to (1) or (3), which comprises using the protein according to (1) or (3). (32) A receptor specifically interacting with the protein according to (1) or (3), obtained by the screening method according to (31). (33) The method for immunologically detecting a protein according to (1) or (3), comprising using the antibody according to any one of (9) to (14). (34) An immunohistochemical staining method comprising detecting the protein according to (1) or (3) using the antibody according to any of (9) to (14). (35) A knockout non-human animal in which expression of the gene encoding the protein according to (1) or (3) is partially or completely suppressed. (36) (1) or (3)
A knockout non-human animal in which the activity of the protein according to 1 is partially or completely suppressed.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The protein of the present invention includes: (a) a protein having the amino acid sequence represented by SEQ ID NO: 2;
A protein having an amino acid sequence of positions 6 to 208; (c) an IL-containing amino acid sequence represented by SEQ ID NO: 1
A protein belonging to one family; (d) a protein having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the protein of (a) or (b), and belonging to the human IL-1 family; A protein having a binding affinity to a receptor higher than Tango-77; (e) an amino acid sequence having 60% or more homology with the amino acid sequence of the protein of (a) or (b),
And a protein having a binding affinity to a receptor of a protein belonging to the human IL-1 family higher than that of Tango-77; and Not recognized by the monoclonal antibody produced by the hybridoma having the number FERM BP-7369,
(D) or the protein described in (e).

The protein of the present invention includes: (a) a protein having the amino acid sequence represented by SEQ ID NO: 1; (b) a protein having the amino acid sequence represented by SEQ ID NO: 1 lacking one or more amino acids. A protein having an amino acid sequence added, substituted and / or added, and having an activity of binding to a receptor of a protein belonging to the human IL-1 family; and (c) an amino acid sequence represented by SEQ ID NO: 1; % Of an amino acid sequence having a homology of at least
A protein having an activity of binding to a receptor of a protein belonging to one family; A protein belonging to the IL-1 family is a protein having affinity for the IL-1 receptor. Specific examples of proteins belonging to the IL-1 family include, for example, IL-1α, IL-1β, IL-1ra, IL-1raβ and
Tango-77 and the like.

Binding affinity of a protein having the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids are deleted, substituted and / or added, and which belongs to the human IL-1 family with a receptor Sex is Tango-
77, a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted and / or added in a protein having the amino acid sequence of positions 46 to 208 of SEQ ID NO: 2, and belonging to the human IL-1 family A protein having a binding affinity to Tango-77 higher than that of Tango-77; and a protein having the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted and / or added, and A protein having an activity of binding to a receptor of a protein belonging to the IL-1 family is Molecu
lar Cloning, A Laboratory Manual, Second Edition,
Cold Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Pr
otocols in Molecular Biology, John Wiley & Sons, 1
987-1997 (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Re
search, 10 , 6487,1982, Proc. Natl. Acad. Sci., US
A, 79 , 6409, 1982, Gene, 34 , 315, 1985, Nucleic Ac
ids Research, 13 , 4431, 1985, Proc. Natl. Acad. Sc
i USA, 82 , 488, 1985, etc., using, for example, a protein having the amino acid sequence of SEQ ID NO: 2, a protein having the amino acid sequence of positions 46 to 208 of SEQ ID NO: 2, or a sequence It can be performed by introducing a site-specific mutation into DNA encoding the protein having the amino acid sequence of No. 1. The number of amino acids to be deleted, substituted and / or added is from 1 to several, and the number is not particularly limited, but can be deleted, substituted or added by well-known techniques such as the site-directed mutagenesis described above. About 1 to several tens, preferably 1 to 20
, More preferably 1 to 10, more preferably 1 to
There are five.

The protein of the present invention contains an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 2 and has a binding affinity with a receptor for a protein belonging to the human IL-1 family. A protein higher than Tango-77, the amino acid sequence of positions 46 to 208 of SEQ ID NO: 2 and 6
A protein comprising an amino acid sequence having 0% or more homology and having a binding affinity to a receptor of a protein belonging to the human IL-1 family higher than that of Tango-77; and 60% or more of the amino acid sequence of SEQ ID NO: 1 Also included are proteins that contain homologous amino acid sequences and that have a higher binding affinity to a receptor for proteins belonging to the human IL-1 family than Tango-77. SEQ ID NO: 2, positions 46 to 208 of SEQ ID NO: 2,
Or homology with the amino acid sequence of SEQ ID NO: 1
ST (J. Mol. Bio., 215 , 403, 1990) and FASTA
(Method in Enzymology, 183 , 63, 1990).
The homology is preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 97% or more. In the present invention, the accession number FERM B
The protein recognized by the antibody produced by the hybridoma P-7368 and not recognized by the antibody produced by the hybridoma having the accession number FERM BP-7369 is detected, for example, by a method such as Western blotting or ELISA using the above antibody. It is a protein that can be identified by whether or not it is performed. Specifically, as the protein, in the amino acid sequence represented by SEQ ID NO: 2, 46
A protein having the amino acid sequence of the -208th position can be mentioned.

The DNA of the present invention includes: (a) DN encoding the protein of the present invention as defined above.
A; (b) In the base sequence represented by SEQ ID NO: 4, 370
(C) a DNA having a nucleotide sequence represented by SEQ ID NO: 4;
(D) a DNA having a nucleotide sequence of the 99th base sequence; and (d) a DNA which hybridizes with any of the DNAs of (a) to (c) under stringent conditions and which is a protein receptor belonging to the human IL-1 family. Tang binding affinity
DNA encoding a protein higher than o-77: Further, the DNA of the present invention includes: (a) DN encoding the protein represented by SEQ ID NO: 1
A; (b) DNA having a base sequence represented by SEQ ID NO: 3;
And (c) a DNA that hybridizes with the DNA of (a) or (b) under stringent conditions and encodes a protein having a binding affinity for a receptor of a protein belonging to the human IL-1 family. be able to.

The DNA that hybridizes under stringent conditions includes, for example, SEQ ID NO: 3 or SEQ ID NO: 4.
DN of the present invention such as DNA having a base sequence represented by
A or a partial fragment thereof was used as a probe to
DNA obtained by using a hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like,
Specifically, after performing hybridization at 65 ° C. in the presence of 0.7 to 1.0 M sodium chloride using a filter in which colony or plaque-derived DNA is immobilized, 0.1 to 2 × SSC A DNA that can be identified by washing the filter using a solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) at 65 ° C. can be mentioned. Hybridization is performed using Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A
Practical Approach, Second Edition, Oxford Univers
, 1995, etc. Specifically, as a hybridizable DNA,
When calculated using analysis software such as BLAST or FASTA, DNA having at least 60% or more homology with the base sequence represented by SEQ ID NO: 3, preferably 70%
Or more, more preferably 80% or more, and still more preferably 9% or more.
DNAs having a homology of 0% or more, particularly preferably 95% or more, most preferably 98% or more can be mentioned. The type of the “activity of the protein having the amino acid sequence of SEQ ID NO: 1” referred to in the present specification is not particularly limited, and examples thereof include a binding activity of a protein belonging to the human IL-1 family to a receptor. But not limited to this.

Hereinafter, the present invention will be described in detail. 1. Preparation of DNA of the Present Invention Human bone marrow mRNA is commercially available (for example, manufactured by Clontech)
May be used, or may be prepared from human bone marrow tissue as follows. As methods for preparing total RNA from bone marrow tissue, guanidine thiocyanate-cesium trifluoroacetate method (Methods in Enzymology, 154 , 3,1987), guanidine acid thiocyanate / phenol / chloroform (A
GPC) method (Analytical Biochemistry, 162 , 156, 198)
7; and Experimental Medicine, 9 , 1937, 1991).
In addition, mR is converted to poly (A) ⁺ RNA from total RNA.
As a method for preparing NA, an oligo (dT) -immobilized cellulose column method (Molecular Cloning II
Edition) and the like. Furthermore, Fast Track mRNA Isolat
ion Kit (Invitrogen), Quick Prep mRNA Purifi
mRNA can be prepared by using a kit such as a cation Kit (Pharmacia).

From the prepared human bone marrow tissue mRNA, cDN
Create A library. Methods for preparing a cDNA library include Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, A Laboratory Manual, 2 ed Ed., 1989, or a commercially available kit such as SuperScript
Plasmid System for cDNA Synthesis and Plasmid Clon
ing (Life Technologies), ZAP-cDNA Synthesis K
and a method using it (manufactured by STRATAGENE).
As a cloning vector for preparing a cDNA library, any phage vector, plasmid vector, or the like can be used as long as it can replicate autonomously in E. coli K12 strain. Specifically, ZAP Express (STRATA
GENE, Strategies, 5 , 58, 1992), pBluescript II
SK (+) (Nucleic Acids Research, 17 , 9494, 1989), La
mbda ZAP II (STRATAGENE), λgt10, λgt11 (DNA
cloning, A Practical Approach, 1 , 49, 1985), λTripl
Ex (Clontech), λExCell (Pharmacia), pT7
T318U (Pharmacia), pcD2 (Mol. Cell. Biol., 3 , 2
80, 1983) and pUC18 (Gene, 33 , 103, 1985).

As the host microorganism, any microorganism belonging to Escherichia coli can be used. Specifically, Escherichia coli XL1-Blue MRF '(STRATAGENE, Strategies, 5 , 81, 1992), Escherichia coli C60
0 (Genetics, 39 , 440, 1954), Escherichia coli Y1088
(Science, 222 , 778, 1983), Escherichia coli Y1090
(Science, 222 , 778, 1983), Escherichia coli NM522
(J. Mol. Biol., 166 , 1, 1983), Escherichia coli K8
02 (J. Mol. Biol., 16 , 118, 1966) and Escherichiac
oli JM105 (Gene, 38 , 275, 1985) and the like are used. The cDNA library may be used as it is in the following analysis, decreasing the ratio of incomplete length cDNA, as much as possible a full-length cDNA In order to efficiently retrieve, oligo-capping method Kanno et al developed (Gene, 138 , 171, 1994; Gene,
200 , 149, 1997; protein nucleic acid enzyme, 41 , 603, 1996; experimental medicine, 11 , 2491, 1993; cDNA cloning, Yodosha, 1996; and a method for preparing a gene library, Yodosha,
1994) may be used for the following analysis.

Each clone is isolated from the prepared cDNA library, and the base sequence of the cDNA of each clone is determined from the end by a conventional nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. (Proc.
Natl. Acad. Sci. USA, 74 , 5463, 1977) or AB
IPRISM377 DNA sequencer (PE Biosyst
The base sequence of the DNA is determined by analysis using a base sequence analyzer (e.g., ems). Each c
Whether or not the DNA base sequence has a new sequence can be determined by using a homology search program such as BLAST.
By searching a base sequence database such as enBank, EMBL, and DDBJ, it can be confirmed that there is no base sequence having a clear homology that is considered to completely match the base sequence of an existing gene in the database.

C containing the novel sequence obtained by the above method
The base sequence of DNA includes, for example, the base sequence of SEQ ID NO: 4. The protein obtained by translating the DNA consisting of the nucleotide sequence of SEQ ID NO: 4 is a protein belonging to the IL-1 family, human IL, in homology analysis using BLAST2.
-1α, human IL-1β and human IL-1ra, respectively, 33%, 2
It has 8% and 29% homology. Also, from the nucleotide sequence comparison between the nucleotide sequence of SEQ ID NO: 4 and Tango-77, the nucleotide sequence of SEQ ID NO: 4
It turns out that it has a sequence to which a new base sequence of bases has been added. It is known that a gene of a protein belonging to the IL-1 family forms a cluster on the long arm of human chromosome 2. That is, human IL-1α, human
The gene for IL-1β and human IL-1ra is human chromosome 2q13-1
4 and located within 450 kb of each other. Human IL-1raβ and Tango-77 genes are also located near human chromosome 2q13. In order to confirm whether the nucleotide sequence of SEQ ID NO: 3 exists in the cluster region of the long arm of human chromosome 2, the human genomic library was subjected to SEQ ID NO: 4
Screening using a sequence specific to the base sequence of
It can be determined by determining the nucleotide sequence of the obtained genomic clone.

As the human genomic library, a commercially available one (for example, a BAC library manufactured by Research Genetics) may be used, or a method known from human cells or tissues (for example, Genomics, 29 , 413, 1995; and Genomics). , 24 , 5
27, 1994 etc.). As a method for screening a human genomic library using a sequence specific to the nucleotide sequence of SEQ ID NO: 4, a PCR method using primers specific to the nucleotide sequence of SEQ ID NO: 4 (PCR Protocols, Academic Press, 1990)
And colony hybridization and plaque hybridization (molecular cloning second edition) using an oligonucleotide specific to the nucleotide sequence of SEQ ID NO: 4. Human IL-1ra by the above method
A genomic DNA clone (for example, a human genomic BAC clone) containing both the nucleotide sequence of SEQ ID NO: 4 and the nucleotide sequence of SEQ ID NO: 4 is obtained. Therefore, it can be seen that the gene consisting of the nucleotide sequence of SEQ ID NO: 4 exists near the cluster region where the gene belonging to the IL-1 family of the long arm of human chromosome 2 exists.

In the base sequence of the obtained genomic DNA clone, the base sequence containing the base sequence of SEQ ID NO: 3 includes the base sequence of SEQ ID NO: 5. The nucleotide sequence of SEQ ID NO: 3 is surrounded by AG, which is a splicing acceptor sequence, and GT, which is a splicing donor sequence, indicating that it forms an exon of the gene consisting of the nucleotide sequence of SEQ ID NO: 4. The two-dimensional structure of the protein obtained by translating the DNA consisting of the nucleotide sequence of SEQ ID NO: 4 was compared with the known Chofasman, Robson, DSC
(Http://www.bmm.icnet.uk/software.htm
l, http://www.gh.wits.ac.za/crystal/w3vlc/misc.anc
003.html), it can be seen that 12 β-sheet structures common to factors belonging to the IL-1 family are retained. On the other hand, Tango-77 does not have three N-terminal β-sheets. Therefore, one splice form of the protein obtained by translating the DNA consisting of the nucleotide sequence of SEQ ID NO: 4 is Tango-77, and the protein encoded by the nucleotide sequence of SEQ ID NO: 4 belongs to a novel IL-1 family. It is clear that it has an essential activity as a factor.

[0032] Factors belonging to the IL-1 family are known to be involved in various diseases. The following is an example. IL-1 is induced by cytokines such as TNF-α, IFN-α, IFN-β, IFN-γ, bacterial endotoxins, viruses, and various mitogens, and is important as a mediator of inflammatory responses, especially acute inflammatory responses Plays an important role.
That is, if the endotoxin concentration in the blood increases due to bacterial infection, etc., IL-1 is released by monocytes, macrophages, neutrophils, etc., inflammatory reactions are triggered, and biological reactions such as fever and induction of acute phase proteins are caused. Caused (Bloo
d, 87 , 2095, 1996). In the immune system, IL-1 enhances IL-2 production in the presence of antigens and mitogens,
It is known to activate T cells by promoting receptor expression. In addition, IL-2, IFN-
It is known to promote the production of cytokines such as γ, CSF and IL-6 and to increase the expression of cytokine genes such as TNF, IFN-β and CSF (J. Biol. Chem., 26
9 , 19021, 1994). Increased production of these cytokines stimulates B cells, promotes proliferation and activation, and enhances immunoglobulin production (J. Immunol. 130 , 2708,
1983). Furthermore, IL-1 is known to be involved in the process of monocyte maturation into inflammatory macrophages (J.
Exp. Med., 175 , 1793, 1992). As described above, since IL-1 expresses various actions as a mediator of the inflammatory response, it can be used for chronic rheumatoid arthritis, diabetes, microbial infection, HI
It has been pointed out that many diseases associated with infection and inflammation, such as V infection, chronic hepatitis B, etc., have been shown to enhance IL-1 production in these diseases (Virus Re
s., 32 , 253, 1994; and N. Eng. J. Med., 328 , 106, 19
93). Therefore, inhibitors of IL-1 action are expected to have anti-inflammatory action. IL-1ra is being developed for clinical applications targeting inflammation and rheumatism.

When IL-1 is constitutively expressed in smooth muscle,
It is known to promote smooth muscle proliferation (Am. J. P.
hysiol., 45 , H901, 1999). In addition, it is known that the action of IL-1 on vascular smooth muscle increases the expression of adhesion molecules such as E-selectin and promotes leukocyte adhesion (Atherosclerosis, 115 , 89, 1995). . For these reasons, attention has also been paid to the association with the onset of arteriosclerosis, and IL-1
Inhibitors of action may also be useful as therapeutic or prophylactic agents for arteriosclerosis.

It is known that IL-1 stimulates fibroblasts and releases proteolytic enzymes such as collagenase and prostaglandins at the site of rheumatoid arthritis (Annu. Rev. Immunol). ., 3 , 263, 1985). In addition, it has been reported that the synovial tissue of a patient with rheumatoid arthritis produces IL-1, and that the amount of production is correlated with the degree of arthritis (Arthritis Rheum., 31 , 480, 1988). These facts strongly suggest that IL-1 has an effect on the onset and progression of rheumatoid arthritis. Therefore, an inhibitor of IL-1 action may be useful as a therapeutic or prophylactic agent for rheumatoid arthritis.

IL-1 has a damaging effect on the islets of Langerhans of the pancreas (Science, 232 , 1545, 198).
6) IL-1 released by macrophages infiltrating into islets of Langerhans damages β-cells and is strongly involved in the development of insulin-dependent diabetes mellitus (IDDM) (Autoimmunit
y, 10 , 241, 1991). For these reasons, inhibitors of IL-1 action may be promising as therapeutic or preventive drugs for diabetes.

IL-1 has been reported to promote osteoclast activation and bone resorption (J. BoneMiner. Res., 4 , 11).
3, 1989). Therefore, an inhibitor of IL-1 action is expected to have a bone resorption inhibiting action, that is, it may be promising as a therapeutic or preventive drug for osteoporosis. In fact, it has been reported that IL-1ra suppressed bone resorption (J. Immunol., 145 , 4181, 1990), and it is considered that the possibility of such a drug is high.

It has been reported that the administration of IL-1 to mice weakens the radiosensitivity of bone marrow cells (Blood, 74 , 74) .
2257, 1989), it has been found that when administered before an anticancer drug or the like, the lethal effect due to suppression of bone marrow function is reduced. Thus, IL-1 is expected to have a protective effect on bone marrow cells in cancer radiotherapy and chemotherapy.

It has been reported that IL-1 has cytotoxicity against several types of cancer cells including the melanoma cell line A375 (J. Immunol., 136 , 3098, 1986; and J. Immunol. , 135 , 3962, 1985). T cells, B
Promotes cell proliferation for cells, NK cells, fibroblasts, and vascular endothelial cells;
It has been reported to inhibit the growth of certain tumor cells, such as breast cancer cell MCF-7 or mouse T cell line Eb (J. Immunol., 136 , 340, 1986). For these reasons, IL-1 may be useful as a therapeutic or prophylactic agent for diseases based on abnormal cell proliferation, for example, malignant tumors. In addition, IL-1α has a growth promoting activity on gastric cancer cell lines (Cancer Res., 53 , 4102, 1993). From this, IL-
An agent that inhibits the action of 1α may be useful as a therapeutic or prophylactic agent for diseases based on abnormal cell proliferation, such as malignant tumors.

It has been reported that IL-1 acts on nerve cells to increase the expression of nerve growth factor (NGF) (Nature, 330 , 658, 1987). In addition, it acts on fibroblasts to enhance NGF expression, and
Is known to increase the stability of (J. Biol. Che
m., 263 , 16348, 1988). NGF, which is a neurotransmitter, has an action of promoting proliferation of nerve cells and progression of neurites, and is known to function as a regeneration factor and a protective factor for damaged nerve cells. Therefore, IL-1 enhances the expression of NGF, by diseases based on neuronal disorders,
For example, it may be useful as a therapeutic or prophylactic for Alzheimer's disease, Parkinson's disease, and ischemic allopathies. In addition, an effect of promoting regeneration after nerve transection can be expected. Furthermore, it has been reported that it increases cAMP in the hypothalamus to cause prostaglandin E ₂ (PGE ₂ ) production and promotes secretion of hypothalamic hormone and pituitary hormone (Biochem. Biophys. Res. . Commun.,
164 , 1262, 1989), and its effect on improving brain function has attracted attention.

IL-1ra is a type I and type II of IL-1
Inhibits the biological activity of IL-1 by binding to the receptor and competitively inhibiting the binding of IL-1α and IL-1β to the receptor (Annu. Rev. Immunol., 16 , 27, 1998). IL-
It has been reported that administration of 1ra to various disease model animals can suppress inflammation. For example, IL-1ra prevents septic shock caused by LPS administration and suppresses inflammatory cell infiltration, edema, necrosis, etc. in tissues (Nature, 348 , 550,
1990). The results of these pathological models include sepsis, rheumatoid arthritis, graft-versus-host disease, asthma,
IL-1ra in stroke, ischemic heart disease, myeloid leukemia, etc.
Is expected to have a therapeutic effect, and clinical trials have been made (Int. Rev. Immunol., 16 , 457, 1998).

The sIL-1 receptor binds to IL-1β,
Inhibits the binding of β to the receptor (J. Exp. Med., 174 , 125
1, 1991). It is also known to have an effect of promoting engraftment of the transplanted heart and suppressing the growth of acute myeloid leukemia cells (Blood, 79 , 1938, 1992).

As described above, factors belonging to the IL-1 family include microbial infection, HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes, and traumatic brain disease. Diseases associated with infection or inflammation such as injury, inflammatory bowel disease, diseases involving abnormal proliferation and proliferation of smooth muscle cells such as arteriosclerosis and restenosis, abnormal fibroblasts or synovial tissue activity such as rheumatoid arthritis Diseases associated with pancreatic β-cell disorders such as diabetes, diseases involving abnormal activation of osteoclasts such as osteoporosis, allergies, atopy, asthma, hay fever, airway hypersensitivity, autoimmune diseases, etc. It has been shown to be involved in diseases involving abnormal cell proliferation, such as diseases associated with the activation of various immune cells, acute myeloid leukemia, and malignant tumors. Also, an IL-1 antagonist, an antibody, an antisense D capable of inhibiting the IL-1 activity of a factor belonging to the IL-1 family.
Inhibitors such as NA include infections such as microbial infection, HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes mellitus, traumatic brain injury, inflammatory bowel disease, etc. Diseases involving inflammation, diseases involving abnormal proliferation and proliferation of smooth muscle cells such as arteriosclerosis, restenosis, abnormal fibroblasts such as rheumatoid arthritis and diseases involving activation of synovial tissue, and pancreas β such as diabetes Diseases involving cell damage, diseases involving abnormal activation of osteoclasts such as osteoporosis, allergies,
Diseases involving abnormal activation of immune cells such as atopy, asthma, hay fever, airway hyperreactivity, and autoimmune diseases, diseases involving abnormal cell proliferation such as acute myeloid leukemia and malignant tumors, and nerves such as ischemic disease Activity has been shown to treat diseases based on cell damage. In addition, a factor having IL-1 activity, which belongs to the IL-1 family, is caused by administration of a protein or a gene encoding the same. It has been shown to be effective in the treatment of diseases based on disorders of nerve cells such as diseases and in the reduction of side effects in cancer radiotherapy or chemotherapy by suppressing bone marrow function and promoting the therapeutic effect.

Once the DNA consisting of the nucleotide sequence of SEQ ID NO: 4 has been obtained and its nucleotide sequence has been determined, primers based on the nucleotide sequences at the 5 'and 3' ends of the nucleotide sequence are prepared, and human Or cDNA or c synthesized from mRNA contained in tissues or cells such as bone marrow of a non-human animal
Using the DNA library as a template, the PCR method (PCR Prot
ocols, Academic Press, 1990) to obtain the DNA of the present invention. Also, colony hybridization or plaque hybridization (cDNA or cDNA library) synthesized from mRNA contained in tissues or cells such as bone marrow of a human or non-human animal using the full length or a part of the DNA of SEQ ID NO: 4 as a probe. By performing molecular cloning (2nd edition), the DNA of the present invention can be obtained. Determined DN
The DNA of the present invention is chemically synthesized on the basis of the nucleotide sequence of A using a DNA synthesizer such as a DNA synthesizer model 392 manufactured by Perkin-Elmer Co., Ltd. using the phosphoramidite method.
A can also be obtained.

The obtained DNA is expressed by expressing a protein using a transformant obtained by introducing a recombinant vector containing the DNA into a host cell, or
Amino acid sequence encoded by NA and human IL-1α, human IL-1
By comparing the homology and the two-dimensional structure with the amino acid sequences of β, human IL-1ra, human IL-1raβ, and Tango-77,
It can be confirmed that the DNA is a DNA encoding a protein having an activity as an IL-1 family. Further, a protein obtained using a transformant obtained by introducing a recombinant vector containing the obtained DNA into a host cell, and a transformant obtained by introducing a recombinant vector containing Tango-77 into a host cell By comparing the activities of the proteins obtained using the above, it can be seen that Tango-77 does not necessarily have the same activity as the protein having the amino acid sequence of SEQ ID NO: 2. Furthermore, it has been reported that partial peptides of factors belonging to the IL-1 family have various activities (Blood, 177 , 1627, 19).
91, Am. J. Physiol., 259 , R439, 1990, Biochem. Bio
phys. Res. Comm., 147 , 204, 1987, Scand. J. Immuno.
l., 30 , 549, 1989, Adv. Exp. Med. Biol., 251 , 153,
1989; Eur. Cytokine Net., 1 , 21, 1990). It can be seen that the protein having the amino acid sequence of SEQ ID NO: 1, which is a partial peptide of the protein having the amino acid sequence of SEQ ID NO: 2, also has a unique activity. From the examples described below, since the protein is an important part involved in binding to the IL-1 receptor, the protein can be used as an antagonist of another IL-1 family, and the amino acid sequence of the protein can be changed. The binding to the IL-1 receptor can be enhanced by introducing the sequence into a protein having no IL-1 family. Introduction of the amino acid sequence of the protein into another IL-1 family can be achieved, for example, by inserting a DNA having the nucleotide sequence of SEQ ID NO: 3 into DNA encoding another IL-1 family. Expression of the protein encoding SEQ ID NO: 1 in vivo can be achieved, for example, by using a virus vector incorporating the nucleotide sequence of SEQ ID NO: 3, or the like. Furthermore, the expression of the gene containing the nucleotide sequence can be inhibited by introducing the antisense DNA having the nucleotide sequence of SEQ ID NO: 3 into cells.

Based on the information on the nucleotide sequence of SEQ ID NO: 4 or a fragment thereof, the DNA sequence of the present invention, for example, Oligonucleotide having a sequence corresponding to 5 to 60 bases, preferably 10 to 40 bases or an oligonucleotide corresponding to a sequence complementary to the oligonucleotide (hereinafter, also referred to as antisense oligonucleotide) can be prepared. . Oligonucleotides of the present invention include oligo D
Oligonucleotides such as NA and oligoRNA, and derivatives of the oligonucleotides (hereinafter referred to as derivative oligonucleotides), and the like. As the oligonucleotide or antisense oligonucleotide, for example, in the base sequence of a part of mRNA to be detected, 5 ′
Examples include a sense primer corresponding to the base sequence on the terminal side, an antisense primer corresponding to the base sequence on the 3 ′ end side, and the like. However, the base corresponding to uracil in mRNA becomes thymidine in the oligonucleotide primer. Examples of the sense primer and the antisense primer include oligonucleotides whose melting temperature (Tm) and the number of bases thereof do not extremely change, and have 5 to 60 bases, preferably 10 to 50 bases.

Derivative oligonucleotides include those in which the phosphodiester bond in the oligonucleotide has been converted to a phosphorothioate bond, and those in which the phosphodiester bond in the oligonucleotide has been converted to an N3'-P5 'phosphoramidate bond. The ribosil and the phosphodiester bond in the oligonucleotide were converted into the peptide nucleic acid bond, and the uracil in the oligonucleotide was C
-5 propynyl uracil substituted, uracil in the oligonucleotide substituted with C-5 thiazole uracil, cytosine in the oligonucleotide substituted with C-
Cytosine substituted with 5-propynylcytosine, in which the cytosine in the oligonucleotide is phenoxazine-modified cytosine (ph
enoxazine-modified cytosine), those in which the ribose in the oligonucleotide is substituted with 2′-O-propyl ribose, those in which the ribose in the oligonucleotide is substituted with 2′-methoxyethoxy ribose, etc. (Cell Engineering, 16 , 1463, 199
7).

2. Production of the protein of the present invention The protein of the present invention can be prepared by the method described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, etc., for example, by the following method. Can be expressed and expressed in host cells. Based on the full-length cDNA, if necessary, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared. A recombinant vector is prepared by inserting the DNA fragment or the full-length cDNA downstream of the promoter of an appropriate expression vector. By introducing the recombinant vector into a host cell suitable for the expression vector, a transformant producing the protein of the present invention can be obtained. As a host cell,
Any bacteria, yeast, animal cells, insect cells, plant cells and the like can be used as long as they can express the target gene. As an expression vector, it is capable of autonomous replication in the host cell or integration into a chromosome,
Those containing a promoter at a position where the DNA encoding the protein of the present invention can be transcribed are used.

When a prokaryote such as a bacterium is used as a host cell, the recombinant vector containing the DNA encoding the protein of the present invention is capable of autonomous replication in the prokaryote and has a promoter and a ribosome binding sequence. And a vector comprising a gene encoding the protein of the present invention and a transcription termination sequence. A gene that controls the promoter may be included. Examples of expression vectors include pBTrp2 (Boehringer Mannheim), pBTac1 (Boehringer Mannheim), pBTac2
(Boehringer Mannheim), pKK233-2 (Pharmaci
a), pSE280 (Invitrogen), pGEMEX-1 (Prome
ga), pQE-8 (QIAGEN), pKYP10 (JP-A-58-11)
0600), pKYP200 (Agricultural Biological Chemist
ry, 48 , 669, 1984), pLSA1 (Agric. Biol. Chem., 5
3 , 277, 1989), pGEL1 (Proc. Natl. Acad. Sci. USA,
82 , 4306, 1985), pBluescript II SK (-) (Stratagen
e), pTrs30 [ Escherichia coli JM109 / pTrS30 (FE
RM BP-5407)], pTrs32 [ Escherichia coli J
Prepared from M109 / pTrS32 (FERM BP-5408)], pGHA2 [ Esch
prepared from E. coli IGHA2 (FERM BP-400);
No. 0-221091], pGKA2 [ Escherichia coli IGKA2 (FERM
BP-6798), JP-A-60-221091], pTerm2 (US Pat. No. 4,686,191, US Pat. No. 4,939,094, and US Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC19
4, pEG400 (J. Bacteriol., 172 , 2392, 1990), pGEX
(Pharmacia), pET system (Novagen), pSu
pex and so on.

The promoter may be any promoter as long as it can be expressed in a host cell. For example, tr
p promoter (P _trp), lac promoter, P _L promoter, P _R promoter, it can be mentioned promoters derived from such T7 promoter, E. coli or phage, or the like. Two series is not the promoter P _trp also (P _trp ×
2) Artificially designed and modified promoters such as the 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). By substituting the nucleotide sequence of the portion encoding the protein of the present invention so that the codon is optimal for host expression, the production rate of the target protein can be improved. In the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for expression of the DNA of the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.

As the host cell, microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., 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,
Serratia ficaria , Serratia fonticola , Serratia liq
uefaciens , Serratia marcescens , Bacillus subtili
s , Bacillus amyloliquefacines , Brevibacterium imma
riophilum ATCC14068, Brevibacterium saccharolyticu
m ATCC14066, Brevibacterium flavum ATCC14067, Brev
ibacterium lactofermentum ATCC13869, Corynebacteri
um glutamicum ATCC13032, Corynebacterium acetoacid
ophilum ATCC13870, Microbacterium ammoniaphilum AT
CC15354, Pseudomonas sp. D-0110 and the like.

As a method for introducing a recombinant vector, any method for introducing DNA into the above host cells can be used. For example, a method using calcium ions (Proc. Natl. Acad. Sci. USA, 69) , 2110, 1972), the protoplast method (JP-A-63-248394), or Gene, 1
7 , 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) and the like. As the promoter, any promoter can be used as long as it can be expressed in yeast strains. For example, a promoter for glycolytic genes such as hexose kinase, PHO5
Promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10
Promoter, heat shock protein promoter, MF
α1 promoter, CUP1 promoter and the like can be mentioned.

Examples of the host cell include microorganisms belonging to the genera Saccharomyces, Kluyveromyces, Trichosporone, Schwaniomyces, and the like, for example, Saccharomyces cerevisiae.
visiae , Schizosaccharomyces pombe , Kluyveromyces l
actis , Trichosporon pullulans , Schwanniomyces allu
vius and the like. As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast. For example, an electroporation method (Methods. Enzymol., 194 , 182, 1990), a spheroplast method ( Proc. Natl. Acad. Sci. USA, 84 , 192
9, 1978), lithium acetate method (J. Bacteriology, 153 , 1
63, 1983; and Proc. Natl. Acad. Sci. USA, 75 , 192.
9, 1978).

When an animal cell is used as a host, examples of the expression vector include pcDNAI and pcDM8.
(Manufactured by Funakoshi), pAGE107 (JP-A-3-22979; and Cy)
totechnology, 3 , 133, 1990), pAS3-3 (JP-A-2-22707)
No. 5), pCDM8 (Nature, 329, 840, 1987), pcDNAI / A
mp (Invitrogen), pREP4 (Invitrogen), pAG
E103 (J. Biochemistry, 101 , 1307, 1987), pAGE210
Etc. can be given. Any promoter can be used as long as it can be expressed in animal cells. For example, the promoter of cytomegalovirus (CMV)
ediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, S
Ra promoter and the like can be mentioned. Further, an enhancer of the IE gene of human CMV may be used together with the promoter. Host cells include Namalwa cells, human cells, COS cells, monkey cells,
HBT56, a CHO cell that is a Chinese hamster cell
37 (JP-A-63-299). As a method for introducing a recombinant vector, any method for introducing DNA into animal cells can be used. For example, electroporation (Cytotechnology, 3 , 133, 19)
90), calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075), lipofection method (Proc. Natl. Acad. Sci. USA, 84 , 74).
13, 1987).

When an insect cell is used as a host, for example, the current protocol in molecular.
Biology Baculovirus Expression Vectors, A Labo
ratory Manual, WH Freeman and Company, New York
(1992), Bio / Technology, 6 , 47 (1988) and the like, and the protein can be expressed.
That is, after the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain the recombinant virus in the insect cell culture supernatant, the recombinant virus is further infected into insect cells to express the protein. it can. Examples of the gene transfer vector used in the method include, for example, pVL1392, pVL1393, and pBlueBacIII (Invitor
ogen). Examples of the baculovirus include Autographa californica nucl (Autographa californica nucl), which is a virus that infects night-moth insects.
ear polyhedrosis virus) and the like can be used.

As insect cells, Spodoptera frugiperd
Sf9 is a ovarian cells, Sf21 (Baculovirus Expression
Vectors, A Laboratory Manual, WH Freeman and C
ompany, New York, 1992), High 5 (Invitrogen), which is an ovarian cell of Trichoplusia ni , and the like can be used. Examples of a method for co-transferring the above-described recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method (Proc.
Acad. Sci. USA, 84 , 7413, 1987).

When a plant cell is used as a host cell, examples of the expression vector include a Ti plasmid and a tobacco mosaic virus vector. Any promoter may be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and rice actin 1 promoter. Host cells include tobacco, potato, tomato,
Carrot, soybean, rape, alfalfa, rice,
Plant cells such as wheat and barley can be mentioned. As a method for introducing a recombinant vector, D
Any method can be used as long as it introduces NA. For example, Agrobacterium
(JP-A-59-140885, JP-A-60-70080, International Publication WO9
4/00977), electroporation method (JP-A-60-2518)
No. 87), a method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), and the like.

As a method for expressing the gene, secretory production, fusion protein expression and the like can be carried out according to the method described in Molecular Cloning, 2nd edition, etc., in addition to direct expression. When expressed by yeast, animal cells, insect cells, or plant cells, a protein having a sugar or a sugar chain added thereto can be obtained. The protein of the present invention can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the protein of the present invention in the culture, and collecting from the culture. The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.

A culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant. The carbon source may be any as long as the organism can assimilate,
Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, organic acids such as propionic acid, ethanol,
Alcohols such as propanol can be used. Nitrogen sources include ammonia, ammonium chloride,
Ammonium salts of inorganic or organic acids such as ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal water Decomposed products, various fermented cells and digested products thereof can be used. As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, man sulfate, copper sulfate, calcium carbonate, and the like can be used.

The culture is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture. Culture temperature is 15-40
C is good, and the culture time is usually 16 hours to 7 days.
During the culture, the pH is maintained at 3.0 to 9.0. 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 as necessary. For example, when culturing a microorganism transformed with a recombinant vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used for culturing a microorganism transformed with a recombinant vector using a trp promoter. Indole acrylic acid or the like may be added to the medium.

As a medium for culturing the transformant obtained 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, 1950)
Alternatively, a medium or the like obtained by adding fetal calf serum or the like to these mediums can be used. Culture is usually performed at pH 6-8, 30-4
The reaction is carried out for 1 to 7 days under conditions such as 0 ° C. and 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 a transformant obtained by using an insect cell as a host, generally used TNM-FH
Medium (Pharmingen), Sf-900 II SFM medium (Life Te
chnologies), ExCell400, ExCell405 (JR
H Biosciences), Grace's Insect Medium (Grace,
TCC, Nature, 195 , 788, 1962) can be used. The cultivation is usually performed under conditions of pH 6 to 7, 25 to 30 ° C, and the like for 1 to 5 days. If necessary, an antibiotic such as gentamicin may be added to the medium during the culture.

A 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. As a medium for culturing the transformant, a commonly used Murashige and Skoog (MS) medium, a white (White) medium, or an auxin, cytokinin, or the like to these mediums, a medium to which a plant hormone is added, or the like is used. be able to. Culture is usually performed at pH
The reaction is carried out at 5 to 9, 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 culture.

As described above, a transformant derived from a microorganism, animal cell, or plant cell having a recombinant vector into which a DNA encoding the protein of the present invention has been incorporated,
The protein can be produced by culturing according to a usual culture method to produce and accumulate the protein, and collecting the protein from the culture. As a method for expressing a gene, besides direct expression, molecular cloning
Secretory production, fusion protein expression, etc. can be performed according to the method described in the edition. The method for producing the protein of the present invention includes a method of producing the protein in a host cell, a method of secreting the protein out of the host cell, and a method of producing the protein on the host cell outer membrane. , The method can be selected.

When the protein of the present invention is produced in a host cell or on a host cell outer membrane, the method of Paulson et al.
Biol. Chem., 264 , 17619, 1989);
oc. Natl. Acad. Sci., USA, 86 , 8227, 1989; and Gen.
es Develop., 4 , 1288, 1990) or JP-A-5-336963.
The protein can be positively secreted out of the host cell by applying the method described in JP-A-6-223021 or the like. That is, the protein of the present invention is expressed in a form in which a signal peptide is added in front of the protein containing the active site of the protein of the present invention, thereby actively secreting the protein of the present invention out of the host cell. be able to. Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the transgenic animal or plant, an individual animal (transgenic non-human animal) or individual plant (transgenic plant) into which the gene has been introduced is created. The protein of the invention can also be produced.

When the transformant is an individual animal or plant, the protein is bred or cultivated according to a conventional method to produce and accumulate the protein, and the protein is collected from the individual animal or plant to obtain the protein. Can be manufactured. As a method for producing the protein of the present invention using an animal individual, for example, a known method (American Journal of
Clinical Nutrition, 63 , 639S, 1996, American Journ
al of Clinical Nutrition, 63 , 627S, 1996, and Bio /
Technology, 9 , 830, 1991). In the case of an animal individual, for example, a transgenic non-human animal into which a DNA encoding the protein of the present invention has been introduced is bred, and the protein is produced and accumulated in the animal.
By collecting the protein from the animal, the protein can be produced. Examples of the production / accumulation site in the animal include milk of the animal (JP-A-63-30919).
2) Eggs etc. can be given. Any promoter that can be used in this case can be used as long as it can be expressed in animals. For example, α casein promoter, β casein promoter, β lactoglobulin promoter, whey acid A protein promoter or the like is preferably used.

A method for producing the protein of the present invention using a plant individual includes, for example, D-encoding the protein of the present invention.
Transgenic plants into which NA has been introduced can be prepared by known methods (tissue culture, 20, 1994; tissue culture, 21, 1995; and Trends
in Biotechnology, 15 , 45, 1997)
A method of producing the protein by producing and accumulating the protein in the plant, and collecting the protein from the plant. The protein produced by the transformant of the present invention is, for example, when the protein of the present invention is expressed in a dissolved state in cells, after completion of the culture, the cells are collected by centrifugation, and suspended in an aqueous buffer. Thereafter, the cells are crushed with an ultrasonic crusher, a French press, a Mantongaulin homogenizer, a Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation and purification method, 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
Anion exchange chromatography using a resin such as (Mitsubishi Kasei), cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia),
Hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing Used in combination, a purified preparation can be obtained.

When the protein is expressed by forming an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to collect the insoluble form of the protein as a precipitate fraction. The insoluble form of the recovered protein is solubilized with a protein denaturant. After diluting or dialyzing the solubilized solution to return the protein to a normal tertiary structure, a purified sample of the protein can be obtained by the same isolation and purification method as described above. When the protein of the present invention or its derivative such as a modified sugar is secreted extracellularly, the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant. That is, the culture is treated by a method such as centrifugation as described above to obtain a soluble fraction, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to. Examples of the protein thus obtained include a protein having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

The protein of the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Also, Advanced ChemTec
h, Perkin-Elmer, Pharmacia, Protein Te
chnology Instrument, Synthecell-Vega, PerSepti
Chemical synthesis can also be performed using a peptide synthesizer such as ve or Shimadzu Corporation.

3. Preparation of an antibody recognizing the protein of the present invention The purified protein of the protein of the present invention or a partial fragment polypeptide of the protein, or by using a peptide having a partial amino acid sequence of the protein of the present invention as an antigen,
Antibodies that recognize the protein of the present invention, such as polyclonal antibodies and monoclonal antibodies, can be prepared. (1) Preparation of polyclonal antibody A purified preparation of the protein of the present invention or a partial fragment polypeptide of the protein, or a peptide having a partial amino acid sequence of the protein of the present invention is used as an antigen and administered to an animal. Antibodies can be made. As animals to be administered, rabbits, goats, rats, mice, hamsters and the like aged 3 to 20 weeks can be used. The dose of the antigen is 50-100 μg per animal.
Is preferred. When a peptide is used, it is desirable that the peptide be covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin as the antigen. A peptide serving as an antigen can be synthesized by a peptide synthesizer.

The administration of the antigen is performed 1-2 times after the first administration.
Perform 3 to 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 determined by enzyme immunoassay [enzyme immunoassay (ELIS)].
A), published by Medical Shoin (1976); and Antibodies-A
Laboratory Manual, Cold Spring Harbor Laboratory,
1988]. 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. Methods for separation and purification include centrifugation, salting out with 40-50% saturated ammonium sulfate, and caprylic acid precipitation (Antibodies, A Labora
tory manual, Cold SpringHarbor Laboratory, 198
8) or a method in which a chromatography using a DEAE-Sepharose column, an anion exchange column, a protein A or G-column, a gel filtration column or the like is used alone or in combination.

(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 the protein of the present invention used for immunization were used for antibody-producing cells. Serve as a source of The spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer. The spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded. The spleen cells of the obtained sedimentation fraction were washed with a Tris-ammonium chloride buffer (pH 7.65).
For 1 to 2 minutes to remove red blood cells, followed by washing three times with MEM medium, and using the obtained splenocytes as antibody-producing cells.

(B) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine resistant mouse (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (hereinafter, referred to 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, 1978), P
3-X63-Ag8653 (653) (J. Immunol., 123 , 1548, 197
9), P3-X63-Ag8 (X63) (Nature, 256 , 495, 1975) and the like can be used. These cell lines were prepared in 8-azaguanine medium [RPMI-1640 medium in glutamine (1.
5 mmol / l), 2-mercaptoethanol (5 × 1
0 ^-5 mol / l), gentamicin (10 μg / m
l) and fetal calf serum (FCS) (CSL, 10
%) To a medium (hereinafter referred to as a normal medium) supplemented with 8-azaguanine (15 μg / ml). Use 2 × 10 ⁷ or more of the cells.

(C) Preparation of hybridoma The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b)
With MEM medium or PBS (1.8% disodium phosphate)
3 g, monopotassium phosphate 0.21 g, salt 7.65 g,
Wash well with 1 liter of distilled water, pH 7.2) and count cells
But antibody producing cells: myeloma cells = 5-10: 1
And centrifuged at 1200 rpm for 5 minutes.
Discard the supernatant. Dissolve the cells in the precipitate fraction
Then, the cells were stirred at 37 ° C. for 10 hours.⁸antibody
Polyethylene glycol-1000 per producer cell
2 g of (PEG-1000), 2 ml of MEM and dimethyl
Solution mixed with 0.7 ml of lusulfoxide (DMSO)
Is added, and MEM is further added every 1 to 2 minutes.
Add 1-2 ml of medium several times. After the addition, add the MEM medium
In addition, adjust the total volume to 50 ml. The mixture
After centrifugation at 900 rpm for 5 minutes, the supernatant is discarded. Profit
Gently loosen the cells in the sedimented fraction
HAT medium gently with suction and blowing by pipette
[Hypoxanthin (10 in normal medium)^-Fourmol / l),
Midin (1.5 × 10^-Fivemol / l) and Aminopte
Phosphorus (4 × 10 ^-7mol / l)] 100m
Suspend in l.

The suspension was added to a 96-well culture plate in an amount of 100
aliquots per well and in a 5% CO ₂ incubator
Incubate at 37 ° C for 7-14 days. After culturing, a part of the culture supernatant is removed and used for antibodies (Antibodies, A Laboratory
manual, Cold Spring Harbor Laboratory, Chapter 14,
1988) and the like, a hybridoma that specifically reacts with the partial fragment polypeptide of the protein of the present invention is selected. The following method can be mentioned as a specific example of the enzyme immunoassay. At the time of immunization, a partial fragment polypeptide of the protein of the present invention used for the antigen is coated on a suitable plate, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below is reacted as a primary antibody, and further reacted with a secondary antibody. Biotin, enzymes,
After reacting with an anti-rat or anti-mouse immunoglobulin antibody labeled with a chemiluminescent substance or a radioactive compound, a reaction corresponding to the labeling substance is carried out, and a substance which specifically reacts with the protein of the present invention is converted into a monoclonal antibody of the present invention. Select as a hybridoma to produce. Using the hybridoma, cloning was repeated twice by the limiting dilution method [the first time, an HT medium (a medium obtained by removing aminopterin from the HAT medium), and the second time, a normal medium was used]. Those whose valencies are recognized are selected as hybridoma strains producing the monoclonal antibody of the present invention.

(D) Preparation of Monoclonal Antibody 8 to 10-week-old mouse or pristane-treated [0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane) is intraperitoneally administered and bred for 2 weeks] 5-20 × 10 5 hybridoma cells producing the protein monoclonal antibody of the present invention obtained in (c)
⁶ cells / animal are injected intraperitoneally. In 10 to 21 days, the hybridoma becomes ascites cancer. Ascites is collected from the mouse with ascites tumor and centrifuged at 3000 rpm for 5 minutes to remove solids. From the obtained supernatant, a monoclonal antibody was purified in the same manner as in the polyclonal method,
Can be obtained. The subclass of the antibody is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The protein amount is calculated by the Lowry method or from the absorbance at 280 nm.

4. Use of the DNA, protein or antibody of the present invention (1) Using the DNA or oligonucleotide of the present invention, a hybridization method or a polymerase.
Method for detecting gene expression by chain reaction (PCR) method Using the DNA or oligonucleotide of the present invention, Northern hybridization method (Molecular Cloning 2nd edition), PCR method and RT (reverse-transc
ribed) -PCR method (both PCR Protocols, Academic P
ress (1990)] (hereinafter, also referred to as PCR) to obtain mRNA encoding the protein of the present invention.
Can be detected. Among them, RT-PC
Since the R method is a simple method, it is particularly useful as a detection method. The detection method is also used for quantifying the expression level of the gene, and is also used for microbial infection, HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes, traumatic brain Diseases associated with infection or inflammation such as injury, inflammatory bowel disease, diseases involving abnormal proliferation and proliferation of smooth muscle cells such as arteriosclerosis and restenosis, abnormal fibroblasts or synovial tissue activity such as rheumatoid arthritis Diseases associated with pancreatic β-cell disorders such as diabetes, diseases associated with abnormal activation of osteoclasts such as osteoporosis, allergies, atopy,
Genes encoding the protein of the present invention, such as diseases involving abnormal cell activation such as asthma, hay fever, airway hypersensitivity, and autoimmune diseases, and diseases involving abnormal cell proliferation such as acute myeloid leukemia and malignant tumors It can be used for diagnosis of diseases caused by changes in expression of.

(2) Hybridization or PCR using the DNA or oligonucleotide of the present invention
Method for Detecting Mutations in the Gene Encoding the Protein of the Present Invention Using the DNA or Oligonucleotide of the Present Invention as a Probe for Southern Hybridization (Molecular Cloning, 2nd Edition) on Genomic DNA, PCR
By performing the method or the like, mutation of the gene encoding the protein of the present invention can be detected. The method of detection involves infection or inflammation of microbial infection, HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes, traumatic brain injury, inflammatory bowel disease, etc. Diseases associated with abnormal proliferation and differentiation of smooth muscle cells such as arteriosclerosis and restenosis, abnormal fibroblasts such as rheumatoid arthritis and diseases associated with activation of synovial tissue, and pancreatic β-cells such as diabetes. Diseases associated with disorders, diseases involving abnormal activation of osteoclasts such as osteoporosis, diseases involving abnormal activation of immune cells such as allergies, atopy, asthma, hay fever, airway hypersensitivity, autoimmune diseases, acute bone marrow The present invention can be used for diagnosing diseases caused by mutations in the gene encoding the protein of the present invention, such as diseases associated with abnormal cell proliferation such as leukemia and malignant tumor.

(3) A method for suppressing the transcription or translation of the gene encoding the protein of the present invention using the DNA or oligonucleotide of the present invention. 50 , 322, 199
2; Chemistry, 46 , 681, 1991; Biotechnology, 9 , 358, 199
2; Trends in Biotechnology, 10 , 87, 1992; Trends
in Biotechnology, 10 , 152, 1992; and cell engineering, 16 ,
1463, 1997), Triple helix technology (Trends in
Biotechnology, 10 , 132, 1992) can be used to suppress the transcription or translation of the gene encoding the protein of the present invention.

For example, by administering the DNA or oligonucleotide of the present invention, the production of the protein of the present invention can be suppressed. That is, the use of the DNA or the oligonucleotide of the present invention can suppress the transcription of the gene encoding the protein of the present invention or the translation of mRNA encoding the protein of the present invention, respectively.

[0080] The control method includes microbial infection, HIV infection,
Chronic hepatitis B, rheumatoid arthritis, sepsis, grafts vs.
Diseases associated with infection and inflammation, such as host diseases, insulin-dependent diabetes mellitus, traumatic brain injury, inflammatory bowel disease, diseases associated with abnormal smooth muscle cell differentiation and proliferation, such as arteriosclerosis and restenosis, rheumatoid arthritis, etc. Diseases associated with abnormal fibroblast or synovial tissue activation, diseases associated with pancreatic β-cell damage such as diabetes, diseases associated with abnormal activation of osteoclasts such as osteoporosis, allergies, atopy, asthma, pollen Diseases associated with abnormal activation of immune cells, such as illness, airway hyperreactivity, autoimmune diseases, diseases associated with abnormal cell proliferation such as acute myeloid leukemia, malignant tumors, and neuronal disorders such as ischemic dysfunction. The present invention can be used for treating or preventing a disease such as a disease caused by a mutation in a gene encoding the protein of the present invention.

As a method for treating a disease caused by a mutation in the gene encoding the protein of the present invention, a recombinant vector of the present invention appropriately prepared for gene therapy was introduced into cells taken from a patient. Thereafter, the cells are returned to the living body, and are administered to the living body by carrying them on appropriate viral vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and lentivirus. A method is used in which the protein of the present invention is expressed in a living body of a patient by being encapsulated in a vesicle structure and administered to the living body.

(4) Method for Obtaining the Promoter Region of the Gene Encoding the Protein of the Present Invention Using the DNA or Oligonucleotide of the Present Invention Laboratory Cancer Research Division, New Cell Engineering Protocol, Shujunsha, 1993
Year) can be used to obtain the promoter region of the gene. Examples of the promoter region include all promoter regions involved in transcription of a gene encoding the protein of the present invention in mammalian cells.
For example, in human bone marrow, a promoter region involved in transcription of a gene encoding the protein of the present invention can be mentioned. The promoter can be used in the screening method described below.

(5) Pharmaceuticals Containing the Protein of the Present Invention The protein of the present invention is, for example, a protein having the amino acid sequence of positions 46 to 208 of SEQ ID NO: 2 or SEQ ID NO: 2.
Although showing high homology to IL-1ra, IL-1 expresses various actions as a mediator of the inflammatory response, and is used for infections such as rheumatoid arthritis, diabetes, microbial infection, HIV infection, chronic hepatitis B, etc. Since it has been reported that the protein is associated with many diseases associated with inflammation, SEQ ID NO: 2 or a protein having the amino acid sequence of positions 46 to 208 of SEQ ID NO: 2 can be used for microbial infection, HIV infection, chronic hepatitis B, chronic hepatitis B, It can be a therapeutic or preventive agent for diseases associated with infection or inflammation, such as rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes, traumatic brain injury, and inflammatory bowel disease.

When IL-1 is constitutively expressed in smooth muscle,
It has been reported that the promotion of smooth muscle proliferation and the effect of IL-1 on vascular smooth muscle increase the expression of adhesion molecules such as E-selectin and promote the adhesion of leukocytes. , SEQ ID NO: 2 or 46-2 of SEQ ID NO: 2
The protein having the 08th amino acid sequence can be a therapeutic or preventive agent for arteriosclerosis.

IL-1 stimulates fibroblasts to release proteolytic enzymes such as collagenase and prostaglandins locally at rheumatoid arthritis. Synovial tissue of rheumatoid arthritis patients -1 has been reported, and the amount of production is reported to correlate with the degree of arthritis. Can be a therapeutic or prophylactic agent.

IL-1 has a damaging effect on islets of Langerhans of the pancreas, and IL-1 released by macrophages infiltrating into islets of Langerhans damages β-cells. ) Has been reported to be strongly involved in the pathogenesis of SEQ.

Since it has been reported that IL-1 promotes osteoclast activation and bone resorption, SEQ ID NO: 2 or a protein having the amino acid sequence of positions 46 to 208 of SEQ ID NO: 2 is used for osteoporosis. Can be a therapeutic or prophylactic agent. IL
Since -1α has a growth-promoting activity against gastric cancer cell lines, SEQ ID NO: 2 or a protein having the amino acid sequence of positions 46 to 208 of SEQ ID NO: 2 is a therapeutic agent for diseases based on abnormal cell growth, such as malignant tumors. Or it can be a prophylactic.

(6) Method for Screening a Receptor That Specifically Binds to a Protein Using the Protein of the Present Invention The method for identifying a substance that specifically binds to a protein of the present invention by using a method for identifying a substance that specifically binds to the protein, etc. Receptors that specifically bind to can be screened. As an example of such a method, C. Vries et al. Used VEGF protein,
An expression cloning method that identified the VEGF receptor is mentioned (Science, 255 , 989, 1992). The receptor can be used as a therapeutic agent for a disease associated with the protein of the present invention, or for research on a signal transduction system or biological function involving the protein of the present invention.

(7) Method for Analyzing Expression of Gene Encoding Protein of the Present Invention Using Transformant of the Present Invention The transformant of the present invention is allowed to coexist with various test substances, and the gene in the transformant is used. By analyzing the expression level of the gene, a substance that regulates the transcription of a gene encoding the protein of the present invention, a substance that participates in the transcription control function of the protein of the present invention, or a gene that is transcriptionally regulated by the protein of the present invention is screened. can do.

(8) Method for Immunologically Detecting the Protein of the Present Invention Using the Antibody of the Present Invention The protein of the present invention or a protein containing the protein of the present invention is obtained by carrying out an antigen-antibody reaction using the antibody of the present invention. The tissue can be detected immunologically. The detection method includes microbial infection,
Diseases associated with infection or inflammation, such as HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes, traumatic brain injury, inflammatory bowel disease, etc.
Diseases involving abnormal proliferation and differentiation of smooth muscle cells such as arteriosclerosis and restenosis, diseases involving abnormal activation of fibroblasts and synovial tissue such as rheumatoid arthritis, and impairment of pancreatic β cells such as diabetes Diseases associated with abnormal activation of osteoclasts such as diseases, osteoporosis, diseases associated with abnormal activation of immune cells such as allergies, atopy, asthma, hay fever, airway hypersensitivity, autoimmune diseases, acute myeloid leukemia, It can be used for diagnosis of diseases caused by mutation of the gene encoding the protein of the present invention, such as diseases accompanied by abnormal cell proliferation such as malignant tumors. The detection method is also used for protein quantification. Examples of the method for immunological detection include an ELISA method using a microtiter plate, a fluorescent antibody method, a Western blot method, and an immunohistological staining method. As a method for immunological quantification, sandwich ELISA using two kinds of monoclonal antibodies having different epitopes among antibodies reacting with the protein of the present invention in a liquid phase,
Radioimmunoassay using the protein of the present invention labeled with a radioisotope such as ¹²⁵ I and an antibody recognizing the protein of the present invention can be mentioned.

(9) Pharmaceutical Containing Antibody of the Present Invention The protein of the present invention, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 2 shows high homology to IL-1ra,
Has cytotoxicity against several types of cancer cells including melanoma cell line A375, human myeloid cell line K562, breast cancer cell MCF-7, or mouse T
Antibodies against a protein consisting of the amino acid sequence of SEQ ID NO: 2 have been reported to inhibit the growth of certain types of tumor cells such as the cell line Eb. Or it can be a prophylactic.

Further, IL-1 acts on nerve cells to increase the expression of nerve growth factor (NGF); it acts on fibroblasts to enhance the expression of NGF,
Reported to increase RNA stability, increased cAMP in hypothalamus, PGE2
It has been reported that they cause production and promote secretion of hypothalamic hormones and pituitary hormones. Therefore, antibodies against a protein consisting of the amino acid sequence of SEQ ID NO: 2 are used for diseases based on nerve cell disorders, such as neurological disorders. It can be a therapeutic or prophylactic agent for amputation, Alzheimer's disease, Parkinson's disease, ischemic disease.

Further, it has been reported that administration of IL-1 before administration of an anticancer agent or the like reduces the lethal effect due to suppression of bone marrow function. It can be a therapeutic or prophylactic agent for cancer in therapy or chemotherapy.

The pharmaceutical containing the protein, DNA or antibody of the present invention as an active ingredient can be administered alone, but usually the active ingredient is pharmacologically acceptable. Alternatively, it may be admixed with more than one carrier and provided as a pharmaceutical preparation manufactured by any of the methods well-known in the art of pharmacy. Preferably, a sterile solution dissolved in water or an aqueous carrier such as an aqueous solution of salt, glycine, glucose, human albumin or the like is used. Also, pharmacologically acceptable additives such as buffering agents and tonicity agents for bringing the formulation solution closer to physiological conditions, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, citric acid Sodium and the like can be added. Alternatively, it can be stored after being freeze-dried and dissolved in an appropriate solvent before use.

The administration route is preferably the one that is most effective in the treatment, and may be oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous. it can. Administration forms include sprays, capsules, tablets, granules, syrups, emulsions,
Suppositories, injections, ointments, tapes and the like can be mentioned. Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as, for example, emulsions and syrups include water, sucrose, sorbitol, sugars such as fructose, polyethylene glycol,
It can be produced using glycols such as propylene glycol, oils such as sesame oil, olive oil and soybean oil, preservatives such as p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint 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.

Examples of preparations suitable for parenteral administration include injections, suppositories, sprays and the like. For example, injections
It is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both. Suppositories are prepared using carriers such as cocoa butter, hydrogenated fats or carboxylic acids. Also,
Sprays are prepared using the compound itself or a carrier which does not irritate the oral and respiratory tract mucosa of the recipient and which disperses the compound as fine particles to facilitate absorption. Specific examples of the carrier include lactose and glycerin. Formulations such as aerosols and dry powders are possible depending on the properties of the compound and the carrier used. Also, in these parenteral preparations, the components exemplified as additives in the oral preparation can be added. The dose or frequency of administration depends on the desired therapeutic effect, administration method, treatment period, age,
Normally 10 μg / kg per adult per day, depending on body weight
８8 mg / kg.

(10) Preparation of Knockout Non-Human Animal Using the DNA of the Present Invention
Non-human animals of interest, preferably non-human mammals, such as cows, sheep, goats, pigs, horses, mice, chickens and other embryonic stem cells (embryonic stem cells), encoding the protein of the present invention on the chromosome The gene can be obtained by a known homologous recombination technique (for example, Nature, 326 , 6110, 295, 198).
7; and Cell, 51 , 3, 503, 1987, etc.) to produce a mutant clone inactivated or substituted with an arbitrary sequence [for example, Nature, 350 , 6315, 243, 1991]. Blastcyst of fertilized egg of animal using mutant clone of embryonic stem cell
By injection chimera method or assembly chimera method, etc.
Chimeric individuals consisting of embryonic stem cell clones and normal cells can be prepared. By crossing the chimeric individual with a normal individual, an individual having an arbitrary mutation in the gene encoding the protein of the present invention on the chromosome of cells of the whole body can be obtained. A knockout non-human animal can be obtained as an individual in which the expression of the gene encoding the protein of the present invention is partially or completely suppressed from among homozygous individuals having the above-mentioned mutation.

Further, by introducing a mutation into any position of the gene encoding the protein of the present invention on the chromosome,
It is also possible to produce knockout non-human animals.
For example, the activity of the product can be modified by introducing a mutation by substituting, deleting, and / or inserting a base into the translation region of the gene encoding the protein of the present invention on the chromosome. Further, by introducing a similar mutation into the expression control region, the degree, timing, tissue specificity and the like of the expression can be altered. Cre-
In combination with the loxP system, the expression timing, expression site, expression amount, and the like can be more positively controlled. Examples of such cases include the use of a promoter that is expressed in a specific region of the brain and deletion of the target gene only in that region (Cell, 87 , 7, 1317, 1996) or expression of Cre. It is known that an adenovirus is used to delete a target gene in an organ-specific manner at a target time (Science, 278 , 5335, 1997).

Accordingly, the expression of the gene encoding the protein of the present invention on the chromosome can be controlled at any time or in any tissue, or any insertion, deletion, or substitution can be performed in the translation region or expression control region. , A knockout non-human animal can be produced. A knockout non-human animal can be produced at any time, at any degree, or at any site,
Various disease symptoms caused by the protein of the present invention can be induced. Thus, the knockout non-human animal of the present invention becomes an extremely useful animal model in the treatment and prevention of various diseases caused by the protein of the present invention. In particular, it is very useful as a therapeutic drug, a preventive drug, and a model for evaluating functional foods, health foods and the like. The present invention will be described in more detail with reference to the following Examples, which are merely illustrative of the present invention and do not limit the scope of the present invention.

[0100]

EXAMPLES Example 1: New growth belonging to the IL-1 family
Identification of Factors A human cDNA library purchased from Clontech (Hu
For the base sequence of each clone of man Bone Marrow 5'-STRETCH PLUS cDNA), the base sequences at the 5 'end and 3' end of the cDNA were converted to a DNA sequencing reagent (Dye Terminator C).
ycle Sequencing FS Ready Reaction Kit; PE Biosyste
ms) according to the attached manual, followed by a DNA sequencer (AB
The base sequence was determined using I PRISM377 (manufactured by PE Biosystems). The determined nucleotide sequence of each clone was identified as a known protein belonging to the IL-1 family registered in the protein amino acid sequence database SWISSPROT or the nucleotide sequence database GenBank.
IL-1α (SWISSPROT accession number: P14778), human IL-1β (SWISSPROT)
T accession number: P01584), human IL-
1ra (SWISSPROT accession number:
P18510), a cDNA clone having homology to the amino acid sequence of these molecules was selected, and the protein encoded by the clone was translated. The amino acid sequence of the protein translated into SEQ ID NO: 2 is shown in SEQ ID NO: 4.

The protein having the amino acid sequence of SEQ ID NO: 2 was found to be 28% by a homology analysis using BLAST2 at a P value of 0.009 and 28% with human IL-1β and human IL-1ra belonging to the IL-1 family. , P value 1 × 10 ^-8 and 29
% Significant homology. Further, from the comparison of the nucleotide sequence with Tango-77 belonging to the same IL-1 family (International Publication WO99 / 06426), the nucleotide sequence of SEQ ID NO: 4 is the same as the nucleotide sequence of Tango-77 having 120 nucleotides represented by SEQ ID NO: 3. It was found that it had a sequence to which a new base sequence was added. In addition, a comparison with the nucleotide sequence of a human genomic BAC clone encoding human IL-1α, human IL-1β, and Tango-77 revealed a genomic DNA nucleotide sequence containing the nucleotide sequence of SEQ ID NO: 3. The nucleotide sequence of the genomic DNA containing the nucleotide sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 5. The nucleotide sequence of SEQ ID NO: 3 was surrounded by AG as a splicing acceptor sequence and GT as a splicing donor sequence, and was found to form an exon of the gene consisting of the nucleotide sequence of SEQ ID NO: 4. Therefore, SEQ ID NO: 4
Has been found to be a splice form of a novel IL-1 antagonist obtained for the first time according to the present invention. Escherichia coli Es containing a plasmid containing the cDNA encoded by the nucleotide sequence of SEQ ID NO: 4
cherichia coli DH5a was designated as FERM BP-6943 on November 22, 1999 by the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, 1-3-1 Higashi, Tsukuba, Ibaraki, Japan (zip code 305-8566). Has been deposited.

Example 2: The nucleotide sequence of SEQ ID NO: 4 is a code
Amino acid sequence is the major protein in bone marrow
Confirmation of expression product Human cDNA library Mara purchased from Clontech
thon-Ready ^™ cDNA (Human Bone Marrow: Cat.
416-1) as a template, the attached AP1 primer and a 27-mer primer 1 (5′-CAAATTAGCGCG containing a nucleotide sequence common to the nucleotide sequence of Tango-77 and SEQ ID NO: 4)
AGGAAGGCGTTCAATGG-3 ': Once using PC
R reaction, and using the reaction product as a template, AP
2 primer and 24-mer primer 2 (5 ′ containing the nucleotide sequence common to the nucleotide sequence of Tango-77 and SEQ ID NO: 4 and containing the nucleotide sequence closer to the AP1 primer than primer 1.
PCR reaction was performed using -CTCCTTCTTCAGCTGAAGGGATGG-3 ′: SEQ ID NO: 7). Human bone marrow cDNA library Ma
Using rathon-Ready ^™ cDNA as material, AP1 primer and Primer 1 and AmpliTaq Gold ^™ (PE Applied Biosystems
The reaction solution was prepared at 94 ° C. for 9 minutes, and then the reaction was repeated 38 times at 94 ° C. for 30 seconds and at 68 ° C. for 2 minutes. This reaction solution was diluted 50-fold with sterile water, and the diluted DNA solution was used as a material to prepare AP2 primer and Primer 2 and AmpliTaq Gold ^™ (PEApplied Biosyst.
The reaction mixture was further prepared at 94 ° C for 9 minutes, then the reaction at 94 ° C for 30 seconds and 68 ° C for 2 minutes was repeated 30 times, and finally the reaction was performed at 72 ° C for 10 minutes. I let it. After the reaction solution was subjected to agarose gel electrophoresis, the amplified DNA
The fragment was recovered and its nucleotide sequence was determined. In agarose electrophoresis, two bands, about 800 bp in size and about 400 bp in size, were observed. DN of each band
A was recovered and its nucleotide sequence was determined.
The sequence completely matched the nucleotide sequence of SEQ ID NO: 4. About 400b
The DNA sequence of the band of the size p was a partial sequence of the band of the size of about 800 bp. Therefore, in human bone marrow, it was found that the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4 was the main expression product as compared with Tango-77.

Example 3: The nucleotide sequence of SEQ ID NO: 4 is a code
Based on the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4 shown in SEQ ID NO: 2, Chofusman, Robson, DSC (http://www.bmm.icnet.uk/software. htm
l, http://www.gh.wits.ac.za/crystal/w3vlc/misc.anc
003.html), and analyze the two-dimensional structure of the protein.
α, human IL-1β, human IL-1ra, human IL-1raβ (Japanese
-304888), compared to Tango-77, as shown in FIG. FIG.
In the description, a protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4 is referred to as IL-1raγ for convenience (hereinafter, the protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4 of the present invention is referred to as “IL-1raγ” ). The amino acid sequence having a β-sheet structure is surrounded by a frame, and hydrophobic amino acids in the β-sheet structure are indicated by white letters. The protein consisting of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 4 retains a 12 β-sheet structure commonly found in human IL-1α, human IL-1β, human IL-1ra, and human IL-1raβ. I knew it was. On the other hand, Tango-77 does not contain the nucleotide sequence of SEQ ID NO: 3, and thus lacks three β-sheet structures. This part is important for binding to the IL-1 receptor, which is evident from the results of analysis using site-specific mutants of IL-1 and IL-1 receptor and X-ray crystallography. (E.
L-Tompkins et al .; Protein Engineering, 6 , 535, 1993, h
ttp: //www.rcsb.org/pdb/). Therefore, it was found that the biological activity of Tango-77 was partial as compared with the protein encoded by the nucleotide sequence of SEQ ID NO: 4.

Example 4 Human IL-1raγ Using RT-PCR
Gene tissue distribution analysis Human organs purchased from Clontech (brain, heart, lung, liver, kidney, small intestine, bone marrow, spleen, thymus, lymph nodes, mammary
4 μg of total RNA prepared from 4 μg of polyA ⁺ RNA derived from uterus, prostate, testis) by the AGPC method [Analytical Biochemistry, 162, 156 (1987), Experimental Medicine, 9 , 1937 (1991)] as a template, and commercially available SUPER SCRIPT Preamplification System f
cDNA was synthesized using or first strand cDNA synthesis (GIBCOBRL) according to the attached manual. Synthesized
Using a 50-fold diluted solution of cDNA in sterile water as a material, primers containing a base sequence specific to human IL-1raγ or human β-actin and AmpliTq Gold ^™ (PE Applied
After preparing a reaction solution using a conventional method using Biosystems,
The reaction was performed at 94 ° C. for 9 minutes, and then a cycle of 94 ° C. for 30 seconds, 62.5 ° C. for 1 minute, and 72 ° C. for 1 minute was repeated 36 cycles,
Finally, PCR was performed under the condition of reacting at 72 ° C. for 10 minutes. The reaction solution was subjected to agarose gel electrophoresis, and the expression level was compared semi-quantitatively by comparing the concentration of a DNA band specific to the primer used. The results are shown in FIG. IL-1
raγ has the strongest expression detected in the bone marrow,
Weak expression was observed in lymph nodes and spleen.

The primer used was a primer having the nucleotide sequence represented by SEQ ID NO: 7 or SEQ ID NO: 8 used in Example 2.
PCR was performed using NA as a primer set specific to human IL-1raγ (Tango-77 was not amplified) and DNA having the nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 as a primer set specific to human β-actin. Was done. In the above RT-PCR reaction using these primers,
A primer-specific DNA band was observed, the size of which was human IL-1raγ and human β-actin, respectively.
Approximately 200 bp and 800 bp.

Example 5: Human IL-1raγ using RT-PCR
Analysis of gene expression in lymphoid cells Human lymphoid cell lines and activated human peripheral cells shown below
Expression analysis by RT-PCR was performed using T cells as a material in the same manner as in Example 4. As a human lymphoid cell line, T
Cell line [Jurkat cells (Riken Cell Bank; RCB 0806), Mo
lt-3 cells (ATCC CRL 1552), Molt-4 (ATCC CRL 158)
2), HuT78 (ATCC TIB-161)], B cell line [Namalwa KJ
M-1 (J. Biol. Chem., 268 , 22782, 1993), Daudi (ATCC
CCL-213), Raji (ATCC CCL 86)], granulocyte / monocyte cell line [HL-60 (ATCC CCL 240), U-937 (ATCC CCL 159)
3), THP-1 (ATCC CCL 1593)]. Peripheral T cells
Polymorphprep ^TM (Nycomed Pha
rma) to obtain polymorphonuclear leukocytes and mononuclear cells, and then obtained T cells from the obtained mononuclear cells using Nyron Fiber (manufactured by Wako Pure Chemical Industries, Ltd.). The method is Nyron Fibe
I followed the instructions of r. Further, J. Immunol., 150 , 1122
(1993).
Add 10 ng / ml of RPMI1640 medium containing 10% FCS to a concentration of ⁶ cells / ml, and add 50 ng / ml of interleukin 2 (IL-2), 1 μg / ml
Phytohemagglutinin-P (PHA-P) and 5 ng / ml transforming growth factor β (TGF-P
Activated T cells were obtained by adding β) and culturing for 2, 4, 6, or 8 days and then collecting. The results for the human lymphoid cell line are shown in FIG.
The results for T cells are shown in FIG. In the lymphoid cell line, expression was detected only in the B cell line (Namalwa KJM-1, Daudi, Raji). On the other hand, in peripheral T cells, almost no expression was observed without treatment, whereas IL-2, PHA-P, TGFβ
It was clarified that the expression was induced by the addition of E. These results suggest that IL-1raγ is a functioning factor in lymphoid cells and is involved in inflammatory and immune responses.

Example 6: Human IL-1 using insect cells as a host
The production of recombinant proteins by insect cells requires the production of a recombinant virus incorporating the gene of interest, which involves the process of producing a special vector having a cDNA encoding the protein of interest, a process called baculovirus. Insect cells are co-transfected with the viral DNA and the transfer vector, a recombinant virus is produced by homologous recombination and further propagated, and the cells are infected with the recombinant virus to express a target protein. This will be described in detail below.

(1) Preparation of Transfer Vector First, in order to obtain the nucleic acid sequence of the entire translation region of IL-1raγ, the human bone marrow cDNA library Marathon-Rea described in Example 2 was obtained.
A primer having the nucleotide sequence represented by SEQ ID NO: 6, a primer having the nucleotide sequence represented by SEQ ID NO: 11, and a KOD DNA Polyme used in Example 2 using dy ^™ cDNA as a template.
After preparing a reaction solution by a conventional method using rase (manufactured by TOYOBO), a cycle of 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, 72 ° C. for 1 minute is repeated 36 cycles, and finally at 72 ° C. for 10 minutes. PCR was performed under the conditions for reaction. Next, the obtained amplification product of about 800 bp was placed at the Sma I site of pBluescriptII SK- (manufactured by Stratagene), and Kpn I of pBluescriptII SK-
On the site side, 3 'downstream of IL-1raγ is S of pBluescriptII SK-
pSK-IL-1ra by inserting it into the ac I site
γ was prepared. Next, the Pst I- Xba I fragment of pSK-IL-1raγ (840
kb) was inserted into the Pst I- Xba I site of an insect cell transfer vector pVL1392 (manufactured by Pharmingen), and pVL-IL-
1raγ was prepared.

(2) Preparation of Recombinant Virus A linear baculovirus DNA [baculogold baculovirus DNA, manufactured by Pharmingen) was applied to insect cells Sf9 (manufactured by Iwaki Glass) cultured on ESF921 medium (manufactured by Protein Expression). And the transfer vector prepared in (1) above was introduced by a lipofectin method (protein nucleic acid enzyme, 37 , 2701, 1992) to prepare a recombinant baculovirus. This will be described in detail below. 4 μg of pVL-IL-1raγ and linear baculovirus
15 ng of DNA was dissolved in 12 μl of sterilized distilled water, a mixture of 6 μl of lipofectin and 6 μl of sterilized distilled water was added, and the mixture was allowed to stand at room temperature for 15 minutes. On the other hand, Sf9 cells 1 × 10 ⁶
The cells were suspended in 2 ml of ESF921 medium and placed in a 50 mm diameter plastic culture dish for cell culture. The above plasmid DNA, linear baculovirus DNA, and lipofectin mixed solution were all added thereto, and cultured at 27 ° C. for 3 days. Then, 1 ml of a culture supernatant containing the recombinant virus was collected. Add 1 ml of ESF921 medium to the Petri dish and culture at 27 ° C for 3 days.Add 1.5 ml of culture supernatant containing the recombinant virus.
I got it.

Next, the recombinant virus carrying the IL-1raγ gene was propagated by the following procedure. The Sf9 cells were cultured in a 50 ml ESF921 medium to a concentration of 5 × 10 ⁵ / ml in a 125 ml Erlenmeyer flask at 27 ° C. with shaking at 125 rpm. When the cells grew to 2 × 10 ⁶ / ml, the recombinant virus was infected at MOI = 10 and cultured for further 3 days. The culture was centrifuged at 1,200 × g for 10 minutes to remove the cells, and a recombinant virus solution used for protein expression was obtained.
The titer of the recombinant virus solution was measured by the following method. 6 × 10 ⁵ Sf9 cells were suspended in 4 ml of ESF921 medium, placed in a cell culture plastic dish having a diameter of 50 mm, and allowed to stand at room temperature for 1 hour to allow the cells to adhere to the dish. Remove the supernatant, add 400 μl of ESF921 medium and 100 μl of the recombinant virus solution diluted with ESF921 medium, and add
After standing for a period of time, the medium was removed, and a medium containing 5 ml of 1% low melting point agarose (Agarplaque agarose, manufactured by Pharmingen) [sterile, 1 ml of 5% agarplaque plus agarose aqueous solution and 4 ml TMN-FH Insect Medium (manufactured by Pharmingen) and kept at 42 ° C.] was poured into the petri dish. After standing at room temperature for 15 minutes, a vinyl tape was wrapped around a Petri dish to prevent drying, the Petri dish was placed in a sealable plastic container, and cultured at 27 ° C for 5 days. 1 ml of PBS containing 0.01% neutral red was added to the petri dish, and after further culturing for 1 day, the number of plaques appeared was counted. As a result, 0.5-2x
A 10 ⁸ / ml recombinant virus solution could be prepared.

(3) Expression of protein Sf9 cells were cultured in a 250 ml Erlenmeyer flask at 27 ° C. with shaking at 125 rpm in 100 ml of ESF921 medium at 5 × 10 ⁵ / ml. When the cells grew to 3-4 × 10 ⁶ / ml, the cells were subcultured to 3 × 10 ⁷ cells in a 182 cm ² bottom flask pre-added with 25 ml of ESF921 medium. After allowing the cells to adhere at room temperature for 1 hour, the medium was removed, and the recombinant virus carrying the IL-1raγ gene was
It was added so that I = 5, and ESF921 medium was further added to make 10 ml, and the cells were infected at room temperature for 1 hour. ESF921 medium for 20
ml was added and the cells were cultured at 27 ° C. for 3 days to express the target recombinant protein. A culture supernatant of cells infected with the recombinant virus carrying the IL-1raγ gene was used as a sample, subjected to 15% SDS-polyacrylamide gel electrophoresis, and analyzed by gel staining using Coomassie brilliant blue R250.
As a result, as shown in FIG. 5, a band having a molecular weight of about 25 kDa, which was not found in non-infected cells, was detected in the culture supernatant of cells infected with the IL-1raγ gene.

(4) Preparation of Insect Cell-Expressed Human IL-1raγ Protein Standard In order to mainly isolate low molecular contaminants derived from the culture medium or Sf9 cells, the insect cell-expressed human IL-1raγ protein was prepared as follows. Was partially purified. 20 ml of the culture supernatant of Sf9 cells infected with the recombinant virus carrying the IL-1raγ gene was concentrated to 500 μl using Centricon-10 (manufactured by Amicon).
Next, this concentrated solution is added to Phosphate-buffered saline (PBS).
Through a 20 ml column of Sephacryl S-300 (manufactured by Amersham Pharmacia Biotech) equilibrated with, and gel filtration column chromatography using PBS as a solvent at 0.07 ml / mi.
n, 1 ml / fraction conditions. About each elution fraction
The fraction containing IL-1raγ was confirmed by performing 15% polyacrylamide gel electrophoresis.
IL-1raγ protein standard was used.

Example 7: Antigen for producing anti-IL-1raγ antibody
Analyze the protein sequence of the prepared IL-1raγ, from the portion having a high hydrophilicity, N-terminal, C-terminal, turn structure on the secondary structure, random coil structure, as a partial sequence considered suitable as an antigen, Compound 1 and Compound 2 (a polypeptide having the amino acid sequence represented by SEQ ID NO: 12 and a polypeptide having the amino acid sequence represented by SEQ ID NO: 13) were selected. (About abbreviations) The abbreviations for amino acids and their protecting groups used in the present invention are IUPA for biochemical nomenclature.
C-IUB Committee (IUPAC-IUB Joint Commission on Biochem
ical Nomenclature] [European Journal of Biochemistry
Biochemistry), volume 138, page 9 (1984)].

The following abbreviations represent the corresponding amino acids below unless otherwise specified. Ala: L-alanine Asn: L-asparagine Asp: L-aspartic acid Asx: L-aspartic acid or L-asparagine Cys: L-cysteine Gln: L-glutamine Glu: L-glutamic acid Glx: L-glutamic acid or L-glutamine Gly: glycine His: L-histidine Ile: L-isoleucine Leu: L-leucine Lys: L-lysine Phe: L-phenylalanine Pro: L-proline Ser: L-serine Met: L-methionine

The following abbreviations represent the corresponding amino acid protecting groups and side chain protected amino acids shown below. Fmoc: 9-fluorenylmethyloxycarbonyl tBu: t-butyl Trt: trityl Boc: t-butyloxycarbonyl Fmoc-Asn (Trt) -OH: Nα-9-fluorenylmethyloxycarbonyl-Nγ-trityl-L -Asparagine Fmoc-Asp (OtBu) -OH: Nα-9-Fluorenylmethyloxycarbonyl-L-aspartic acid-β-t-butyl ester Fmoc-Cys (Trt) -OH: Nα-9-Fluorenylmethyl Oxycarbonyl-S-trityl-L-cysteine Fmoc-Gln (Trt) -OH: Nα-9-Fluorenylmethyloxycarbonyl-Nε-trityl-L-glutamine Fmoc-Glu (OtBu) -OH: Nα-9- Fluorenylmethyloxycarbonyl-L-glutamic acid-γ-t-butyl ester Fmoc-His (Trt) -OH: Nα-9-Fluorenylmethyloxycarbonyl-N ^im -trityl-L-cysteine Fmoc-Lys (Boc ) -OH: Nα-9-fluorenylmethyloxycarbonyl-Nε-t-butyloxycarbonyl-L-lysine Fmoc-Ser (tBu) -OH: Nα-9-fluor Oleynylmethyloxycarbonyl-Ot-butyl-L-serine

The following abbreviations correspond to the following reaction solvents:
Represents a reaction reagent and the like. PyBOP: benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate HOBt: N-hydroxybenzotriazole NMM: N-methylmorpholine HOBt: N-hydroxybenzotriazole DMF: N, N-dimethylformamide TFA: trifluoro Acetic acid

In the following examples, the physicochemical properties of the compounds were measured by the following methods. Mass spectrometry, JEOL
The measurement was performed by the FAB-MS method using JMS-HX110A or the MALDI-TOFMS method using a Bruker mass spectrometer REFLEX. Amino acid analysis is performed according to the method of Cohen, SA et al. [Analytica Biochemistry (Analytica
Biochemistry), 222 , 19, 1994]. Hydrolysis is performed in hydrochloric acid vapor at 110 ° C for 20 hours, and the amino acid composition of the hydrolyzate is determined by Waters Acu
(ccQ-Tag) amino acid analyzer (Waters) was used for analysis.

(1) Compound 1 (SEQ ID NO: 12) (H-Cys-
Pro-Lys-Val-Lys-Asn-Leu-Asn-Pro-Lys-Lys-Phe-Ser-Il
Synthesis of e-His-Asp-Gln-Asp-His-Lys-Val-NH ₂ ) Carrier resin (Rink amide MB) with 22 μmol of Fmoc-NH
Place 40 mg of HA resin resin (Novabiochem) in a reaction vessel of an automatic synthesizer (Shimadzu Corporation), add 900 μl of DMF, stir for 1 minute, drain the solution, and perform the following operations according to the synthesis program of Shimadzu Corporation Was. (a) 900 μl of 30% piperidine-DMF solution was added and the mixture was
After stirring for minutes, the solution was drained and this operation was repeated once more. (b) The carrier resin was washed with 900 μl of DMF for 1 minute, the solution was discharged, and this operation was repeated 5 times. (c) Fmoc-Val-OH (220 μmol), PyBOP (220 μmol), HOBt
Monohydrate (220 μmol) and NMM (330 μmol) were added to DMF (7
(70.2 μl) for 3 minutes, the resulting solution was added to the resin, the mixture was stirred for 30 minutes, and the solution was drained. (d) After washing the carrier resin with 900 μl of DMF for 1 minute, the solution was discharged, and this was repeated five times. Thus, Fmoc-Val-NH 2 was synthesized on the support.

Next, after the steps (a) and (b), in the step (c), F
A condensation reaction was carried out using moc-Lys (Boc) -OH, and through the washing step (d), Fmoc-Lys (Boc) -Val-NH 2 was synthesized on the carrier. Hereinafter, in the step (c), Fmoc-His (Trt) -OH, Fmo
c-Asp (OtBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Asp (OtBu) -O
H, Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Ser (tBu) -O
H, Fmoc-Phe-OH, Fmoc-Lys (Boc) -OH, Fmoc-Lys (Boc) -O
H, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Leu-OH, Fmo
c-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc
-Lys (Boc) -OH, Fmoc-Pro-OH, and Fmoc-Cys (Trt) -OH were used sequentially to repeat (a) to (d), followed by deprotection and washing of (a) and (b). After the process, the resultant was washed with methanol and butyl ether in that order, and dried under reduced pressure for 12 hours to obtain a carrier resin to which the side-chain protected peptide was bound. To this was added 1 ml of a mixed solution consisting of TFA (90%), thioanisole (5%), and 1,2-ethanedithiol (5%), and the mixture was allowed to stand at room temperature for 2 hours to remove the side-chain protecting groups. The peptide was cut out from the resin.
After filtering the resin, about 10 ml of ether was added to the obtained solution, and the resulting precipitate was collected by centrifugation and decantation to obtain 49.4 mg as a crude peptide. The crude product was dissolved in 5 mL of a 2M acetic acid aqueous solution, passed through a reverse-phase silica gel-filled cartridge (YMC Dispo SPE C18) to adsorb the peptide, washed with 0.1% TFA, 10% acetonitrile aqueous solution, and then washed with 0.1% acetonitrile aqueous solution. The fraction was eluted with TFA and 25% acetonitrile aqueous solution to obtain a fraction containing Compound 1. This was freeze-dried to obtain 37.9 mg of compound 1. Mass spectrometry [FABMS]; m / z = 2475.0 (M + H ⁺ ) amino acid analysis; Asx 4.2 (4), Ser 1.0 (1), Glx 1.1
(1), His 1.9 (2), Pro 2.1 (2), Val 1.8 (2), Ile 0.
8 (1), Leu 1.0 (1), Phe 1.0 (1), Lys 5.1 (5), Cys
1.3 (1)

(2) Compound 2 (SEQ ID NO: 13) (H-Cys-
Synthesis of Lys-Ala-Glu-Met-Ser-Pro-Ser-Glu-Val-Ser-Asp-OH) Fmoc-Asp (OtBu), carrier resin (Wang
Resin (Novabiochem) 30 mg as a starting material
Similarly, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-
Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-
Ser (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Thr (tBu))-O
H, Fmoc-Arg (Pmc) -OH, Fmoc-Met-OH, Fmoc-Glu (OtBu) -O
H, Fmoc-Ala-OH, Fmoc-Lys (Boc), Fmoc-Cys (Trt) -OH
Are sequentially condensed, followed by Fmoc group removal, washing and drying,
A carrier resin to which the side chain protected peptide was bound was obtained. to this,
Add 1 ml of a mixed solution consisting of TFA (90%), thioanisole (5%), and 1,2-ethanedithiol (5%), and let stand at room temperature for 2 hours to remove side-chain protecting groups and remove peptide from resin. Was cut out. After filtering off the resin, about 10 ml of ether was added to the obtained solution, and the resulting precipitate was collected by centrifugation and decantation to obtain a crude peptide.
14.4 mg was obtained. This crude product was dissolved in 5 mL of a 2 M aqueous acetic acid solution, and the reverse phase silica gel-filled cartridge (YMC
spo SPE C18) to adsorb the peptide, 0.1% TF
A, After washing with 5% acetonitrile aqueous solution, elution was performed with 0.1% TFA, 20% acetonitrile aqueous solution to obtain a fraction containing compound 2. This was freeze-dried to obtain 19.8 mg of Compound 2. Mass spectrometry [TOFMS]; m / z = 1282.5 (M + H ⁺ ) amino acid analysis; Asx 1.0 (1), Ser 2.9 (3), Glx 2.1
(2), Ala 1.0 (1), Pro 1.0 (1), Val 1.0 (1), Met 0.
9 (1), Lys 1.0 (1), Cys 1.2 (1)

Example 8: Monochrome recognizing IL-1raγ
Preparation of Null Antibody (1) Preparation of Immunogen Compounds 1 and 2 obtained in Example 7 were conjugated with KLH (Calbiochem) by the following method in order to enhance immunogenicity. The original. That is,
KLH was dissolved in PBS and adjusted to 10 mg / ml, and 1/10 volume of 25 m
g / ml MBS (N- (m-Maleimidobenzoyloxy) succinimide;
Nacalai Tesque] was added dropwise, and the mixture was stirred and reacted for 30 minutes.
K obtained by removing free MBS on a gel filtration column such as a Sephadex G-25 column equilibrated with PBS in advance.
LH-MB 2.5 mg in 0.1 M sodium phosphate buffer (pH
The resulting mixture was mixed with 1 mg of the peptide dissolved in 7.0), and reacted with stirring at room temperature for 3 hours. After the reaction, one dialyzed with PBS was used as an immunogen.

(2) Animal immunization and preparation of antibody-producing cells 100 μg of the KLH conjugates of compound 1 and compound 2 prepared in (1) above were each added to an aluminum hydroxide adjuvant [Antibodies-A Laboratory Manual, Cold Sp.
ring Harbor Laboratory, p99, 1988] and 3 × 10 ⁹ cells of pertussis vaccine (manufactured by Chiba Prefectural Serum Research Institute) were administered to 6 to 8 week old female BALB / c mice. Two weeks after the administration, 100 μg of each KLH conjugate is administered once a week.
Four times in total. Blood was collected from the carotid artery of the mouse, and its serum antibody titer was examined by the enzyme immunoassay shown below. From the mouse showing a sufficient antibody titer, the spleen was extracted 3 days after the final immunization. The spleen was shredded in MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, and centrifuged (250 × g, 5 minutes). Tris-ammonium chloride buffer (pH 7.6) was added to the obtained precipitate fraction,
Erythrocytes were removed by treating for 1-2 minutes. The obtained precipitate fraction (cell fraction) was washed three times with MEM medium,
Used for cell fusion.

(3) Enzyme immunoassay (Binding EL
ISA) As the antigen for the assay, those obtained by conjugated with the respective compounds obtained in Example 7 and thyroglobulin (hereinafter abbreviated as THY) were used. The preparation method was as described in Example 8 (1), except that SMCC [4- (NM
aleimidomethyl) -cyclohexane-1-carboxylic acid Nh
ydroxysuccinimido ester; Sigma]. The conjugate prepared as described above was dispensed into a 96-well EIA plate (Greiner) at 10 μg / ml and 50 μl / well, and allowed to stand at 4 ° C. overnight for adsorption. After washing the plate, 1% bovine serum albumin (BSA) / Dulbecco phosphate buffer (Phosphate buffered saline: PBS) was added.
100 ml / well was added, and the mixture was left at room temperature for 1 hour to block the remaining active groups. After standing, 1% BSA / PBS was removed, the antiserum of the mouse to be immunized, and the culture supernatant of the monoclonal antibody were dispensed into the plate at 50 μl / well, and left for 2 hours. The plate was treated with 0.05% polyoxyethylene (20) sorbitan monolaurate (equivalent to the trade name of Tween 20, trademark of ICI, manufactured by Wako Pure Chemical Industries, Ltd.) /
After washing with PBS (hereinafter referred to as Tween-PBS), peroxidase-labeled rabbit anti-mouse immunoglobulin (manufactured by DAKO) was added.
50 μl / well was added and left at room temperature for 1 hour. After washing the plate with Tween-PBS, ABTS substrate solution [2.2-azinobis (3
Ammonium-ethylbenzothiazole-6-sulfonate), 1 mmol / l ABTS / 0.1 mol / l citrate buffer (pH
4.2)] was added, and the absorbance at 415 nm was measured using a plate reader (Emax; Molecular Devices).

(4) Preparation of mouse myeloma cells 8-Azaguanine-resistant mouse myeloma cell line P3X63Ag8U.1 (P
3-U1: purchased from ATCC) was cultured in a normal medium (RPMI medium supplemented with 10% fetal bovine serum) to secure 2 × 10 ⁷ or more cells at the time of cell fusion and used as a parent strain for cell fusion.

(5) Preparation of hybridoma The mouse spleen cells obtained in Example 8 (2) and Example 8
The myeloma cells obtained in (4) were mixed at a ratio of 10: 1, and centrifuged (250 × g, 5 minutes). After thoroughly loosening the cells of the obtained precipitate fraction, while stirring, at 37 ° C,
Polyethylene glycol -1000 (PEG-1000) 2 g , MEM medium 2 ml and dimethyl sulfoxide 0.7ml mixture was added 0.5 ml per 10 ⁸ mouse spleen cells, MEM medium 1 every 1-2 minutes to the suspension After adding ml several times, a MEM medium was added to adjust the total volume to 50 ml. The suspension was centrifuged (900 rpm, 5 minutes), and the cells in the obtained precipitate fraction were loosened gently. Then, the cells were gently sucked and aspirated with a mespipet to HAT medium [10% fetal bovine serum]. Additive R
PMI medium supplemented with HAT Media Supplement (Boehringer Mannheim)]. The suspension was dispensed at 200 μl / well into a 96-well culture plate and cultured at 37 ° C. for 10 to 14 days in a 5% CO ₂ incubator.

After culturing, the culture supernatant was examined by the enzyme immunoassay described in Example 8 (3), and a well which did not react with the control peptide in response to the antigen peptide was selected. Was repeated twice to establish a hybridoma producing an anti-human IL-1raγ monoclonal antibody. The anti-human IL-1raγ monoclonal antibody obtained using Compound 1 as an antigen was named KM2779, and the anti-human IL-1raγ monoclonal antibody obtained using Compound 2 as an antigen was named KM2780. As shown in FIG.
2779 and KM2780 showed specific reactivity to each antigenic peptide. The KM2779-producing hybridoma cell line and the KM2780-producing hybridoma cell line
MBP-7368 and FERM BP-7369, dated November 17, 2000, National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan)
No., postal code 305-8566).

(6) Purification of Monoclonal Antibody The hybridoma strain obtained in Example 8 (5) was used for 5 to 20 × 1 in pristane-treated 8-week-old nude female mice (BALB / c).
0 ⁶ cells / mouse were respectively injected intraperitoneally. After 10 to 21 days, ascites was collected (1 to 8 ml / animal) from the mice that had accumulated ascites due to ascites cancer of the hybridoma. The ascites was centrifuged (1200 × g, 5 minutes) to remove solids. Purification
IgG monoclonal antibody is obtained by the caprylic acid precipitation method (Antibod
ies-A Laboratory Manual, Cold Spring Harbor Labo
ratory, 1988). By ELISA subclass of monoclonal antibodies using a subclass typing kit, KM2779 is IgG _1, KM2780
Was determined to be IgG _2a , respectively.

Example 9: Anti-human IL-1raγ monoclonal
Western blot of human IL-1raγ protein using antibodies
Detection of the insect cell-expressed IL-1raγ protein prepared in Example 6 by 10 n
g / lane 15% SDS-polyacrylamide gel electrophoresis (Antibodies-A Laboratory Manual, Cold Spring Ha
rbor Laboratory, 1988), and then blotted on a PVDF membrane (Millipore). 5% skim mi
After blocking with lk-PBS, culture supernatants of anti-human IL-1raγ monoclonal antibodies KM2779 and KM2780 were added as stock solutions to the membrane, and left at room temperature for 2 hours. After thoroughly washing the membrane with Tween-PBS, a 1,000-fold diluted peroxidase-labeled rabbit anti-mouse immunoglobulin (manufactured by DAKO) was added as a secondary antibody, and the mixture was allowed to stand at room temperature for 1 hour. Tween-PB
After washing well with S, detection was performed using an ECL kit (Amersham Pharmacia Biotech). As shown in FIG. 7, anti-human IL-1raγ monoclonal antibodies KM2779 and KM
2780 specifically recognized the insect cell expressed IL-1raγ described in Example 6.

Example 10: Anti-human IL-1 as neutralizing antibody
raγ monoclonal antibody Anti-human IL-1raγ monoclonal antibody described in Example 8
KM2779 was found to be a neutralizing antibody that inhibits the binding of human IL-1raγ to its receptor based on the following facts. (1) Homology model of IL-1raγ and IL-18 receptor FIG. 8 shows that the recognition site of KM2779 is IL-1raγ (the part of the protein having the amino acid sequence of positions 46 to 208 of SEQ ID NO: 2) and IL-1. For the amino acid numbers 20 to 324 of the amino acid sequence (Genbank Accession no. HSU43672) of the IL-18 receptor (J. Biol. Chem., 271 , 3967-3970, 1996) which is a member of the receptor family, human IL- Crystal structure of complex between 1ra and IL-1 receptor (HA Schreuder et. Al., Natur
e, 194 , 386, 1997) in a dark color on a three-dimensional structure model prepared by a known method. IL-1raγ
Is shown by a surface model, and that of IL-18 receptor is shown by a ribbon model. In addition, Quanta (Molecular Simulation Inc.), Insi
ghtII (Molecular Simulation Inc.) software was used. As shown in FIG. 8, the recognition site of KM2779 was IL-18
The complex was found to be three-dimensionally adjacent to the IL-18 receptor at the time of complex formation with the receptor, and significantly contributed to noncovalent bonding for complex formation between the two.

In this embodiment and the following embodiments,
In the amino acid sequence represented by SEQ ID NO: 2, a protein having the amino acid sequence of SEQ ID NOs: 46 to 208 may be used, but from the following facts, the protein having the amino acid sequence represented by SEQ ID NO: 2 After being expressed and translated as the protein, the N-terminal 45
2 between the amino acids at positions 46 and 46 and at the C-terminus
It was presumed that the amino acid between the 08th and 209th amino acids was cleaved by a protease or the like to form mature IL-1raγ. That is, the N-terminal of the mature IL-1ra
It is presumed to be valine, which is the 46th amino acid of SEQ ID NO: 2 which almost matches the cleavage position of the family of molecules and can be generated as a result of cleavage by chymotrypsin-like protease. As a result of verification by a three-dimensional structure model of a complex consisting of IL-18 receptor, which is a member of the body family, valine, which is the 46th amino acid of SEQ ID NO: 2, was located at the N-terminus of mature IL-1raγ. It was determined that there was. Further, in (3) of Example 11 described later, when IL-1raγ was expressed using an animal cell transformed with the IL-1raγ expression plasmid prepared in Example 6, the culture supernatant was expressed as follows. IL-1raγ in which 10 to 25 amino acids at the C-terminal side were deleted (considered to be a mature form) was detected. In the protein belonging to the IL-1 family, the β-sheet structure existing in its internal structure is important. If a protease cleavage point exists after the last amino acid (proline 205) constituting β12 (see FIG. 1), Since it is considered, it is highly probable that the amino acid is cleaved by the protease at the carboxyl group side of lysine at position 209 which is a basic amino acid. This was supported by the three-dimensional structure model of Example 12 (1) described later. Therefore, the mature IL-1raγ C
The end was determined to be the 209th lysine. From the above, in the amino acid sequence represented by SEQ ID NO: 2, it is considered that the protein having the amino acid sequence of SEQ ID NOs: 46 to 208 is mature IL-1raγ, and the present Example and Example 12 were performed. Demonstrated.

(2) Surface exposed area of each residue of IL-1raγ FIG. 9 shows IL-1raγ based on the three-dimensional structure model shown in FIG.
The solvent exposed area of each amino acid residue of γ (the portion of the protein having the amino acid sequence at positions 46 to 208 of SEQ ID NO: 2) is calculated and plotted by a known method. Quanta (Molecular Simulation Inc.), InsightII
(Molecular Simulation Inc.) software was used.
As shown in FIG. 9, the recognition site of KM2779 contains a plurality of amino acid residues having a large solvent exposed area, and it was revealed that the recognition site can be recognized by an antibody.

(3) Binding energy of non-covalent bond between IL-1raγ and IL-18 receptor FIG. 10 shows KM27 based on the three-dimensional model shown in FIG.
The non-covalent binding energy between the 79 recognition sites and the amino acid residues (all 20-324 residues) of the IL-18 receptor was calculated by known methods and calculated for IL-1raγ (46- of SEQ ID NO: 2). 20
(The part of the protein having the eighth amino acid sequence). To make it,
CHARMm (Molucular Simulation Inc.), Discover (Mol
ecularSimulation Inc.), AMBER (University of Cali)
fornia Sanfrancisco) was used. As shown in FIG. 10, the recognition site of KM2779 contains a plurality of amino acid residues having a large non-covalent binding energy to the IL-18 receptor, and has a large non-covalent bond for forming a complex between the two. It became clear that the contribution ratio was high.

Example 11: Human using animal cells as host
IL-1raγ expression of proteins (1) of pSK-IL-1raγ described in Preparation Example 6 of the vector Hind III-Not I fragment (870 kb) and Namalwa KJM-1 cell expression plasmid pAMo
(J.Biol.Chem., 268, 22782, 1993) was inserted into the Hind III-Not I sites of was prepared pAMo-IL-1raγ.

(2) Introduction of vector into cells [0134] The control plasmid pAMo and the plasmid for human IL-1raγ expression pAMo-IL-1raγ described in (1) above were dissolved in TE so as to be 1 µg / µl each. The cells were introduced into Namalwa KJM-1 cells by electroporation (Cytotechnology, 3 , 133, 1990) to obtain transformed cells. After introducing 4 μg of plasmid per 1.6 × 10 ⁶ cells, 8 ml of RPMI1640 / ITPSG medium [7.5% NaHCO ₃ was added to 1/40
Volume, 200 mmol / l L-glutamine solution (GIBCO)
3%, penicillin-streptomycin solution (GIBCO, 5000 units / ml penicillin, 5000 μg / ml streptomycin) 0.5%, N-2-hydroxyethylpiperazine-
N'-2-hydroxypropane-3-sulfonic acid (N-2-hydroxyethylpiperazine-N'-2-hydroxypropane-3
-sulfonic acid; HEPES) (10 mmol / l), insulin (3 μg / ml), transferrin (5 μg / ml), sodium pyruvate (5 mmol / l), sodium selenite (125 nmol / l), galactose (1mg / ml) was added
RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.)], and cultured in a CO ₂ incubator at 37 ° C. for 24 hours. After that, G418 (manufactured by Gibco) was added to a concentration of 0.5 mg / ml, and
After culturing for a day, a stable transformant was obtained. The transformant was subcultured in RPMI1640 • ITPSG medium containing 0.3 mg / ml G418. Hereinafter, the resistant strains prepared by introducing pAMo and pAMo-IL-1raγ are referred to as KJM-1 / mock strain and KJM-1 / IL-1raγ strain, respectively.

(3) Confirmation of Expression The KJM-1 / mock strain and the KJM-1 / IL-1raγ strain prepared in the above (2) were cultured in RPMI1640 / ITPSG medium until they became confluent. After completion of the culture, the culture supernatant was recovered, and the cells were suspended in PBS containing 1/10 volume of a protease inhibitor cocktail for animal cells (manufactured by Sigma, product number P8340), followed by sonication. The prepared culture supernatant and cell lysate were prepared using the anti-IL-1raγ obtained in Example 8.
Western blotting was performed using antibodies (KM2779, KM2780). As a result, as shown in FIG. 11, for the cell lysate, both KM2779 and KM2780 have the same molecular weight (about 25 kDa) as the insect cell-expressed IL-1raγ described in Example 9.
KJM-1 / IL-1 prepared above
It was revealed that the raγ strain produced IL-1raγ protein. On the other hand, KM2779 was about 24 kDa
No signal was detected with KM2780. As described in Example 7, since KM2779 and KM2780 recognize the internal amino acid sequence and the C-terminal amino acid sequence in the IL-1raγ sequence, respectively, the results in FIG. 11 show that the IL-1raγ protein is in Namalwa KJM-1 cells. When secreted from E. coli, C-terminal sequence is cleaved. Considering the difference in molecular weight from the band detected in the cell lysate, IL-1ra
It was expected that about 25 amino acids had been deleted.

(4) Preparation of Mature Human IL-1raγ Protein Standard IL-1raγ in the culture supernatant of the KJM-1 / IL-1raγ strain was reduced to about 1,000-fold using Centriplus-10 (manufactured by Amicon). Concentrate,
A mature human IL-1raγ protein sample having an IL-1raγ concentration of several μg / ml was used.

Example 12: Namalw as mature protein
a KJM-1 cell-produced IL-1raγ As described in Example 5, Namalwa KJM-1 cells originally show endogenous IL-1raγ expression (FIG. 3).
It is considered that malwa KJM-1 cells have an original IL-1raγ secretion mechanism. Therefore, the IL-1raγ which has been cleaved at the C-terminal sequence detected in the culture supernatant of the KJM-1 / IL-1raγ strain described in Example 11 (3) is a mature protein, and is described in Example 6. Insect cell produced IL-1raγ is expected to be its precursor form. KM2780 is a monoclonal antibody that recognizes only the precursor IL-1raγ, while KM2779 is a precursor,
It can be said that the monoclonal antibody recognizes both mature forms of IL-1raγ. Namalwa KJM-1 cell production IL-1r
It was further confirmed that aγ was a mature protein in the following two points.

(1) Homology model of IL-1raγ and IL-18 receptor FIG. 12 shows the C-terminal amino acid which is a recognition site of KM2780, which is cleaved when IL-1raγ is secreted from Namalwa KJM-1 cells. It is a three-dimensional structure model obtained by predicting the three-dimensional structure of the sequence (SEQ ID NO: 13) by a known method and adding it to the three-dimensional structure model in FIG. As shown in FIG. 12, the recognition site of KM2780 to be cleaved is three-dimensionally IL- during complex formation with the IL-18 receptor.
It was shown that it was located away from the 18 receptor and did not contribute to non-covalent binding for complex formation.

(2) Surface exposed area of each residue of IL-1raγ FIG. 13 shows, based on the three-dimensional structure model shown in FIG. 12, IL-1raγ containing the recognition site of KM2780 to be cleaved (SEQ ID NO: 2).
A known method for determining the binding energy by non-covalent bond between each amino acid residue of the protein having the amino acid sequence of positions 46 to 208) and the amino acid residue of the IL-18 receptor (amino acids 20 to 324). And plotted for each amino acid residue of IL-1raγ. As shown in FIG. 13, the recognition site of KM2780 to be cleaved was IL-18.
The small binding energy of the non-covalent bond to the receptor revealed that the sequence was unnecessary for the non-covalent bond to form a complex between the two.

Example 13 Human IL-1raγ Precursor
Effect on fibroblasts In order to examine the effect of human IL-1raγ on the IL-1 receptor, the precursor type human IL-1raγ obtained in Example 6 was converted to IL-1 dependent prostaglandin E of human fibroblasts. ₂ (PGE ₂ ) production (Po
USA, 88 , 3681, 1991) was examined as follows. 10% fetal bovine serum, penicillin (GIBCO BRL) 50 unit / ml, streptomycin (GIBCO BRL) 50 μg / ml, HEPES [2- [4- (2-Hydroxy
ethyl) -1-piperazinyl] ethanesulfonic acid] (manufactured by Nakarai Tesque) in DMEM medium (GIBCO B
Normal human neonatal foreskin dermal fibroblasts (NHDF cells) (Kurabo) adjusted to 1 × 10 ⁶ cells / ml with RL (NHDF medium) were dispensed at 100 μl / well into a 96-well plate. , Overnight in a CO ₂ incubator at 37 ° C. After replacing the medium with fresh NHDF medium, the precursor human IL-1ra
γ, or human IL-1ra (R & D Systems
Was added to a final concentration of 100-200 ng / ml.
The cells were cultured in a CO ₂ incubator at ₂ ° C for 2 hours. The wells were divided into two groups. One group was added with human IL-1β (manufactured by PEPROTECH EC) to a final concentration of 1 ng / ml, and the other group was added with the same volume of NHDF medium as a control. CO ₂ at 37 ° C
After incubation for one day in an incubator, and the PGE ₂ content in the culture supernatant was measured by enzymeimmunoassay system (Amersham Pharmacia Biotech).

As a result, as shown in FIG. 14, in the precursor type human IL-1raγ, the effect of inducing PGE ₂ production by NHDF cells and the effect of suppressing the production of PGE ₂ by human IL-1β were not detected. Therefore, it was indicated that the precursor type human IL-1raγ does not have an agonistic activity and an antagonistic activity on the IL-1 receptor, or even if present, it may be so weak that it cannot be measured by the present system.

Example 14: Human IL-1raγ in human peripheral blood
Effect on Numperocytes The effects of the precursor and mature human IL-1raγ obtained in Examples 6 and 10 on human peripheral blood lymphocytes were examined. Lymphoprep ^™ (Nycomed Ph) from peripheral blood of healthy adults
mononuclear cells were separated and obtained by using the following method (manufactured by arma) and adjusted to 3 × 10 ⁶ cells / ml using RPMI1640 medium containing 10% FCS.
Concanavalin A (Con.A, final concentration 500 ng / ml) and I
In the presence of L-18 (final concentration 10 ng / ml, MBL), precursor human IL-1raγ (final concentration 200 ng / ml) or mature human I
After adding L-1raγ (final concentration 150 ng / ml) and culturing in a CO ₂ incubator at 37 ° C for 2 days, IL-
The amount of 4 was measured by ELISA (Pharmingen's OptEIA ELISA se
t). As a positive control, phorbor 12-myri
state 13-acetate (PMA, manufactured by Sigma, final concentration 50 ng / m
l) and ionomycin (Sigma, final concentration 1 μg / ml) were used. As a result, as shown in FIG.
No activity was detected on IL-1raγ, whereas mature human IL-1raγ was expressed on lymphocytes IL-4 by Con.A and IL-18.
Production was suppressed.

[0143]

According to the present invention, microbial infection, HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, grafts
-Versus host disease, insulin-dependent diabetes mellitus, traumatic brain injury, inflammatory bowel disease and other diseases involving infection and inflammation, arteriosclerosis, restenosis and other abnormal smooth muscle cell differentiation and proliferation, rheumatic diseases Diseases associated with abnormal activation of fibroblasts and synovial tissue such as arthritis, diseases associated with impairment of pancreatic β-cells such as diabetes, diseases associated with abnormal activation of osteoclasts such as osteoporosis, allergies, atopy, Asthma, hay fever, airway irritability,
Diseases associated with abnormal activation of immune cells, such as autoimmune diseases,
Search for diagnostic, prophylactic and / or therapeutic agents for diseases associated with abnormal cell proliferation such as acute myeloid leukemia, malignant tumors, and diseases based on neuronal disorders such as Alzheimer's disease, Parkinson's disease, and ischemic disease. And a protein having activity as a factor belonging to the IL-1 family useful for development, a DNA encoding the protein, an antibody recognizing the protein, and a method of using these.

[0144]

[Sequence List Free Text] SEQ ID NO: 6-Description of Artificial Sequence: Artificially Synthesized Primer Sequence SEQ ID NO: 7-Description of Artificial Sequence: Artificially Synthesized Primer Sequence SEQ ID NO: 8-Description of Artificial Sequence: Artificially Synthesized primer sequence SEQ ID NO: 9-Description of artificial sequence: Primer sequence synthesized artificially SEQ ID NO: 10-Description of artificial sequence: Primer sequence synthesized artificially SEQ ID NO: 11-Description of artificial sequence: Synthesized artificially Primer sequence SEQ ID NO: 12-Description of artificial sequence: amino acid sequence of artificially synthesized peptide SEQ ID NO: 13-Description of artificial sequence: amino acid sequence of artificially synthesized peptide

[0145]

[Sequence List] SEQUENCE LISTING <110> KYOWA HAKKO KOGYO CO., LTD. <120> An IL-1 family protein <130> A01515MA <160> 13

<210> 1 <211> 39 <212> PRT <213> Homo sapiens <400> 1 Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp Gln 1 5 10 15 Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile Ala Val Pro 20 25 30 Asp Lys Asn Tyr Ile Arg Pro 35

<210> 2 <211> 218 <212> PRT <213> Homo sapiens <400> 2 Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp 1 5 10 15 Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro Ala Val Ser 20 25 30 Pro Leu Glu Pro Gly Pro Ser Leu Pro Ala Met Asn Phe Val His Thr 35 40 45 Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp 50 55 60 Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile Ala Val 65 70 75 80 Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala Leu Ala Ser 85 90 95 Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile Leu Leu Gly 100 105 110 Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys Gly Gln 115 120 125 Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys Leu Ala 130 135 140 Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gln 145 150 155 160 Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro Gly Trp Phe 165 170 175 Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys 180 185 190 Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys 195 200 205 Ala Glu Met Ser Pro Ser Glu Val Ser Asp 210 215

<210> 3 <211> 120 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (3) .. (119) <400> 3 gt cca aag gtg aag aac tta aac ccg aag aaa ttc agc att cat gac 47 Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp 1 5 10 15 cag gat cac aaa gta ctg gtc ctg gac tct ggg aat ctc ata gca gtt 95 Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile Ala Val 20 25 30 cca gat aaa aac tac ata cgc cca g 120 Pro Asp Lys Asn Tyr Ile Arg Pro 35

<210> 4 <211> 1104 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (370) .. (1023) <400> 4 ctgatctatc acaagtacct tgaaatgtgt tgaataggtg tggcacagtc cttagcagag 60 tggcactacc cccacaggaa tttgtttata cctttggcat gggaaatagc aggaaatgag 120 tgatcactga taactgagga tgctatttat tattggccaa aggaatactt gtgttgtatt 180 tgcataacca ctcacaaact gttgattaca aatgagtacc agacctagct ccttcaagta 240 aaggatcctg agaactgaag gcaaacagag ctccaggagt ccaagacaga gccacagacc 300 acgaggatcc ctggcccagg tcttggactt cattccattt tctgttgagt aataaactca 360 acgttgaaa atg tcc ttt gtg ggg gag aac tca gga gtg aaa atg ggc tct 411 Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser 1 5 10 gag gac tgg gaa aaa gat gaa ccc cag tgc tgc tta gaa gac ccg gct 459 Glu Asp Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro Ala 15 20 25 30 gta agc ccc ctg gaa cca ggc cca agc ctc ccc gcc atg aat ttt gtt 507 Val Ser Pro Leu Glu Pro Gly Pro Ser Leu Pro Ala Met Asn Phe Val 35 40 45 cac aca agt cca aag gtg aag aac tta aac ccg aag aaa ttc agc att 555 His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile 50 55 60 cat gac cag gat cac aaa gta ctg gtc ctg gac tct ggg aat ctc ata 603 His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu Ile 65 70 75 gca gtt cca gat aaa aac tac ata cgc cca gag atc ttc ttt gca tta 651 Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe Ala Leu 80 85 90 gcc tca tcc ttg agc tca gcc tct gcg gag aaa gga agt ccg att ctc 699 Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile Leu 95 100 105 110 ctg ggg gtc tct aaa ggg gag ttt tgt ctc tac tgt gac aag gat aaa 747 Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp Lys 115 120 125 gga caa agt cat cca tcc ctt cag ctg aag aag gag aaa ctg atg aag 795 Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met Lys 130 135 140 ctg gct gcc caa aag gaa tca gca cgc cgg ccc ttc atc ttt tat agg 843 Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr Arg 145 150 155 gct cag gtg ggc tcc tgg aac atg tcg gcg gc t cac ccc gga 891 Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro Gly 160 165 170 tgg ttc atc tgc acc tcc tgc aat tgt aat gag cct gtt ggg gtg aca 939 Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val Thr 175 180 185 190 gat aaa ttt gag aac agg aaa cac att gaa ttt tca ttt caa cca gtt 987 Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe Gln Pro Val 195 200 205 tgc aaa gct gaa atg agc ccc agt gag gtc agc gat tag gaaactgccc 1036 Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp 210 215 cattgaacgc cttcctcgct aatttgaact aattgtataa aaaccccaaa cctgctcact 1096 aaaaaaaa 1104

<210> 5 <211> 140 <212> DNA <213> Homo sapiens <400> 5 attgatctag gtccaaaggt gaagaactta aacccgaaga aattcagcat tcatgaccag 60 gatcacaaag tactggtcct ggactctggg aatctcatag cagttccaga taacactca 120gt

<210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 6 caaattagcg aggaaggcgt tcaatgg 27

<210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 7 ctccttcttc agctgaaggg atgg 24

<210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 8 gtcctggact ctgggaatct catagca 27

<210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 9 gatatcgccg cgctcgtcgt cgac 24

<210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 10 caggaaggaa ggctggaaga gtgc 24

<210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic DNA <400> 11 cctgagaact gaaggcaaac agagctc 27

<210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic PRT <220> <221> AMIDATION <222> 21 <223> Xaa represents L -Valine amide <400> 12 Cys Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp 1 5 10 15 Gln Asp His Lys Xaa 20

<210> 13 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic PRT <400> 13 Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp 1 5 10

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence (IL-1Raγ) encoded by the nucleotide sequence of SEQ ID NO: 4 and the sequences of human IL-1α, human IL-1β, human IL-1ra, human IL-1raβ, and Tango-77. It is a figure which shows the comparison with an amino acid sequence.

FIG. 2 shows the results of analyzing the expression of IL-1raγ and β-actin in human-derived tissues by RT-PCR.

FIG. 3 shows the results of analyzing the expression of IL-1raγ and β-actin in a lymphoid cell line by RT-PCR.

FIG. 4. IL-1r associated with activation of human peripheral blood T cells.
The result of having analyzed the fluctuation | variation of expression of a (gamma) and (beta) -actin by RT-PCR method is shown. Activated T cells were 50 ng / ml interleukin 2 (IL-2), 1 μg / ml phytohemagglutinin.
It was obtained by adding in-P (PHA-P) and 5 ng / ml of transforming growth factor β (TGF-β), and culturing for 2, 4, 6, or 8 days. .

FIG. 5 shows IL-1 expressed using insect cells as a host.
1 shows an electrophoresis image of raγ protein. Culture supernatant of Sf9 cells infected with recombinant virus carrying IL-1raγ gene 2.5μ
g (corresponding to 16 μl) was used as a sample, subjected to SDS-PAGE under reducing conditions, and stained with Coomassie Brilliant Blue R250 for detection.

FIG. 6 shows anti-human IL-1raγ monoclonal antibody KM
2 shows the results of analyzing the reaction specificity of KM2780 and 2676. The reaction specificity for the peptide used for immunization and the control peptide was examined by binding ELISA. The outline shows the assay results when the peptide is a negative control peptide, the black shows the antigen peptide used when preparing KM2779, and the hatched lines show the antigen peptide used when preparing KM2780.

FIG. 7 shows the results of Western blotting detection using KM2679 and KM2780. SDS-PAGE of Sf9 cell lysate infected with recombinant virus carrying IL-1raγ gene
And Western blotting was performed using an anti-human IL-1raγ monoclonal antibody KM2679 or KM2780.

FIG. 8 shows a three-dimensional structural model diagram of a complex of IL-1raγ and IL-18 receptor. IL-1raγ is shown by a surface model,
IL-18R is shown in a ribbon structure. The recognition site of KM2779 is shown in dark color.

FIG. 9 shows a distribution diagram in which the value of the solvent exposed area of each amino acid residue of IL-1raγ is plotted. The recognition site of KM2779 is indicated by an arrow.

FIG. 10 is a distribution plotting non-covalent binding energy values between each amino acid residue of IL-1raγ and IL-18 receptor in a complex of IL-1raγ and IL-18 receptor. The figure is shown. The recognition site of KM2779 is indicated by an arrow.

FIG. 11 shows human B-cell lymphoma-derived Namalwa KJM-1 cells into which human IL-1raγ gene has been introduced (IL-1ra in the figure).
cell lysate (shown as cells in the figure) obtained by culturing Namalwa KJM-1 cells (shown as mock in the figure) derived from human B-cell lymphoma into which pAMo was introduced 10
FIG. 4 is a diagram showing the results of Western blotting of μg and 1.8 ml of a culture supernatant (shown as supernatant in the figure) using anti-human IL-1raγ antibodies KM2779 and KM2780.

FIG. 12 shows a three-dimensional structural model of a complex of IL-1raγ and IL-18 receptor. IL-1raγ is indicated by a surface model, and IL-18R is indicated by a ribbon. The recognition site of KM2780 that is cleaved with maturation is shown in dark color.

FIG. 13 shows a distribution diagram in which the value of the solvent exposed area of each amino acid residue of IL-1raγ is plotted. The recognition site of KM2780 that is cleaved with maturation is indicated by an arrow.

FIG. 14 shows human foreskin fibroblasts (NHDF cells)
3 shows the results of examining the effect of insect cell-produced precursor IL-1raγ protein on IL-1 dependent PGE ₂ (prostaglandin E ₂ ) production in E. coli. The agonist activity of IL-1β is 1n
g / ml test group, IL-1ra of agonist activity was IL
-1ra represents a test group to which 1 μg / ml was added. Also,
The antagonist activity was measured by adding human IL-1β to a final concentration of 1 ng / ml.

FIG. 15 shows the results of examining the effect of human IL-1raγ protein on IL-4 production of human peripheral blood lymphocytes. PMA represents phorbor 12-myristate 13-acetate, ConA represents concanavalin A, and IL-18 represents interleukin 18.
PMA + ionomycin is 50 ng / ml PMA and ionomyci
The test group to which n was added to a concentration of 1 μg / ml was
18 + ConA represents a test group to which IL-18 was added to a concentration of 10 ng / ml and ConA to a concentration of 500 ng / ml.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61P 1/04 48/00 5/48 A61P 1/04 5/50 5/48 9/10 5/50 11/06 9/10 19/08 11/06 19/10 19/08 25/00 19/10 29/00 25/00 31/00 29/00 31/04 31/00 31/10 31 / 04 31/12 31/10 31/18 31/12 31/20 31/18 35/00 31/20 35/02 35/00 37/06 35/02 37/08 37/06 43/00 37/08 105 43/00 C07K 14/545 105 16/24 C07K 14/545 C12N 1/15 16/24 1/19 C12N 1/15 1/21 1/19 C12P 21/02 K 1/21 21/08 5/10 C12Q 1/68 A C12P 21/02 G01N 33/53 P 21/08 33/577 B C12Q 1/68 C12R 1:91) G01N 33/53 C12N 15/00 ZNAA 33/577 A61K 37/02 // (C12N 5 / 10 C12N 5/00 A C12R 1:91) C12R 1:91) (72) Inventor Susumu Neji 3-6-6 Asahicho, Machida-shi, Tokyo Kyowa Hakko Kogyo Co., Ltd. (72) Inventor Mitsuo Sato 3-6-6 Asahicho, Machida-shi, Tokyo Kyowa Hakko Kogyo Co., Ltd. 72) Inventor Jin Sakurai 3-6-6 Asahimachi, Machida-shi, Tokyo Kyowa Fermentation Industry Co., Ltd. Tokyo Research Laboratory (72) Inventor Akiko Furuya 3-6-6 Asahimachi, Machida-shi, Tokyo Kyowa Fermentation Industry Co., Ltd. Tokyo Research Laboratory

Claims (36)

[Claims] 1. A protein selected from the following (a) to (f): (a) a protein having the amino acid sequence represented by SEQ ID NO: 2; (b) a protein having the amino acid sequence represented by SEQ ID NO: 2;
A protein having an amino acid sequence of positions 6 to 208; (c) an IL-containing amino acid sequence represented by SEQ ID NO: 1
A protein belonging to one family; (d) a protein having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the protein of (a) or (b), and belonging to the human IL-1 family; A protein having a binding affinity to a receptor higher than Tango-77; (e) an amino acid sequence having 60% or more homology with the amino acid sequence of the protein of (a) or (b),
A protein belonging to the human IL-1 family and having a binding affinity to a receptor higher than that of Tango-77; and The protein according to the above (d) or (e), which is not recognized by the monoclonal antibody produced by (1). 2. A DN selected from the following (a) to (d):
A: (a) DNA encoding the protein of claim 1; (b) 370 amino acids in the nucleotide sequence represented by SEQ ID NO: 4;
(C) a DNA having a nucleotide sequence represented by SEQ ID NO: 4;
(D) a DNA having a nucleotide sequence of the 99th base sequence; and (d) a DNA which hybridizes with any of the DNAs of (a) to (c) under stringent conditions and which is a protein receptor belonging to the human IL-1 family. Tang binding affinity
DNA encoding a protein higher than o-77. 3. A protein selected from the following (a) to (c): (a) a protein having the amino acid sequence represented by SEQ ID NO: 1; (b) a protein having the amino acid sequence represented by SEQ ID NO: 1 A protein having an amino acid sequence in which one or more amino acids have been deleted, substituted and / or added, and having an activity of binding to a receptor of a protein belonging to the human IL-1 family; and (c) SEQ ID NO: 1. A human IL- having an amino acid sequence having 60% or more homology with the represented amino acid sequence;
A protein having a binding activity of a protein belonging to one family to a receptor. 4. A DN selected from the following (a) to (c):
A: (a) a DNA encoding the protein of claim 3; (b) a DNA having the nucleotide sequence of SEQ ID NO: 3;
And (c) a DNA that hybridizes with the DNA of (a) or (b) under stringent conditions and encodes a protein having a binding affinity for a receptor of a protein belonging to the human IL-1 family. 5. A recombinant DNA obtained by incorporating the DNA according to claim 2 into a vector. 6. A transformant having the recombinant DNA according to claim 5. 7. The transformant according to claim 6, wherein the accession number is FERM BP-6943. 8. A method for culturing the transformant according to claim 6 or 7 in a medium, producing and accumulating the protein according to claim 1 in the culture, and collecting the protein from the culture. The method for producing a protein according to claim 1 or 3, characterized in that: 9. An antibody that recognizes the protein according to claim 1 or 3. 10. An antibody having an activity of inhibiting the activity of the protein according to claim 1 or 3,
The antibody according to claim 9. 11. The antibody according to claim 1, wherein the antibody recognizes a protein having the amino acid sequence represented by SEQ ID NO: 1.
The antibody according to 0. 12. The antibody according to claim 10, wherein the antibody is an antibody that recognizes an amino acid sequence at positions 1 to 20 in the amino acid sequence represented by SEQ ID NO: 1 as an antigenic epitope. 13. A monoclonal antibody produced by a hybridoma having an accession number of FERM BP-7368. 14. The deposit number is FERM BP-7369.
A monoclonal antibody produced by a hybridoma. 15. A sequence consisting of 5 to 60 consecutive nucleotides in the nucleotide sequence of the DNA encoding the region of the amino acid sequence of SEQ ID NO: 1 or the region corresponding to the amino acid sequence of SEQ ID NO: 1 in the protein according to claim 1. Or an oligonucleotide having a sequence complementary to the oligonucleotide. 16. A method comprising performing hybridization using the DNA according to claim 2 or 4 or the oligonucleotide according to claim 15 as a probe.
A method for detecting the expression of a gene encoding the protein according to claim 1 or 3. 17. A polymerase chain using the oligonucleotide according to claim 15 as a primer.
4. A method for detecting expression of a gene encoding a protein according to claim 1 or 3, which comprises performing a reaction. 18. A method for detecting a mutation in a gene encoding the protein according to claim 1 or 3 by a hybridization method using the DNA according to claim 2 or 4 or the oligonucleotide according to claim 15. . 19. A method for detecting a mutation in the gene encoding the protein according to claim 1 or 3, which comprises performing a polymerase chain reaction using the oligonucleotide according to claim 15. 20. A disease associated with infection or inflammation, a disease associated with abnormal differentiation and proliferation of smooth muscle cells, a disease associated with abnormal fibroblast activation, a disease associated with abnormal synovial tissue activation, and pancreatic β. 20. Use for detecting a disease associated with cell damage, a disease associated with abnormal activation of osteoclasts, a disease associated with abnormal activation of immune cells, or a disease associated with abnormal cell proliferation. The method according to any one of claims 1 to 4. 21. A disease associated with infection or inflammation is a microbial infection,
HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes, traumatic injury or inflammatory bowel disease with abnormal smooth muscle cell differentiation and proliferation Is arteriosclerosis or restenosis, a disease with abnormal fibroblast activation or a disease with abnormal synovial tissue activation is rheumatoid arthritis, and a disease with pancreatic β-cell damage is diabetes. Yes, the disease with abnormal activation of osteoclasts is osteoporosis, and the disease with abnormal activation of immune cells is allergy, atopy, asthma, hay fever, airway hyperreactivity or autoimmune disease, and abnormal cells 21. The method according to claim 20, wherein the disease associated with proliferation is acute myeloid leukemia or malignancy. 22. Transcription of a gene encoding the protein of claim 1 or 3 or translation of mRNA, wherein the DNA of claim 2 or 4 or the oligonucleotide of claim 15 is used. How to control. 23. A promoter region of a gene encoding the protein according to claim 1 or 3, wherein the DNA according to claim 2 or 4 or the oligonucleotide according to claim 15 is used. Method. 24. A medicament comprising the protein according to claim 1 or 3. 25. A medicament comprising the DNA according to claim 2 or 4. 26. A medicament comprising the antibody according to any one of claims 9 to 14. 27. A medicament comprising the oligonucleotide according to claim 15. 28. A disease associated with infection or inflammation, a disease associated with abnormal smooth muscle cell differentiation and proliferation, a disease associated with abnormal fibroblast activation, a disease associated with abnormal synovial tissue activation, pancreatic β Treatment of a disease associated with cell damage, a disease associated with abnormal activation of osteoclasts, a disease associated with abnormal activation of immune cells, a disease associated with abnormal cell proliferation, or a disease based on nerve cell damage and / or 3. A medicament for prevention.
28. The medicament according to any one of 4 to 27. 29. A disease associated with infection or inflammation, a disease associated with abnormal smooth muscle cell differentiation and proliferation, a disease associated with abnormal fibroblast activation, a disease associated with abnormal synovial tissue activation, pancreatic β 25. A medicament for diagnosis of a disease associated with cell damage, a disease associated with abnormal activation of osteoclasts, a disease associated with abnormal activation of immune cells, or a disease associated with abnormal cell proliferation. 28. The medicament according to any one of 27. 30. A disease associated with infection or inflammation is a microbial infection,
HIV infection, chronic hepatitis B, rheumatoid arthritis, sepsis, graft-versus-host disease, insulin-dependent diabetes, traumatic injury or inflammatory bowel disease with abnormal smooth muscle cell differentiation and proliferation Is arteriosclerosis or restenosis, a disease with abnormal fibroblast activation or a disease with abnormal synovial tissue activation is rheumatoid arthritis, and a disease with pancreatic β-cell damage is diabetes. Yes, the disease with abnormal activation of osteoclasts is osteoporosis, and the disease with abnormal activation of immune cells is allergy, atopy, asthma, hay fever, airway hyperreactivity or autoimmune disease, and abnormal cells The medicament according to claim 28 or 29, wherein the disease associated with proliferation is acute myeloid leukemia or malignant tumor, and the disease based on neuronal cell disorder is Alzheimer's disease or ischemic brain disease. 31. A method for screening a receptor that specifically interacts with the protein according to claim 1 or 3, wherein the method uses the protein according to claim 1. 32. A receptor that specifically interacts with the protein according to claim 1 or 3, which is obtained by the screening method according to claim 31. 33. The method for immunologically detecting a protein according to claim 1 or 3, wherein the antibody according to any one of claims 9 to 14 is used. 34. An immunohistological staining method comprising detecting the protein according to claim 1 or 3 using the antibody according to any one of claims 9 to 14. 35. A knockout non-human animal in which the expression of the gene encoding the protein according to claim 1 or 3 is partially or completely suppressed. 36. A knockout non-human animal in which the activity of the protein according to claim 1 or 3 is partially or completely suppressed.