JPH11152300A - New receptor protein and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new receptor protein containing a specific amino acid sequence, originating from a dendritic cell belonging to TNF receptor family, and useful for screening agonists, antagonists and the like which are used as medicines. SOLUTION: This new receptor protein (salt) contains the same or substantially the same amino acid sequence as an amino acid sequence of the formula and originats from a dendritic cell belonging to TNF receptor family. The receptor protein is useful as a reagent for screening ligands, agonists, antagonists, and so on. The agonists are effective as agents or treating and preventing cancers, AIDS, infectious diseases, and so on, and the antagonists are effective as agents for preventing and treating allergic diseases, inflammation, autoimmune diseases, AIDS, glomerulonephritis, bronchial asthma, and so on. the protein is obtained by subjecting a human dendritic cell cDNA library to a PCR using an oligo DNA comprising the partial sequence of a receptor protein gene, inserting the obtained gene into a vector, and subsequently expressing in a host cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel receptor protein derived from human dendritic cells and its use.

[0002]

2. Description of the Related Art Dendritic cells mature and differentiate from CD34-positive progenitor cells present in bone marrow and are distinguished from other monocyte cells by their morphology, motility, and degree of phagocytosis. Cells. These cells migrate into peripheral blood, and are widely distributed in non-lymphoid tissues such as skin tissue, heart and lungs, and in lymphoid tissues such as lymph nodes and thymus. It plays an important role as an antigen presenting cell to T cells such as virus antigens and tumor-associated antigens. For example, Langerhans cells, a kind of cells belonging to the dendritic cell family, are widely distributed in skin tissues, particularly in the epidermal cell layer, and have allergic inflammatory skin diseases (eg, allergic contact dermatitis, atopic dermatitis) It is known to be deeply involved as an effector cell in the onset of the disease [Clinical Immunity, Vol. 27, pp. 1013-1018 (1995)]. In addition, because of its strong antigen-presenting ability, it has recently attracted attention as a new material to replace macrophages when applying antitumor immunotherapy or immunostimulation therapy for various infectious diseases [Immunology Today, 18 , Pp. 102-104 (199
7)]. However, until recently, a large-scale isolation / purification technology of the cells, which is essential for examining the cell functions and various biological activities in detail, has not been established. In comparison, the study was significantly delayed.

At present, maturation of dendritic cells is considered in a so-called two-step model, and foreign antigens such as microorganisms and cytokines (IL-1β, GM-CSF, T
NF-α) via initial maturation (Initial Maturation) and cell surface molecules (C
D40L) and a new cytokine signal (IFN-
Terminal Maturation via γ)
Maturation) [Immunology Today, 18, 102-104 (1
997)]. Further, when the aforementioned Langerhans cells activate helper T cells as antigen presenting cells, a signal mediated by a Class II antigen and a T cell receptor, B7-1,
It has also been reported that two signals from so-called costimulatory molecules such as B7-2 are required [Immunology Today, 15, 464.
-469 (1994)]. By the way, TNF receptor type 1 and type 2, low affinity NGF receptor, Fas antigen,
A group of type 1 membrane proteins such as CD27, CD30 and CD40 is called a so-called TNF receptor family, and although their homology at the amino acid level is low,
All have a ligand-binding region rich in cysteine residues characteristic of the extracellular region, and bind to and cross-link with receptor-specific ligands belonging to trimerized type 2 membrane proteins. It is thought to induce signal transduction leading to diverse biological activities of individual receptors. Some intracellular regions also have a common motif structure. For example, a receptor such as TNF receptor type 1 or Fas antigen which has apoptosis-inducing ability has a region called a death domain. (Cell, Volume 81, 479-4
82 (1995)).

[0004] CD40 is a member of the receptor family having a molecular weight of about 50 kD, which was reported in 1986 as an antigen that transmits an activation signal for B lymphocytes. Further, the ligand CD40L (CD40 ligand) of this receptor was isolated in 1993, and is known to be expressed on the surface of T cells as a type 2 membrane protein of about 35 kD. More recently, signals via CD40 have been detected in germinal center B lymphocytes and B lymphocyte cell lines and normal B lymphocytes.
It has been shown that it inhibits lymphocyte apoptosis, and that this molecule has various effects such as proliferation of B lymphocytes and induction of class switch, and plays an important role in various steps of T lymphocyte-dependent antibody reaction. Is thought to play. Furthermore, a signal is also transmitted via CD40L, and it has been revealed that this signal is involved in the activation of T lymphocytes. Such a CD40
Since the expression of the molecule is also observed on the dendritic cell surface described above, it is suggested that CD40 controls apoptosis similarly to B cells, activates T lymphocytes, and the like in the present cells. However, when CD40L is deficient at the individual level, serum IgM shows normal to high levels,
It is recognized that the signal through CD40 is important for the activation and differentiation of B lymphocytes, since the production of antibodies of other isotypes is remarkably reduced, and immunodeficiency called so-called sex-related high IgM syndrome is exhibited. However, the physiological significance of dendritic cells is still unknown. Therefore, it is not possible to clearly explain the original physiological functions of dendritic cells, and furthermore, the relationship with various diseases, using only the existing models of the functions of these existing genes and the aforementioned maturation of cells and the activation mechanism of T cells. It is difficult at present.
Therefore, we will not only clarify the function of dendritic cells in more detail, but also develop drugs for treating and preventing various diseases targeting dendritic cells, or effectively develop immunotherapy using dendritic cells. In doing so, it has been desired to isolate new cell function regulatory genes derived from dendritic cells and elucidate their physiological functions.

[0005]

The present invention relates to a novel receptor protein derived from a human dendritic cell, a partial peptide thereof or a salt thereof, a method for producing the receptor protein or a salt thereof, the receptor protein, a partial peptide thereof or Using an antibody against a salt of the above, a method for determining a ligand for the receptor, a method for screening a ligand for the receptor, a compound that changes the binding property between the ligand and the receptor protein, the screening kit, the screening method or the screening kit. A compound which changes the binding property between the ligand and the receptor protein obtained as described above; and a medicament comprising a compound which changes the binding property between the ligand and the receptor protein.

[0006]

Means for Solving the Problems In view of the above problems, the present inventors have conducted intensive studies and, as a result, have isolated a cDNA encoding a novel receptor protein derived from human dendritic cells,
The entire nucleotide sequence and the entire amino acid sequence of the receptor protein encoded thereby were successfully analyzed. The present inventors have further studied based on these findings, and have completed the present invention.

That is, the present invention provides (1) SEQ ID NO: 1.
(Preferably SEQ ID NO: 1)
(2) a dendritic cell belonging to the TNF receptor family, which comprises an amino acid sequence identical or substantially identical to the amino acid sequence of positions 30 to 616 of the amino acid sequence represented by The receptor protein according to (1), which is a receptor protein derived from the protein. (3) a transformant transformed with a partial peptide of the receptor protein described in (1) or a salt thereof, and (4) a recombinant vector containing a DNA encoding the receptor protein described in (1). (1) a method for producing a receptor protein or a salt thereof according to (1), wherein the receptor protein is produced and accumulated, and collected; (3) an antibody against the partial peptide or the salt thereof according to (6), (6) the receptor protein according to (1), or the partial peptide or salt thereof according to (3). 1) A method for determining a ligand for the receptor protein or a salt thereof according to the above,
(7) Altering the binding between a ligand characterized by using the receptor protein described in (1), the partial peptide described in (3) or a salt thereof and the receptor protein described in (1). A method of screening for a compound or a salt thereof, (8) a ligand characterized by containing the receptor protein described in (1), the partial peptide described in (3) or a salt thereof, and (1) A kit for screening a compound or a salt thereof that changes the binding property to the receptor protein or a salt thereof according to the above item,
(9) Altering the binding between the ligand and the receptor protein or the salt thereof according to (1), which is obtained using the screening method according to (7) or the screening kit according to (8). A compound or a salt thereof,
And (10) altering the binding between the ligand and the receptor protein or the salt thereof according to (1), which is obtained by using the screening method according to (7) or the screening kit according to (8). Provided is a medicament comprising a compound or a salt thereof.

More specifically, (11) substantially the same as the amino acid sequence represented by SEQ ID NO: 1 (preferably the 30th to 616th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1) The amino acid sequence is SEQ ID NO:
1 (preferably SEQ ID NO:
The amino acid sequence at positions 30 to 616 of the amino acid sequence represented by 1) and about 40% or more (preferably about 50% or more, more preferably 70% or more, and still more preferably about 8% or more;
(1) the amino acid sequence having a homology of 0% or more, more preferably about 90% or more, and most preferably about 95% or more. (12) SEQ ID NO: 1
(Preferably SEQ ID NO: 1)
The amino acid sequence substantially the same as the amino acid sequence at positions 30 to 616 of the amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence represented by SEQ ID NO: 1 (preferably, the amino acid sequence represented by SEQ ID NO: 1). 1 to 616 amino acid sequences) (preferably,
Amino acid sequence in which about 1 to 30 amino acids have been deleted,
The amino acid sequence represented by SEQ ID NO: 1 (preferably the 30th to
An amino acid sequence in which one or more (preferably about 1 to 30) amino acids are added to the amino acid sequence at position 616 (the amino acid sequence at position 616); the amino acid sequence represented by SEQ ID NO: 1 (preferably represented by SEQ ID NO: 1) An amino acid sequence in which one or more (preferably about 1 to 30) amino acids in the amino acid sequence at positions 30 to 616 in the amino acid sequence have been substituted with another amino acid, or an amino acid sequence obtained by combining them. (1) the receptor protein described in (1) or a salt thereof; (13) the partial peptide described in (3) having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2,
4) the partial peptide of (13), which is soluble;

(15) A DN containing a DNA having a base sequence encoding the receptor protein described in (1).
A, (16) The base sequence represented by SEQ ID NO: 4 (preferably, the base sequence represented by SEQ ID NO: 4
(1814) the DNA of (14), (17) the recombinant vector containing the DNA of (16), or (18) the recombinant vector of (17). The transformed transformant, (19) the base sequence represented by SEQ ID NO: 4 (preferably SEQ ID NO: 4)
A DNA containing a DNA having a base sequence that hybridizes under high stringent conditions with the base sequence at positions 88 to 1848 of the base sequence represented by No. 4 (2)
0) a recombinant vector containing the DNA of (19), (21) a transformant transformed with the recombinant vector of (20), (22) a transformant of (21). (19) The method for producing a receptor protein encoded by DNA or a salt thereof according to (19), wherein the transformant is cultured to produce and accumulate the receptor protein.
(23) a receptor protein produced by the production method described in (22) or a salt thereof, (24) a DNA containing a DNA encoding the partial peptide described in (3) or (13), ( 25) The DNA according to (24) having the base sequence of SEQ ID NO: 5, (26) a recombinant vector containing the DNA according to (25),
(27) A transformant transformed with the recombinant vector according to (26), (28) A transformant according to (27) is cultured to produce and accumulate the peptide, and this is collected. A method for producing the partial peptide or the salt thereof according to the above (3) or (13), wherein (29) a signal peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 or a salt thereof, (30) a DNA containing a DNA encoding the signal peptide according to (29); (31) the DNA according to (30), having the base sequence represented by SEQ ID NO: 6;

(32) The ligand according to item (6), wherein the receptor protein according to item (1), the partial peptide according to item (3) or a salt thereof is brought into contact with a test compound. (33) No. (6)
Ligand obtained by the method for determining a ligand described in (3), (3
4) TNF (eg, TNF-α), lymphotoxin (eg,
Lymphotoxin-α, Lymphotoxin-β), Fas ligand, NGF, CD40 ligand, CD27 ligand, CD30 ligand, OX-40 ligand, 4-1B
The ligand according to (33), which is B ligand or Apo-2L (TRAIL); (35) (i) the receptor protein according to (1), the partial peptide according to (3) or a salt thereof; And (ii) the receptor protein described in (1), the partial peptide described in (3) or a salt thereof, and a ligand and a test compound. (7) the screening method according to (7), wherein the labeled ligand is obtained by comparing the labeled ligand with the receptor protein according to (1), the partial peptide according to (3) or a partial peptide thereof; (Ii) when the labeled ligand and test compound are brought into contact with the receptor protein described in (1), the partial peptide described in (3), or a salt thereof. Measuring the amount of the labeled ligand bound to the receptor protein according to item (1), the partial peptide according to item (3), or a salt thereof,
(37) a method for screening a compound or a salt thereof that changes the binding property between the ligand and the receptor protein or the salt thereof according to (1), wherein
(I) when the labeled ligand is brought into contact with cells containing the receptor protein described in (1), and (ii) when the labeled ligand and the test compound are brought into contact with cells containing the receptor protein described in (1). Measuring the amount of binding of the labeled ligand to the cell when contacting with the compound, and comparing the ligand with the receptor protein or the salt thereof according to item (1), or Screening method of the salt, (3
8) (i) contacting the labeled ligand with the membrane fraction of the cell containing the receptor protein described in (1), and (ii) combining the labeled ligand and the test compound according to (1). The ligand and the receptor protein according to item (1), wherein the amount of binding of the labeled ligand to the cell membrane fraction when contacted with the membrane fraction of the cell containing the receptor protein is measured and compared. Or a method for screening a compound or a salt thereof that changes the binding property with a salt thereof,

(39) (i) The labeled ligand is replaced with (1)
Culturing the transformant according to item 8) or (21) to contact with the receptor protein expressed on the cell membrane of the transformant; and (ii) labeling the ligand and test compound with (18) )) Or measuring the amount of labeled ligand bound to the receptor protein when the transformant according to item (21) is cultured and brought into contact with the receptor protein expressed on the cell membrane of the transformant; A method for screening a compound or a salt thereof that alters the binding property between a ligand and the receptor protein or a salt thereof according to item (1), wherein (40) (i) the receptor according to item (1); Contacting a substance that activates a protein or a salt thereof with a cell containing the receptor protein according to item (1);
(Ii) A cell mediated by the receptor protein when the substance activating the receptor protein or the salt thereof according to (1) and a test compound are brought into contact with a cell containing the receptor protein according to (1). (4) a method for screening a compound or a salt thereof that changes the binding property between a ligand and a receptor protein or a salt thereof according to item (1), wherein the stimulating activity is measured and compared;
1) A substance that activates the receptor protein described in (1) or a salt thereof was expressed in the cell membrane of the transformant by culturing the transformant described in (18) or (21). When contacted with a receptor protein,
A substance that activates the receptor protein or a salt thereof according to item (1) and a test compound are expressed on the cell membrane of the transformant by culturing the transformant according to item (18) or (21). A compound that changes the binding property between a ligand and the receptor protein or a salt thereof according to item (1), wherein the cell stimulating activity mediated by the receptor protein is measured and compared when brought into contact with the receptor protein. Or a method of screening for a salt thereof,

(42) The substance that activates the receptor protein described in (1) is TNF (eg, TNF-α), lymphotoxin (eg, lymphotoxin-α, lymphotoxin-β), Fas ligand, NGF, CD40. Ligand, CD27 ligand, CD30 ligand, OX-40
Ligand, 4-1BB ligand or Apo-2L (T
(38) or the screening method according to the above (39), which is (RAIL);
A compound or a salt thereof, which alters the binding property between the ligand and the receptor protein or the salt thereof according to item (1), obtained by any of the screening methods according to item 1);
(44) A compound or a salt thereof, which alters the binding between the ligand and the receptor protein or the salt thereof according to the item (1), which is obtained by the screening method according to any one of the items (35) to (41). (45) a screening kit according to (8), which comprises a cell containing the receptor protein according to (1), (46) a pharmaceutical kit comprising: The screening kit according to item (8), which comprises a membrane fraction of a cell containing the receptor protein according to item 1), or the kit according to item (47) or item (18). (48) the screening kit according to (8), which comprises a receptor protein expressed on the cell membrane of the transformant by culturing the transformant;
Ligand and (1) obtained using the screening kit according to any one of (45) to (47).
(49) A compound or a salt thereof which alters the binding property to the receptor protein or a salt thereof according to the above (49), wherein
A compound obtained by using any one of the screening kits according to the above (47), which comprises a compound capable of changing the binding property between the ligand and the receptor protein according to the above (1) or a salt thereof, or a salt thereof. Medicine,

(50) Any of the screening methods of (7) or (35) to (41), or any of (8) or (45) to (47) Obtained using such a screening kit,
(5) a medicament comprising an agonist for the receptor protein or a salt thereof according to (1),
1) Cancer (eg, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer with p53 mutation, brain tumor, bladder cancer, cervical cancer, colorectal cancer (colon / rectal cancer), non-small cell lung cancer, small cell Lung cancer, gastric cancer, etc., AIDS, infectious diseases (eg, herpes virus infection, adenovirus infection, box virus infection, Helicobacter pylori infection, varicella-zoster virus infection, human papillomavirus infection, invasive grape Staphylococcal infections, influenza infections, severe systemic fungal infections, etc.), acute bacterial meningitis, acute viral encephalitis, adult respiratory distress syndrome, bacterial pneumonia, chronic lymphocytic leukemia, chronic myelogenous leukemia, insulin-dependent diabetes Treatment and prognosis of (type I), malignant melanoma, multiple myeloma, non-Hodgkin's lymphoma, peptic ulcer, sepsis, septic shock, tuberculosis, etc. (50) The medicament according to (50), which is an inhibitor, (52) the screening method according to any one of (7) or (35) to (41), or (8) or (45). (5) a medicament obtained by using the screening kit according to any one of the items (47) to (47), which comprises an antagonist to the receptor protein or the salt thereof according to the item (1);
3) Allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, hay fever, etc.), inflammation (eg, arthritis, hepatitis, etc.), autoimmune diseases (eg, rheumatoid arthritis, systemic lupus erythematosus) , Sjogren's disease), the medicament according to (52), which is a therapeutic / prophylactic agent for AIDS, glomerulonephritis, bronchial asthma, etc.

(54) The antibody according to item (5), wherein the antibody according to item (5) is brought into contact with the receptor protein according to item (1), the partial peptide according to item (3), or a salt thereof. (1) A method for quantifying the receptor protein of (1), the partial peptide of (3) or a salt thereof, (55) the antibody of (5), a test solution and labeled (1) The receptor protein according to item (1), wherein the receptor protein according to item (1) is allowed to competitively react with the receptor protein according to item (3) or a salt thereof, and is bound to the antibody. A method for quantifying the receptor protein according to (1), the partial peptide according to (3) or a salt thereof in a test solution, wherein the ratio of the partial peptide or the salt thereof is measured. (56) The test solution and the insolubilized ( )) And reacting the labeled antibody with another antibody described in (5) simultaneously or continuously, and then measuring the activity of the labeling agent on the insolubilized carrier. (57) A method for quantifying the receptor protein according to (1), the partial peptide according to (3) or a salt thereof in a liquid, (57) a medicament comprising the antibody according to (5), (58) ) A pharmaceutical comprising the antibody according to item (5), which has an agonist-like activity;
Cancer (eg, breast, prostate, ovarian, follicular lymphoma, p
Cancers with 53 mutations, brain tumors, bladder cancers, cervical cancers, colorectal cancer (colorectal / rectal cancer), non-small cell lung cancer, small cell lung cancer, gastric cancer, etc.), AIDS, infectious diseases (eg, herpes virus infection, Adenovirus infection, box virus infection,
Helicobacter pylori infection, varicella-zoster virus infection, human papillomavirus infection, invasive staphylococcal infection, influenza infection, severe systemic fungal infection, etc.), acute bacterial meningitis, acute viral encephalitis, Adult respiratory distress syndrome, bacterial pneumonia, chronic lymphocytic leukemia, chronic myelogenous leukemia, insulin-dependent diabetes mellitus (type I), malignant melanoma, multiple myeloma, non-Hodgkin's lymphoma, peptic ulcer, sepsis, septic shock, The medicament according to (58), which is an agent for treating or preventing tuberculosis or the like,

(60) a medicament comprising the antibody according to (5) having an antagonist-like activity, (61) an allergic immune disease (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, Hay fever), inflammation (eg, arthritis, hepatitis, etc.), autoimmune diseases (eg, rheumatoid arthritis,
(60), which contains a medicament according to (60), which is a therapeutic / prophylactic agent for treating systemic lupus erythematosus, Sjogren's disease, AIDS, glomerulonephritis, bronchial asthma, etc., and (62) a soluble partial peptide according to (14). Medicine, (63) allergic immune diseases (eg, atopic dermatitis, contact dermatitis,
Allergic rhinitis, hay fever, etc.), inflammation (eg, arthritis,
Hepatitis), autoimmune diseases (eg, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's disease, etc.), AIDS,
The medicament according to (62), which is a therapeutic / prophylactic agent for glomerulonephritis, bronchial asthma, etc., or (64) complementary to or substantially complementary to the DNA according to (15) or (19). (65) a base sequence substantially complementary to the DNA according to (65), (15) or (19), which has a base sequence and has an action of suppressing expression of the DNA; About 70% or more (preferably about 80% or more, more preferably about 90% or more, and most preferably about 9% or more) of the total base sequence or partial base sequence of the base sequence complementary to DNA.
(64%) having a homology of at least 5%).
The antisense DNA described in (6) and (66) (6)
4) a medicine comprising the antisense DNA according to the above,
And (67) allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, hay fever, etc.), inflammation (eg, arthritis, hepatitis, etc.), autoimmune diseases (eg, rheumatoid arthritis, systemic (66), which is a therapeutic or prophylactic agent for AIDS, glomerulonephritis, bronchial asthma, and the like.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The receptor protein of the present invention comprises an amino acid sequence represented by SEQ ID NO: 1 [FIG. 1] (preferably, a 30th amino acid sequence represented by SEQ ID NO: 1).
(The 616th amino acid sequence). The receptor protein of the present invention includes, for example, humans and mammals (eg, guinea pigs, rats, mice, rabbits,
All cells of pigs, sheep, horses, cows, monkeys, etc. (eg, dendritic cells (eg, Langerhans cells), spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermis) Cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, Acidocytes, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors, stem cells or cancer cells of these cells, etc. And preferably, dendritic cells) and blood cell lines (eg, MEL, M1, CTLL-2, HT-
2, WEHI-3, HL-60, JOSK-1, K56
2, ML-1, MOLT-3, MOLT-4, MOLT
-10, CCRF-CEM, TALL-1, Jurka
t, CCRT-HSB-2, KE-37, SKW-3,
HUT-78, HUT-102, H9, U937, TH
P-1, HEL, JK-1, CMK, KO-812, M
EG-01), or any tissue in which these cells are present or derived, for example, the brain, each part of the brain (eg, olfactory bulb, nucleus pulposus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus) , Cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gall bladder,
Bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testis, ovary, placenta, uterus, bone, It may be a protein derived from joints, skeletal muscle, etc. (particularly, brain or various parts of the brain), or may be a synthetic protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 (preferably the 30th to 616th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1) includes, for example, SEQ ID NO: : About 40% or more, preferably about 50%
% Or more, more preferably about 70% or more, still more preferably about 80% or more, further preferably about 90% or more, and most preferably about 95% or more. Specific examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 (preferably, the amino acid sequence at positions 30 to 616 of the amino acid sequence represented by SEQ ID NO: 1) include, for example, SEQ ID NO: No. 30 to No. 21 of the amino acid sequence represented by 1:
Having an amino acid sequence of the 0th position and having an amino acid sequence represented by SEQ ID NO: 1 (preferably the 30th to 616th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1) of about 40% or more, preferably Is preferably about 50% or more, more preferably about 70% or more, further preferably about 80% or more, further preferably about 90% or more, and most preferably about 95% or more amino acid sequence. The receptor protein of the present invention having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 (preferably the 30th to 616th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1) ,
For example, it has an amino acid sequence substantially the same as the above-mentioned amino acid sequence represented by SEQ ID NO: 1 (preferably, the 30th to 616th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1), and Preferred is a receptor protein having substantially the same activity as a receptor protein having the amino acid sequence represented by SEQ ID NO: 1 (preferably, the 30th to 616th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1). Examples of substantially the same activity include ligand binding activity and signal transduction. Substantially the same means that their activities are qualitatively (eg, physiochemically or pharmacologically) the same. Therefore, the activities such as ligand binding activity and signal information transmission activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). However, quantitative factors such as the degree of these activities and the molecular weight of the receptor protein may be different. The measurement of the activity such as the ligand binding activity and the signal information transduction can be carried out according to a method known per se. For example, the activity can be measured according to a ligand determination method and a screening method described later.

The receptor protein of the present invention includes an amino acid sequence represented by SEQ ID NO: 1 (preferably a 30th amino acid sequence represented by SEQ ID NO: 1).
One to two or more (preferably about 1 to 30, more preferably 1 to 1)
About 0 amino acids, more preferably several (eg, 1 to 5) amino acids are deleted, the amino acid sequence represented by SEQ ID NO: 1 (preferably the amino acid sequence represented by SEQ ID NO: 1) One or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably several (eg, 1 to 5)) amino acids in the 30th to 616th amino acid sequence) , The amino acid sequence represented by SEQ ID NO: 1 (preferably the third amino acid sequence represented by SEQ ID NO: 1).
1 or 2 or more (preferably about 1 to 30, more preferably 1 to 1)
A protein containing an amino acid sequence in which about 0, more preferably several (eg, 1 to 5) amino acids are substituted with another amino acid, or an amino acid sequence obtained by combining them is also used. When the amino acid sequence is deleted, added, or substituted as described above, the position of the deletion, addition, or substitution is not particularly limited. For example, the 30th position of the amino acid sequence represented by SEQ ID NO: 1 And positions other than the amino acid sequence from the 210th to the 210th amino acid sequence. In addition, the 30th to 210th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 (that is, the amino acid sequence represented by SEQ ID NO: 2) contains the amino acid sequence represented by SEQ ID NO: 1. 2 shows the amino acid sequence of the extracellular region of the receptor protein of the present invention, and preferably shows the ligand binding site. In addition, the 30th to 616th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 corresponds to the amino acid sequence represented by SEQ ID NO: 3 (described later) And the amino acid sequence of the mature receptor protein of the present invention.

The receptor protein used in the present specification has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end in accordance with the convention of peptide labeling. The receptor proteins of the present invention, including the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, usually have a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ) at the C-terminus. May be an amide (—CONH ₂ ) or an ester (—COOR). Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl A C _6-12 aryl group such as α-naphthyl, for example, a phenyl-C _1-2 alkyl group such as benzyl and phenethyl or a C _7- alkyl group such as α-naphthyl-C _1-2 alkyl group such as α-naphthylmethyl. _In addition to ₁₄ aralkyl groups, a pivaloyloxymethyl group widely used as an oral ester is used. When the receptor protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the receptor protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Further, in the receptor protein of the present invention, the amino group of the N-terminal methionine residue in the receptor protein described above is protected by a protecting group (for example, a C _1-6 acyl group such as a formyl group and an acetyl group). , A glutamyl group formed by cleavage of the N-terminal side in vivo and a pyroglutamate, a substituent on the side chain of an amino acid in the molecule (for example, -OH, -COOH, an amino group, an imidazole group, an indole group) , A guanidino group or the like) protected with a suitable protecting group (eg, a C _1-6 acyl group such as a formyl group or an acetyl group), or a complex receptor protein such as a so-called sugar receptor protein to which a sugar chain is bound. Also included. Specific examples of the receptor protein of the present invention include, for example, a human dendritic cell-derived receptor protein containing the amino acid sequence represented by SEQ ID NO: 1 (FIG. 1) and the 30th amino acid sequence represented by SEQ ID NO: 1. A human dendritic cell-derived receptor protein containing the -616th amino acid sequence is used.

Partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as the partial peptide of the present invention)
As may be any as long as it is a partial peptide of the above-described receptor protein of the present invention, for example,
Of the receptor protein molecules of the present invention, those which are exposed outside the cell membrane and preferably have ligand binding activity are used. Further, the partial peptide of the present invention is preferably soluble. Specifically, as a partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1, the extracellular region (hydrophilic (Hydrophi
lic) site). Further, a peptide partially containing a hydrophobic (Hydrophobic) site can also be used. A peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used. The number of amino acids of the partial peptide of the present invention may be at least about 20 or more, preferably about 50 or more, more preferably about 100 or more, of the amino acid sequences constituting the receptor protein of the present invention.
Or more, more preferably about 200 or more and about 300
Peptides having less than or equal to the amino acid sequence are preferred. Specifically, for example, a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 and preferably having substantially the same activity as the receptor protein of the present invention, or the like. Preference is given to peptides which are more preferably soluble. SEQ ID NO: 2
Is substantially the same as the amino acid sequence represented by SEQ ID NO: 2.
An amino acid sequence having a homology of 0% or more, preferably about 50% or more, more preferably about 70% or more, still more preferably about 80% or more, still more preferably about 90% or more, and most preferably about 95% or more. Show. Here, “substantially the same activity” has the same meaning as described above. The “substantially equivalent activity” can be measured according to a ligand determination method and a screening method described later.

The partial peptide of the present invention may comprise one or more (preferably 1 to 10)
About, more preferably several (eg, 1 to 5) amino acids are deleted, or one or more (preferably about 1 to 20, more preferably 1 to 2) About 10 pieces, more preferably several pieces (eg, 1 to 5
)), Or one or more (preferably about 1 to 10)
More preferably, several (eg, 1 to 5) amino acids may be substituted with another amino acid. In the partial peptide of the present invention, the C-terminus usually has a carboxyl group (—COO).
H) or carboxylate (—COO ⁻ ),
As the receptor protein of the present invention described above, C-terminal, the amide (-CONH ₂₎ or an ester (-COOR)
It may be. Furthermore, the partial peptide of the present invention includes
Similar to the above-described receptor protein of the present invention, those in which the amino group of the N-terminal methionine residue is protected with a protecting group, those in which the glutamyl group formed by cleavage of the N-terminal side in vivo is pyroglutamylated, And those in which a substituent on the side chain of the amino acid in the above is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound. The C-terminal of the partial peptide of the present invention is usually a carboxyl group (—COOH) or a carboxylate (—COO ⁻ ).
H ₂ ) or an ester (—COOR).
Since the partial peptide of the present invention can be used as an antigen for producing an antibody, it is not necessary to have receptor activity.

As the signal peptide of the present invention, for example, a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3 is used. The signal peptide of the present invention includes, for example, cells of the above-mentioned humans and warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, pigs, sheep, cows, monkeys, etc.), and any tissues in which those cells are present. Or a synthetic peptide. An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3 is about 70% or more, preferably about 80% or more, more preferably about 90% or more, as the amino acid sequence represented by SEQ ID NO: 3. Most preferably, it shows an amino acid sequence having about 95% or more homology. More specifically, any peptide may be used as long as it has an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 and can exert a function as a signal peptide. Thus, quantitative factors such as the molecular weight of the receptor protein may be different.

The signal peptide of the present invention may contain one or more (preferably 1 to 2)
About 10, preferably 1 to 5, more preferably 1
~ 3 amino acids are deleted, or 1 or 2 or more (preferably about 1 to 10)
(Preferably about 1 to 5, more preferably 1 to 3)
Amino acid is added or 1 in the amino acid sequence
Alternatively, two or more (preferably about 1 to 10, preferably about 1 to 5, and more preferably 1 to 3) amino acids are replaced with another amino acid (for example, about 1 to 10, preferably 1 to 5). , More preferably 1 to 3 amino acids). In the signal peptide of the present invention, the C-terminus usually has a carboxyl group (—COOH)
Or carboxylate (—COO ⁻ ), but the C-terminal may be an amide (—CONH ₂ ) or an ester (—COOR) as in the receptor protein of the present invention described above. Further, the signal peptide of the present invention includes
As in the receptor protein of the present invention described above, the amino group of the N-terminal methionine residue is protected with a protecting group, and the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group. Or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound. As a specific example of the signal peptide of the present invention, for example, a peptide having the amino acid sequence represented by SEQ ID NO: 3 (that is, the 1st to 29th amino acids of the amino acid sequence represented by SEQ ID NO: 1) and the like are used. . The signal peptide of the present invention can efficiently secrete various extracellular secretory receptor proteins, including the receptor protein of the present invention, out of cells.

The salt of the receptor protein, partial peptide or signal peptide of the present invention is preferably a physiologically acceptable acid addition salt. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) ,
Salts with tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used. The receptor protein, partial peptide, signal peptide or a salt thereof of the present invention can be produced from human or mammalian cells or tissues according to the method for purifying a receptor protein or peptide known per se, or the present invention described below. Can be also produced by culturing a transformant containing a DNA encoding the receptor protein, partial peptide or signal peptide. Alternatively, it can be produced according to the peptide synthesis method described below or a method analogous thereto. When these receptor proteins or peptides are produced from human or mammalian tissues or cells, the homogenized human or mammalian tissues or cells are extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange. Purification and isolation can also be performed by combining chromatography such as chromatography.

For the synthesis of the receptor protein, partial peptide, signal peptide or their salts, or their amides of the present invention, commercially available resins for synthesis of receptor proteins can be used. Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,
-Benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-
(Hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin and the like. Using such a resin, α-
An amino acid having an amino group and a side chain functional group appropriately protected is condensed on a resin in accordance with the sequence of a target receptor protein according to various condensation methods known per se. At the end of the reaction, the receptor protein is cleaved from the resin, various protecting groups are removed, and an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution. To obtain the amide form of For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used.
Activation by these includes racemization inhibiting additives (e.g.,
(HOOBt, HOOBt) together with the protected amino acid directly to the resin or symmetrical anhydride or HOBt ester or
HOOBt ester can be added to the resin after activation of the protected amino acid in advance.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol,
Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; and an appropriate mixture thereof are used. The reaction temperature is appropriately selected from the range known to be usable for the receptor protein bond formation reaction, and is usually about -20 ° C to 50 ° C.
The temperature is appropriately selected from the range of ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When a sufficient condensation cannot be obtained by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole so as not to affect the subsequent reaction.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2
-Nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used. The carboxyl group is
For example, alkyl esterification (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl,
Cyclohexyl, cycloheptyl, cyclooctyl, 2
Linear, branched or cyclic alkyl esterification such as adamantyl), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification) , Phenacyl esterification, benzyloxycarbonyl hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like. The hydroxyl group of serine is
For example, it can be protected by esterification or etherification. Examples of the group suitable for the esterification include a lower alkanoyl group such as an acetyl group, an aroyl group such as a benzoyl group, a benzyloxycarbonyl group,
A group derived from carbonic acid such as an ethoxycarbonyl group is used. Also, as a group suitable for etherification,
For example, a benzyl group, a tetrahydropyranyl group, a t-butyl group, etc. As the protecting group for the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used. As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, para-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used. As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd black or Pd-carbon, or anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethane Acid treatment with sulfonic acid, trifluoroacetic acid, or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is as follows:
Generally, it is carried out at a temperature of about -20 ° C to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethane The addition of a cation scavenger such as dithiol is effective. In addition, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is 1,2-ethanedithiol, 1,4-butane described above. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means. . As another method for obtaining the amide form of the receptor protein or its peptide of the present invention, for example, first, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, a peptide chain (protein chain) is added to the amino group side. After extending to the desired chain length, a protein was prepared by removing only the protecting group of the N-terminal α-amino group of the peptide chain, and a protein was obtained by removing only the protecting group of the C-terminal carboxyl group. In a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. The crude protein or crude peptide is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein or its peptide. In order to obtain an ester of the receptor protein or its peptide of the present invention, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, in the same manner as the amide of the receptor protein, An ester of the desired receptor protein or its peptide can be obtained.

The partial peptide, signal peptide or salt thereof of the present invention can be produced according to a peptide synthesis method known per se, or by cleaving the receptor protein of the present invention with an appropriate peptidase.
As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the target peptide with the remaining portion, and removing the protective group when the product has a protective group. Known methods for condensation and elimination of protecting groups include, for example, the methods described in (1) to (4) below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Chemistry of Receptor Proteins IV, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis, Hirokawa Shoten The target peptide can be purified and isolated by a combination of conventional purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the peptide is obtained as a salt, a known method or analogous method To the free form or other salts.

D encoding the receptor protein of the present invention
The NA may be any as long as it contains the above-described nucleotide sequence encoding the receptor protein of the present invention. In addition, genomic DNA, genomic DNA library, cDNA derived from the aforementioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, synthetic DN
Any of A may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse transcriptase polymerase was prepared directly by using a total RNA or mRNA fraction prepared from the cells and tissues described above.
It can also be amplified by Chain Reaction (hereinafter abbreviated as RT-PCR method). Specifically, as the DNA encoding the receptor protein of the present invention, for example,
DNA containing the base sequence represented by SEQ ID NO: 4,
Alternatively, it has a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 4 under high stringent conditions, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal signal transduction action) Such)
Any DNA may be used as long as it encodes a receptor protein having Examples of the DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 4 include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably the nucleotide sequence represented by SEQ ID NO: 4. Preferably, a DNA containing a base sequence having about 95% or more homology is used.

Hybridization is carried out by a method known per se or a method analogous thereto, for example, molecular
Cloning (Molecular Cloning) 2nd (J. Sambrook
et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions. High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 40 mM.
At 20 mM, the temperature is about 50-70 ° C., preferably about 60 ° C.
The condition of ~ 65 ° C is shown. In particular, when the sodium concentration is about 19
Most preferably, the temperature is about 65 ° C. in mM. More specifically, as the DNA encoding the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, a DNA having the base sequence represented by SEQ ID NO: 2 or the like is used. As the DNA encoding the receptor protein containing the amino acid sequence at positions 30 to 616 of the amino acid sequence to be used, a DNA having the base sequence at positions 88 to 1848 of the base sequence represented by SEQ ID NO: 2 or the like is used.

DNA encoding the partial peptide of the present invention
May be any as long as it contains a base sequence encoding the above-described partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, RT-P was directly prepared using an mRNA fraction prepared from the cells and tissues described above.
It can also be amplified by the CR method. Specifically, the DNA encoding the partial peptide of the present invention includes, for example, DN having the base sequence represented by SEQ ID NO: 4.
DNA having the partial base sequence of A, or SEQ ID NO:
4. It has a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by No. 4 and has substantially the same activity (eg, ligand binding activity, signal information transduction action, etc.) as the receptor protein peptide of the present invention. For example, a DNA having a partial nucleotide sequence of a DNA encoding the receptor protein may be used. Examples of the DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 4 include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% with the nucleotide sequence represented by SEQ ID NO: 4.
As described above, DNA containing a nucleotide sequence having a homology of about 95% or more is most preferably used. More specifically, the DNA encoding the partial peptide having the amino acid sequence represented by SEQ ID NO: 2 includes SEQ ID NO: 2.
DNA having a base sequence represented by 5, or a base sequence that hybridizes with the base sequence under stringent conditions is used. Examples of the DNA that can hybridize with the base sequence represented by SEQ ID NO: 5 include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% with the base sequence represented by SEQ ID NO: 5.
As described above, DNA containing a nucleotide sequence having a homology of about 95% or more is most preferably used. The same hybridization method and high stringency conditions as described above are used.

D encoding the signal peptide of the present invention
The NA may be any as long as it contains the above-described nucleotide sequence encoding the signal peptide of the present invention. In addition, genomic DNA, genomic DNA library, cDNA derived from the above-described cells / tissue, cDNA library derived from the aforementioned cells / tissue, synthetic DNA
Either may be used. As the DNA encoding the signal peptide of the present invention, for example, a DNA having the nucleotide sequence represented by SEQ ID NO: 6 or a nucleotide sequence hybridizing under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 6 And a DNA encoding a peptide capable of exhibiting a function as a signal peptide. Examples of the DNA capable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 6 include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably the nucleotide sequence represented by SEQ ID NO: 6. Preferably, a DNA containing a base sequence having about 95% or more homology is used. The same hybridization method and high stringency conditions as described above are used. More specifically, SEQ ID NO: 3
As a DNA encoding a signal peptide having the amino acid sequence represented by SEQ ID NO: 6, a DNA containing the DNA having the base sequence represented by SEQ ID NO: 6 or the like is used.

Means for cloning a DNA that completely encodes the receptor protein of the present invention or a partial peptide thereof (hereinafter sometimes abbreviated as the receptor protein of the present invention) include (1) the receptor protein of the present invention and the like. Amplification by PCR using a synthetic DNA primer having a partial nucleotide sequence of (1) or (2) DNA incorporated into an appropriate vector and a DNA fragment encoding a partial or entire region of the receptor protein or the like of the present invention Selection by hybridization with a labeled synthetic DNA. Hybridization methods are described, for example, in Molecular Cloning 2nd (J. Sambrook et al.).
al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. The DNA encoding the signal peptide of the present invention can be produced according to a known oligonucleotide synthesis method. DN
Conversion (deletion / addition / substitution) of the nucleotide sequence of A can be performed by a known kit, for example, Mutan ^™ -G (Takara Shuzo), Mutan ^™ -K
(Takara Shuzo Co., Ltd.), using the Gapped duplex method or Kun
It can be performed according to a method known per se such as the kel method or a method analogous thereto. The DNA encoding the cloned receptor protein or the like can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA has ATG at the 5 'end as a translation initiation codon and TA at the 3' end as a translation stop codon.
It may have A, TGA or TAG. These translation initiation codons and translation stop codons are appropriately synthesized DNA
It can also be added using an adapter. The expression vector of the receptor protein or the like of the present invention can be prepared, for example, by (A) cutting out a DNA fragment of interest from DNA encoding the receptor protein or the like of the present invention, and (B) converting the DNA fragment into a promoter of an appropriate expression vector. It can be manufactured by connecting downstream.

As a vector, a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC12) is used.
UC13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as hosts, the SRα promoter, SV40 promoter, L
TR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter and the like can be mentioned. Among them, it is preferable to use the CMV promoter, SRα promoter and the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter,
When the host is a bacterium belonging to the genus Bacillus, an SPO1 promoter, an SPO2 promoter, a penP promoter, etc., and when the host is a yeast, a PHO5 promoter, a PGK promoter, a GA
P promoter, ADH promoter and the like are preferable.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

[0037] In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a poly-A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Can be used. As a selection marker, for example, dihydrofolate reductase (hereinafter, dhfr)
Gene (sometimes abbreviated as “methotrexate (M
TX) resistance], an ampicillin resistance gene (hereinafter referred to as Amp
^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo, G418 resistance)
And the like. In particular, when the dhfr gene is used as a selection marker by using dhfr gene-deficient Chinese hamster cells CHO, cells transformed with the target gene can be selected using a thymidine-free medium. If necessary, a signal sequence suitable for the host is added to the amino terminal side of the receptor protein of the present invention. When the host is Escherichia, PhoA.
When the host is Bacillus, an α-amylase signal sequence, a subtilisin signal sequence, or the like is used. When the host is yeast, an MFα signal sequence, an SUC2 signal, or the like is used. When the host is an animal cell such as a sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule / signal sequence, etc. can be used, respectively. Using the vector containing the DNA encoding the receptor protein or the like of the present invention thus constructed,
Transformants can be produced.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include
Escherichia coli K12 DH
1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160]
(1968)], JM103 [Nucleic Acids
Research, (Nucleic Acids Research), 9, 309
(1981)], JA221 [Journal of Molecular Biolog
y)], 120, 517 (1978)], HB101
[Journal of Molecular Biology, 4
1, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of Bacillus bacteria include, for example, Bacillus
Bacillus subtilis MI114 [Gene (G
ene), 24, 255 (1983)], 207-21.
[Journal of Biochemistry
Biochemistry), 95, 87 (1984)]. As yeast, for example, Saccharomyces
Saccharomyces cerevisiae AH22, A
^{H22R -, NA87-11A, DKD-5D} , 20B
-12, Schizosacchamyces pombe
romyces pombe) NCYC1913, NCYC203
6, Pichia pastoris and the like are used.

As insect cells, for example, virus is A
In the case of cNPV, a cell line derived from night larvae of moth (Spodop
tera frugiperda cell (Sf cell), Trichoplusia ni
MG1 cells from the midgut, H from the eggs of Trichoplusia ni
igh Five ^TM cells, cells derived from Mamestra brassicae or
Estigmena acrea-derived cells and the like are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mor
iN; BmN cells). Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf21
Cells (Vaughn, JL et al., In Viv
o), 13, 213-217, (1977)). As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (198).
5)]. As animal cells, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dhf)
r ⁻ ) cells), mouse L cells, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells, 293 cells, C127 cells, BALB3T3 cells,
Sp-2 cells and the like are used. Among them, CH
O cells, CHO (dhfr ⁻ ) cells, 293 cells and the like are preferred.

For transformation of Escherichia, for example, Processing of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), Vol. 69 , 21
10 (1972) and Gene, 17, 107 (1)
982). For transformation of Bacillus sp., For example, Molecular and General Genetics (Mo
lecular & General Genetics), 168, 111 (1
979) and the like. To transform yeast, see, for example, Methods in Enzymology, 19
Vol. 4, 182-187 (1991), Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad.
Sci. USA), vol. 75, 1929 (1978). Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1)
973). As a method for introducing an expression vector into cells, for example, the calcium phosphate method (Graham, FL and van der Eb, A.
J. Virology 52, 456-467 (1973)],
Electroporation [Neumann, E. et al. Embo J. (EMBO J.) 1, 841-845 (1982)] and the like. Thus, a transformant transformed with the expression vector containing the DNA encoding the receptor protein or the like of the present invention is obtained.

As a method for stably expressing the receptor protein or the like of the present invention using animal cells, a method in which the above-described expression vector introduced into the animal cells is integrated into the chromosome by clonal selection is selected. There is.
Specifically, a transformant is selected using the above-mentioned selection marker as an index. Furthermore, a stable animal cell line having a high expression ability of the protein of the present invention can be obtained by repeatedly performing clonal selection on the animal cells obtained using the selection marker. Also, dhf
When the r gene is used as a selection marker, MTX concentration is gradually increased, and culture is performed.
A DNA encoding the receptor protein or the like of the present invention can be amplified in a cell together with the dhfr gene to obtain an animal cell strain with higher expression. The protein of the present invention or a salt thereof is produced by culturing the above transformant under conditions capable of expressing DNA encoding the receptor protein or the like of the present invention to produce and accumulate the receptor protein or the like of the present invention. be able to. When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the cultivation. Nitrogen sources, inorganic substances, etc. are contained. As the carbon source, for example, glucose, dextrin, soluble starch,
As a nitrogen source such as sucrose, for example, inorganic salts or organic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, inorganic substances such as calcium chloride , Sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. PH of the medium is about 5-8
Is desirable.

As a medium for culturing the genus Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa)
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York 1
972] is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration and stirring may be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant whose host is yeast, for example, a Burkholder minimum medium [Bostian, KL
Processing of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 77, 450
5 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., Processings of the.
National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. US
A), 81, 5330 (1984)].
Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and / or stirring are added.

When culturing an insect cell or a transformant whose host is an insect, the medium used is Grace's Insect Mediu.
m (Grace, TCC, Nature, 195,788 (196
Those to which an additive such as 10% bovine serum inactivated as described in 2)) is appropriately added are used. The pH of the medium is about 6.2-6.
Adjustment to 4 is preferred. Culture is usually performed at about 27 ° C for about 3
５5 days, with aeration and / or agitation as needed. When culturing a transformant in which the host is an animal cell, the medium may be, for example, MEM containing about 5 to 20% fetal bovine serum.
Medium [Science, Vol. 122, 501 (19)
52)], DMEM medium [Virology,
8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medica
l Association) 199, 519 (1967)], 19
9 Medium [Proceeding of the Society for the Biological Medicine (Proceeding o
fthe Society for the Biological Medicine), 73
Vol., 1 (1950)]. pH is about 6-8
It is preferred that Culture is usually performed at about 30-40 ° C for about 1 hour.
Perform for 5 to 70 hours, adding aeration and stirring as needed. In particular, when CHO (dhfr ⁻ ) cells and the dhfr gene are used as selection markers, it is preferable to use a DMEM medium containing dialyzed fetal bovine serum containing almost no thymidine. As described above, the transformant can produce the receptor protein or the like of the present invention.

The receptor protein and the like of the present invention can be separated and purified from the above culture by, for example, the following method. When extracting the receptor protein or the like of the present invention from cultured cells, insects or cells, the cells or cells are collected by a known method after culture, suspended in an appropriate buffer, and then subjected to ultrasound, lysozyme and / or lysozyme. Alternatively, a method of disrupting cells, insects, or cells by freeze-thawing or the like, and then obtaining a crude extract such as a receptor protein by centrifugation or filtration is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the partial peptide is secreted into the culture solution,
After the culture, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. Purification of the receptor protein or the like contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods. These known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SD.
A method using mainly a difference in molecular weight such as S-polyacrylamide gel electrophoresis, a method using a difference in charge such as ion exchange chromatography, a method using specific affinity such as affinity chromatography,
A method using a difference in hydrophobicity such as reversed phase high performance liquid chromatography, a method using a difference in isoelectric point such as isoelectric focusing, and the like are used. When the receptor protein or the like thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained as a salt, a method known per se or The compound can be converted into a free form or another salt by an analogous method. The receptor protein or the like produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification.
As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The presence or activity of the receptor protein or the like of the present invention thus produced or a salt thereof can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

The receptor protein of the present invention, its partial peptide or a salt thereof (such as the receptor protein of the present invention)
May be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the receptor protein of the present invention. Furthermore, the antibody against the receptor protein or the like of the present invention may have an activity of binding to the receptor protein or the like of the present invention and neutralizing the activity of the receptor protein or the like (antagonist-like activity), or It may have an activity (agonist-like activity) of binding to the receptor protein of the present invention or the like to induce the activity of the receptor protein or the like. An antibody against the receptor protein or the like of the present invention can be produced by using the receptor protein or the like of the present invention as an antigen according to a method for producing an antibody or antiserum known per se.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell
Administered with diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually 2-
This is performed once every six weeks, for a total of about two to ten times. Examples of the mammal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. A monoclonal antibody-producing hybridoma can be prepared by fusing the contained antibody-producing cells with myeloma cells of the same or different species. The antibody titer in the antiserum can be measured, for example, by reacting a labeled receptor protein or the like described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be performed by known methods, for example, the method of Kohler and Milstein [Nature
re), 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2
/ 0, myeloma cells of warm-blooded animals such as AP-a, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells to be used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is 10 to 80.
%, And incubated at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes, so that cell fusion can be carried out efficiently.

Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which an antigen such as a receptor protein is adsorbed directly or together with a carrier. Then, an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (an anti-mouse immunoglobulin antibody is used when the cells used for cell fusion are mice) or protein A is added, and the monoclonal antibody bound to the solid phase is added. Detection method: Add hybridoma culture supernatant to solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, add receptor protein etc. labeled with radioactive substances, enzymes, etc., and detect monoclonal antibody bound to solid phase And the like. The selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, 1-2
RP containing 0%, preferably 10-20% fetal calf serum
MI 1640 medium, G containing 1-10% fetal calf serum
IT medium (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.)
Etc. can be used. Culture temperature is usually 20-4
0 ° C., preferably about 37 ° C. Culture time is usually 5
Days to 3 weeks, preferably 1 to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of monoclonal antibodies can be carried out by the same method as that for ordinary polyclonal antibodies such as immunoglobulin separation and purification [eg, salting out, alcohol precipitation, isoelectric precipitation, , Electrophoresis, adsorption / desorption with an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or an active adsorbent such as protein A or protein G to collect only antibodies and bind Specific Purification Method for Obtaining Antibody by Dissociation].

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immunizing antigen (an antigen such as a receptor protein) itself or a complex thereof with a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting the antibody-containing substance against the antibody and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used to immunize a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten should be such that the antibody can be efficiently produced against the hapten immunized by crosslinking the carrier. Any one may be cross-linked at any ratio, for example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc., in a weight ratio of about 0.1 to 1 hapten.
A method of coupling at a rate of 1 to 20, preferably about 1 to 5 is used. Various coupling agents can be used for coupling the hapten and the carrier. For example, glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times. The polyclonal antibody can be collected from blood, ascites, or the like, preferably from blood, of the mammal immunized by the above method. The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

Antisense DNA having a base sequence complementary to or substantially complementary to DNA encoding the receptor protein or partial peptide of the present invention (hereinafter sometimes abbreviated to DNA of the present invention) includes: Any antisense DNA may be used as long as it has a base sequence complementary or substantially complementary to the DNA of the present invention and has an action capable of suppressing the expression of the DNA.
As the base sequence substantially complementary to the DNA of the present invention,
For example, about 70% or more, preferably about 80% or more, of the total nucleotide sequence or the partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention),
More preferably about 90% or more, most preferably about 95%
Base sequences having the above homology are exemplified. In particular, of the entire base sequence of the complementary strand of the DNA of the present invention, the complementary sequence of the base sequence of the portion encoding the N-terminal site of the receptor protein of the present invention or a partial peptide thereof (for example, the base sequence near the start codon). Antisense D having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more.
NA is preferred. These antisense DNAs can be produced using a known DNA synthesizer or the like.

The receptor protein of the present invention is characterized by its TNF receptor family (eg, TNF
Receptor type 1, TNF receptor type 2, Fas, CD
40, CD30, etc.). Although the physiological functions of each protein belonging to this family are extremely diverse such as cell differentiation, proliferation, activation or cell death (Cell, Vol. 76, 959-962 (1994)), the receptor of the present invention It plays an important role in the expression of proteins in dendritic cells and signal transduction related to physiological functions (eg, cell differentiation, proliferation, activation, etc.) in cells with antigen presenting ability represented by the same cells. From these facts, it is considered that the receptor protein of the present invention is involved in diseases caused by abnormal immune functions such as allergic diseases and autoimmune diseases and diseases accompanied by abnormal immune functions. The receptor protein or the like of the present invention or a DNA encoding the same may be used to determine a ligand for the receptor protein of the present invention, obtain an antibody and an antiserum, construct a recombinant receptor protein expression system, and bind to a receptor using the expression system. Assay system development and drug candidate screening,
It can be used for drug design based on comparison with structurally similar ligands / receptors, preparation of probes and PCR primers in gene diagnosis, preparation of transgenic animals, and gene preventive / therapeutic agents. In particular, compounds (eg, agonists, antagonists, etc.) that change the binding of ligands to human or mammalian specific receptors by using a receptor binding assay system using the recombinant receptor protein expression system of the present invention. ) Can be screened, and the agonist or antagonist can be used as an agent for preventing or treating various diseases.
The use of the DNA of the present invention, such as the receptor protein of the present invention, and the antibody against the receptor protein, etc. of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) will be specifically described below.

(1) Method for Determining Ligand for Receptor Protein of the Present Invention The receptor protein of the present invention is useful as a reagent for determining or searching for a ligand (agonist) for the receptor protein of the present invention. That is, the present invention provides a method for determining a ligand for the receptor protein of the present invention, which comprises contacting the receptor protein or the like of the present invention with a test compound. Specifically, the ligand determination method of the present invention uses the receptor protein or the like of the present invention or constructs an expression system for a recombinant receptor protein or the like and uses a receptor binding assay system using the expression system. Binds to the receptor protein of the present invention, and activates cell stimulating activities (eg, NF-κB activation, TRAF molecule binding activity, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, Compounds having an activity of promoting or inhibiting intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (for example, peptides, proteins, Peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof. In the ligand determination method of the present invention, when the test compound is brought into contact with the receptor protein or the like of the present invention, for example, the amount of the test compound bound to the receptor protein or the like, or the cell to the cell containing the receptor protein or the like It is characterized by measuring stimulating activity and the like.

More specifically, the present invention is characterized in that when a labeled test compound is brought into contact with the receptor protein or the like of the present invention, the amount of binding of the labeled test compound to the receptor protein or the like is measured. A method for determining a ligand for the receptor protein of the present invention or a salt thereof, the method comprising the steps of: contacting a labeled test compound with a cell containing the receptor protein or the like of the present invention or a membrane fraction thereof; A method for determining a ligand for a receptor protein of the present invention or a salt thereof, which comprises measuring the amount of binding to a cell or a membrane fraction thereof, and a method for preparing a labeled test compound comprising a DNA encoding the receptor protein of the present invention. When the transformant is brought into contact with the receptor protein expressed on the cell membrane by culturing Determining the amount of binding of the labeled test compound to the receptor protein or a salt thereof, wherein the method comprises the steps of: contacting the test compound with a cell containing the receptor protein of the present invention; Cell stimulating activity via receptor protein (eg, NF-κB activation, TR
AF molecule binding activity, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activity , P
Activity that promotes or suppresses the reduction of H, etc.)
And a method for determining a ligand for the receptor protein of the present invention or a salt thereof.
When a transformant containing NA is cultured and brought into contact with a receptor protein expressed on the cell membrane, cell stimulating activity via the receptor protein (for example, NF-κB activation, TRAF molecule binding activity, arachidonic acid Release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. A method for determining a ligand for the receptor protein of the present invention or a salt thereof. In particular, it is preferable to perform the above-mentioned tests after performing the above-mentioned tests and confirming that the test compound binds to the receptor protein of the present invention.

First, the receptor protein preparation used in the method for determining a ligand may be any of the above-described receptor proteins and the like of the present invention or cells containing the receptor protein and the like of the present invention (eg, dendritic cells). However, a receptor protein expressed in large amounts using animal cells is suitable. The above-mentioned expression method is used for producing the receptor protein and the like of the present invention, but it is preferably carried out by expressing the DNA encoding the receptor protein in mammalian cells or insect cells. DNA fragments encoding the target receptor protein portion include:
Usually, cDNA is used, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. In order to introduce a DNA fragment encoding the receptor protein or the like of the present invention into host animal cells and to express them efficiently, the DNA fragment is converted into a nuclear polyhedrosis virus (nucl
It is preferably incorporated into the downstream of a polyhedrin promoter of ear polyhedrosis virus (NPV), a promoter derived from SV40, a retrovirus promoter, a metallothionein promoter, a human heat shock promoter, a cytomegalovirus promoter, and an SRα promoter. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P .; Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19
559, 1992].

Therefore, the receptor protein or the like of the present invention used in the method for determining the ligand of the present invention may be a receptor protein or the like purified according to a method known per se, a cell containing the receptor protein or the like, or a cell containing the same. A membrane fraction may be used. When cells containing the receptor protein or the like of the present invention are used in the ligand determination method of the present invention, the cells may be immobilized with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se. The cell containing the receptor protein or the like of the present invention refers to a host cell expressing the receptor protein or the like of the present invention. As the host cell, Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like are used. . The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. Potting method for cells
a method of crushing cells with an er-Elvehjem type homogenizer,
Waring blender and Polytron (Kinematica)
Crushing by ultrasonic waves, crushing by ultrasonic waves, crushing by ejecting cells from a thin nozzle while applying pressure by a French press or the like. For fractionation of cell membranes, fractionation by centrifugal force, such as fractionation centrifugation and density gradient centrifugation, is mainly used. For example, the cell lysate is slowed down (5
Centrifugation at 00 rpm to 3000 rpm for a short time (usually about 1 to 10 minutes).
m-30,000 rpm) for 30 minutes to 2 hours,
The resulting precipitate is used as the membrane fraction. The membrane fraction contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the receptor protein or the like in the cells containing the receptor protein or the like or the membrane fraction thereof is preferably 10 ³ to 10 ⁸ molecules per cell, and more preferably 10 ⁵ to 10 ⁷ molecules per cell. . The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .

In order to carry out the above-mentioned method for determining a ligand for the receptor protein of the present invention,
Appropriate receptor protein fractions and labeled test compounds are required. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor fraction having an activity equivalent thereto is desirable. here,
Equivalent activities are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 20 times).
2 times) ligand binding activity, signal transduction action, etc. Test compounds include known ligands (eg, TNF (eg, TNF-α), lymphotoxin (eg,
Lymphotoxin-α, Lymphotoxin-β), Fas ligand, NGF, CD40 ligand, CD27 ligand, CD30 ligand, OX-40 ligand, 4-1B
B ligand or Apo-2L (TRAIL)) or those obtained by preparing the extracellular region or a part thereof as a recombinant, for example, human or mammal (eg, mouse, rat, pig, cow, sheep) , Monkeys, etc.)
Tissue extract, cell culture supernatant and the like are used. For example, the tissue extract, cell culture supernatant, or the like is added to the receptor protein or the like of the present invention or cells containing the same, and fractionated while measuring ligand binding activity, cell stimulating activity, and the like. Can be obtained. [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [
Radioactive substances such as ³⁵ S] are used.

Specifically, in order to carry out the method for determining a ligand for the receptor protein or a salt thereof of the present invention, for example, cells containing the receptor protein of the present invention or a membrane fraction thereof are suitable for the determination method. A receptor standard is prepared by suspending in a buffer. The buffer has a pH of about 4 to 10 (preferably, a pH of about 6 to 8).
Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer and Tris-HCl buffer. For the purpose of reducing non-specific binding, surfactants such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin, deoxycholate and various proteins such as bovine serum albumin and gelatin may be added to the buffer. it can. further,
A protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories) and pepstatin can also be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. And 0.01ml-10
Add a fixed amount (5000 cpm-
500000 cpm) [ ³ H], [ ¹²⁵ I],
A test compound labeled with [ ¹⁴ C], [ ³⁵ S] or the like is allowed to coexist. A reaction tube containing a large excess of unlabeled test compound is also prepared to determine the amount of non-specific binding (NSB).
The reaction is carried out at about 0 to 50 ° C, preferably about 4 to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. The count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) is 0 cpm.
More than one test compound can be selected as a ligand (agonist) for the receptor protein of the present invention or a salt thereof.

In order to carry out the above methods (1) and (2) for determining a ligand for the receptor protein or a salt thereof of the present invention, a cell stimulating activity (for example, NF-κB activation, TRAF molecule binding activity) Arachidonic acid release, acetylcholine release, intracellular C
a ²⁺ release, intracellular cAMP production, intracellular cGMP production,
Inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.) using a known method or a commercially available measurement kit. Can be measured. Specifically, first, cells containing a receptor protein or the like are cultured in a multiwell plate or the like. Prior to ligand determination, replace the cells with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, and incubate for a certain period of time. The quantified product is quantified according to each method. When the production of a substance (for example, arachidonic acid or the like) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor against the degrading enzyme.

As a kit for determining a ligand to the receptor protein of the present invention, a kit containing a cell containing the receptor protein of the present invention, a cell containing the receptor protein of the present invention, or a membrane fraction thereof is used.
Examples of the kit for determining a ligand of the present invention include the following. 1. Ligand determination reagent (i) Measurement buffer and washing buffer Hanks' Balanced Salt Solution (Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. (ii) Receptor protein preparation: CHO cells expressing the receptor protein of the present invention,
Passage into a 12-well plate at 5 × 10 ⁵ / well, 37 ° C.
Cultured at 5% CO ₂ for 2 days. (iii) Labeled test compound A commercially available compound labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], or the like, or labeled by an appropriate method. Alternatively, store at −20 ° C. and dilute to 1 μM with a measuring buffer before use. Test compounds that are poorly soluble in water are dissolved in dimethylformamide, DMSO, methanol and the like. (iv) Unlabeled test compound The same as the labeled compound is prepared at a concentration 100 to 1000 times higher.

2. Assay method (i) CHO cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate were mixed with 1 ml of assay buffer.
After washing twice, 490 μl of measurement buffer is added to each well. (ii) 5 μl of the labeled test compound is added and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, add 5 μl of unlabeled test compound. (iii) Remove the reaction solution and wash three times with 1 ml of washing buffer. The labeled test compound bound to cells was 0.2N N
Dissolve in aOH-1% SDS and mix with 4 ml of liquid scintillator A (Wako Pure Chemical Industries). (iv) Radioactivity is measured using a liquid scintillation counter (manufactured by Beckman). (v) When the radioactivity decreases, the test compound can be selected as a candidate ligand compound. (vi) Using this ligand candidate compound,
And a cell having cell stimulating activity can be selected as a ligand (agonist). Examples of ligands that can bind to the receptor protein of the present invention include TNF (eg, TNF-α), lymphotoxin (eg, lymphotoxin-α, lymphotoxin-β), Fas ligand, NGF, CD40 ligand, CD27 ligand, CD30 ligand, OX-40
Ligand, 4-1BB ligand, Apo-2L (TRA
IL) and the like.

(2) Therapeutic / prophylactic agent for the receptor protein deficiency of the present invention The DNA encoding the receptor protein of the present invention or its partial peptide (the DNA of the present invention) is used for the treatment or prevention of the receptor protein deficiency of the present invention. It can be used as a medicine such as an agent. For example, when there is a patient in whom the physiological action of the ligand cannot be expected because the receptor protein of the present invention is reduced in the living body, (a) administering the DNA of the present invention to the patient and expressing it; B) After inserting and expressing the DNA of the present invention into target cells and then transplanting the cells into the patient, the amount of receptor protein in the patient's body is increased, and the effect of the ligand is fully exerted. Can be done. Therefore, the DNA of the present invention is safe and low toxic, and the receptor protein deficiency of the present invention, for example, cancer (eg, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, p5
Cancer with three mutations, brain tumor, bladder cancer, cervical cancer, colorectal cancer (colorectal cancer), non-small cell lung cancer, small cell lung cancer, gastric cancer, etc., AIDS, infectious disease (eg, herpes virus infection,
Adenovirus infection, box virus infection, Helicobacter pylori infection, varicella-zoster virus infection, human papillomavirus infection, invasive staphylococcal infection, influenza infection, severe systemic fungal infection, etc.), Acute bacterial meningitis, acute viral encephalitis, adult respiratory distress syndrome, bacterial pneumonia, chronic lymphocytic leukemia, chronic myelogenous leukemia, insulin-dependent diabetes mellitus (type I), melanoma, multiple myeloma, non-Hodgkin's lymphoma It is useful as a medicament such as an agent for treating or preventing peptic ulcer, sepsis, septic shock, tuberculosis and the like.

When the DNA of the present invention is used as an agent for treating or preventing the above-mentioned diseases, the DNA of the present invention is used alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. After administration, it can be administered to humans or warm-blooded animals according to conventional means. The DNA of the present invention can be administered as it is or together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake by a gene gun or a catheter such as a hydrogel catheter. The vector containing the DNA of the present invention can be, for example, orally coated as sugar-coated tablets, capsules, elixirs, microcapsules, and the like, if necessary.
Alternatively, it can be used parenterally in the form of an injection such as a sterile solution with water or another pharmaceutically acceptable liquid, or a suspension. For example, the vector containing the DNA of the present invention may be combined with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc., in a unit dosage form required for generally accepted pharmaceutical practice. And can be produced by mixing. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

Examples of additives that can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. And a lubricating agent such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, reddish oil or cherry, and the like. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. For example, it may be used in combination with an alcohol (eg, ethanol), a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant (eg, polysorbate 80 (TM), HCO-50).
As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. In addition, the above-mentioned therapeutic / prophylactic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, ,
Human serum albumin, polyethylene glycol, etc.),
Preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like may be added. The prepared pharmaceutical composition such as an injection solution is usually filled in a suitable ampoule.

Since the preparation thus obtained is safe and has low toxicity, it is administered, for example, to humans and mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). be able to. The dosage of the DNA of the present invention varies depending on the target disease, the administration subject, the administration route, and the like.
When oral administration of is generally adult (60kg)
In about 0.1 mg to 10 mg of the DNA per day.
0 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose of the DNA varies depending on the administration subject, target disease, and the like. )), The DNA should be administered in an amount of about 0.01 to 30 per day.
It is convenient to administer about mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(3) Genetic Diagnostic Agent The DNA of the present invention can be used as a probe to produce the DNA of the present invention in humans or mammals (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). DNA or m encoding a receptor protein or the like
Since it can detect RNA abnormality (gene abnormality), it can be used as a gene diagnostic agent for detecting, for example, damage, mutation, or decreased expression of the DNA or mRNA, or increased or excessive expression of the DNA or mRNA. Useful. The above-mentioned genetic diagnosis using the DNA of the present invention can be performed, for example, by the known Northern hybridization or PCR-SSCP method (Genomics (Genomics)).
s), Volume 5, pp. 874-879 (1989), Proceedings of the National Academy of Sciences of USA (Proceeding)
s of the Natinal Academy of Sciences of the United
States of America), Vol. 86, 2766-2770
(1989)).
For example, when a decrease in the expression of mRNA encoding the receptor protein or the like of the present invention is detected by Northern hybridization, for example, cancer (eg, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer with p53 mutation, Brain tumor, bladder cancer, cervical cancer, colorectal cancer (colorectal / rectal cancer),
Non-small cell lung cancer, small cell lung cancer, gastric cancer, etc., AIDS, infection (eg, herpes virus infection, adenovirus infection, box virus infection, Helicobacter pylori infection, varicella-zoster virus infection, human papilloma) Viral infections, invasive staphylococcal infections, influenza infections, severe systemic fungal infections, etc.), acute bacterial meningitis, acute viral encephalitis, adult respiratory distress syndrome, bacterial pneumonia, chronic lymphocytic leukemia, chronic bone marrow Diseases such as leukemia, insulin-dependent diabetes mellitus (type I), malignant melanoma, multiple myeloma, non-Hodgkin's lymphoma, peptic ulcer, sepsis, septic shock, and tuberculosis.
Alternatively, it can be diagnosed that the possibility of illness is high in the future.

On the other hand, by hybridization,
When overexpression of RNA is detected, for example, allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, hay fever, etc.), inflammation (eg, arthritis, hepatitis, etc.), Immune disorders (eg, rheumatoid arthritis,
Systemic lupus erythematosus, Sjogren's disease, etc.), AIDS, glomerulonephritis, bronchial asthma, and the like, or can be diagnosed as having a high possibility of contracting in the future. When a DNA mutation is detected by the PCR-SSCP method, for example, cancer (eg, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer with p53 mutation, brain tumor, bladder cancer, cervix) Cancer, colorectal (colorectal / rectal), non-small cell lung cancer, small cell lung cancer, gastric cancer, etc., AIDS, infectious disease (eg,
Herpes virus infection, adenovirus infection, box virus infection, Helicobacter pylori infection,
Varicella-zoster virus infection, human papillomavirus infection, invasive staphylococcal infection, influenza infection, severe systemic fungal infection, etc.), acute bacterial meningitis, acute viral encephalitis, adult respiratory distress syndrome, bacteria Pneumonia, chronic lymphocytic leukemia, chronic myelogenous leukemia,
Insulin-dependent diabetes mellitus (type I), malignant melanoma, multiple myeloma, non-Hodgkin's lymphoma, peptic ulcer, sepsis, septic shock, tuberculosis, allergic immune disease (eg, atopic dermatitis, contact dermatitis) , Allergic rhinitis, hay fever, etc.), inflammation (eg, arthritis, hepatitis, etc.),
Autoimmune diseases (eg, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's disease, etc.), AIDS, glomerulonephritis,
It can be diagnosed as having a disease such as bronchial asthma or having a high possibility of suffering in the future.

(4) Method for Quantifying Ligands for the Receptor Protein of the Present Invention Since the receptor protein and the like of the present invention have a binding property to a ligand, the ligand concentration in a living body can be quantified with high sensitivity. . The quantification method of the present invention can be used, for example, by combining it with a competition method. That is, the ligand concentration in the test sample can be measured by bringing the test sample into contact with the receptor protein of the present invention. Specifically, for example, it can be used according to the method described below or the like or a method analogous thereto. Edited by Hiro Irie "Radio Immunoassay" (Kodansha, Showa 4)
(Published in 1997) edited by Hiroshi Irie, "Radio Immunoassay" (Kodansha, published in 1979)

(5) Screening method for compounds that alter the binding between the receptor protein of the present invention and a ligand The receptor protein or the like of the present invention is used, or an expression system for a recombinant receptor protein or the like is constructed and expressed. By using a system-based receptor binding assay system,
Compounds (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof that change the binding between the ligand and the receptor protein of the present invention can be efficiently screened. Such compounds include (a) cell stimulating activity via receptor (eg, NF-κB activation, TRAF molecule binding activity, arachidonic acid release, acetylcholine release, intracellular C
a ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress the activity of pH reduction, etc. ), A compound having no cell-stimulating activity (a so-called antagonist to the receptor protein of the present invention), (c) a ligand and the receptor protein of the present invention. Or (d) a compound that decreases the binding force between the ligand and the receptor protein of the present invention, and the like.
Screening is preferably performed by the above-described ligand determination method). That is, the present invention provides a comparison between (i) the case where the receptor protein or the like of the present invention is brought into contact with a ligand and (ii) the case where the receptor protein or the like of the present invention is brought into contact with a ligand or a test compound. And a method for screening a compound or a salt thereof that alters the binding property between the ligand and the receptor protein of the present invention. In the screening method of the present invention, in the cases (i) and (ii), for example, the amount of ligand binding to the receptor protein of the present invention and the cell stimulating activity on cells containing the receptor protein of the present invention are measured. And comparing them.

More specifically, the present invention relates to (i) the case where a labeled ligand is brought into contact with the receptor protein or the like of the present invention; and (ii) the case where the labeled ligand and a test compound are contacted with the receptor protein or the like of the present invention. A method for screening a compound or a salt thereof that changes the binding property between a ligand and a receptor protein of the present invention, comprising measuring and comparing the amount of binding of a labeled ligand to the receptor protein or the like when contacted with (I) when the labeled ligand is brought into contact with a cell containing the receptor protein or the like of the present invention or a membrane fraction thereof, and (ii) when the labeled ligand and test compound contain the receptor protein or the like of the present invention. The amount of labeled ligand bound to the cell or its membrane fraction when contacted with the cell or its membrane fraction is measured. And a method for screening for a compound or a salt thereof that alters the binding property between the ligand and the receptor protein of the present invention, wherein (i) using a labeled ligand to transform the transformant containing the DNA of the present invention. (Ii) a method in which a labeled ligand and a test compound are expressed on a cell membrane by culturing a transformant containing the DNA of the present invention by contacting with a receptor protein or the like expressed on the cell membrane by culturing; A compound which changes the binding property between the ligand and the receptor protein of the present invention, wherein the amount of the labeled ligand bound to the receptor protein or the like when contacted with the receptor protein or the like of the present invention is measured and compared. A screening method for the salt,

(I) a case where a substance which activates the receptor protein or the like of the present invention (eg, a ligand for the receptor protein or the like of the present invention) is brought into contact with a cell containing the receptor protein or the like of the present invention; A) a receptor-mediated cell stimulating activity (for example, activation of NF-κB, when a substance that activates the receptor protein or the like of the present invention and a test compound are brought into contact with cells containing the receptor protein or the like of the present invention; TRAF molecule binding activity, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activity Ligands characterized by measuring and comparing the activities of promoting or inhibiting the activation or reduction of pH, etc.) A method for screening a compound or a salt thereof that alters the binding of the receptor protein of the present invention to a compound of the present invention, and (i) a substance that activates the receptor protein of the present invention (eg, a ligand to the receptor protein of the present invention) Culturing a transformant containing the DNA of the present invention to bring it into contact with the receptor protein or the like of the present invention expressed on the cell membrane; and (ii) a substance or test compound that activates the receptor protein or the like of the present invention. Is contacted with the receptor protein or the like of the present invention expressed on the cell membrane by culturing a transformant containing the DNA of the present invention, resulting in receptor-mediated cell stimulating activity (for example, activation of NF-κB, TRAF molecule binding activity, arachidonic acid release, acetylcholine release, intracellular C
a ²⁺ release, intracellular cAMP production, intracellular cGMP production,
Inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.), and compare with a ligand characterized by measuring The present invention provides a method for screening a compound or a salt thereof that alters the binding to the receptor protein of the present invention.

In particular, prior to obtaining the human-derived receptor protein or the like of the present invention, when screening for receptor agonists or antagonists, first, a candidate compound is identified by using a cell or tissue containing the receptor protein such as a rat or its cell membrane fraction. After that (primary screening), a test (secondary screening) for confirming whether or not the candidate compound actually inhibits the binding between a human receptor protein and a ligand was required. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins will be mixed, and it has been difficult to actually screen for an agonist or antagonist for the target receptor protein. However, for example, by using the human-derived receptor protein of the present invention, primary screening is not required, and a compound that inhibits binding between a ligand and a receptor protein can be efficiently screened. Furthermore, it can be easily evaluated whether the screened compound is an agonist or an antagonist.

The specific description of the screening method of the present invention is shown below. First, the receptor protein preparation of the present invention used in the screening method of the present invention may be any of the above-described receptor proteins of the present invention and the like or cells containing the same. Cell membrane fractions of mammalian organs containing proteins and the like are preferred. However, since it is extremely difficult to obtain human-derived organs, human-derived receptor proteins and the like that are expressed in large amounts using recombinants are suitable for screening. To produce the receptor protein or the like of the present invention, the above-described method is used,
It is preferred to carry out by expressing the DNA of the present invention in mammalian cells or insect cells. CDNA is used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. In order to introduce a DNA fragment encoding the receptor protein or the like of the present invention into a host animal cell and express them efficiently, the DNA fragment is converted into a nuclear polyhedrosis virus (nuclear polyhedrosis virus belonging to baculovirus using an insect as a host). virus; NPV)
Polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, C
It is preferable to incorporate into downstream such as MV (cytomegalovirus) promoter and SRα promoter. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P .; Et al., The Journal of Biological Chemistry (J. B
iol. Chem.), 267, 19555-19559, 1992]. Therefore, the receptor protein sample of the present invention used in the screening method of the present invention may be a receptor protein or the like purified according to a method known per se, or may be a cell containing the receptor protein or the like. Alternatively, a membrane fraction of cells containing the receptor protein or the like may be used.

When cells containing the receptor protein or the like of the present invention are used in the screening method of the present invention, the cells may be immobilized with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se. Cells containing the receptor protein or the like of the present invention refer to host cells that express the receptor protein or the like.
Bacillus subtilis, yeast, insect cells, animal cells and the like are preferred. The cell membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. Cell crushing methods include crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or Polytron (Kinematica), crushing with ultrasonic waves, and blowing cells from a thin nozzle while applying pressure with a French press. Crushing, etc. For fractionation of cell membranes, fractionation by centrifugal force, such as fractionation centrifugation and density gradient centrifugation, is mainly used. For example, the cell lysate is prepared at a low speed (about 500 rpm to about 3000 rpm) for a short time (usually about 1 minute to about 10 minutes).
Centrifuge and centrifuge the supernatant at higher speed (about 15,000 rpm to about 3
(0000 rpm) for about 30 minutes to about 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the receptor protein in the cells containing the receptor protein and the like and the membrane fraction thereof is as follows:
1 is preferably from about 10 ³ to about 10 ⁸ molecules per cell, in particular, it is suitably from about ¹⁰⁵ to about ¹⁰⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .

In order to carry out the above (1) to screen for a compound that alters the binding property between the ligand and the receptor protein of the present invention, for example, an appropriate receptor protein fraction and a labeled ligand are required. As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto is desirable. Here, “equivalent activity” refers to equivalent ligand binding activity, signal transduction action, and the like. As the ligand, a ligand obtained by using the above-described method for determining a ligand or an analog compound thereof is used. As a labeled ligand, for example, [
^The above-mentioned ligands labeled with radioactive substances such as [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], and [ ³⁵ S] are used. Specifically, to screen for a compound that alters the binding between the ligand and the receptor protein of the present invention, first, a cell containing the receptor protein of the present invention or a cell membrane fraction thereof is placed in a buffer suitable for screening. A receptor protein standard is prepared by suspending. The buffer contains about pH 4 to 10 (preferably about pH 6 to 10).
Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as the phosphate buffer and Tris-HCl buffer of 8). Further, in order to reduce non-specific binding, CHAPS, Tween-80 ^™
Surfactants such as (Kao-Atlas), digitonin and deoxycholate can also be added to the buffer.
In addition, PMSF, leupeptin, E-64
(Manufactured by Peptide Research Laboratories) and protease inhibitors such as pepstatin can also be added. About 0.01ml-1
A fixed amount (about 5000 cp) is added to 0 ml of the receptor solution.
(m to about 500,000 cpm) of the labeled ligand, and at the same time about 10 ^-4 M to about 10 ^-1 M of the test compound. A reaction tube containing a large excess of unlabeled ligand is also prepared to determine the amount of non-specific binding (NSB).
The reaction is carried out at about 0 to about 50C, preferably at about 4 to about 37C,
It is carried out for about 20 minutes to about 24 hours, preferably for about 30 minutes to about 3 hours. After the reaction, the mixture is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. When the count (B ₀ -NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance is defined as 100%, the specific binding amount (B-NSB) is, for example, , About 80% or less, preferably 70% or less, more preferably about 50% or less, can be selected as candidate substances having competitive inhibitory activity.

In order to carry out the above-mentioned methods (1) and (2) for screening a compound that alters the binding property between a ligand and the receptor protein of the present invention, for example, a cell stimulating activity via receptor protein (for example, activation of NF-κB) , TRAF molecule binding activity, arachidonic acid release, acetylcholine release, intracellular Ca release, intracellular cAMP production,
A known method or a commercially available method for measuring intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity for promoting or suppressing pH reduction, etc.) It can be measured using a kit for use. Specifically, first, cells containing the receptor protein of the present invention are cultured in a multiwell plate or the like. When conducting screening,
Replace in advance with a fresh medium or an appropriate buffer that is not toxic to cells, add the test compound, etc., incubate for a certain period of time, then extract the cells or collect the supernatant, and use the resulting product for each method. Quantify according to When the production of a substance (for example, arachidonic acid or the like) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor against the degrading enzyme. In order to perform screening by measuring cell stimulating activity, cells expressing an appropriate receptor protein are required. As the cells expressing the receptor protein of the present invention, a cell line having a natural type of the receptor protein of the present invention, a cell line expressing the above-mentioned recombinant receptor protein, and the like are preferable. Test compounds used in the screening method of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. May be a novel compound or a known compound.

A screening kit for a compound or a salt thereof that alters the binding property between a ligand and the receptor protein of the present invention can be used for a cell containing the receptor protein of the present invention such as the receptor protein of the present invention or a cell membrane fraction thereof. It contains. Examples of the screening kit of the present invention include the following. 1. Screening reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. CHO cells expressing the receptor protein of the present invention
Passage into a 12-well plate at 5 × 10 ⁵ / well, 37 ° C.
Cultured at 5% CO ₂ for 2 days. Labeled ligands Commercially available ligands labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc., stored at 4 ° C. or -20 ° C. Dilute the solution to 1 μM. Ligand standard solution 0.1% bovine serum albumin (Sigma)
And dissolved at 1 mM in PBS, and stored at -20 ° C.

2. Assay Method CHO cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate are washed twice with 1 ml of assay buffer, and then 490 μl of assay buffer is added to each well. After adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M, 5 μl of a labeled ligand is added, and the mixture is reacted at room temperature for 1 hour. To determine the amount of non-specific binding, 5 μl of 10 ⁻³ M ligand is added instead of the test compound. Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled ligand bound to the cells was 0.2N NaOH
1% SDS, 4 ml of liquid scintillator A
(Made by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Radioactivity was measured using Percent Maximum Binding
(PMB) is obtained by the following equation (Equation 1).

[0078]

(Equation 1) PMB: Percent Maximum Binding B: Value at the time of adding the sample NSB: Non-specific Binding (non-specific binding amount) B ₀ : Maximum binding amount

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between the ligand and the receptor protein of the present invention.
(A) Cell stimulating activity (eg, NF
-ΚB activation, TRAF molecule binding activity, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation , C
-A compound having an activity of promoting or suppressing the activation of fos, a decrease in pH, etc. (a so-called agonist for the receptor protein of the present invention); (b) a compound not having the cell stimulating activity (a so-called present (C) a compound that enhances the binding force between the ligand and the receptor protein of the present invention, or (d) a compound that decreases the binding force between the ligand and the receptor protein of the present invention. The compound is obtained from the test compound described above, and includes peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like.These compounds may be novel compounds or may be known compounds. It may be a compound. Since the agonist for the receptor protein of the present invention has the same activity as the physiological activity of the ligand for the receptor protein of the present invention, it is useful as a safe and low-toxic drug according to the ligand activity. The agonist may be a cancer (eg, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer with p53 mutation, brain tumor, bladder cancer, cervical cancer, colorectal cancer (colon / rectal cancer), non-small cell lung cancer, Small cell lung cancer, gastric cancer, etc., AIDS, infectious diseases (eg, herpes virus infection, adenovirus infection, box virus infection, Helicobacter pylori infection, varicella-zoster virus infection, human papillomavirus infection, invasion Staphylococcal infection, influenza infection, severe systemic fungal infection, etc.), acute bacterial meningitis, acute viral encephalitis, adult respiratory distress syndrome, bacterial pneumonia, chronic lymphocytic leukemia, chronic myelogenous leukemia, insulin dependence Diabetes mellitus (type I), malignant melanoma, multiple myeloma, non-Hodgkin's lymphoma, peptic ulcer, sepsis, septic shock, tuberculosis It is useful as a medicine such as any therapeutic or prophylactic agent.

The antagonist to the receptor protein or the like of the present invention can suppress the physiological activity of the ligand for the receptor protein of the present invention, and thus is useful as a safe and low-toxic drug for suppressing the ligand activity. Such antagonists include, for example, allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, hay fever, etc.), inflammation (eg, arthritis, hepatitis, etc.), autoimmune diseases (eg, rheumatoid arthritis) , Lupus erythematosus, Sjogren's disease, etc.), AIDS, glomerulonephritis, bronchial asthma and the like. The compound that enhances the binding force between the ligand and the receptor protein of the present invention is useful as a safe and low-toxic drug for enhancing the physiological activity of the ligand for the receptor protein of the present invention. The compound can be used, for example, in cancer (eg, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer with a p53 mutation, brain tumor, bladder cancer, cervical cancer, colorectal cancer (colon / rectal cancer), non-small cell Lung cancer, small cell lung cancer, gastric cancer, etc., AIDS, infectious diseases (eg, herpes virus infection, adenovirus infection, box virus infection, Helicobacter pylori infection, varicella-zoster virus infection, human papillomavirus infection , Invasive staphylococcal infections, influenza infections, severe systemic fungal infections), acute bacterial meningitis, acute viral encephalitis, adult respiratory distress syndrome, bacterial pneumonia, chronic lymphocytic leukemia, chronic myeloid leukemia, Insulin-dependent diabetes mellitus (type I), malignant melanoma, multiple myeloma, non-Hodgkin's lymphoma, peptic ulcer, sepsis, septic shock, It is useful as a medicament such as an agent for treating or preventing nuclei. The compound that decreases the binding force between the ligand and the receptor protein of the present invention is useful as a safe and low-toxic drug for reducing the physiological activity of the ligand for the receptor protein of the present invention. The compound is, for example,
Allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, hay fever, etc.), inflammation (eg,
Arthritis, hepatitis, etc.), autoimmune diseases (eg, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's disease, etc.),
It is useful as a medicament such as an agent for treating or preventing AIDS, glomerulonephritis, bronchial asthma and the like.

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned medicine, it can be carried out according to a conventional method. For example, the DNA of the present invention described above
And tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions and the like. Since the thus obtained preparation is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). .
The dose of the compound or a salt thereof varies depending on the target disease, the subject to be administered, the administration route, and the like. (As 60 kg), the antagonist is added at about 0.5 mg / day.
1 mg to 100 mg, preferably about 1.0 to 50 mg,
More preferably, about 1.0 to 20 mg is administered. When administered parenterally, a single dose of the compound should be administered to the subject,
For example, when an agonist for the receptor protein or the like of the present invention is usually administered to an adult (as 60 kg) in the form of an injection for the purpose of treating cancer, the amount of the agonist is about 0. 01-30mg
It is convenient to administer intravenously, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg. In the case of other animals, the dose can be administered in terms of 60 kg. Also, for example,
When orally administering an antagonist to the receptor protein or the like of the present invention for the purpose of treating allergic immune diseases,
In general, for an adult (as 60 kg), the antagonist is administered in a daily dose of about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 50 mg.
Administer 20 mg. When administered parenterally, a single dose of the compound may vary depending on the administration subject, target disease, and the like. When administered to a normal adult (as 60 kg) in the form, the antagonist is administered in an amount of about 0.01-30 m / day.
It is convenient to administer about g, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(6) Quantification of the Receptor Protein of the Present Invention, Its Partial Peptide, or a Salt Thereof Since the antibody of the present invention can specifically recognize the receptor protein of the present invention, etc. It can be used for quantification of the receptor protein and the like of the invention, particularly for quantification by sandwich immunoassay. That is, the present invention, for example,
(I) reacting the antibody of the present invention with a test solution and a labeled receptor protein or the like competitively, and measuring the ratio of the labeled receptor protein or the like bound to the antibody; A method for quantifying the receptor protein of the present invention,
(Ii) After simultaneously or continuously reacting the test solution with the antibody of the present invention insolubilized on the carrier and another labeled antibody of the present invention, the activity of the labeling agent on the insolubilized carrier is measured. A method for quantifying the receptor protein of the present invention in a test solution is provided. In the above (ii), it is preferable that one antibody is an antibody that recognizes the N-terminal of the receptor protein of the present invention and the other antibody is an antibody that recognizes the C-terminal of the receptor protein of the present invention. .

In the present invention, the receptor protein and the like of the present invention can be measured using a monoclonal antibody against the receptor protein and the like of the present invention (hereinafter abbreviated as the monoclonal antibody of the present invention), and can be detected by tissue staining or the like. Can also be performed. For these purposes, the antibody molecule itself may be used, and the F (a
b ′) ₂ , Fab ′, or Fab fraction may be used. The assay method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of an antigen (eg, the amount of the receptor protein) in a test solution. Any measurement method may be used as long as the amount is detected by chemical or physical means and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, a competitive method, an immunometric method and a sandwich method are preferably used, but in terms of sensitivity and specificity,
It is particularly preferable to use a sandwich method described later. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [
¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing proteins or enzymes may be used. As the carrier, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used. In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the receptor protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. You may. In the method for measuring a receptor protein or the like by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the receptor protein or the like. That is, when the antibody used in the primary reaction and the secondary reaction recognizes the C-terminal of the receptor protein, for example, the antibody used in the primary reaction is preferably the C-terminal. For example, an antibody recognizing the N-terminal other than the N-terminal is used.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephelometry. In the competition method, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of any of B and F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid phase antibody is used as the first antibody, or
A solid-phase method using a soluble first antibody and a solid-phased antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then a solid phase and a liquid phase are separated. And an excess amount of the labeled antibody, and then immobilized antigen is added to bind unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. next,
The amount of label in either phase is measured to determine the amount of antigen in the test solution. In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying each of these immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the receptor protein of the present invention or a salt thereof by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For more information on these common technical means,
Review articles, books, etc. can be referred to [for example, Hiroe Irie, edited by "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Continued Radio Immunoassay" (Kodansha, published in 1974), Eiji Ishikawa et al. "Enzyme immunoassay" (ed. Medical Shoin, published in 1978), Eiji Ishikawa et al., "Enzyme immunoassay" (second edition) (ed.), Eiji Ishikawa et. Law (3rd edition) (Medical Shoin, published in 1987), "Methods in ENZYMOLOGY" Vol. 70 (Immunoch)
emical Techniques (Part A)), ibid.Vol. 73 (Immunoch
emical Techniques (Part B)), ibid.Vol. 74 (Immunoch
emical Techniques (Part C)), ibid.Vol. 84 (Immunoch
emical Techniques (Part D: Selected Immunoassays)),
Ibid. Vol. 92 (Immunochemical Techniques (Part E: Mon
oclonal Antibodies and General Immunoassay Method
s)), ibid.Vol. 121 (Immunochemical Techniques (Part
I: Hybridoma Technology and Monoclonal Antibodie
s)) (see Academic Press). As described above, the receptor protein and the like of the present invention can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, by quantifying the receptor protein or the like of the present invention using the antibody of the present invention, various diseases involving the receptor protein of the present invention can be diagnosed.

Specifically, when a decrease in the concentration of the receptor protein or the like of the present invention is detected, for example, cancer (eg, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer with p53 mutation, brain tumor) , Bladder cancer, cervical cancer, colon cancer (colon /
Rectal cancer), non-small cell lung cancer, small cell lung cancer, gastric cancer, etc.), AIDS, infections (eg, herpes virus infection, adenovirus infection, box virus infection, Helicobacter pylori infection, varicella-zoster virus) Infectious disease, human papillomavirus infection, invasive staphylococcal infection, influenza infection, severe systemic fungal infection, etc.), acute bacterial meningitis, acute viral encephalitis, adult respiratory distress syndrome, bacterial pneumonia, chronic lymphocytic Leukemia, chronic myeloid leukemia, insulin-dependent diabetes (I
Type), malignant melanoma, multiple myeloma, non-Hodgkin's lymphoma, peptic ulcer, sepsis, septic shock, tuberculosis, or other diseases, or can be diagnosed as having a high possibility of contracting in the future. On the other hand, when an increase in the concentration of the receptor protein or the like of the present invention is detected, for example, allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, pollinosis, etc.), inflammation (eg, , Arthritis, hepatitis, etc.), autoimmune diseases (eg, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's disease, etc.), AIDS, glomerulonephritis, bronchial asthma, etc., or diagnosed as likely to be affected in the future be able to. Thus, the antibodies of the present invention are useful as diagnostic agents for the above diseases. Further, the antibody of the present invention can be used for detecting the receptor protein of the present invention or the like present in a subject such as a body fluid or a tissue. Further, preparation of an antibody column used to purify the receptor protein of the present invention, detection of the receptor protein of the present invention in each fraction during purification,
It can be used for analysis of the behavior of the receptor protein of the present invention in test cells.

(7) Pharmaceutical Containing Soluble Partial Peptide of the Present Invention The soluble partial peptide of the present invention binds to a ligand and can inhibit the binding of the ligand to the receptor protein of the present invention. Physiological activity can be suppressed. Therefore, the soluble partial peptide of the present invention can be used for, for example, allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, hay fever, etc.), inflammation (eg, arthritis, hepatitis, etc.), autoimmunity It can be used as a medicament such as an agent for treating or preventing various diseases such as diseases (eg, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's disease), AIDS, glomerulonephritis, bronchial asthma and the like. When the soluble partial peptide of the present invention is used as the above medicine, it can be produced in the same manner as the above-mentioned medicine containing the DNA of the present invention, and can be administered to humans or mammals.

The preparation thus obtained is safe and has low toxicity, and is thus administered to, for example, humans and mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). be able to. The dosage of the soluble partial peptide of the present invention varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the soluble partial peptide of the present invention is orally administered for the purpose of treating allergic immune diseases, it is generally used. In adults (as 60 kg), about 0.1 mg of the soluble partial peptide per day is used.
Administer 100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the soluble partial peptide varies depending on the administration subject, target disease, and the like. For example, the soluble partial peptide of the present invention is administered in the form of an injection for the purpose of treating an autoimmune disease. When usually administered to an adult (as 60 kg), the soluble partial peptide is administered in an amount of about 0.01 to 3 per day.
It is convenient to administer about 0 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. 60 kg for other animals
Per-converted amounts can be administered.

(8) Pharmaceutical Containing Antibody of the Present Invention The antibody of the present invention having an action of eliciting receptor activity of the receptor protein of the present invention (agonist-like activity) is
For example, cancer (eg, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer with p53 mutation, brain tumor, bladder cancer, cervical cancer, colorectal cancer (colon / rectal cancer), non-small cell lung cancer, small cell Lung cancer, gastric cancer, etc., AIDS, infectious diseases (eg, herpes virus infection, adenovirus infection, box virus infection, Helicobacter pylori infection, varicella-zoster virus infection, human papillomavirus infection, invasive grape Staphylococcal infections, influenza infections, severe systemic fungal infections, etc.), acute bacterial meningitis, acute viral encephalitis, adult respiratory distress syndrome, bacterial pneumonia, chronic lymphocytic leukemia, chronic myelogenous leukemia, insulin-dependent diabetes (Type I), malignant melanoma, multiple myeloma, non-Hodgkin's lymphoma, peptic ulcer, sepsis, septic shock, tuberculosis and other diseases It is useful as a medicament such as a therapeutic / prophylactic agent. On the other hand, the antibody of the present invention having an action of neutralizing receptor activity such as the receptor protein of the present invention (antagonist-like activity) can be used, for example, for allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic Rhinitis, hay fever, etc.), inflammation (eg, arthritis, hepatitis, etc.),
Autoimmune diseases (eg, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's disease, etc.), AIDS, glomerulonephritis,
It can be used as a medicament such as an agent for treating or preventing various diseases such as bronchial asthma.

Treatment of the above diseases containing the antibody of the present invention
The prophylactic agent can be orally or non-orally administered to humans or mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) as a liquid as it is or as a pharmaceutical composition in an appropriate dosage form. It can be administered orally.
The dose varies depending on the administration subject, target disease, symptoms, administration route and the like.For example, when used for treatment or prevention of cancer in adults, an antibody that elicits the receptor activity of the receptor protein of the present invention As a single dose, usually about 0.01 to 20 mg / kg body weight, preferably 0.1 to 20 mg / kg body weight.
About 10 mg / kg body weight, more preferably 0.1 to 5
It is convenient to administer about mg / kg body weight by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. For example, when used for the treatment or prevention of an allergic immune disease in an adult, the amount of the antibody that neutralizes the receptor activity of the receptor protein of the present invention is usually 0.01 to 20 mg / kg as a single dose. A body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, is intravenously injected about 1 to 5 times a day, preferably about 1 to 3 times a day. It is convenient to administer. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The antibody of the present invention having agonist-like activity or antagonist activity can be administered by itself or as a suitable pharmaceutical composition. The pharmaceutical composition used for the above administration contains the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration. That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). Including)
Syrups, emulsions, suspensions and the like can be mentioned. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate and the like.

[0092] Compositions for parenteral administration include, for example, injections, suppositories, and the like. In the dosage form. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and a suitable solubilizing agent,
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80,
HCO-50 (polyoxyethylene (50mol) adduct of
hydrogenated castor oil)].
As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used in combination as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. Suppositories to be used for rectal administration are prepared by mixing the above antibody or a salt thereof with a conventional suppository base. The above-mentioned oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form so as to be compatible with the dosage of the active ingredient.
Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like, and usually 5 to 500 mg, especially 5 to 100 mg for each dosage unit, 10-2 for other dosage forms
Preferably, it contains 50 mg of the above antibody.
Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.

(9) Pharmaceuticals Containing Antisense DNA Antisense DNA that can complementarily bind to the DNA of the present invention and suppress the expression of the DNA is a receptor protein or the like of the present invention or DNA in vivo. (MRN
Since the function of A) can be suppressed, for example, allergic immune diseases (eg, atopic dermatitis, contact dermatitis, allergic rhinitis, hay fever, etc.), inflammation (eg, arthritis, hepatitis, etc.), Autoimmune diseases (eg, rheumatoid arthritis,
Treatment of various diseases such as systemic lupus erythematosus, Sjogren's disease), AIDS, glomerulonephritis, bronchial asthma
It can be used as a medicine such as a prophylactic agent. When the above-mentioned antisense DNA is used as the above-mentioned medicine, it can be produced in the same manner as the above-mentioned medicine containing the DNA of the present invention, and can be administered to humans or mammals.

Since the preparation thus obtained is safe and has low toxicity, it is administered, for example, to humans and mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). be able to. The dose of the DNA of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like. ), The DNA is used in an amount of about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg per day.
mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose of the DNA varies depending on the administration subject, target disease, and the like. For example, for the purpose of treating an autoimmune disease, the DNA of the present invention is usually administered in the form of an injection to an adult ( (As 60 kg), about 0.01 to 30 mg of the DNA per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day.
Conveniently, about mg is administered by intravenous injection.
In the case of other animals, the dose can be administered in terms of 60 kg.

(10) Preparation of Non-Human Animal Having DNA of the Present Invention Using the DNA of the present invention, a transgenic non-human animal expressing the receptor protein or the like of the present invention can be prepared. Non-human animals include mammals (eg,
Rats, mice, rabbits, sheep, pigs, cows, cats,
Dogs, monkeys, etc.) (hereinafter abbreviated as animals), and mouse, rat, rabbit and the like are particularly preferable. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells. For example, the DN of the present invention derived from rabbits
In the case of transferring A, a gene construct linked to downstream of various promoters which are highly homologous thereto and which can express the DNA of the present invention in animal cells can be microinjected into, for example, rabbit fertilized eggs. By doing so, a DNA-transferred animal that highly produces the receptor protein of the present invention can be produced. As this promoter, for example, a ubiquitous expression promoter such as a virus-derived promoter and metallothionein can be used.

Transfer of the DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the subject animal. The presence of the receptor protein or the like of the present invention in the germ cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the receptor protein or the like of the present invention in all of its germ cells and somatic cells. The progeny of this type of animal that has inherited the gene have the receptor protein of the present invention in all of its germinal and somatic cells. After confirming that the DNA-transferred animal of the present invention stably retains the gene by breeding, it can be subcultured in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, a homozygous animal having the transgene on both homologous chromosomes is obtained, and by crossing the male and female animals, all progeny have the DNA. Breeding can be subcultured. Since the animal into which the DNA of the present invention has been transferred has high expression of the receptor protein of the present invention, it is useful as an animal for screening an agonist or antagonist for the receptor protein of the present invention. The DNA transgenic animal of the present invention can also be used as a cell source for tissue culture. For example, the receptor protein of the present invention can be analyzed by directly analyzing DNA or RNA in the tissue of the DNA-transferred mouse of the present invention or by analyzing the tissue in which the receptor protein of the present invention expressed by a gene is present. can do. Cells of a tissue having the receptor protein or the like of the present invention are cultured by standard tissue culture techniques, and these are used, for example, for brain and peripheral tissues (eg, skin, etc.).
The function of cells from tissues that are generally difficult to culture, such as their origin, can be studied. In addition, by using the cells, for example, a drug that enhances the function of various tissues can be selected. In addition, if there is a high expression cell line, the receptor protein of the present invention can be isolated and purified therefrom.

In the present specification and drawings, when bases and amino acids are indicated by abbreviations, IUPAC-IUB
Abbreviations based on Commision on Biochemical Nomenclature or common abbreviations in the field,
An example is given below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly (or G): glycine Ala (or A): alanine Val (or V): valine Leu (or L): leucine Ile (or I): isoleucine Ser (or S): serine Thr (or T) : Threonine Cys (or C): Cysteine Met (or M): Methionine Glu (or E): Glutamate Asp (or D): Aspartic acid Lys (or K): Lysine Arg (or R): Arginine His (or H) : Histidine Phe (or F): Phenylalanine Tyr (or Y): Tyrosine Trp (or W): Tryptophan Pro (or P): Proline Asn (or N): Asparagine Gln (or Q): Glutamine pGlu: Pyroglutamic acid M e: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine-4 (R) -carboxamide group

Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols. Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenyl Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBT: 3,4-dihydro-3-hydroxy -4-oxo-1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

[0100] The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of the human dendritic cell-derived receptor protein of the present invention. [SEQ ID NO: 2] This shows the amino acid sequence of the soluble partial peptide of the present invention. [SEQ ID NO: 3] This shows the amino acid sequence of the signal peptide of the present invention. [SEQ ID NO: 4] This shows the base sequence of the DNA encoding the human dendritic cell-derived receptor protein of the present invention having the amino acid sequence represented by SEQ ID NO: 1. [SEQ ID NO: 5] D encoding the soluble partial peptide of the present invention having the amino acid sequence represented by SEQ ID NO: 2
Shows the base sequence of NA. [SEQ ID NO: 6] DN encoding the signal peptide of the present invention having the amino acid sequence represented by SEQ ID NO: 3
1 shows the nucleotide sequence of A.

[SEQ ID NO: 7] In Example 1, c coding for the human dendritic cell-derived receptor protein of the present invention
The base sequence of a synthetic primer used for DNA amplification is shown. [SEQ ID NO: 8] This shows the base sequence of the synthetic primer used in Example 1 for amplification of cDNA encoding the human dendritic cell-derived receptor protein of the present invention. [SEQ ID NO: 9] This shows the base sequence of the primer used in Example 3 and Example 4 for amplification of cDNA encoding the human dendritic cell-derived receptor protein of the present invention. [SEQ ID NO: 10] This shows the base sequence of the primer used in Example 3 for amplification of cDNA encoding the human dendritic cell-derived receptor protein of the present invention. [SEQ ID NO: 11] This shows the base sequence of primer used in Example 3 for amplification of cDNA encoding the human dendritic cell-derived receptor protein of the present invention. [SEQ ID NO: 12] This shows the base sequence of the primer used in Example 4 for amplification of cDNA encoding the human dendritic cell-derived receptor protein of the present invention. [SEQ ID NO: 13] This shows the amino acid sequence of peptide synthesized in Example 2 for use as an antigen peptide. [SEQ ID NO: 14] This shows the amino acid sequence of peptide synthesized in Example 2 to be used as an antigen peptide. [SEQ ID NO: 15] This shows the base sequence of the primer used in Example 5 for amplification of cDNA encoding the protein of the present invention. [SEQ ID NO: 16] This shows the base sequence of the primer used in Example 5 for amplification of cDNA encoding the protein of the present invention. [SEQ ID NO: 17] In Example 5, genomic PC
This shows the base sequence of the primer used for R. [SEQ ID NO: 18] In Example 5, genomic PC
This shows the base sequence of the primer used for R.

The transformant Escherichia coli DH10B / pT obtained in Example 1 described later.
B1984 has been deposited with the National Institute of Advanced Industrial Science and Technology (NIBH) as a deposit number FERM BP-6102 from September 16, 1997 as a deposit number FERM BP-6102 from September 17, 1997. IFO) and the deposit number IFO
No. 16113 has been deposited.

[0103]

The present invention will be described in more detail with reference to the following Examples, which do not limit the scope of the present invention. In addition, the genetic manipulation method using Escherichia coli followed the method described in Molecular cloning.

[0104]

Example 1 Cloning of cDNA Encoding Receptor Protein Derived from Human Dendritic Cells and Determination of Base Sequence DNA encoding the receptor protein of the present invention was obtained by the following PCR method. First, a cryopreserved human dendritic cell cDNA library (Human Genome Sciences, Lib.
rary ID H0521) E. coli DH10B at 100 μg / ml
Includes ampicillin sodium salt (Wako Pure Chemical Industries, Ltd.)
Terrific Broth (12 g / l bacto-tryptone (Difco), 24 g / l bacto-yeast extract (Difco),
2.3 g / l potassium dihydrogenphosphate, 12.5
g / l potassium monohydrogen phosphate) at 30 ℃
After culturing with shaking for 16 hours, the plasmid DNA was amplified.
Next, the plasmid DNA was purified using a Qiagen plasmid kit (Qiagen) to obtain a DNA solution. The D
Two types of oligo DNA synthesized with 0.63 μg NA and a DNA synthesizer (Oligo 1000M, Beckman): 5′-GCCACGCCTGTCCCGCCGCATCAT-
3 ′ (SEQ ID NO: 7) 5′-CAGAATATGAAAGTCCCACCACAC
AT-3 ′ (SEQ ID NO: 8) 3.2 μmol each, 8 μl of dNTP Mixture, 10 × contained in Takara LA PCR ^™ Kit Ver. 2 (Takara Shuzo Co., Ltd.)
LA PCR Buffer II (Mg ²⁺ plus) 5μl, and Takara
LA The mixture 50μl containing Taq ^TM 5.0Unit, using a thermal cycler GeneAmp ^R PCR System 2400 (Perkin Elmer), 94 ° C. · 1 minute, followed by 98 ° C. · 20 seconds → 68 ° C. · 15 minutes cycle Was repeated 30 times to react. When the reaction-completed solution was subjected to electrophoresis using 0.8% agarose, a single band amplified by PCR was confirmed at a position of 2.8 kb.

The DNA fragment was recovered using a Chiaquick Gel Extraction Kit (Qiagen), and subcloned into the T-cloning site of pT7Blue T-vector (Novagen) to determine the nucleotide sequence. The obtained plasmid DNA (pTB1984)
ABI PRISM ^™ , using two types of primer DNA (PRM-007, PRM-008) and oligo DNA synthesized with a DNA synthesizer (Oligo1000M, Beckman) as primer DNA Dye Termi
After performing in GeneAmp ^R PCR System 2400 according nator Cycle Sequencing FS Ready Reaction Kit conditions Appendix sequencing reaction using (Perkin Elmer), a sample was analyzed by DNA sequencer 373A (Perkin-Elmer). The obtained nucleotide sequence was analyzed using a gene analysis software Laser Gene (Lasergene, DNASTAR). As a result, at the T-cloning site of the clone, an open reading frame consisting of the 1848 nucleotide sequence represented by SEQ ID NO: 2 encoding a novel receptor protein consisting of 616 amino acids represented by SEQ ID NO: 1 (Open
DNA of 2,781 base sequence including reading frame)
Fragments were included [Fig. 1] and [Fig. 2]. In the present receptor protein, the region from the hydrophobicity plot analysis to the 29th (Gln) is a secretion signal, and the 211th (Le)
u) to 234 (Cys) hydrophobic region were predicted to be transmembrane regions, so-called type 1 membrane proteins (FIG. 3). This receptor protein had the highest homology with known proteins in the human CD40 precursor protein, but the homology was 29% at the amino acid level. A cysteine-rich repeat that is characteristic of the TNF receptor family of proteins is present in a region predicted to be the extracellular region from No. 30 (Ile) to No. 210 (Tyr) of the present protein. TNF is a new protein
It was revealed that the protein was a receptor family protein. DNA encoding the receptor protein of the present invention derived from human dendritic cells (hereinafter sometimes abbreviated as DC2)
Plasmid pTB1984 carrying Escherichia coli (Escher
(Escherichia coli) DH10B to obtain a transformant: Escherichia coli DH10B / pTB1984.

Example 2 Preparation of anti-DC2 antiserum First, SEQ ID NO: 1
185 (His) to 206 (G
lu) was added to the carboxyl group of cysteine by adding the amino acid sequence up to SEQ ID NO: 13 (CHGTEKSDVVC
SSSPARKPPNE) (A-789), and 64
SEQ ID NO: 14 wherein the amino acid sequence from position (Ser) to position 85 (Lys) is added to the carboxyl group of cysteine
(CSDSVCLPCGPDEYLDSWNEEDK)
The peptide represented by (A-782) was chemically synthesized by a method known per se. This peptide was converted to KLH (ke
After binding to yhole limpet hemocyanin), rabbits (SPF, Japanese White Rabbi) were added together with FCA (complete Freund's adjuvant) in an amount equivalent to 1 mg of the antigen peptide.
t) Two birds were injected subcutaneously for primary immunization. Thereafter, immunization was repeated 6 times every other week, and after 3 months, whole blood was collected, and a serum fraction was obtained by a known method to obtain anti-DC2 antiserum. The antibody titer was confirmed by dot blot analysis of the synthetic peptide used as the immunogen (the secondary antibody was an alkaline phosphatase-labeled anti-rabbit IgG antibody) and ELISA.
The method A was used (the secondary antibody was a peroxidase-labeled anti-rabbit IgG antibody). Table 1 shows the results of dot blot analysis performed using each antiserum (1000-fold diluted solution). Both samples were able to specifically detect up to 5 ng of each synthetic peptide of the immunogen.

[Table 1]

[0106]

Example 3 Expression of the Recombinant Receptor Protein of the Present Invention and Soluble Recombinant Receptor Protein in COS Cells The expression of the receptor protein encoded by the DNA obtained in Example 1 in animal cells was determined by using an expression vector. Plasmid p
cDNA 3.1 (-) / Myc-His B (Invitrogen) is used. This vector has a cytomegalovirus (CMV) enhancer promoter, a multiple cloning site downstream thereof, and a base sequence encoding a histidine tag downstream thereof.
It can be produced as a tagged fusion protein. By using this plasmid, a high expression level of the target receptor protein in COS cells is expected. Plasmid pTB1984 carrying the cDNA encoding the receptor protein of the present invention was transformed into E. coli (Escheric
hia coli) Transformant obtained by introduction into DH10B:
A plasmid was recovered from Escherichia coli DH10B / pTB1984 by a known method, and the following PCR was carried out using this plasmid as a template to obtain a full-length receptor protein of the present invention and a DNA fragment encoding a soluble receptor protein. Let go.

First, a DNA fragment encoding the full-length receptor protein was prepared using plasmid pTB1984 as a template.
The synthetic oligonucleotide of the present invention, ie, the synthetic oligonucleotide corresponding to the amino-terminal region of the open reading frame having a sequence cleavable with EcoRI upstream of the start codon: 5′-GAATTCATGGCCCCGCCGCGCC-
3 ′ (SEQ ID NO: 9) and a synthetic oligonucleotide complementary to the carboxyl-terminal region of the open reading frame having a sequence that can be cleaved with XbaI in the sequence: 5′-TTCTAGAGCCTTGGCCCCGCC-
3A (SEQ ID NO: 10) as a primer and thermal cycler GeneAmp ^R PCR Sy using Takara LAPCR ^™ Kit Ver.2 (Takara Shuzo Co., Ltd.)
94 ° C, 1 with stem 2400 (PerkinElmer)
Amplification is carried out by repeating a PCR reaction 30 times for 30 minutes at 98 ° C. for 20 seconds → 15 minutes at 68 ° C. The amino and carboxyl termini of the obtained DNA fragment are restricted with restriction enzymes Ec
After digestion using oRI and XbaI, electrophoresis was performed using a 1% agarose gel to confirm the target DNA fragment, and then a Qiaquick Gel Extraction Kit (Qiagen)
Collect and purify in. The DNA fragment thus obtained is designated as F1.

The DNA fragment corresponding to the soluble receptor protein was cleaved with XbaI into a sequence complementary to the base sequence encoding the carboxyl terminus of the extracellular region of the receptor protein of the present invention using the plasmid pTB1984 as a template. 5′-TTCTAGAAAGTAAACATGGGG-
Using 3 ′ (SEQ ID NO: 11) and the above synthetic oligonucleotide (SEQ ID NO: 9) as primers, a PCR reaction is carried out under the same conditions as above to amplify. After the amino and carboxyl termini of the obtained DNA fragment are digested with restriction enzymes EcoRI and XbaI, the DNA fragment is subjected to electrophoresis using 1% agarose gel to confirm the target DNA fragment, and then a Qiaquick Gel Extraction Kit (Qiagen) Company). The DNA fragment thus obtained is designated as F2. On the other hand, pc
DNA3.1 (-) / Myc-His B is a restriction enzyme Eco
After linearization using RI and XbaI, alkaline phosphatase treatment is performed by a known method to dephosphorylate the terminal phosphate group. The plasmid D after the reaction
NA is electrophoresed using 1% agarose, and after confirming the target DNA fragment, it is recovered and purified using a Qiaquick gel extraction kit (Qiagen). The vector DN
Let A be V1.

The DNA fragment F1 or F2 was
After insertion and ligation into T.1 using T4 DNA ligase by a known method, the target plasmid DNA is selected and isolated from an ampicillin-resistant strain obtained by introducing into E. coli DH5α. The nucleotide sequence of the cloned DNA fragment is confirmed by performing DNA sequencing in the same manner as described in Example 1. The D thus obtained
The recombinant plasmid into which the NA fragment F1 has been inserted is designated as P1. The recombinant plasmid into which the DNA fragment F2 has been inserted is designated as P2. COS cells (Fermentation Research Institute, Osaka)
The cells are cultured in a DMEM medium (Bio Wicker) containing 10% fetal bovine serum (FBS) until they reach 50% confluence. The recombinant plasmid P1 or P2 is introduced into the COS cells using Transfectam (Nippon Gene Co., Ltd.). After culturing the COS cells at 37 ° C. for 4 hours in 5% CO _{2 in} the absence of FBS,
Add 10% final concentration of FBS to OS cells. After further culturing for 20 hours, the medium is replaced with a serum-free DMEM medium. After 3 days of culture, the culture supernatant was collected, and the cells were washed with RIPA buffer (150 mM NaCl, 1% NP-4).
Dissolve in 0.1% SDS, 0.5% DOC, 40 mM Tris-HCl (pH 7.5). The obtained cell lysate is centrifuged at 10,000 × g for 10 minutes, and the supernatant is collected as a crude extract. The expression of the target protein in the culture supernatant or the crude extract was carried out using the anti-DC2 antiserum obtained in Example 2 or Anti-His (C-term), a specific antibody against the histidine tag (Invitrogen). Confirm by Western blot analysis. Purification of the target protein is performed by affinity chromatography using a nickel chelate column.

[0110]

Example 4 Expression of the Recombinant Receptor Protein of the Present Invention Using a Baculovirus Expression System
It can be performed using the c-To-Bac baculovirus expression system (Gibco BRL).
This system includes the following four steps. (1) The target gene was replaced with the donor plasmid pFastBac.
1 and the resulting recombinant plasmid was inserted into Escherichia coli DH1.
0Bac. (2) the target gene inserted into the recombinant plasmid,
It is translocated to a baculovirus vector (Bacmid) carried by DH10Bac. (3) After selecting an Escherichia coli strain carrying the recombinant bacmid into which the gene of interest has been inserted, the bacmid DN
A is isolated. (4) Infect insect cells with the obtained bacmid DNA to express the target gene.

D encoding the receptor protein of the present invention
The NA fragment was the recombinant plasmid P1 constructed in Example 3.
Is used as a template, and is complementary to the carboxyl-terminal region of the open reading frame of the present gene designed to have a sequence that can be cleaved by HindIII in the synthetic oligonucleotide used in Example 3 (SEQ ID NO: 9). Synthetic oligonucleotide: 5'-AAGCTTTCAATGATGATGATG-
GeneAmp ^R PCR using Takara LAPCR ^™ Kit Ver.2 (Takara Shuzo Co., Ltd.) with 3 ′ (SEQ ID NO: 12) as a primer
94 ° C / 1 with System 2400 (PerkinElmer)
Amplification is carried out by repeating a PCR reaction 30 times for 30 minutes at 98 ° C. for 20 seconds → 15 minutes at 68 ° C. The obtained DNA fragment was digested with amino and carboxyl ends using restriction enzymes EcoRI and HindIII, and then subjected to electrophoresis using 1% agarose gel to confirm the target DNA fragment. Collect and purify using an extraction kit (Qiagen). The D thus obtained
The NA fragment is designated as F3. On the other hand, pFastBac1 used as a donor plasmid is linearized with restriction enzymes EcoRI and HindIII, and then treated with alkaline phosphatase by a known method to dephosphorylate the terminal phosphate group. After the reaction, the plasmid DNA is subjected to electrophoresis using 1% agarose, and the target DNA
After confirming the fragment, it is recovered and purified using a Qiaquick Gel Extraction Kit (Qiagen). The vector DNA is
Let it be 2. DNA fragment F3 was added to plasmid V2 by T4
After insertion and ligation by a known method using DNA ligase, the target plasmid DNA is selected and isolated from an ampicillin-resistant strain obtained by introducing into Escherichia coli DH5α.
The nucleotide sequence of the cloned DNA fragment and the inserted form thereof are confirmed by performing DNA sequencing in the same manner as described in Example 1. The thus obtained recombinant plasmid into which the DNA encoding the receptor protein of the present invention has been inserted is designated as P3.

Next, F3 inserted into plasmid P3
The plasmid is introduced into Escherichia coli DH10Bac in order to transfer the DNA into the bacmid. First, plasmid P
31 ng was added to 100 μl of DH10Bac competent E. coli suspension, left on ice for 30 minutes,
For 45 seconds, and further left on ice for 2 minutes. Thereafter, 900 μl of SOC medium is added, and the cells are cultured at 37 ° C. for 4 hours. After culturing, dilute E. coli to each concentration and
g / ml kanamycin, 7 μg / ml gentamicin,
10 μg / ml tetracycline, 100 μg / ml Bl
uo-gal (Gibco BRL) and 40 μg /
ml of IPTG on Luria agar medium, 37 ° C
And culture overnight. A white colony is selected from the colonies that have appeared. Escherichia coli having the obtained recombinant bacmid was 50 μg / ml kanamycin, 7 μg / ml.
Inoculate 2 ml of LB medium containing ml gentamicin and 10 μg / ml tetracycline and incubate at 37 ° C. overnight. Next, bacmid is isolated and purified from the E. coli by the following method. First, 0.3 ml of a solution I (15
mM Tris-HCl buffer pH 8.0, 10 mM EDT
A, 100 μg / ml RNase A) and 0.3 ml
Solution II (0.2N sodium hydroxide, 1% SDS)
And mix gently and leave at room temperature for 5 minutes. Thereafter, 0.3 ml of 3M potassium acetate (pH 5.5) is added, stirred, and left on ice for 10 minutes. 14, at room temperature
After centrifugation at 000 × g for 15 minutes, the supernatant is recovered and 0.8 ml of isopropanol is added thereto. Stir several times and leave in ice for 10 minutes, then 14,000 xg at room temperature
And centrifuge for 15 minutes. The supernatant was removed, 0.5 ml of ethanol was added, and the mixture was stirred several times.
Centrifuge at 00 × g for 15 minutes. After removing the supernatant and leaving the mixture at room temperature for 10 minutes to dry the pellet, 40 μl of T
Dissolve in E buffer to make a recombinant bacmid DNA solution.

Next, the recombinant bacmid DNA is transduced into insect cells Sf9 by the method described below to express the target protein. First, antibiotics (50 units / ml
After thoroughly mixing a cellfectin reagent (Gibco BRL) diluted with 100 μl of SF-900II SFM medium (Gibco BRL) without penicillin and 50 μg / ml streptomycin), the mixture was mixed well at room temperature.
Minutes, and add SF-900II SFM medium to it for 0.8 minutes.
Add 1 ml to prepare 1 ml lipid-DNA solution. On the other hand, 9 × 10 ⁵ Sf9 cells were transferred to a 35 m plastic
S containing the antibiotics (50 units / ml penicillin, 50 μg / ml streptomycin)
After culturing for 1 hour at 27 ° C. in 2 ml of f-900II SFM medium, 2 m of Sf-900II SFM medium without antibiotics was added.
Wash cells once with l. The lipid-D previously prepared was added to the cells.
After adding 1 ml of NA solution and culturing at 27 ° C. for 5 hours, the mixture was removed, and Sf-containing antibiotics (50 units / ml penicillin, 50 μg / ml streptomycin) was used.
Add 2 ml of 900II SFM medium and culture for 2 days.
After the cells thus obtained were collected by centrifugation, 5 ml of a cell lysis solution (50 mM Tris-HCl (pH
8.5), 10 mM 2-mercaptoethanol, 1 mM
Dissolve in PMSF, 1% NP-40) and 10,000
Centrifuge at × g for 10 minutes, and collect the supernatant as a crude extract. The expression of the target protein in the obtained crude extract was determined by the anti-DC2 antiserum obtained in Example 2 and the specific antibody Anti-His (Ct) against the histidine tag.
erm) (Invitrogen)
Confirm by blot analysis. In addition, purification of the target protein
It is performed by affinity chromatography using a nickel chelate column.

Example 5 Expression of Recombinant Receptor Protein of the Present Invention in CHO Cells (1) Construction of Expression Vector First, DN encoding the full-length receptor protein of the present invention
A fragment is obtained as follows. First, the plasmid pTB1984 containing the DNA encoding the receptor protein obtained in Example 1 was used as a template, and SEQ ID NO: 15 (A
GGATCCCCTGTCCCGCGCCATGGC
C), SEQ ID NO: 16 (AAAGCTTAAGCCTT
GGCCCCGCCTTGCTCC) as primers and Takara LA
PCR ^TM Kit Ver.2 at (Takara Shuzo) thermal cycler GeneAmp ^R PCR System 2400 using a (Perkin Elmer), 94 ° C. · 1 minute, followed by 98 ° C. · 20 seconds → 55
After repeating 25 cycles of 20 ° C. for 20 seconds → 72 ° C. for 4 minutes, a PCR reaction is performed by a program at 72 ° C. for 5 minutes to obtain an amplification product. Next, both ends are digested with restriction enzymes BamHI and HindIII, followed by electrophoresis using 1% agarose gel. After confirming the target DNA fragment, the DNA fragment is recovered and purified using a Qiaquick gel extraction kit. . On the other hand, pcDNA3.1 (-) (Invitrogen (I
nvitrogen) also cuts and linearizes with the restriction enzymes BamHI and HindIII, and then inserts and ligates the above purified DNA with T4 DNA ligase. The desired plasmid DNA is selected and isolated from an ampicillin-resistant strain obtained by introduction into Escherichia coli DH5α, and the base sequence of the inserted DNA is confirmed by performing a sequence reaction in the same manner as described in Example 1. (2) Acquisition of CHO cells expressing recombinant receptor protein First, CHO cells were placed on a 60 mm culture dish for 10 hours.
% -FBS-containing α-MEM medium containing nucleic acid (NIKKEN BIO MEDICAL LABORATORY)
Incubate until the number reaches × 10 ⁵ . The expression vector plasmid obtained in the above (1) is introduced into the CHO cells using the transfectam used in Example 3. 5% C
After culturing for 24 hours at 37 ° C. in O ₂ , G418 was added to a final concentration of 5
It is added to a concentration of 00 μg / ml, and the culture is continued, whereby drug-resistant strains capable of growing there are selected. After the resulting drug-resistant strain was cloned into a single clone by the limiting dilution method, each chromosomal DNA was prepared, and using them as templates, SEQ ID NO:
17 (TTGCCCGGTTTAATAATTCTGC
TTCTCTTC), SEQ ID NO: 18 (TGTATTC)
Genomic PCR is performed using two synthetic oligonucleotides represented by ATCTTCTGTGGGCATCTGCTCG as primers, and a strain in which an amplified DNA fragment of 418 base pairs derived from DNA encoding the receptor protein is selected as a candidate strain. The expression of the target protein is confirmed by Western blot analysis of the crude cell extract or its membrane fraction using an anti-DC2 antibody (for example, the antiserum obtained in Example 2). The cell line obtained here is effective for searching for a ligand of the receptor protein, screening for a substance that promotes or suppresses the function of the receptor protein, and the like.

[0114]

The receptor protein of the present invention, its partial peptide or a salt thereof is useful as a reagent for screening ligands, agonists, antagonists and the like. The agonist is useful as a therapeutic / prophylactic agent for cancer, AIDS, infectious disease, etc., and the antagonist is useful as a therapeutic / prophylactic agent for allergic immune disease, inflammation, autoimmune disease, AIDS, glomerulonephritis, bronchial asthma, etc. is there. By using the antibody of the present invention, the concentration of the receptor protein of the present invention, its partial peptide, or a salt thereof in a test solution can be determined.

[0115]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 616 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Ala Pro Arg Ala Arg Arg Arg Arg Pro Leu Phe Ala Leu Leu leu 1 5 10 15 Leu Cys Ala Leu Leu Ala Arg Leu Gln Val Ala Leu Gln Ile Ala Pro 20 25 30 Pro Cys Thr Ser Glu Lys His Tyr Glu His Leu Gly Arg Cys Cys Asn 35 40 45 Lys Cys Glu Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr Thr Thr Ser 50 55 60 Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu Asp Ser Trp 65 70 75 80 Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp Thr Gly Lys 85 90 95 Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro Arg Arg Cys 100 105 110 Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp Cys Glu Cys Cys Arg 115 120 125 Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly Ala Gln His Pro Leu Gln 130 135 140 Leu Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly Tyr Phe Ser 145 150 155 160 Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr Asn Cys Thr 165 170 175 Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys Ser Asp Val 180 185 190 Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn Glu Pro His 195 200 205 Val Tyr Leu Pro Gly Leu Ile Ile Leu Leu Leu Phe Ala Ser Val Ala 210 215 220 Leu Val Ala Ala Ile Ile Phe Gly Val Cys Tyr Arg Lys Lys Gly Lys 225 230 235 240 Ala Leu Thr Ala Asn Leu Trp His Trp Ile Asn Glu Ala Cys Gly Arg 245 250 255 Leu Ser Gly Asp Lys Glu Ser Ser Gly Asp Ser Cys Val Ser Thr His 260 265 270 Thr Ala Asn Phe Gly Gln Gln Gly Ala Cys Glu Gly Val Leu Leu Leu 275 280 285 Thr Leu Glu Glu Lys Thr Phe Pro Glu Asp Met Cys Tyr Pro Asp Gln 290 295 300 Gly Gly Val Cys Gln Gly Thr Cys Val Gly Gly Gly Pro Tyr Ala Gln 305 310 315 320 Gly Glu Asp Ala Arg Met Leu Ser Leu Val Ser Lys Thr Glu Ile Glu 325 330 335 Glu Asp Ser Phe Arg Gln Met Pro Thr Glu Asp Glu Tyr Met Asp Arg 340 345 350 350 Pro Ser Gln Pro Thr Asp Gln Leu Leu Phe Leu Thr Glu Pro Gly Ser 355 360 365 Lys Ser Thr Pro Pro Phe Ser Glu Pro Leu Glu Val Gly Glu Asn Asp 370 375 380 Ser Leu Ser Gln Cys Phe Thr Gly Thr Gln Ser Thr Val Gly Ser Glu 385 390 395 400 400 Ser Cys Asn Cys Thr Glu Pro Leu Cys Arg Thr Asp Trp Thr Pro Met 405 410 415 Ser Ser Glu Asn Tyr Leu Gln Lys Glu Val Asp Ser Gly His Cys Pro 420 425 430 His Trp Ala Ala Ser Pro Ser Pro Asn Trp Ala Asp Val Cys Thr Gly 435 440 445 Cys Arg Asn Pro Pro Gly Glu Asp Cys Glu Pro Leu Val Gly Ser Pro 450 455 460 Lys Arg Gly Pro Leu Pro Gln Cys Ala Tyr Gly Met Gly Leu Pro Pro 465 470 475 480 Glu Glu Glu Ala Ser Arg Thr Glu Ala Arg Asp Gln Pro Glu Asp Gly 485 490 495 Ala Asp Gly Arg Leu Pro Ser Ser Ala Arg Ala Gly Ala Gly Ser Gly 500 505 510 Ser Ser Pro Gly Gly Gln Ser Pro Ala Ser Gly Asn Val Thr Gly Asn 515 520 525 Ser Asn Ser Thr Phe Ile Ser Ser Gly Gln Val Met Asn Phe Lys Gly 530 535 540 Asp Ile Ile Val Val Tyr Val Ser Gln Thr Ser Gln Glu Gly Ala Ala 545 550 555 560 Ala Ala Ala Glu Pro Met Gly Arg Pro Val Gln Glu Glu Thr Leu Ala 565 570 575 Arg Arg Asp Ser Phe Ala Gly Asn Gly Pro Arg Phe Pro Asp Pro Cys 580 585 590 590 Gly Gly Pro Glu Gly Leu Arg Glu Pro Glu Lys Ala Ser Arg Pro Val 595 600 605 Gln Glu Gln Gly Gly Ala Lys Ala 610 615

[0116]

[SEQ ID NO: 2] Sequence length: 181 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ile Ala Pro Cys Thr Ser Glu Lys His Tyr Glu His Leu Gly Arg 1 5 10 15 Cys Cys Asn Lys Cys Glu Pro Gly Lys Tyr Met Ser Ser Lys Cys Thr 20 25 30 Thr Thr Ser Asp Ser Val Cys Leu Pro Cys Gly Pro Asp Glu Tyr Leu 35 40 45 Asp Ser Trp Asn Glu Glu Asp Lys Cys Leu Leu His Lys Val Cys Asp 50 55 60 Thr Gly Lys Ala Leu Val Ala Val Val Ala Gly Asn Ser Thr Thr Pro 65 70 75 80 Arg Arg Cys Ala Cys Thr Ala Gly Tyr His Trp Ser Gln Asp Cys Glu 85 90 95 Cys Cys Arg Arg Asn Thr Glu Cys Ala Pro Gly Leu Gly Ala Gln His 100 105 110 Pro Leu Gln Leu Asn Lys Asp Thr Val Cys Lys Pro Cys Leu Ala Gly 115 120 125 Tyr Phe Ser Asp Ala Phe Ser Ser Thr Asp Lys Cys Arg Pro Trp Thr 130 135 140 Asn Cys Thr Phe Leu Gly Lys Arg Val Glu His His Gly Thr Glu Lys 145 150 155 160 Ser Asp Val Val Cys Ser Ser Ser Leu Pro Ala Arg Lys Pro Pro Asn 165 170 175 Glu Pro His Val Tyr 180

[0117]

[SEQ ID NO: 3] Sequence length: 29 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Ala Pro Arg Ala Arg Arg Arg Arg Pro Leu Phe Ala Leu Leu leu 1 5 10 15 Leu Cys Ala Leu Leu Ala Arg Leu Gln Val Ala Leu Gln 20 25

[0118]

[SEQ ID NO: 4] Sequence length: 1848 Sequence type: Nucleic acid number of strands: Double strand Topology: Linear Sequence type: cDNA sequence ATGGCCCCGC GCGCCCGGCG GCGCCGCCCG CTGTTCGCGC TGCTGCTGCT CTGCGCGCTG 60 CTCGCCCGGC TGCAGGTGGC TTTGCAGACAT GCTCCTCCATCATGCATC GACGGTGCTG TAACAAATGT GAACCAGGAA AGTACATGTC TTCTAAATGC 180 ACTACTACCT CTGACAGTGT ATGTCTGCCC TGTGGCCCGG ATGAATACTT GGATAGCTGG 240 AATGAAGAAG ATAAATGCTT GCTGCATAAA GTTTGTGATA CAGGCAAGGC CCTGGTGGCC 300 GTGGTCGCCG GCAACAGCAC GACCCCCCGG CGCTGCGCGT GCACGGCTGG GTACCACTGG 360 AGCCAGGACT GCGAGTGCTG CCGCCGCAAC ACCGAGTGCG CGCCGGGCCT GGGCGCCCAG 420 CACCCGTTGC AGCTCAACAA GGACACAGTG TGCAAACCTT GCCTTGCAGG CTACTTCTCT 480 GATGCCTTTT CCTCCACGGA CAAATGCAGA CCCTGGACCA ACTGTACCTT CCTTGGAAAG 540 AGAGTAGAAC ATCATGGGAC AGAGAAATCC GATGTGGTTT GCAGTTCTTC TCTGCCAGCT 600 AGAAAACCAC CAAATGAACC CCATGTTTAC TTGCCCGGTT TAATAATTCT GCTTCTCTTC 660 GCGTCTGTGG CCCTGGTGGC TGCCATCATC TTTGGCGTTT GCTATAGGAA AAAAGGGAAA 720 GCACTCACAG CTAATTTGTG GCACTGGATC AATGAGGCTT GTGGCCGCCT AAGTGGAGAT 780 AAGGAGTCCT CAGGTGACAG TTGTGTCAGT ACACACACGG CAAACTTTGG TCAGCAGGGA 840 GCATGTGAAG GTGTCTTACT GCTGACTCTG GAGGAGAAGA CATTTCCAGA AGATATGTGC 900 TACCCAGATC AAGGTGGTGT CTGTCAGGGC ACGTGTGTAG GAGGTGGTCC CTACGCACAA 960 GGCGAAGATG CCAGGATGCT CTCATTGGTC AGCAAGACCG AGATAGAGGA AGACAGCTTC 1020 AGACAGATGC CCACAGAAGA TGAATACATG GACAGGCCCT CCCAGCCCAC AGACCAGTTA 1080 CTGTTCCTCA CTGAGCCTGG AAGCAAATCC ACACCTCCTT TCTCTGAACC CCTGGAGGTG 1140 GGGGAGAATG ACAGTTTAAG CCAGTGCTTC ACGGGGACAC AGAGCACAGT GGGTTCAGAA 1200 AGCTGCAACT GCACTGAGCC CCTGTGCAGG ACTGATTGGA CTCCCATGTC CTCTGAAAAC 1260 TACTTGCAAA AAGAGGTGGA CAGTGGCCAT TGCCCGCACT GGGCAGCCAG CCCCAGCCCC 1320 AACTGGGCAG ATGTCTGCAC AGGCTGCCGG AACCCTCCTG GGGAGGACTG TGAACCCCTC 1380 GTGGGTTCCC CAAAACGTGG ACCCTTGCCC CAGTGCGCCT ATGGCATGGG CCTTCCCCCT 1440 GAAGAAGAAG CCAGCAGGAC GGAGGCCAGA GACCAGCCCG AGGATGGGGC TGATGGGAGG 1500 CTCCCAAGCT CAGCGAGGGC AGGTGCCGGG TCTGGAAGCT CCCCTGGTGG CCAGTCCCCT 1560 GCATC TGGAA ATGTGACTGG AAACAGTAAC TCCACGTTCA TCTCCAGCGG GCAGGTGATG 1620 AACTTCAAGG GCGACATCAT CGTGGTCTAC GTCAGCCAGA CCTCGCAGGA GGGCGCGGCG 1680 GCGGCTGCGG AGCCCATGGG CCGCCCGGTG CAGGAGGAGA CCCTGGCGCG CCGAGACTCC 1740 TTCGCGGGGA ACGGCCCGCG CTTCCCGGAC CCGTGCGGCG GCCCCGAGGG GCTGCGGGAG 1800 CCGGAGAAGG CCTCGAGGCC GGTGCAGGAG CAAGGCGGGG CCAAGGCT 1848

[0119]

[SEQ ID NO: 5] Sequence length: 543 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATCGCTCCTC CATGTACCAG TGAGAAGCAT TATGAGCATC TGGGACGGTG CTGTAACAAA 60 TGTGAACCAG GAAAGTACAT GTCTTCTACTTCTCTGCAG CGGATGAATA CTTGGATAGC TGGAATGAAG AAGATAAATG CTTGCTGCAT 180 AAAGTTTGTG ATACAGGCAA GGCCCTGGTG GCCGTGGTCG CCGGCAACAG CACGACCCCC 240 CGGCGCTGCG CGTGCACGGC TGGGTACCAC TGGAGCCAGG ACTGCGAGTG CTGCCGCCGC 300 AACACCGAGT GCGCGCCGGG CCTGGGCGCC CAGCACCCGT TGCAGCTCAA CAAGGACACA 360 GTGTGCAAAC CTTGCCTTGC AGGCTACTTC TCTGATGCCT TTTCCTCCAC GGACAAATGC 420 AGACCCTGGA CCAACTGTAC CTTCCTTGGA AAGAGAGTAG AACATCATGG GACAGAGAAA 480 TCCGATGTGG TTTGCAGTTC TTCTCTGCCA GCTAGAAAAC CACCAAATGA ACCCCATGTT 540 TAC 543

[0120]

[SEQ ID NO: 6] Sequence length: 87 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence ATGGCCCCGC GCGCCCGGCG GCGCCGCCCG CTGTTCGCGC TGCTGCTGCT CTGCGCGCTG60 CTCGCCCGGC TGCAGGTGGC TTTGCAG 87

[0121]

[SEQ ID NO: 7] Sequence length: 21 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GCCAGCCTGT CCCGCGCCAT G 21

[0122]

[SEQ ID NO: 8] Sequence length: 24 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CAGAATATGA AAGTCCCACC ACAT 24

[0123]

[SEQ ID NO: 9] Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GAATTCATGG CCCCGCGCGC C 21

[0124]

[SEQ ID NO: 10] Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TTCTAGAGCC TTGGCCCCGC 21

[0125]

[SEQ ID NO: 11] Sequence length: 21 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TTCTAGAAAAAG TAAACATGGGG
21

[0126]

[SEQ ID NO: 12] Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AAGCTTTCAA TGATGATGAT G
21

[SEQ ID NO: 13] Sequence length: 23 Sequence type: amino acid Topology: linear Sequence type: protein sequence Cys His Gly Thr Glu Lys Ser Asp Val Val Cys Ser Ser Ser Leu Pro 1 5 10 15 Ala Arg Lys Pro Pro Asn Glu 20

[SEQ ID NO: 14] Sequence length: 23 Sequence type: amino acid Topology: linear Sequence type: protein sequence Cys Ser Asp Ser Val Cys Leu Pro Cys
Gly Pro Asp Glu Tyr Leu Asp 15
10 15 Ser Trp Asn Glu Glu Asp Lys 20

[SEQ ID NO: 15] Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AGGATCCCCT GTCCCGCGCC ATG
GCC 26

[SEQ ID NO: 16] Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AAAGCTTAAG CCTTGGCCCC GCCTTGCTCC 30

[SEQ ID NO: 17] Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TTGCCCGGTT TTATAATTCT GCTTCTCTTC 30

[SEQ ID NO: 18] Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence TGTATTCATC TTCTGTGGGC ATCTGTCTG 30

[0127]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence (1st to 1320th) of the DNA encoding the novel receptor protein derived from a human dendritic cell of the present invention obtained in Example 1 and the amino acid sequence deduced therefrom (FIG. 1) (Continued to FIG. 2).

FIG. 2 shows the nucleotide sequence (1321 to 2781) of the DNA encoding the novel human dendritic cell-derived receptor protein of the present invention obtained in Example 1 and the amino acid sequence deduced therefrom (FIG. 2) (Continuation of FIG. 1).

FIG. 3 shows a hydrophobicity plot of the human dendritic cell-derived receptor protein of the present invention prepared based on the amino acid sequences shown in FIGS. 1 and 2. Extracellular Domain is extracellular domain, Cytoplasmic Domain is intracellular domain, Signal
Sequence indicates a signal sequence, and Transmembrane Region indicates a transmembrane region.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12Q 1/02 A61K 48/00 // A61K 48/00 C12N 15/00 ZNAA C12N 5/10 5/00 B (C12N 15/09 ZNA C12R 1:91) (C12P 21/02 C12R 1:91)

Claims (10)

[Claims] 1. A receptor protein or a salt thereof comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. 2. The receptor protein according to claim 1, which is a receptor protein derived from dendritic cells belonging to the TNF receptor family. 3. A partial peptide of the receptor protein according to claim 1, or a salt thereof. 4. A method of culturing a transformant transformed with a recombinant vector containing the DNA encoding the receptor protein according to claim 1 to produce and accumulate the receptor protein, and collecting the receptor protein. A method for producing the receptor protein or a salt thereof according to claim 1. 5. An antibody against the receptor protein according to claim 1, the partial peptide according to claim 3, or a salt thereof. 6. The method for determining a ligand for a receptor protein or a salt thereof according to claim 1, wherein the receptor protein according to claim 1 or the partial peptide or a salt thereof according to claim 3 is used. 7. The binding property between a ligand characterized by using the receptor protein according to claim 1, the partial peptide according to claim 3 or a salt thereof, and the receptor protein according to claim 1 or a salt thereof is changed. A method for screening a compound or a salt thereof. [8] The binding property between a ligand comprising the receptor protein according to [1], the partial peptide according to [3] or a salt thereof and the receptor protein according to [1] or a salt thereof is changed. Kit for screening a compound or a salt thereof. 9. A compound or a salt thereof, obtained by using the screening method of claim 7 or the screening kit of claim 8, which alters the binding property between the ligand and the receptor protein of claim 1 or a salt thereof. . 10. A compound or a salt thereof, which is obtained by using the screening method according to claim 7 or the screening kit according to claim 8, which alters the binding property between the ligand and the receptor protein according to claim 1 or a salt thereof. A medicament comprising: