JP2009060787A - Screening method and identification method for substance capable of inhibiting degranulation reaction in mast cell through rec168, and therapeutic agent for inflammatory disease concerned with mast cell containing rec168 antagonist - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening a substance which regulates degranulation and a method for identification of a substance capable of inhibiting the degranulation in a mast cell, for developing a therapeutic agent for an allergic disease or an autoimmune disease based on a new mechanism vi a G-protein-conjugated receptor Rec168. <P>SOLUTION: The method for screening of a substance capable of inhibiting the signal transduction capability or a substance capable of regulating the degranulation in a mast cell via Rec168, includes a step for determining the signal transduction capability of a ligand, having a Rec168-agonistic activity toward Rec168 in the presence or absence of a substance to be tested; and a step for comparing a measurement value obtained, in the presence of the substance with a measurement value obtained in the absence of the substance. Also disclosed is a method for identification of a substance capable of inhibiting degranulation in a mast cell. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a G protein-coupled receptor (G-protein
The present invention relates to a screening method for a substance having an action of suppressing the signal transduction ability mediated by Rec168 protein (more specifically, GPCR), more specifically, a substance that suppresses degranulation of mast cells via Rec168. Furthermore, a method of identifying a substance that suppresses degranulation of mast cells via Rec168, a degranulation inhibitor comprising a substance identified by the method, or a therapeutic agent for inflammatory diseases involving mast cells It relates to a therapeutic agent.

  GPCRs exist on the surface of each functional cell in living cells and organs and play a physiologically important role as targets for molecules that regulate the functions of these cells and organs, such as hormones, neurotransmitters, and physiologically active substances. I'm in charge. The receptor transmits a signal into the cell through binding with a physiologically active substance, and various signals such as activation and suppression of the cell are induced by this signal. GPCR has seven transmembrane portions and plays a role of transmitting ligand information to an effector system via a GTP (guanosine 5'-triphosphate) -binding regulatory protein (G protein). G protein is a trimer composed of three subunits of α, β and γ, and is roughly divided into Gs, Gi, Go, Gq, etc. depending on the type of α subunit, and each has receptor specificity (conjugate G protein). Receptors) and effector systems (enzymes and ion channels) are different.

For example, glucagon-like peptide 1 receptor and β2 adrenergic receptor are coupled to Gs to promote the effector system adenylate cyclase system and increase cyclic AMP (cAMP). In addition, α2 adrenergic receptor is coupled with Gi to suppress the adenylate cyclase system and reduce cAMP. The H1 histamine receptor couples with Gq to promote the phospholipase C system, which is an effector system, increases diacylglycerol and inositol triphosphate, and increases intracellular Ca ²⁺ . The main types of effector systems are adenylate cyclase system, phospholipase C system, and cGMP phosphodiesterase system.

The β1 adrenergic receptor is localized in the heart, adipose tissue, cerebral cortex and the like, and the adenylate cyclase system is promoted by Gs, resulting in effects such as increased heart rate, increased cardiac contractility, and lipolysis.

  In the G protein, GDP (guanosine 5'-diphosphate) is bound to the α subunit, and is associated with the β subunit and the γ subunit. α, β and γ are in an inactive state. When a ligand binds to the receptor, GDP bound to α is replaced with GTP, and α-GTP having GTP bound to the α subunit , Dissociates from the β-γ conjugate. α-G TP and β-γ act on the effector system to transmit signals. During this time, when GTP is decomposed to GDP by GTPase activity of the α subunit, α-GDP leaves the effector system and associates with β and γ, and becomes inactive again. By repeating this reaction, information is amplified and transmitted.

The biodistribution of GPCRs is often localized in specific organs, but G proteins are widely distributed in the living body. As described above, G protein mainly triggers a series of intracellular phosphorylation reactions through GPCR and controls cell and organ functions widely from transcriptional regulation to muscle contraction at the gene level.

  Thus, the information transmission system controlled by GPCR / G protein is an indispensable mechanism for the control of physiological functions of living organisms. In other words, the information transmission system is widely involved in the development of various diseases. is there. Therefore, once a GPCR involved in the development of a certain disease is identified, the disease can be treated with the drug by developing a drug (agonist, antagonist, agonist action inhibitor, etc.) that modulates the physiological function of the receptor. It becomes possible to do.

Upon completion of the Human Genome Project, analysis of the database suggests the existence of many new GPCRs, some of which have already been isolated (see, for example, Patent Documents 1 and 2). Although it is suggested that GPCR may exist in the whole human genome from 700 to nearly 800, there are about 250 receptors with known ligands and the rest are orphan receptors with unknown ligands. Therefore, it can be said that most of the physiological phenomena involving GPCRs are undeveloped areas. As mentioned above, GPCRs are deeply involved in the control of physiological functions in the body and the onset of various diseases. Therefore, clarifying the physiological functions of the receptors can elucidate the cause of the onset of various diseases. Is extremely important. The key to elucidating the physiological function of GPCRs is to identify ligands for the receptor, and many scientists are actively researching. On the other hand, so-called GPCR drug discovery for creating drugs that regulate the signal transduction ability of GPCRs whose physiological functions have been clarified has been attempted by many approaches centered on US venture companies and pharmaceutical companies. The method is based on bioinformatics, structural biology, etc.
The approach from DRY BIOLOGY and the so-called “approach from WET BIOLOGY” at the protein, cell, and individual level based on conventional molecular biology and cell biology research, but these methods are not separate. Aiming for drug discovery goals by complementing each other, what is important in any case is that by quickly acquiring new useful knowledge such as identifying ligand binding sites, agonists and antagonists useful as drugs Many pharmaceutical companies have accelerated the development of new drugs by introducing approaches to predict the three-dimensional structure of receptors, etc. In this way, drug treatment via GPCRs is remarkable. Successful, more than 50% of the drugs currently used are targeted to GPCRs, and the world of the drugs Among them, sales account for more than the top 25%, and sales are about to reach $ 2 trillion (see Non-Patent Document 1, for example).

The importance of GPCR is related not only to drug development but also to many genetic diseases (see, for example, Non-Patent Document 1). At present, GPCR mutations have been detected in diseases such as color blindness, night blindness, and retinitis pigmentosa. In addition, it is said that there are many GPCRs related to taste and smell, and the elucidation of abnormal taste and smell may progress rapidly. Since GPCRs are receptors that receive extracellular stimuli, it is well understood that they are closely related to these five senses. Also in hormone secretion
Many GPCR mutations have been reported, suggesting their importance in physiological roles

  However, even when a plurality of substances have been revealed as GPCR agonists, it is only possible to input a signal through the receptor in an artificial screening system. There are many things that are not clear whether or not they are reflected.

  Rec168 according to the present invention is a receptor belonging to the Mrg family and is one of GPCRs called MrgX2. The gene sequence of Rec168 is already known (see, for example, Patent Documents 3 to 5). In these documents, there is no disclosure of information about the endogenous ligand of Rec168. Regarding the same protein as Rec168, Miwa et al. Have already disclosed the amino acid sequence and gene sequence thereof as hTGR-12 (see Patent Document 6). However, there is no specific disclosure about its physiological function and physiological ligand. Other similar information is disclosed, but it does not specifically disclose the physiological function or physiological ligand of Rec168.

Regarding the ligand substance of Rec168, Janssen
Pharmaceutical Buist Arjan et al., MrgX2 (Rec168), one of the human dorsal root receptors (hDRRs), is expressed in lymph nodes, and it is expressed in the N-terminal peptide fragment (4-21) of Chaperonin10 purified from porcine hypothalamus. On the other hand, it was shown to have agonist activity (ED ₅₀ = 354 nM) (see Patent Document 7). ACADIA
The Pharmaceutical group states that the ligand for MrgX2 (Rec168) is STIA, but details regarding STIA are unclear. Nicola Robas et al. Have reported that cortistatin that controls sleep and motor function is a ligand of MrgX2 (Rec168) (see Non-Patent Document 2 and Patent Document 8). It has also been reported that a newly identified family of peptides that activates MrgX1 identified from porcine tissue also acts weakly on MrgX2 (Rec168). The homologues of MrgX1 are rat SNSR1 and mouse MrgC11. Astrazeneca's Lembo and colleagues reported that the ligand for SNSR3 & 4 (MrgX1) is BAM8-22, but γ2-MSH is the most promising peptide in activity against rat SNSR (EC ₅₀ = 37 ± 10 nM) Furthermore, detailed structure-activity relationship analysis reported that the C-terminal sequence of γ2-MSH was an agonist (Kd = 2.7 ± 0.4 nM, Bmax = 350 ± 100 fmol / mg). From the in vivo analysis, the role of SNSR (MrgX1) in analgesia has been analyzed (see Non-Patent Document 3). Also, Substance
P, Substance P2-11, Neuropeptide
It has been reported that FF and the like activate MrgX2 (see Patent Document 9). Furthermore, recently, broadrenomedullin N-terminal peptides have also been reported as a peptide that activates MrgX2 (see Non-Patent Document 14).
As described above, the N-terminal peptide fragment of Chaperonin 10 (4-21), cortistatin, and the like have been disclosed as ligands for Rec168, but the physiological function of Rec168 has not yet been clarified and is described as a suggested function. There are only those inferred from the functions that these ligand substances generally have. For this reason, drug development targeting Rec168 is not progressing, and agonists and antagonists of Rec168 have not yet been put on the market as pharmaceuticals. In order to develop drug development targeting Rec168 in the future, it is necessary to clarify the physiological function of Rec168 and its relationship with diseases.

On the other hand, degranulation of mast cells shows that IgE has high affinity on mast cells
It is broadly classified into those involved in immunity caused by binding to IgE receptor (FcεRI) and those not involved in immunity not via this system, the former being in the early stage of inflammation and the latter being more common in chronic inflammation ( Non-patent document 4). While detailed studies have been conducted on degranulation involving immunity, degranulation not involving immunity has hardly been clarified, including the mechanism of action.

Mast cells are widely distributed in systemic organs, especially in the skin and mucous membrane in contact with the outside world. The cell has a large number of granules, and FcεRI is expressed on the cell surface. When an antigen binds via an IgE molecule, FcεRI is activated, and through various intracellular signal transduction mechanisms, At the same time as histamine is released (degranulation), prostaglandins and leukotrienes are also produced. These substances, called chemical messengers, contract the bronchi, increase vascular permeability, increase sputum secretion, and cause dyspnea attacks of asthma. Since this reaction occurs in a short time, it is called an immediate reaction. More recently, following the release of chemical mediators such as histamine and prostaglandins, interleukin (IL) -3, IL-4, IL-5, IL-6, macrophage colony stimulating factor (GM-CSF), tumor necrosis It has become known that cytokines such as factors (Tumor necrosis factor-α: TNF-α) are produced and released, and that FcεRI association stimulation is transmitted into the nucleus.

Chemical mediators such as histamine, enzymes, and lipid mediators cause smooth muscle contraction, increased vascular permeability, tissue edema, etc. On the other hand, various cytokines released after a few hours are skin, lung, It has been revealed that it is involved in the development of clinical symptoms such as late-onset allergic reactions in the nose, eyes, etc., ie, urticaria, atopic dermatitis, bronchial asthma, allergic rhinitis, and allergic conjunctivitis.

So far, it has been known that there is a degranulation reaction not involving IgE, which is involved in chronic inflammation. Moreover, MCD peptides, Substance P, VIP, Somatostatin, compound 48/80, and the like are known as basic secretagogues as non-immune-related mast cell degranulation inducers (see, for example, Non-Patent Documents 5 to 9).
Basic secretagogues such as Substance P, VIP, and Somatostatin were known to cause degranulation at a much higher concentration (2 to 3 μM) than previously known receptors. As for the mechanism, there is a report that there is a possibility that the pathway is not via a receptor (for example, see Non-Patent Documents 10 to 11), but details are unknown. There are no reports on specific receptors.

  Regarding the involvement of the degranulation reaction in which mast cells do not involve IgE, several cases have been reported in connection with inflammation (for example, see Non-Patent Documents 12 to 13). In an autoantibody-induced arthritis model, it does not develop in mast cell-deficient mice (W / Wv, c-kit mutant; Sl / Sld, c-kit ligand mutant), but the onset was confirmed by transplanting normal mast cells, It was proved that the action of mast cells not involving IgE is dominant in the onset of arthritis. The degranulation of mast cells without immune involvement is considered to be directly involved in various chronic inflammations including arthritis.

Under such circumstances, if it is possible to elucidate the mechanism of degranulation of mast cells without immune involvement, it is considered that it can be applied to the treatment of various chronic inflammations, and the elucidation is eagerly desired. It was.
JP-A-9-268 JP-A-9-51795 WO01 / 19983 pamphlet WO01 / 48015 pamphlet WO01 / 16159 pamphlet WO02 / 04641 pamphlet WO03 / 005036 pamphlet WO03 / 073107 pamphlet WO03 / 104818 pamphlet Flower, DR, Biochim. Biophys. Acta (1999) 1422, 207-234 Robas, N. et al., J. Biol. Chem. (2003) 278, 44400-44404. The 3rdjoint meeting of the summer Neuropeptide conference (13th annual meeting) & the European Neuropeptide Club (13th annual meeting), Montauk, NY, June 8-12, 2003. Piliponsky, AM etal., Molecular Immunology (2001) 38, 1369-1372. Buku, A., Peptides (1999) 20, 415-420. Erjavec, F. et al., Naunyn-Schmiedeberg's Arch Pharmacol (1981) 317, 67-70. Theoharides, TC etal., European Journal of Pharmacology (1981) 69, 127-137. Mori, T. et al., Arzneimittelforschung (1994) 44, 1044-6. Odum, L. et al., Inflamm. Res. (1998) 47, 488-492. Aridor, M. et al, J. Cell Biol. (1990) 111 (3), 909-17. Peptides (2002) 23 (8), 1507-15. Lee, DM et al., Science (2002) 297, 1689-92. Mehlhop, PD et al., Proc. Natl. Acad. Sci. USA. (1997) 94, 1344-1349. Kamohara, M. et al., Biochemical and Biophysical Research Communications (2005) 330, 1146-1152.

  An object of the present invention is to elucidate the physiological function of G protein-coupled receptor Rec168 and to provide an application for Rec168 and its ligand that reflects the physiological function. More specifically, the object of the present invention is to provide a screening method for a substance having a signal transduction-inhibiting effect via G protein-coupled receptor Rec168 protein, more specifically, a substance that inhibits degranulation reaction via Rec168. That is. Another object of the present invention is to provide a method for identifying a substance that inhibits Rec168-mediated degranulation reaction, a degranulation inhibitor comprising the substance identified by the same method, or a treatment for inflammatory diseases involving mast cells Is to provide an agent.

  In order to solve the above-mentioned problems, the present inventors have conducted extensive research and as a result, completed the present invention. That is, the present invention is as follows.

1. Characterized in that it comprises a step of measuring a signal transduction ability for a ligand having a Rec168 agonistic action in the presence and absence of a test substance using a cell expressing Rec168, and a step of comparing both measured values. A method for screening for a substance that suppresses degranulation of mast cells via Rec168.
2. 2. The screening method according to 1 above, wherein the degranulation reaction of mast cells via Rec168 is measured and compared using mast cells in the absence and presence of the test substance.
3. Characterized in that it comprises a step of measuring a signal transduction ability for a ligand having a Rec168 agonistic action in the presence and absence of a test substance using a cell expressing Rec168, and a step of comparing both measured values. A method for identifying a substance that inhibits Rec168-mediated mast cell degranulation.
4). 4. The identification method according to 3 above, wherein the degranulation reaction of mast cells via Rec168 is measured and compared using mast cells in the absence and presence of the test substance.
5). 5. A mast cell degranulation inhibitor comprising the degranulation inhibitor identified by the method according to 3 or 4 as an active ingredient.
6). 6. The degranulation inhibitor as described in 5 above, which contains at least one compound selected from the following as an active ingredient.
1- (10H-phenazin-5-yl) -ethanone,
2-azepan-1-yl-7-benzyl-1,7-dihydro-purin-6-one, and 3- (pyridin-2-ylmethyl) -2-thioxo-2,3-dihydro-1H-quinazoline -4-one.
7). 5. A therapeutic agent for inflammatory diseases involving mast cells, comprising a degranulation inhibitor identified by the method according to 3 or 4 as an active ingredient.
8). 8. The therapeutic agent for inflammatory diseases involving mast cells according to 7 above, which contains at least one compound selected from the following as an active ingredient.
1- (10H-phenazin-5-yl) -ethanone,
2-azepan-1-yl-7-benzyl-1,7-dihydro-purin-6-one, and 3- (pyridin-2-ylmethyl) -2-thioxo-2,3-dihydro-1H-quinazoline -4-one.
9. A mast cell degranulation inhibitor comprising a substance having a Rec168 antagonistic action as an active ingredient.
10. A therapeutic agent for inflammatory diseases involving mast cells comprising a substance having Rec168 antagonistic action as an active ingredient.
11. A diagnostic agent for inflammatory diseases involving mast cells, comprising as a reagent, Rec168 protein or a partial peptide thereof and a salt thereof, a polynucleotide encoding Rec168, an antibody against Rec168 or an antibody against Rec168 ligand.

  The background to the completion of the above invention is as follows.

Using the public sequence database GenBank, Rec168 having a sequence characteristic of GPCR was found. Reverse transcription was performed using human testis poly (A) ⁺ RNA as a template to synthesize cDNA. The gene of interest was amplified by PCR and incorporated into an expression vector to produce Rec168 transient expression cells and stable expression cells.

In order to identify ligands of GPCRs whose ligands have not been identified (that is, agonists and antagonists that interact with the receptor), it is necessary to screen a large number of tens of thousands of candidate substances by testing. There is a need for an assay system that can perform the screening rapidly. In addition, the assay quantitatively detects the presence or absence of interaction between the test substance and the receptor, and the increase or decrease in activation or suppression of second messenger induced through the G protein to which the receptor is coupled. Therefore, there is a need for an assay method that can detect increase and decrease of the message with high sensitivity. The present inventors have already obtained a patent for such an assay method that can be detected quickly and with high sensitivity (Japanese Patent No. 3643288), and the assay method was also used in the present invention.

Using the prepared Rec168-expressing cells, screening for a substance showing agonist activity against Rec168 was performed. As a result, porcine
Peptide consisting of N-terminal 20 amino acids of Chaperonin 10, N-terminal 17 amino acids of porcine 2 ', 3'-cyclic nucleotide 3'phosphodiesterase (hereinafter abbreviated as pCNP), Peptide consisting of N-terminal 12 amino acids of porcine tropomyosin, PACAP (6-36), VIP (3-27), Substance P, Somatostatin, Mast cell degranulating (hereinafter abbreviated as MCD) peptide, and the like were obtained.

As described above, MCD peptide, Substance P, VIP, Somatostatin, compound 48/80, etc. are known as basic secretagogues as non-immune-related mast cell degranulation inducers. Substance P, VIP, Somatostatin
Are known to cause degranulation at a much higher concentration (2 to 3 μM) than previously known receptors, but the mechanism of degranulation of non-immune-related mast cells by Basic secretagogues is still It was unknown. However, from a detailed analysis, a part of a group of substances showing agonistic activity against Rec168 and a substance known as a substance for inducing degranulation of non-immune-related mast cells, obtained by our original screening, It was clarified that the action concentration was almost the same. Therefore, we developed a hypothesis that no one had ever predicted that degranulation of non-immune-related mast cells might be mediated by Rec168, and we further studied.

The agonistic substance of Rec168 obtained from screening by reporter gene assay showed almost the same reactivity even when measured by an intracellular free calcium assay system. Moreover, as a result of selecting several types from those showing agonist activity and examining GTP binding activity, all of the examined agonists showed GTP binding activity. Therefore, by binding to Rec168, intracellular binding via G protein occurs. Signaling was strongly suggested.

  Next, it was examined whether the obtained agonist active substance group actually induces degranulation through Rec168 on mast cells. Human cord blood-derived CD34-positive hematopoietic stem cells were isolated, differentiated into mast cells by conventional methods, and each agonist active substance was added in the absence of fetal calf serum (FCS), depending on the concentration of each substance Degranulation reaction was triggered. In contrast, degranulation was not induced in the presence of FCS. When the expression of Rec168 in mast cells in the presence or absence of FCS was examined, it was found that it was expressed in the absence of FCS but hardly expressed in the presence of FCS. From the above, it was strongly suggested that the Rec168 agonist active substance group caused a degranulation reaction of mast cells, and that the reaction occurred via Rec168.

Next, when Rec168 expression organ distribution analysis was performed, this receptor was characterized by high expression in skin and adipose tissue, unlike the expression pattern of other receptors belonging to the MrgX family. It has been confirmed that many mast cells are present in the epidermis. The existence of mast cells in adipose tissue is not clear, but it is fully possible that mast cells are mixed in adipose tissue.

The fact that Rec168 is a receptor that transmits degranulation signals has never been anticipated by anyone, and for the first time one of the mechanisms of non-immune degranulation without IgE involvement is revealed. The present invention is extremely important also in that it has become. Since the onset mechanism was unknown so far, it became possible to proceed with the development of effective therapeutic agents targeting many chronic inflammations for which no effective therapeutic agents or methods were found.

Therefore, the present inventors have further researched and developed a screening method for a drug that can suppress the degranulation reaction from mast cells mediated by Rec168. I succeeded in finding it.

  The present inventors have found a physiological function of Rec168 that has not been clarified so far, developed a screening method and identification method for a new drug based on the function, and actually obtained a plurality of compounds that can be such a drug. Succeeded. According to the method for screening a substance having an inhibitory action on signal transduction mediated by Rec168 protein or the method for identifying a substance having an inhibitory action on signal transduction mediated by Rec168 protein in the present invention, the substance that simply regulates the binding ability of a substance that binds to Rec168 In contrast to obtaining substances that modulate signal transduction ability from substances that transmit signals in artificial cell systems, acquiring substances that actually suppress the degranulation reaction that is the physiological function of Rec168. Became possible. This has made it possible to develop therapeutic agents for inflammatory diseases such as allergic diseases or autoimmune diseases that have been impossible until now, such as inflammatory diseases involving degranulation of mast cells that do not involve immunity. .

Furthermore, the present invention also provides a diagnostic agent for a therapeutic agent for inflammatory diseases involving mast cells such as allergic diseases or autoimmune diseases. This is possible for the first time by finding that Rec168 induces degranulation of mast cells. Increasing or decreasing the expression level of Rec168 protein on the cell surface or gene expression, structural abnormality of the expressed Rec168 protein, or gene mutation may cause chronic inflammation via excessive degranulation reaction, By examining these, it has become possible to diagnose inflammatory diseases involving mast cells, particularly allergic diseases or autoimmune diseases.
Furthermore, it is possible to screen for substances capable of regulating the abnormal expression of Rec168 protein and Rec168 gene that cause inflammatory diseases involving mast cells, particularly allergic diseases or autoimmune diseases, and are obtained by this method. The substance is capable of regulating the degranulation reaction via Rec168. This has made it possible to develop new types of therapeutic agents for inflammatory diseases.

Hereinafter, the present invention will be described in more detail by clarifying the meanings of the terms used in the present invention and specific embodiments of the present invention.

G protein-coupled receptor The "G protein-coupled receptor" in the present invention is a receptor that exists on the cell membrane, and is a regulatory protein that binds to GTP (guanosine 5'-triphosphate) ( G protein) means a protein having a function of transmitting information on a ligand that interacts with the receptor to an effector system. Most G protein-coupled receptors identified so far have a structure that penetrates the cell membrane seven times.

Cells expressing Rec168 In the present invention, “ cells expressing Rec168” are not particularly limited as long as they are cells expressing Rec168 protein on the cell surface.

For example, as a cell expressing Rec168, a cell line in which the Rec168 gene is introduced into an existing cell line and forcedly expressed on the cell surface can also be used. In this case, the parent strain can be used as a cell that does not express Rec168 for comparison. The existing cell line used here is not particularly limited as long as it contains an expression vector and can maintain the replication or expression of the expression vector. For example E.
Prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insects, and amphibians, or mammalian cells such as CHO, HeLa, and HEK-293. Among the cells that can efficiently express Rec168, HEK293 cells are preferred.

  At this time, as the Rec168 forced expression cell, a cell that transiently expresses Rec168 or a cell that stably expresses may be used. Note that Rec168 forced expression cells can be prepared by methods known to those skilled in the art.

  Moreover, as a cell expressing Rec168, a cell derived from a living body and expressing Rec168 natively can also be used. The cells are not particularly limited as long as they express Rec168, but mast cells are particularly preferable.

Here, the mast cell in the present invention may be a primary cultured cell isolated from a living body or an existing cell line that retains the characteristics of a mast cell. For example, as the primary cultured cells, mast cells obtained by induction from human umbilical cord blood-derived CD34-positive hematopoietic stem cells according to a conventional method can be used. Further, the existing cell line is not particularly limited, and examples thereof include P815 and RBL-2H3 cells.

Substances “Substances” identified or screened by the method of the present invention are natural substances that exist in nature (proteins, antibodies, peptides, natural compounds, etc.) or any artificially prepared substances (chemically synthesized compounds). Means.

Specifically, for example, any “synthesized” chemically synthesized can be mentioned. The type and molecular weight of the compound are not particularly limited, but as far as the molecular weight is concerned, the molecular weight of a compound that may be used as a pharmaceutical is about 50 to about 3000, more generally about 100 molecular weight. To about 2000, more typically about 100 to about 1000 molecular weight.

  When the substance is a protein, antibody or peptide, it includes those isolated from biological tissues and cells, and those prepared by genetic recombination or chemical synthesis. Furthermore, those chemical modifications are also included.

  Examples of the peptide include peptides consisting of about 3 to about 500 amino acids, preferably about 3 to about 300 amino acids, and more preferably about 3 to about 200 amino acids.

Protein, peptide, polypeptide Rec168 used in the present invention is a receptor protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. Rec168 is a known protein described in a plurality of documents (for example, WO0116159, WO0119983, WO0172838, etc.).

  Rec168 is, for example, any cell of a human or other mammal (eg, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, etc.) (eg, retinal cell, spleen cell, nerve cell, glial cell, pancreas) β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells) , Mast cells, neutrophils, basophils, eosinophils, monocytes, leukocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or between Or precursor cells of these cells, stem cells or cancer cells (eg, breast cancer cell line (GI-101), colon cancer cell lines (CX-1, GI-112), lung cancer cell lines (LX-1, GI) 117), ovarian cancer cell lines (GI-102), prostate cancer cell lines, etc.), or any tissue in which these cells are present, for example, the brain, each part of the brain (eg, olfactory bulb, skull nucleus, cerebrum) Basal sphere, hippocampus, thalamus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain dye, nigro), spinal cord, pituitary gland, stomach , Pancreas, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells , Prostate, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., or cells of the blood system or cultured cells thereof (eg, MEL, M1, CTLL-2, HT-2, WEHI-3, HL) -60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4 , MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL, JK-1 , CMK, KO-812, MEG-01, etc.) or a synthetic protein.

  In the present specification, the “substantially identical amino acid sequence” means, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably, with respect to the amino acid sequences to be compared. An amino acid sequence having about 95% or more homology. As used herein, Rec168 is a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1, for example, substantially the same as the amino acid sequence represented by SEQ ID NO: 1. A protein containing an amino acid sequence and having substantially the same activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is preferred.

Examples of substantially the same activity include ligand binding activity and signal information transmission action. “Substantially homogeneous” means that their activities are homogeneous in nature. Accordingly, the activities such as ligand binding activity and signal signal transduction 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 activity and the molecular weight of the protein may be different.

Measurement of activities such as ligand binding activity, receptor binding activity and signal information transmission activity can be carried out according to a known method, for example, according to a screening method described later.

As Rec168, (1) 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 2) in the amino acid sequence represented by SEQ ID NO: 1. 5)) amino acid sequence deleted, (2) 1 or 2 (preferably about 1-30, more preferably about 1-10) in the amino acid sequence represented by SEQ ID NO: 1, More preferably, an amino acid sequence to which several (1 to 5) amino acids are added, (3) one or more (preferably about 1 to 30 or more) in the amino acid sequence represented by SEQ ID NO: 1 Preferably about 1 to 10, more preferably several (1 to 5) amino acids are substituted with other amino acids, or (4) a protein containing an amino acid sequence combining them. Used.

Rec168 in the present specification is the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide notation. In Rec168, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), 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, cyclohexyl, for example, phenyl C _6-12 aryl groups such as α-naphthyl, C _7- such as phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl _In addition to the ₁₄ aralkyl group, a pivaloyloxymethyl group, which is widely used as an oral ester, is used.

  When Rec168 has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in Rec168 in the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.

Further, in Rec168, in the above protein, the amino group of the N-terminal methionine residue is protected with a protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl). N-terminal side cleaved in vivo, glutamyl group produced by pyroglutamine oxidation, substituent on amino acid side chain in the molecule (for example, —OH, —SH, amino group, imidazole group, indole) A group, a guanidino group, etc.) protected with an appropriate protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl), or a so-called sugar chain bound Also included are complex proteins such as glycoproteins.

  Specific examples of Rec168 include human Rec168 consisting of the amino acid sequence represented by SEQ ID NO: 1. Rec168 or a salt thereof can be produced from a human or mammalian cell or tissue by the known method for purifying a receptor protein, or a transformant containing DNA encoding Rec168 or Rec168 described later is cultured. Can also be manufactured. Moreover, it can also manufacture according to the protein synthesis method described later or this.

  When producing from human or mammalian tissue or cells, the human or mammalian tissue or cells are homogenized and then extracted with acid, etc., and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.

  The partial peptide of Rec168 (hereinafter sometimes simply referred to as “partial peptide”) may be any peptide as long as it has the above-mentioned partial amino acid sequence of Rec168. Of these, those exposed to the outside of the cell membrane and having substantially the same receptor binding activity as Rec168 are used.

  Specifically, the Rec168 partial peptide having the amino acid sequence represented by SEQ ID NO: 1 is a peptide containing a portion analyzed to be an extracellular region (hydrophilic site) in a hydrophobicity plot analysis. is there. In addition, a peptide partially including a hydrophobic site can also be used. Peptides containing individual domains can also be used, but a peptide containing a portion containing a plurality of domains may be used.

  The number of amino acids of the partial peptide is preferably a peptide having an amino acid sequence of at least 20 or more, preferably 50 or more, more preferably 100 or more of the above-described Rec168 constituent amino acid sequence.

The partial peptide has one or two or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence, or the amino acid sequence thereof. 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids, or in the amino acid sequence One or more amino acids (preferably about 1 to 10, more preferably about several, and more preferably about 1 to 5) may be substituted with other amino acids. The partial peptide may have a carboxyl group (—COOH), carboxylate (—COO ⁻ ), amide (—CONH ₂ ) or ester (—COOR) at the C-terminus.

  Furthermore, in the partial peptide, similar to Rec168 described above, the amino group of the N-terminal methionine residue is protected with a protecting group, and the Gln produced by cleavage of the N-terminal side in vivo is pyroglutamine oxidized. Also included are those in which a substituent on the side chain of an 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.

  Examples of the salt of Rec168 or a partial peptide thereof include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

A partial peptide of Rec168 or a salt thereof can be produced according to a known peptide synthesis method or by cleaving Rec168 with an appropriate peptidase. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute Rec168 is condensed with the remaining portion, and when the product has a protecting group, the target peptide can be produced by removing the protecting group. Examples of known condensation methods and protecting group elimination include the methods described in the following a) to e).
a) M.M. Bodanszky and M.M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
b) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
c) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
d) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
e) Supervision by Yajima Haruaki, Development of follow-up medicines Volume 14: Peptide synthesis Hirokawa Shoten In addition, after the reaction, this method combines ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. The partial peptide of the invention can be purified and isolated. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. Can do.

Polynucleotide The present invention relates to a nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 1 and one or more amino acids of the amino acid sequence represented by SEQ ID NO: 1 substituted, deleted, inserted, and / or A polynucleotide encoding the amino acid sequence of a polypeptide consisting of the added amino acid sequence is also provided.

The polynucleotides of the present invention also include both single-stranded and double-stranded DNA, and RNA complements thereof. The DNA includes, for example, naturally-derived DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. As the polynucleotide of the present invention, DNA is preferred. As is well known, codons are degenerate and some amino acids have multiple base sequences that encode one amino acid, but any base sequence that encodes the above amino acid sequence has any base sequence. Is also included in the scope of the present invention. The base sequence of the cDNA actually cloned in Example 1 below is shown in SEQ ID NO: 2. Therefore, the polynucleotide of the present invention preferably has the base sequence of SEQ ID NO: 2.

The polynucleotide of the present invention can be prepared, for example, by the following method. The base sequence is searched using the base sequence characteristic of Rec168 as a query, and Rec168 is found from the human genome. Using the found genomic sequence or a part thereof, the polynucleotide of the present invention (for example, the DNA of the present invention) is prepared from a cDNA library or the like using basic genetic engineering techniques such as hybridization and nucleic acid amplification reaction. be able to.

Here, the nucleic acid amplification reaction is, for example, polymerase chain reaction (PCR) [Saiki
R. K. et al. , Science, 230, 1350-1354 (1985)], Ligates chain reaction (LCR) [Wu D. et al. Y. et al. Genomics, 4, 560-569 (1989);
Barringer K.M. J. et al. et al. Gene, 89, 117-122 (1990); Barany F .; , Proc. Natl. Acad. Sci. USA, 88, 189-193 (1991)], and transcription-based amplification [Kwoh
D. Y. et al. , Proc. Natl. Acad. Sci. USA, 86, 1173-1177 (1989)] and other reactions that require temperature cycling, as well as strand displacement reactions (SDA) [Walker
G. T.A. et al. , Proc. Natl. Acad. Sci. USA, 89, 392-396 (1992); Walker G. et al. T.A. et al. , Nuc. Acids
Res. , 20, 1691-1696 (1992)], self-retaining sequence replication (3SR) [Guatelli J. et al. C. , Proc. Natl. Acad. Sci. USA, 87, 1874-1878 (1990)], and the Qβ replicase system [Rizildi et al., BioTechnology, 6, 1971-2202 (1988)]. Further, amplification based on nucleic acid sequence by competitive amplification of target nucleic acid and mutant sequence described in European Patent No. 0525882 (Nucleic
An Acid Sequence Based Amplification (NASABA) reaction or the like can also be used. The PCR method is preferred.

  As shown in Example 1, when the primers of SEQ ID NOs: 3 and 4 are used, a 1.0-kbp DNA fragment is obtained as a PCR product, and this is screened with a molecular weight such as agarose gel electrophoresis. The nucleic acid of the present invention can be obtained by isolation according to a conventional method such as separation by a method and cutting out a specific band.

  The homologous nucleic acid cloned using the hybridization, nucleic acid amplification reaction or the like as described above is at least 20% or more, preferably 30% or more, more preferably with respect to the base sequence described in SEQ ID NO: 1 in the Sequence Listing. Has an identity of 50% or more, even more preferably 70% or more, and most preferably 90% or more.

The percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by Devereux et al., Nucl. Acids
Res. 12, 387 (1984) and can be determined by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). Preferred default parameters for the GAP program include: (1) a single unary comparison matrix for nucleotides (including values of 1 for identical and 0 for nonidentical), and supervised by Schwartz and Dayhoff, Atlas
of Protein Sequence and Structure, pp. 353-358, National Biomedical Research Foundation (1979), Gribskov and Burgess, Nucl. Acids
Res. , 14, 6745 (1986); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for the end gap. included. Other programs for sequence comparison used by those skilled in the art can also be used.

  The present invention provides a nucleotide having a chain length of at least 15 nucleotides, which is complementary to the polynucleotide identified by the present inventors (polynucleotide comprising the base sequence described in SEQ ID NO: 2 or its complementary strand). . Here, “complementary strand” refers to the other strand with respect to one strand of a double-stranded nucleic acid consisting of A: T (U in the case of RNA) and G: C base pairs. Further, the term “complementary” is not limited to a case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is just to have the homology on the above base sequences. The algorithm described in the present specification may be used as an algorithm for determining homology. Such a nucleotide can be used as a probe for detecting and isolating the polynucleotide of the present invention and as a primer for amplifying the nucleotide of the present invention. When used as a primer, it usually has a chain length of 15 to 100 nucleotides, preferably 15 to 35 nucleotides. When used as a probe, a polynucleotide having a chain length of at least 15 nucleotides, preferably at least 30 nucleotides, containing at least a part or all of the sequence of the DNA of the present invention is used. Such a polynucleotide preferably hybridizes specifically to a polynucleotide encoding the polypeptide of the present invention. “Specifically hybridize” means to hybridize with the polynucleotide (SEQ ID NO: 1 or 3) identified by the present inventors under normal hybridization conditions, preferably under stringent conditions, This means that it does not hybridize with DNA encoding another polypeptide.

As used herein, “under stringent conditions” means to hybridize under moderate or high stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art based on, for example, the length of DNA. Basic conditions are described in Sambrook, J. et al. Et al, Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, 7.42-7.45 (2001), and for nitrocellulose filters, 5 × SSC, 0.5% SDS, Pre-wash solution of 0 mM EDTA (pH 8.0), about 50% formamide at about 40-50 ° C., Stark solution (Stark in 2 × SSC-6 × SSC (or about 50% formamide at about 42 ° C.) use of other similar hybridization solutions) such as' s solution) and wash conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS. High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA. In general, such conditions include hybridization and / or washing at higher temperatures and / or lower salt concentrations than moderately stringent conditions, eg, hybridization conditions as described above, and approximately 68 ° C. Defined with 0.2 × SSC, 0.1% SDS wash. One skilled in the art will recognize that the temperature and wash solution salt concentration can be adjusted as needed according to factors such as the length of the probe.

  This polynucleotide also includes a polynucleotide that suppresses the expression of the gene encoding Rec168. Such polynucleotides include antisense polynucleotides (antisense DNA / RNA; antisense RNA complementary to the transcript of the gene encoding the polypeptide of the present invention, and DNA encoding the RNA) and ribozymes ( DNA encoding an RNA having ribozyme activity that specifically cleaves the transcript of the gene encoding the polypeptide of the present invention.

There are several factors as described below for the action of an antisense polynucleotide to suppress the expression of a target gene. That is, transcription initiation inhibition by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Of splicing by hybrid formation at the junction with the protein, suppression of splicing by hybridization with the spliceosome formation site, suppression of transition from the nucleus to the cytoplasm by hybridization with mRNA, hybridization with capping sites and poly (A) addition sites Splicing suppression by formation, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, peptization by hybridization with the mRNA translation region and polysome binding site For example, inhibition of gene chain elongation is prevented, and hybridization between nucleic acid and protein interaction sites is performed. They inhibit transcription, splicing, or translation processes and suppress target gene expression (Hirashima and Inoue, "Neurochemistry Experiment Course 2, Nucleic Acid IV Gene Replication and Expression", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin , pp.319-347, 1993).

The antisense polynucleotide used in the present invention may suppress the expression of the target gene by any of the actions described above. As one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene is designed, it is considered effective for inhibiting the translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, a polynucleotide containing an antisense sequence of not only a translation region of a gene but also a sequence of an untranslated region is also included in the antisense polynucleotide used in the present invention. The antisense polynucleotide to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The antisense polynucleotide sequence is preferably a sequence that is complementary to the target gene or a part thereof, but may not be completely complementary as long as it can effectively inhibit the expression of the gene. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene. In order to effectively inhibit the expression of the target gene using the antisense sequence, the antisense polynucleotide is at least 15 nucleotides, preferably 100 nucleotides, more preferably 500 nucleotides or more, in order to cause an antisense effect. The chain length is usually within 3000 nucleotides, preferably within 2000 nucleotides.

The antisense polynucleotide is, for example, a phosphorothioate method (Stein, 1988 Physicochemical properties of phosphorothioate) based on the sequence information of a polynucleotide encoding Rec168 (eg, SEQ ID NO: 2).
oligodeoxynucleotides. Nucleic Acids Res 16, 3209-21 (1988)).

Suppression of endogenous gene expression can also be performed using a polynucleotide encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type and a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, (1990) Protein Nucleic Acid Enzymes, 35: 2191).

For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, it has been shown to be cleaved by A or U (M. Koizumi et al., (1988) FEBS Lett. 228: 225). If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. (M.Koizumi et al., (1988) FEBS Lett. 239: 285, Makoto Koizumi and Eiko Otsuka, (1990)
Protein Nucleic Acid Enzyme, 35: 2191, M. Koizumi et al. (1989) Nucleic Acids Res. 17: 7059).

Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of tobacco ring spot virus satellite RNA (JMBuzayan Nature
323: 349,1986). This ribozyme has also been shown to be designed to cause target-specific RNA cleavage (Y. Kikuchi and N. Sasaki (1992) Nucleic Acids
Res. 19: 6751, Hiroshi Kikuchi, (1992) Chemistry and Biology
30: 112).

Polynucleotides that suppress the expression of the gene encoding Rec168, for example, use viral vectors such as retroviral vectors, adenoviral vectors, and adeno-associated viral vectors, and non-viral vectors such as liposomes when used for gene therapy. Then, it is conceivable to administer to a patient by ex vivo method or in vivo method.

Antibodies The present invention provides antibodies that bind to Rec168. As used herein, “antibodies” include polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and Fab fragments including the products of Fab or other immunoglobulin expression libraries.

Rec168 or fragments or analogs thereof or cells expressing them can also be used as an immunogen to produce antibodies that bind to Rec168. The antibody is preferably immunospecific for Rec168. “Immunospecific” means that the antibody has a substantially higher affinity for Rec168 than its affinity for other polypeptides.

   Examples of the antibody in the present invention include those that can detect Rec168 and have an activity that affects the function of Rec168. Here, the function of Rec168 means a binding function of a ligand substance and a function of inducing a cell degranulation reaction by binding of the ligand substance. Therefore, the antibody as a drug in the present invention is one that partially or completely inhibits the binding between Rec168 and a ligand substance, one that suppresses or enhances part or all of the cell degranulation reaction that occurs through Rec168. Things are included.

Antibodies against Rec168 can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. Serum is obtained by immunizing small animals such as rabbits with Rec168 or a fragment thereof or a fusion protein thereof with GST. This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the polypeptide of the present invention, and the like. In the case of a monoclonal antibody, for example, Rec168 is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate cells and fused with a mouse myeloma cell using a reagent such as polyethylene glycol. From these fused cells (hybridomas), a clone that produces an antibody that binds to Rec168 and affects the function of Rec168 is selected. Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purification using an affinity column coupled with a polypeptide.

The antibody used for detection of Rec168 contained in the inflammatory disease diagnostic agent and inflammatory disease diagnostic kit of the present invention is not particularly limited as long as it is a detectable antibody. It can be prepared by a known method.

Specifically, a polyclonal antibody can be obtained, for example, as follows. Serum is obtained by immunizing small animals such as rabbits with Rec168 or a fragment thereof or a fusion protein thereof with GST. This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the polypeptide of the present invention, and the like. In the case of a monoclonal antibody, for example, Rec168 is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate cells and fused with a mouse myeloma cell using a reagent such as polyethylene glycol. From these fused cells (hybridomas), a clone producing an antibody that binds to Rec168 is selected. Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purification using an affinity column coupled with a polypeptide.

Agent, Drug "Agent" or "Drug", "Pharmaceutical composition" of the present invention means any of the "Compound", "Peptide", "Polynucleotide" or "Antibody" of the present invention as defined above, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.
The “compound” in these “agent” or “drug” and “pharmaceutical composition” means a natural or synthetic compound. Similarly, the “peptide” means a compound in which a plurality of amino acids are bonded to each other, and may be an oligo-peptide or a polypeptide. “Polynucleotide” means a polymer in which about 30 or more nucleotides are polymerized. “Antibody” means so-called immunoglobulin (Ig).
These pharmaceutical compositions of the present invention are effective as therapeutic agents for inflammatory diseases described later, particularly inflammatory diseases involving mast cells.

As used herein, “pharmaceutically acceptable carrier” refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners. , Flavoring agents, solubilizers or other additives. By using one or more of such carriers, pharmaceuticals in the form of tablets, pills, powders, granules, injections, solutions, capsules, trowels, elixirs, suspensions, emulsions or syrups, etc. A composition can be prepared. These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include topical solutions containing one or more active substances and prescribed by conventional methods, suppositories for enteric administration, pessaries, and the like.

The dose varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, or the type of active ingredient (such as the aforementioned polypeptide or antibody) contained in the pharmaceutical composition. The dose can be administered in the range of 10 μg to 1000 mg (or 10 μg to 500 mg) per adult. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be required.

Particularly in the case of an injection, the concentration is 0.1 μg antibody / ml carrier to 10 mg antibody / ml carrier in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection. So that it can be dissolved or suspended. The injection thus produced is 1 μg to 100 mg per kg body weight, preferably 50 μg to 50 mg, per day for a human patient in need of treatment. Can be administered several times to several times. Examples of administration forms include medically suitable administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection. Intravenous injection is preferred.

In addition, injections can be prepared as non-aqueous diluents (for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions or emulsions. .

Such sterilization of the injection can be performed by filtration sterilization through a bacteria-retaining filter, blending of a bactericide, or irradiation. The injection can be produced as a form prepared at the time of use. That is, it can be used as a sterile solid composition by lyophilization, etc., and dissolved in sterile water for injection or other solvents before use.

The “agent” or “drug” to “pharmaceutical composition” of the present invention can be applied to the treatment and prevention of diseases involving degranulation of mast cells, such as inflammatory diseases. It is particularly effective for chronic inflammation such as allergic diseases and autoimmune diseases.
Further details of allergic diseases are as follows.
As is well known, allergic inflammation includes invasion of antigen into the body, macrophagia and antigen presentation by macrophages, Th antigen recognition and IL-4 production, B cell stimulation and IgE antibody production, FcεRI binding and degranulation It develops through the process of progressing to response.
These allergic inflammations are as follows in terms of pathology.
(1) Atopic dermatitis with enhanced IgE production (2) Activation by non-specific stimulation without IgE (Substance P, compound 48/80)
(3) Various inflammation images by mast cell release chemical mediators and accumulation of inflammatory cells by cell migration activity (4) Tissue damage by eosinophils free substance (5) Inflammatory cell function by cytokines from locally infiltrated T cells Modification (Eosinophil migration and activation from Th2 cells by IL-3, IL-5, GM-CSF, etc.)
(6) Induction of adhesion molecules on the surface of inflammatory cells such as vascular endothelial cells and eosinophils by cytokines from T cells and mast cells These inflammations are localized in the body when some harmful inflammatory substance acts on living tissue. It is a reaction and a process of biological defense. Increases vascular permeability, leaches leukocytes locally, leaks protective factors such as plasma locally, promotes blood clotting, occludes blood vessels, reduces local oxygen concentration, and suppresses pathogen growth and toxin diffusion To prevent.
The following phenomena are monitored with these inflammatory reactions.
(1) tissue injury;
This can be caused by pathogenic organisms, physical stimuli (burns) or chemical stimuli (acid corrosion), foreign matter, or antigen-antibody reactions.
(2) Changes in microvessels;
The microvasculature temporarily contracts and then expands. As a result, the tight junction gap between vascular endothelial cells spreads in the venule and plasma infiltrates, and then inflammatory cells migrate out of the blood vessel. Histamine and prostaglandins act on endothelial cells and inflammatory cells.
(3) Restoration;
When macrophages phagocytose, the inflammatory substance is removed, and granulation tissue is formed by the proliferation and angiogenesis of fibroblasts. Damaged parenchymal cells may regenerate.
(4) Chronization;
Chronicity occurs when the substance is not removed during repair.
These inflammations are reactions that a living body exhibits when an inflammatory substance acts, and this is accompanied by an antigen-antibody reaction. Since the reaction of Rec168 is non-immune, allergic inflammation is activated by non-specific stimulation without using IgE (Substance P, compound 48/80) and inflammation caused by burn injury and foreign substances “Mixed” (for example, bee venom, bacterial infection, etc.).
The following diseases can be exemplified as diseases particularly related to mast cells.
(1) Gastritis, gastric ulcer:
A significant increase in mast cells was observed, and an electron microscopic finding of mast cell degranulation was observed.
(2) Cirrhosis:
Increased mass of mast cells around the bile duct, involved in fibrosis of the liver, regulation of intrahepatic microcirculation and angiogenesis (3) Chronic biliary diseases (intrahepatic calculi, primary sclerosing cholangitis):
There is fibrosis of the bile wall of the intrahepatic large bile duct and septal bile duct and fibrosis around the bile duct. Increase in peribile mast cells. Production of cytokines such as bFGF involved in fibrosis, other TNF-α, IL-1, and TGF-β is observed in the mast cells.
(4) Ulcerative colitis:
There are few normal mast cells in the colonic mucosa of patients with ulcerative colitis in the active phase, and the morphology after degranulation is shown.
(5) Chronic inflammatory vascular disease: excessive production of angiotensin II, which is considered to occur due to the increase and degranulation of mast cells and overexpression of mast cell chymase (6) arteriosclerosis:
Mast cells were found in human coronary artery lesions, and many activated mast cells were present in atherosclerotic vascular endothelial plaques.
(7) Cardiac anaphylaxis:
Mast cells are present in the human heart, particularly around blood vessels and in the vicinity of the muscular sheath, and in the arteriosclerotic lesion. Mast cell activation due to type I allergy.
(8) Pulmonary fibrosis:
Mast cells increase in accordance with the location of collagen fiber deposition. Confirmation of the presence of bFGF positive mast cells.
(9) Bronchial asthma:
Increased mast cells, appearance of degranulation.
(10) Nephritis (tubulointerstitial nephritis, glomerulonephritis):
Increased mast cells in the connective tissue around the tubules, renal interstitial blood vessels, and around the glomeruli. Confirmation of the presence of IL-4 positive mast cells.
(11) Interstitial cystitis:
Increase in mast cells. Increase in urine of neuropeptides such as Substance P (12) Atopic dermatitis:
Increase in mast cells, production of TNF-α and IL-4. Substance P secreted from the C fiber nerve ending induces activation and degranulation of new mast cells.
(13) Keloid:
Increased collagen fiber components in the dermis, increased mast cells and degranulation. Effective for treatment and prevention of membrane stabilizer tranilast.
(14) Vascular proliferative disease (Kaposi's sarcoma, malignant vascular endothelial cell type):
Increase in mast cells and confirmation of the presence of bFGF positive mast cells.
(15) Rheumatoid arthritis:
Mast cell increase and degranulation in the synovium. Demonstration of mast cell involvement by mice lacking mast cells.
(16) Allergic rhinitis:
Increase in mast cells (17) Allergic conjunctivitis:
Mast cell proliferation (18) Gastric cancer:
Mast cells infiltrate around stomach cancer, and prognosis is worse when there are many mast cells. Involved in tumor angiogenesis.
(19) Malignant tumors such as colorectal cancer, breast cancer, basal cell carcinoma of the skin, soft tissue tumor, melanoma:
Accumulation of mast cells in the stroma (20) Non-small cell lung cancer:
Accumulation of mast cells in cancer stroma, possible involvement in angiogenesis, expression of VEGF in mast cells, ie, diseases involving mast cells, as described above, gastritis, gastric ulcer: cirrhosis: chronic biliary system Disease (intrahepatic lithiasis, primary sclerosing cholangitis): Ulcerative colitis: Chronic inflammatory vascular disease: Arteriosclerosis: Cardiac anaphylaxis: Pulmonary fibrosis: Bronchial asthma: Nephritis (tubulointerstitial nephritis, glomerulonephritis) ): Interstitial cystitis: Atopic dermatitis: Keloid: Vascular proliferative disorder (Kaposi's sarcoma, malignant vascular endothelial cell type): Rheumatoid arthritis: Allergic rhinitis: Allergic conjunctivitis: Gastric cancer: Colorectal cancer, Breast cancer, Malignant tumors such as basal cell carcinoma of the skin, soft tissue tumors, melanoma:
Non-small cell lung cancer: Others include urticaria, systemic lupus erythematosus, multiple sclerosis, collagen disease, malignant tumor, B lymphoma, B cell leukemia and the like.

Further, the therapeutic effect of various drug symptoms of the “agent” or “drug” of the present invention can be tested and examined by administering it to a known disease model animal according to a conventional method.

Signal transduction ability of ligand having Rec168 agonistic action In the present invention, the term "ligand having Rec168 agonistic action" refers to a substance that can transmit a signal into cells via Rec168, and the type of signal. The type of substance is not particularly limited. The “signal transduction ability” referred to here is expressed by the degree of signal that a ligand substance having a Rec168 agonistic action can transmit into the cell via Rec168. There are no particular limitations on the type, type of ligand substance, and the like.

For example, as the step of measuring the signal transduction ability, an assay using a reporter gene as described below (reporter gene assay) (International Patent Application Publication No. WO92 / 02639, JP 6-502527, etc.) may be used. it can. Here, the reporter gene assay is a method in which a test substance is brought into contact with a cell, and the amount of a reporter protein expressed depending on the action of the compound is indirectly measured by measuring the amount of fluorescence emitted from the protein. This is a method for analyzing the presence or absence of interaction of the test substance with the G protein-coupled receptor. In addition, the reporter gene assay can be performed manually, but a so-called high throughput screening (tissue culture engineering,
Vol. 23, No. 13, p. 521-524; U.S. Pat. No. 5,670,113) can be carried out more quickly and easily.

  For example, the following can be considered as a step of measuring the signal transduction ability via Rec168 of a test substance using a reporter gene assay or a step of selecting a ligand having a Rec168 agonistic action.

Steps in a method for screening a substance to identify a ligand for Rec168, comprising the following steps (a) to (c):
(A) At least exogenous genes (1) and (2) below:
(1) a gene encoding Rec168; and (2) a reporter gene operably linked to the promoter region of a gene whose expression is induced by stimulation via Rec168;
Preparing a plurality of samples consisting of a constant number of PC12-derived cells having: and contacting each of the samples with a different substance;
(B) For each sample contacted with the substance in step (a), the detectable signal generated by the reporter gene expressed in each cell in the sample, and the substance not contacted with any substance Quantitatively determining each of the detectable signals produced by the reporter gene expressed in each cell of the sample; and
(C) A step of comparing the amounts of the respective signals determined in step (b).

In this step, the promoter region of a gene whose expression is induced by stimulation via Rec168 includes the promoter region of an immediate-early gene. Examples of the promoter of the earliest gene used in the present invention include c-fos promoter region and zif268 promoter region (Proc. Natl. Acad. Sci. USA., Vol. 86, p. 377-381, 1989; “EGR -1 "," NGF1-A "," Krox24 "," Tis8 "or" cef5 "). A particularly preferred embodiment is the zif268 (EGR-1) promoter region. In addition, the promoter used in the present invention includes the counterpart of any animal species of the promoter.

  Here, the “promoter region” means any region containing the minimum base sequence essential for expressing promoter activity. For example, it is possible to use a part or all of the region of about 500 bp to about 2 kb upstream from the transcription site of the gene. Further, the title promoter region in the present invention includes a promoter region having a transcription factor binding sequence SRE (serum response element) and / or CRE (cAMP response element) upstream of the minimal promoter.

  The “reporter gene” in this step means a gene encoding a reporter protein that emits detectable fluorescence. Specific examples include luciferase derived from firefly or Renilla, GFP (Green Fluorescence Protein) derived from jellyfish, and the like. Further examples include a gene encoding β-galactosidase, a gene encoding chloramphenicol acetyltransferase, and a gene encoding β-lactamase.

“PC12-derived cells” in this step refers to a rat adrenal pheochromocytoma-derived PC12 cell line (ATCC CRL-1721; Proc. Natl. Acad. Sci. USA., Vol. 73, p.2424-2426,
1976; Science, Vol.229, p.393-395, 1985; EMBO J., Vol.2, p.643-648, 1983) or any cell line subcloned from the PC12 cell line. Preferably, the subcloned cells have the following properties.

  In other words, (1) a gene encoding Rec168 and (2) a “reporter gene operably linked to the promoter region of a gene whose expression is induced by stimulation via G protein” are introduced exogenously into the cells. When the Rec168 ligand is contacted with the recombinant cells obtained in this manner, the amount of the reporter gene product whose expression is induced is larger than the amount of the reporter detected using the PC12 cell line as a host cell. It is a cell from which high values can be obtained.

Preferred examples of the subcloned cells include PC12h cell line (Brain Research, Vol. 222, p.225-233, 1981) and various cells subcloned from the PC12h cell line.

Furthermore, as the step of measuring the signal transduction ability, a method for measuring the amount of intracellular free calcium may be used. Rec168 expressed on the cell surface is brought into contact with a test substance that is expected to contain a compound that binds to it, and intracellular free calcium is measured according to a conventional method, and the activity of transmitting a signal into the cell via Rec168 is measured. The compound having can be selected.

  In this step, a test substance is added to each of Rec168-expressing cells and Rec168 non-expressing cells, and the change in intracellular free calcium concentration is measured. By comparing the results of both, a substance with a large change in intracellular free calcium concentration only in a cell line in which Rec168 is forcibly expressed can be selected as a substance with a high signal transduction ability via Rec168 and selected as a candidate for a Rec168 ligand. .

  The intracellular free calcium concentration can be measured by methods known to those skilled in the art. For example, seed cells in a 96-well or 384-well multi-well plate, load intracellular calcium fluorescence detection reagent (Fura-2, etc.), stimulate with a ligand candidate substance, and use fluorescence spectroscopy for intracellular calcium measurement. Can be measured with the instrument.

Furthermore, as a step of measuring the signal transduction ability, for example, a GTP binding test can be used. In this test, the test substance was reacted with the cell membrane fraction of Rec168-expressing cells and non-expressing cells added to the filter plate, and the amount of GTP bound to the membrane fraction was measured and compared. It is possible to analyze whether or not a specific signal is transmitted through Rec168, which is a type receptor. GTP binding activity can be measured by methods known to those skilled in the art and is not particularly limited. For example, GTP binding activity can be easily measured by using a kit such as DELFIA GTP-binding kit AD0167 manufactured by PerkinElmer.

In the present invention, preferred examples of "ligand having Rec168 agonistic effect", e.g., MCD-P, [D- Pro 2, D-Trp 7,9,10] -Substance P (1-11), [D ^{-Pro 4, D-Trp 7,9,10]} -Substance P (1-11), [D-Trp 7,9,10] -Substance P (1-11), Somatostatin 28, Chaperonin10 (1-20) , Platelet Factor-4, pCNP (1-17), Substance P, Indolicidin, Angiopeptin, PACAP (6-27), Somatostatin, Cortistatin 14 and the like.

Substance that suppresses Rec168-mediated degranulation reactionThe present invention uses a cell expressing Rec168 to measure the signal transduction ability of a ligand having a Rec168 agonistic action in the presence and absence of a test substance, And a method for screening a substance that suppresses the degranulation reaction via Rec168, comprising the step of comparing the two measured values. The present invention also includes a step of measuring the signal transduction ability of a ligand having a Rec168 agonistic action in the presence and absence of a test substance using a cell expressing Rec168, and a step of comparing both measured values. A method for identifying a substance that suppresses the degranulation reaction via Rec168 is also provided.

  The method of identifying a substance that suppresses the degranulation reaction mediated by Rec168 means identifying whether or not the test substance is a substance that suppresses the degranulation reaction mediated by Rec168. Therefore, the “method for identifying a substance that suppresses degranulation reaction mediated by Rec168” conceptually includes “a method for screening a substance that suppresses degranulation reaction mediated by Rec168”.

  The present inventors have found that Rec168 is a protein that induces degranulation of mast cells. That is, when a ligand substance having Rec168 agonist activity binds to Rec168, a degranulation reaction is induced through an intracellular signal transduction process.

  In the present invention, “a substance that suppresses Rec168-mediated degranulation reaction” is a substance that can suppress a cell degranulation reaction caused by binding of a ligand substance having Rec168 agonist activity to Rec168. The substance is not particularly limited as long as it has such an action. In other words, the substance having such an action is “a substance having a Rec168 antagonistic action”.

  Since Rec168 is a protein that induces degranulation of mast cells, measurement of the signal transduction ability of a ligand having Rec168 agonistic activity in the presence and absence of a test substance using cells expressing Rec168 In addition, any substance that suppresses the signal transduction ability selected by the process of comparing both measured values is considered to be a substance that suppresses the degranulation reaction via Rec168. Accordingly, in the present invention, as a screening method or identification method for a substance that suppresses the degranulation reaction mediated by Rec168, a Rec168-expressing cell is used and has a Rec168 agonistic action in the presence and absence of a test substance. Only the step of measuring the signal transduction ability of the ligand and the step of comparing both measured values may be included.

  Furthermore, the method which combined the following processes with the said process is also preferable. Moreover, as a screening method or identification method for a substance that suppresses the degranulation reaction via Rec168, only the steps shown below may be used.

  For example, a substance that suppresses the binding between Rec168 and a ligand substance having Rec168 agonist activity is considered to be a substance that suppresses the degranulation reaction via Rec168. As a method for screening or identifying such a substance, a method for comparing the binding ability of a ligand substance to Rec168 in the presence or absence of a test substance is possible. It should be noted that detection and comparison of ligand substance binding can be performed as follows.

  Here, Rec168 used for screening or identification in the present invention may be a recombinant polypeptide or a naturally derived polypeptide. It may also be a partial peptide. Moreover, the form expressed on the cell surface or the form as a membrane fraction may be sufficient. The test substance is not particularly limited, and examples thereof include cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, purified or crude purified polypeptides, non-peptidic compounds, and synthetic low molecular weight compounds. And natural compounds. The polypeptide of the present invention to be contacted with the test substance is expressed on the cell membrane, for example, as a purified polypeptide, a soluble polypeptide, a form bound to a carrier, or a fusion polypeptide with another polypeptide. As a form to be made, it can be contacted with the test substance as a membrane fraction.

  As a method for detecting and comparing the binding ability of a ligand substance to Rec168 in the presence or absence of a test substance, when the ligand substance is a polypeptide, many methods known to those skilled in the art are as follows. It can be done by a method.

For example, it can be performed by immunoprecipitation. Specifically, it can be performed as follows. A gene encoding Rec168 is inserted into a vector for expressing a foreign gene such as pSV2neo, pcDNA I, or pCD8 to express the gene in animal cells. SV40 early promoter A promoter is a promoter used for expression (Rigby In Williamson (ed.) , Genetic Engineering, Vol.3. Academic Press, London, p.83-141 (1982)), EF-1 α promoter (Kim et al. Gene 91 , P.217-223 (1990)), CAG promoter (Niwa et al. Gene 108 , p.193-200 (1991)), RSV LTR promoter (Cullen Methods in Enzymology 152 , 198-704). SR α promoter (Takebe et al. Mol. Cell. Biol. 8 , p.466 (198 8)), CMV immediate early promoter (Seed and Aruffo Proc. Natl. Acad. Sci. USA 84, p.3365-3369 (1987)), SV40 late promoter (Gheysen and Fiers J. Mol. Appl. Genet. 1, p.385-394 (1982)), Adenovirus late promoter (Kaufman et al. Mol. Cell. Biol. 9 , p. 946 (1989)), HSV TK promoter, etc. It may be used.

In order to express a foreign gene by introducing a gene into an animal cell, electroporation method (Chu, G. et al. Nucl. Acid Res. 15 , 1311-1326 (1987)), calcium phosphate method (Chen, ... C and Okayama, H. Mol Cell Biol 7, 2745-2752 (1987)), DEAE dextran method (Lopata, M. A. et al Nucl Acids Res 12, 5707-5717 (1984...); Sussman , D. J. and Milman, G. Mol Cell Biol 4, 1642-1643 (1985)), lipofectin method (Derijard, B. Cell 7, 1025-1037 (1994);... Lam , B. T. et al Nature Genetics 5 , 22-30 (1993);.. Rabindran, S. K. et al Science 259, there is a method such as 230-234 (1993)), by any of the methods Good.

Rec168 can be expressed as a fusion polypeptide having a monoclonal antibody recognition site by introducing a monoclonal antibody recognition site (epitope) of which specificity is known into the N-terminus or C-terminus of Rec168. A commercially available epitope-antibody system can be used (Experimental Medicine 13 , 85-90 (1995)). Vectors that can express a fusion polypeptide with β-galactosidase, maltose-binding protein, glutathione S-transferase, green fluorescent protein (GFP) and the like via a multicloning site are commercially available.

A method for preparing a fusion polypeptide by introducing only a small epitope consisting of several to a dozen amino acids in order to prevent the properties of Rec168 from changing as much as possible by making it into a fusion polypeptide has also been reported. . For example, polyhistidine (His-tag), influenza agglutinin HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV) -Tag), E-tag (epitope on monoclonal phage) and other monoclonal antibodies that recognize them can be used as an epitope-antibody system for screening for polypeptides that bind to Rec168 (Experimental Medicine 13 , 85). -90 (1995)).

  In immunoprecipitation, an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant in the presence and absence of a test substance. This immune complex consists of Rec168, a polypeptide capable of binding thereto, and an antibody. When the test substance has an activity of suppressing the binding between Rec168 and the ligand substance, the amount of the ligand substance in these immune complexes will be reduced compared to the control. In addition to using an antibody against the above epitope, immunoprecipitation can also be performed using an antibody against the polypeptide of the present invention. The antibody against the polypeptide of the present invention can be obtained by, for example, introducing a gene encoding Rec168 into an appropriate E. coli expression vector and expressing it in E. coli, purifying the expressed polypeptide, and using it for rabbit, mouse, rat, goat, etc. It can be prepared by immunizing chickens and the like. It can also be prepared by immunizing the above animals with the synthesized partial peptide of Rec168.

  For example, when the antibody is a mouse IgG antibody, the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose. In addition, when Rec168 is prepared as a fusion polypeptide with an epitope such as GST, a substance that specifically binds to these epitopes such as glutathione-Sepharose 4B is used and an antibody of Rec168 is used. In the same manner, immune complexes can be formed.

  For general methods of immunoprecipitation, for example, according to the method described in the literature (Harlow, E. and Lane, D .: Antibodies, pp. 511-552, Cold Spring Harbor Laboratory publications, New York (1988)), It may be done according to this.

  Further, as a method for measuring the amount of a polypeptide that is a ligand substance that binds to Rec168, for example, the West Western blotting method (Skolnik, EY et al., Cell (1991) 65, 83-90) is considered. It is done. That is, a cDNA library using a phage vector (λgt11, ZAP, etc.) is prepared from cells, tissues, and organs (eg, testis) expressing a ligand polypeptide that binds to Rec168, and this is prepared on LB-agarose. The polypeptide expressed and immobilized on the filter is immobilized, and the purified and labeled polypeptide of the present invention is reacted with the filter in the presence and absence of the test substance, and the polypeptide bound to Rec168 is reacted with What is necessary is just to detect the plaque which expresses by a label | marker. When the test substance has an activity of inhibiting the binding between Rec168 and the ligand substance, the count is detected to be low. As a method for labeling Rec168, a method using the binding property of biotin and avidin, a method using an antibody that specifically binds to Rec168 or a polypeptide fused to Rec168 (eg, GST), a method using a radioisotope Or the method of utilizing fluorescence, etc. are mentioned.

  As a method for detecting and comparing the binding ability of the ligand substance to Rec168 in the presence or absence of the test substance, a method using affinity chromatography is also conceivable. For example, fix Rec168 to an affinity column carrier, bind a ligand substance having Rec168 agonist activity in the presence or absence of the test substance, wash the column, and compare the amount of ligand substance bound to Rec168. Can do.

  Methods for detecting and comparing the binding of a ligand substance to Rec168 in the presence or absence of a test substance include screening a molecule that binds to Rec168 using a peptide display library, and combinatorial chemistry technology screening method using by high-throughput (Wrighton NC; Farrell FX; Chang R; Kashyap AK; Barbone FP; Mulcahy LS; Johnson DL; Barrett RW; Jolliffe LK;. Dower WJ, Small peptides as potent mimetics of the protein hormone erythropoietin Science (UNITED STATES) Jul 26 1996, 273 p458-64, Verdine GL., The combinatorial chemistry of nature. Nature (ENGLAND) Nov 7 1996, 384 p11-13, Hogan JC Jr., Directed. Are known to those skilled in the art.

  In the present invention, a biosensor using the surface plasmon resonance phenomenon can also be used as a means for detecting or measuring the bound substance. The biosensor using the surface plasmon resonance phenomenon can observe the interaction between Rec168 and the test substance in real time as a surface plasmon resonance signal using a trace amount of polypeptide and without labeling ( For example, BIAcore, manufactured by Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the present invention and the test substance by using a biosensor such as BIAcore.

Further, as a screening method or identification method for a substance that suppresses the degranulation reaction via Rec168, a method of directly measuring and comparing the degranulation reaction of cells is also possible.

A substance capable of bringing a test substance into contact with Rec168-expressing cells and inducing a degranulation reaction of the cell via Rec168 can be said to be one of the “ligands having a Rec168 agonistic action”.

  For example, by adding a test substance to mast cells that express Rec168 and mast cells that do not express Rec168, degranulation is induced in mast cells that express Rec168, but not in cells that do not express Rec168. A method of selecting such a substance can be considered. Mast cells can be induced to differentiate from hematopoietic stem cells according to conventional methods, and Rec168-expressing cells and non-expressing cells can be induced depending on the culture conditions.

  Here, as mast cells, those directly separated from blood or tissue can also be used. Rec168-expressing cells and non-expressing cells can also be separated by sorting using an antibody against Rec168. Moreover, a cultured cell line can also be used as a mast cell.

  A screening method for a substance that suppresses cell degranulation through Rec168 includes a step of comparing the degree of cell degranulation by a ligand substance through Rec168 in the presence or absence of a test substance. If it is, it will not be specifically limited.

Method for screening substance that regulates expression of Rec168 Furthermore, the present invention can also provide a method for screening a substance that regulates the expression of Rec168 protein or a polynucleotide encoding Rec168 in a cell.

  One embodiment of the present invention can be carried out by comparing the levels of Rec168 protein expression or Rec168 gene expression on the cell surface before and after the test substance is allowed to act on the cells.

As a method for detecting a change in Rec168 expression in cells, a method known to those skilled in the art can be used. Examples of such methods include, but are not limited to, northern blotting and RT-PCR. In the Northern blotting method, RNA of a cell to be examined is purified, electrophoresed in an agarose gel, and blotted on a membrane, and a probe labeled with the radioactivity etc. of the gene of the present invention is hybridized, The expression of the polynucleotide of the present invention is measured by the presence / absence of the band that appears specifically and the density. In addition, in the RT-PCR method, RNA of a cell to be examined is purified, converted into cDNA by reverse transcriptase, used as a template, a sequence characteristic of Rec168 as a primer, and derived from the transcription product of the gene of the present invention. The cDNA is amplified by PCR. The amount of cDNA amplified in this way is considered to be proportional to the amount of cDNA used as a template, and hence the amount of transcripts of Rec168. Therefore, the amount of DNA fragment amplified by PCR is measured using electrophoresis or the like. By doing so, the expression level of Rec168 can be measured.

As another method, for example, Rec168 or a cell containing the polypeptide can be immunologically detected by performing an antigen-antibody reaction using the antibody of the present invention. The detection method can also be used for diagnosing a disease that may be caused by mutation or abnormal expression of a gene (genomic DNA or the like) encoding Rec168. As a method for quantitatively determining Rec168, an immunological detection method includes an ELISA method using a microtiter plate, a fluorescent antibody method, a Western blot method, an immunohistochemical staining method, and the like. In addition, the polypeptide of the present invention and the polypeptide of the present invention labeled with a radioisotope such as sandwich ELISA using two monoclonal antibodies having different epitopes among antibodies that react with Rec168 in the liquid phase And a radioimmunoassay method using an antibody to be recognized.

Another method is to contact a transformant transformed with a plasmid containing a reporter gene-linked DNA downstream of a region that controls transcription of Rec168 with a test substance, and to add a compound that controls the expression of Rec168. It can also be done by selecting. The reporter gene is not particularly limited as long as its expression can be detected. For example, a CAT gene, a lacZ gene, a luciferase gene, a β-glucuronidase gene (GUS) and a GFP gene that are generally used by those skilled in the art. Etc. The reporter gene also includes DNA encoding Rec168.
The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of reporter gene used. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of β-glucuronidase gene (GUS), luminescence of Glucuron (ICN) or 5-bromo-4-chloro-3-indolyl- By detecting the color development of β-glucuronide (X-Gluc), and in the case of the GFP gene, the expression level of the reporter gene can be measured by detecting the fluorescence of the GFP protein.

  When Rec168 is used as a reporter, the expression level of the gene can be measured by methods known to those skilled in the art. For example, the transcription level of the gene can be measured by extracting mRNA of the gene according to a standard method and performing Northern hybridization or RT-PCR using the mRNA as a template. Furthermore, the expression level of the gene can be measured using DNA array technology.

  In addition, the fraction containing Rec168 encoded by the gene can be collected according to a standard method, and the translation level of the gene can be measured by detecting the expression of the polynucleotide by electrophoresis such as SDS-PAGE. . Moreover, it is also possible to measure the translation level of a gene by carrying out Western blotting using an antibody against Rec168 and detecting the expression of the polypeptide.

The substance obtained by the screening method or identification method of the present invention regulates the expression of Rec168 protein or a polynucleotide encoding Rec168 or regulates the activity of Rec168 protein (for example, the signal transduction ability via Rec168 protein) ( For example, it can be a candidate for a drug to be suppressed), and can be applied to the treatment of diseases caused by abnormal expression or functional abnormality of Rec168 or diseases that can be treated by controlling the activity of Rec168. Examples of the disease to be treated or prevented include inflammatory diseases, particularly chronic inflammation such as allergic diseases and autoimmune diseases. Substances in which a part of the structure of the substance obtained by the screening method or identification method of the present invention is converted by addition, deletion and / or substitution are also included in the scope of the present invention.

Inflammatory diseases Inflammatory diseases particularly mean inflammatory diseases involving mast cells, especially chronic inflammations such as allergic diseases and autoimmune diseases involving mast cells. These allergic diseases or autoimmune diseases As specific examples, as described above, gastritis, gastric ulcer: cirrhosis: chronic biliary disease (intrahepatic lithiasis, primary sclerosing cholangitis): ulcerative colitis: chronic inflammatory vascular disease: arteriosclerosis: Cardiac anaphylaxis: Pulmonary fibrosis: Bronchial asthma: Nephritis (tubulointerstitial nephritis, glomerulonephritis): Interstitial cystitis: Atopic dermatitis: Keloid: Vascular proliferative disorder (Kaposi sarcoma, malignant vascular endothelial cell type) : Rheumatoid arthritis: Allergic rhinitis: Allergic conjunctivitis: Gastric cancer: Malignant tumors such as colorectal cancer, breast cancer, skin basal cell cancer, soft tissue tumors, melanoma:
Non-small cell lung cancer: Others include urticaria, systemic lupus erythematosus, multiple sclerosis, collagen disease, malignant tumor, B lymphoma, B cell leukemia and the like.
Diagnostic Agent The present invention provides a diagnostic agent for a disease associated with abnormal expression of a gene encoding Rec168 or abnormal activity of Rec168. Since Rec168 has a function of inducing degranulation of mast cells, abnormal expression or function thereof can cause various diseases such as chronic inflammation. Therefore, it is possible to diagnose such diseases by using the inappropriate activity or expression of Rec168 and the in vivo expression level of the ligand molecule as an index.

In the present invention, the “diagnostic agent” is not only a diagnostic agent for establishing a treatment strategy for a subject exhibiting a disease symptom, but also for prevention used to determine whether or not the subject is susceptible to the disease. Diagnostic agents are also included.

One embodiment of the diagnostic agent of the present invention is a diagnostic agent containing the Rec168 protein or a partial peptide thereof used for measuring the in vivo expression level of the Rec168 ligand substance in a subject. A blood sample prepared from a subject is added to a plate pre-coated with Rec168 protein or a partial peptide thereof, and the amount of the ligand substance in the blood sample can be quantified using an antibody against the labeled ligand substance.

One embodiment of the diagnostic agent of the present invention is capable of detecting a mutation in the gene encoding Rec168 or its expression control region in a subject.

One is a diagnostic agent for testing by directly determining the base sequence of the gene encoding Rec168 or its expression control region in a subject, for example, a DNA containing the gene encoding Rec168 or its expression control region It contains a probe or primer that hybridizes. The method of use is not particularly limited as long as a mutation in the gene encoding Rec168 or its expression control region in a subject can be detected. For example, first, a DNA sample is prepared from a subject. A DNA sample can be prepared based on chromosomal DNA or RNA extracted from a subject's cells, such as blood, urine, saliva and the like. In order to prepare a DNA sample of the present method from chromosomal DNA, for example, chromosomal DNA may be cleaved with an appropriate restriction enzyme and cloned into a vector to prepare a genomic library. In order to prepare a DNA sample from RNA, for example, a cDNA library may be prepared from RNA using reverse transcriptase. Subsequently, DNA containing the gene encoding Rec168 or its expression control region is isolated. The DNA can be isolated by screening a genomic library or a cDNA library using a probe that hybridizes to a gene encoding Rec168 or a DNA containing an expression control region thereof. It can also be isolated by genomic DNA library, cDNA library, or PCR using RNA as a template, using a primer that hybridizes to the gene encoding the polypeptide of the present invention or DNA containing its expression control region. it can. Next, the base sequence of the isolated DNA is determined. The base sequence of the selected DNA can be determined by a method known to those skilled in the art. Next, the determined DNA base sequence is compared with a control. “Control” refers to a base sequence of DNA containing a gene encoding normal (wild-type) Rec168 or an expression control region thereof. As a result of such comparison, when the DNA base sequence of the subject is different from that of the control, the subject is determined to be suffering from or at risk of developing the disease.

One embodiment of the diagnostic agent of the present invention is a diagnostic agent containing an antibody against Rec168 or a partial peptide thereof used for measuring the expression level or activity of Rec168 protein in a subject, or a ligand substance of Rec168. Mast cells are isolated from a blood sample prepared from a subject, reacted with an antibody against Rec168 or a partial peptide thereof, or a ligand substance, and the expression level of Rec168 molecule on the mast cells and the ligand binding ability are measured with a flow cytometer. be able to. When the expression level or ligand binding ability is increased, it is determined that there is a risk of onset.
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.

Cloning of Rec168 <br/> human testis poly (A) ⁺
Reverse transcription was performed using RNA as a template to synthesize cDNA. For the reaction, a reagent attached to the High Fidelity RNA PCR Kit (TaKaRa) was used. The reaction system is human testis poly (A) ⁺ RNA (1 μg / μL) 0.01 μL, 2 × Bca 1st
buffer 2 μL, MgSO ₄ (25 mM) 0.8 μL, dNTP Mixture (10 mM) 0.2 μL, RNase
Inhibitor (40 units / μL) 0.1 μL, Bca PLUS RTase
(42 U / μL) 0.2 μL, Random Hexamer (50 μM)
0.2 μL was made up to 4 μL with sterile water. This is 65 ° C 1 minute, 30 ° C 1 minute, 30-65 ° C
The reaction was carried out for 15 minutes, 65 ° C for 30 minutes, 98 ° C for 5 minutes, and 5 ° C for 5 minutes.

Primary PCR is Pyrobest
The reagent attached to DNA Polymerase (TaKaRa) was used. To 4 μL of the reverse transcription reaction solution containing cDNA synthesized above, add 0.4 μL of dNTP Mixture (2.5 mM each), 10
× Pyrobest buffer 2 μL, Pyrobest DNA Polymerase (5 units / μL)
To 0.1 μL, 4 μL of primers 168_PCR_F1 (5 μM) and 4 μL of 168_PCR_R1 (5 μM) having the sequences shown in SEQ ID NOs: 3 and 4, respectively, were added, and the volume was adjusted to 20 μL with sterile water. This is 94 ° C for 30 seconds, (94 ° C for 30 seconds, 72-56 ° C (down 1 ° C per cycle) for 30 seconds, 72 ° C for 2 minutes) × 17 cycles, (94 ° C for 30 seconds, 55 ° C 30 seconds, 72 ° C
(2 minutes) × 13 cycles, 72 ° C. for 3 minutes.

Secondary PCR is Pyrobest
The reagent attached to DNA Polymerase (TaKaRa) was used. Add 1 μL of 100-fold diluted primary PCR reaction solution to 0.8 μL of dNTP Mixture (2.5 mM each), 10 × Pyrobest buffer II 1 μL, Pyrobest
Add 2 μL of primers 168_PCR_F2 (5 μM) and 2 μL of 168_PCR_R2 (5 μL) having the sequences shown in SEQ ID NOs: 5 and 6, respectively, to 0.05 μL of DNA Polymerase (5 units / μL). 10 μL. This is 94 ° C for 30 seconds, (94 ° C for 30 seconds, 72-56 ° C (down 1 ° C for each cycle) for 30 seconds, 72 ° C for 2 minutes) x 17 cycles, (94 ° C for 30 seconds) , 55 ° C 30 seconds, 72 ° C
(2 minutes) × 13 cycles at 72 ° C. for 3 minutes.

Secondary PCR products were separated by electrophoresis using 1% agarose gel, and DNA was extracted from agarose gel fragments. Using T4 Polynucleotide Kinase (TaKaRa), phosphorylation of the terminal is performed and this is treated with TaKaRa Ligation.
Using Kit Ver.2 (TaKaRa), the multicloning site was digested with the restriction enzyme HpaI and reacted with the dephosphorylated vector pENTR1A (Invitrogen Corporation). This reaction solution was introduced into E. coli DH5α strain and transformed, and selected on LB agar medium containing kanamycin. From the obtained Escherichia coli colonies, clones into which the gene had been inserted were selected, and plasmid DNA was recovered. Using this plasmid DNA as a template, base sequence analysis was performed using six types of Rec168 primers. Primers are 168_PCR_F2 (SEQ ID NO: 5), 168_PCR_R2 (SEQ ID NO: 6), 168_SEQ_F1 (SEQ ID NO: 7), 168_SEQ_F2 (SEQ ID NO: 8), 168_SEQ_R1 (SEQ ID NO: 9), 168_SEQ_R2 (SEQ ID NO: 10) It was used. The reaction was performed by the dye terminator method using DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences) and analyzed using the sequencer MegaBace1000 (Amersham Biosciences). The plasmid DNA into which the target sequence was inserted was named “pENTR-Rec168”.

Construction of expression vector
pENTR-Rec168 cloned into pENTR1A (entry vector) was replaced with the target expression vector (destination vector) by the homologous recombination technique using Gateway System technology (Invitrogen Corporation).

attL × attR Enzyme Gateway that mediates recombination reaction
LR Clonase Enzyme Mix (Invitrogen Corp. 11791-019) was used. The reagent is attached to the enzyme. This time, we used pME18S.rfA with pME18S as its destination as the expression vector. 100 ng of the entry vector and 100 ng of the destination vector were made up to 6 μL with TE buffer and mixed with 2 μL of 5 × LR Clonase Reaction Buffer. 2 μL of LR Clonase Enzyme Mix was added and incubated at room temperature (25 ° C) for 1 hour. Proteinase K Solution (2 μg / μL) to stop the reaction
1 μL was added and incubated at 37 ° C. for 10 minutes. After completion of the reaction, it was cooled on ice. Competent cell JM109 (TOYOB) thawed on ice with 2.5 μL of this reaction solution was transformed and selected on LB agar medium containing ampicillin. From the obtained Escherichia coli colonies, clones into which the gene had been inserted were selected, and plasmid DNA was recovered. Using this plasmid DNA as a template, nucleotide sequence analysis was performed using gene-specific primers. The reaction was performed by a dye terminator method using BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems Japan Co., Ltd.), and analyzed using a sequencer ABI3100 (Applied Biosystems Japan Co., Ltd.). The plasmid DNA into which the target sequence was inserted was named “pME-Rec168”.

Changes in reporter activity in Rec168 transiently expressed PC12h cells
96-well method
10% horse serum (Invitrogen 26050-088) and 5% fetal calf serum (JRH 12103-78P) and 0.5% penicillin / streptomycin (Sigma
P0906) containing D-MEM (High-glucose) ( Nikken biological CM4402) the rat kidney-derived PC12h cells cultured in collagen type I coated flasks (Iwaki glass 4143-010) in a medium, 225 cm ² flask (1 × 10 After rinsing from ⁷ cells) with PBS (-) (Nikken Biological CM6201), stripping with trypsin / EDTA solution (Invitrogen 5300-054), 0.5% horse serum (Invitrogen 26050-088) and 0.25% Prepared at 1.5 × 10 ⁵ cells / mL in D-MEM (High-glucose) phenol red-free (Niken Biological CM4410) medium containing fetal bovine serum (JRH 12103-78P) and 0.5% penicillin / streptomycin (Sigma P0906) did. The cell suspension per 1 mL, firefly luciferase -zif reporter plasmid DNA (pGL2-zif) and ₁₀₀ ng, G α16 -protein plasmid DNA (pME-neo-G16) and 50 ng, receptor plasmid DNA (pME-MRGX2) 1 μg was dissolved in 60 μL of D-MEM (High-glucose) phenol red-free (Nikken Biological CM4410) medium, and the transfection reagent SuperFect (QIAGEN 301307) was mixed with 2 μL. A mixture of this and the above cell suspension was dispensed 100 μL each into a collagen type I-coated 96-well plate (Iwaki Glass 4860-010) and cultured at 37 ° C. for 48 hours in a CO ₂ incubator.

After removing 50 μL of the culture supernatant, add 50 μL of the test substance prepared using D-MEM (High-glucose) phenol red-free (Nikken Biological CM4410) medium to the twice concentrated solution. _The cells were cultured in an incubator at 37 ° C for 6 hours. Remove all culture supernatant and use Luciferase Cell Culture Lysis Reagent (Promega
E1531) was added at 25 μL / well to lyse the cells.

10 μL of cell lysate was dispensed into a 96-well white plate (Costar 3912), 50 μL of luciferase substrate flash type (Promega E4550) was added using a luminometer LB96V (Bertold), and luciferase activity (unit: RLU) was measured . A concentration-dependent curve was created by plotting the luciferase activity value on the Y-axis and the test substance concentration on the X-axis, and the EC ₅₀ value was derived.

384 well method
Transfect the cell suspension into the PEI as in the 96-well method.
40 μL each was dispensed onto a (Polyethyleneimine) -coated 384-well white plate (Falcon 3988) and cultured at 37 ° C. for 48 hours in a CO ₂ incubator. PEI coating is PEI
(Sigma P3143) was diluted 1000 times with sterilized water, added at 25 μL / well, allowed to stand for 1 hour or more, and then washed twice with 70 μL sterilized water.

After removing 50 μL of the culture supernatant, add 10 μL of the test substance prepared using D-MEM (High-glucose) (Nikken Biological CM4402) medium to a 5-fold concentrated solution, and use a CO ₂ incubator. The cells were cultured at 37 ° C for 6 hours. After the reaction, Steady Glo Luciferase Assay
Reagent (Promega E2550) was added at 25 μL / well, and luciferase activity (unit: CCPS) was measured with 1450 MICROBETA (Wallac). A concentration-dependent curve was created by plotting the luciferase activity value on the Y-axis and the test substance concentration on the X-axis, and the EC ₅₀ value was derived.

The results are shown in FIGS.
As shown in FIG. 1, an increase in luciferase activity was observed in all PC12h cells into which Rec168 was introduced.
As shown in FIG. 2, an increase in luciferase activity was observed in all PC12h cells into which Rec168 was introduced.
As shown in FIG. 3, an increase in luciferase activity was observed in PC12h cells into which Rec168 had been introduced.

Production of stable expression cells
Expression vector construction
Rec168 cDNA was subcloned into the expression vector pLHCXI4 to construct pLHC-Rec168-I4. After culturing E. coli DH5α transformed with pLHC-Rec168-I4, DNA of pLHC-Rec168-I4 was prepared using EndoFree Plasmid Mega Kit (Qiagen).

293 cell transfection
Seed 293 cells (ATCC CRL-1573) in D-MEM medium (Nikken Biomedical Research Institute CM4402) supplemented with 10% fetal calf serum in a Falcon 6-well plate 7.5 × 10 ⁵ cells in a 5% CO ₂ incubator Cultured overnight at 37 ° C. The expression plasmid pLHC-Rec168-I4 DNA 2.5 μg was transfected with 8 μL of LipofectAMINE 2000 Reagent (Invitrogen) according to the description in the attached instruction manual, and the medium was changed after 6 hours. After 2 days of culture, replace with selective medium [D-MEM medium (Nikken Biomedical Research Institute CM4402) supplemented with 200 μg / mL hygromycin B (Invitrogen 10687-010) and 10% fetal calf serum]. did. The cells were cultured for 4 weeks while changing the selective medium every 3 to 4 days to obtain hygromycin resistant cells.

Selection of CD4 positive cells by MACS From the hygromycin resistant cells obtained above, CD4 positive cells were selected as MACSelect 4 transfected cells.
Collected with a selection kit (Miltenyi Biotec). Hygromycin-resistant cells obtained above were selected in a selective medium [D-MEM medium (Nikken Biomedical Research Institute CM4402) supplemented with 200 μg / mL hygromycin B (Invitrogen 10687-010) and 10% fetal calf serum]. The cells were seeded in a 75 cm ² flask and cultured to 1 × 10 ⁷ cells. The medium was discarded, and the cells were washed with 10 mL of D-PBS (Nikken Biomedical Research Institute CM6201). 5 mL of D-PBS supplemented with 5 mM EDTA was added and left at room temperature for 10 minutes to detach the cells. The cells were collected by centrifugation at 210 × g for 3 minutes and suspended in 720 μL of PBE (D-PBS / 5 mM EDTA / 0.5% BSA). 80 μL of MACSelect4 MicroBeads (Miltenyi Biotec) was added and allowed to bind at 4 ° C. for 15 minutes, and then diluted by adding 1.2 mL of PBE. According to the description of the attached instruction manual, the cell suspension combined with MACSelect4 MicroBeads was added to the LS column (Miltenyi Biotec) washed with 3 mL of PBE and bound. The column was washed 4 times with 3 mL of PBE, then removed from the MACS separator magnet, and CD4 positive cells were recovered with 5 mL of PBE. The collected CD4 positive cells were centrifuged at 210 × g for 3 minutes and then seeded in a 75 cm ² flask with a selective medium. Cultivation was performed for 10 days while changing the selection medium every 3-4 days to obtain CD4-positive hygromycin-resistant cells. The obtained CD4-positive hygromycin resistant cells were designated as Rec168 stably expressing 293 cells.

Measurement of intracellular free calcium
Changes in intracellular free Ca2 ⁺ concentration in 293 cells stably expressing Rec168
10% fetal bovine serum (JRH 12103-78P) and 0.5% penicillin / streptomycin (Sigma
After rinsing Rec168 stably expressing 293 cells and 293 cells (parent strain) in D-MEM (High-glucose) (Niken Biological CM4402) medium containing P0906) with PBS (-) (Niken Biological CM6201) The cells were peeled off by hitting the flask. Ca ²⁺ assay buffer (20 mM HEPES (Nacalai Code 17514-15); pH 7.4; prepared with 1 M KOH (Nacalai Code 286-16)), 115 mM NaCl (Sigma Cat. S5150), 5.4 mM KCl (Nacalai Code 285-14), 0.8 mM MgCl ₂ (Nacalai Code
209-09), 1.8 mM CaCl ₂ (Nacalai Code 067-29), 13.8 mM Glucose (Nacalai Code 168-06), 0.2% BSA (Sigma Cat. A8806), 2.5 mM Probenicid (Sigma Cat. P8761) After washing, Fura2 loading buffer (2 μM Fura2-AM (Dojindo 343-05401), 0.02% Pluronic F127 (Molecular
The suspension was suspended in 10 mL of the above Ca ²⁺ assay buffer containing Probe P6866) and incubated at room temperature for 1 hour. The cells were washed twice with Ca ²⁺ assay buffer and adjusted to 1 × 10 ⁶ cells / mL. 384 well plate (Nunc.
In 142761), 40 μL was seeded at 40,000 cells / well.

The intracellular free Ca ²⁺ concentration was measured over time using FDSS3000 (Hamamatsu Photonics). Specifically, the fluorescence intensity of a 384-well plate seeded with cells was measured every 1.5 seconds for 3.5 minutes. 10 μL of the ligand prepared using Ca ²⁺ assay buffer in a 5-fold concentrated solution of final concentration was added 30 seconds after the start of measurement and allowed to stir for 20 seconds.

The measured values are the ratio values of the fluorescence intensities at the fluorescence wavelength of 500 nm obtained for two types of excitation wavelengths of 340 nm (Ca ²⁺ binding type) and 380 nm (Ca ²⁺ non-binding type) [340 nm excitation (Ca ^{2 +} Bond) ÷ 380 nm excitation (Ca ²⁺ non-bonded type)]. This Ratio value was plotted on the Y axis and time on the X axis, and changes in intracellular free Ca ²⁺ concentration for various ligands were measured. In addition, a concentration dependence curve was created by plotting the maximum ratio value for various concentrations of various test substances on the Y axis and the test substance concentration on the X axis, and the EC ₅₀ value was derived. The assay was performed 4 times, and each EC ₅₀ value was calculated using the calculation software “XLfit”, and the average value was obtained.

As a test substance, Angiopeptin (American
Peptide 68-1-45), Bradykinin (American Peptide 18-1-10), Granuliberin R (American Peptide 87-0-72), Indolicidin (American Peptide 72-2-28), Magainin 2 (American
Peptide 72-2-26), PACAP (6-27) (American Peptide 34-0-45), Somatostatin 28 (American Peptide 68-1-50), VIP (1-28) (American Peptide 48-1-10) ), Compound 48/80 (Sigma
C2313), Atrial Natriuretic Peptide (1-28), human (Peptide Institute 4135-v), cortistatin 14 (Peptide Institute 4329-v), Histatin 5 (Peptide Institute 4270-s), MCD-P (Peptide Institute)
4258-v), Neurotensin (Peptide Institute)
4029-v), Platelet Factor-4
4305-v), Somatostatin (Peptide Institute)
4023-v), Substance P (Peptide Laboratories)
4014-v), and the following were obtained by synthetic methods ^{(A) [D-Pro 2} , D-Trp 7,9,10] -Substance P (1-11), [D-Pro 4, D-Trp 7 ^{, 9,10} ] -Substance P (1-11), BAM (8-22), Catestatin, cathelicin (LL-37), chaperonin 10 (1-20), eosinophil peroxidase peptide, histamine
releasing peptide, human HRP-1, human
HRP-2, PACAP (6-38) , pCNP (1-17), was used ^{[D-Trp 7,9,10] -Substance P} (1-11), Tropomyosin (1-12), 31 kinds of . Ionomycin (BIOMOL CA201) was used as a positive control.
Synthesis method (A)
The above peptides were synthesized using an automatic peptide synthesizer (Model 431A, manufactured by Applied Biosystems). Peptide synthesis and protection were performed according to the manufacturer's manual. Impurities contained in the obtained peptide preparation were analyzed using a high-performance liquid chromatograph (Waters), reverse phase C18 column (5 μm, 2 × 250 mm, YMC-Pac ODS-A
, YMC) and separated with a gradient of 0.1% trifluoroacetic acid (solution A) and acetonitrile (solution B). The structure and purity of the obtained synthetic peptide preparation were confirmed by molecular weight measurement using a laser ionization mass spectrometer (KOMPACT MALDI III, manufactured by Shimadzu Corporation).

result
Thirty-one ligands were stimulated on 293 cells that stably expressed Rec168. As a result, Tropomyosin (1-12), Platelet Factor-4, Cortistatin 14, Angiopeptin, HRP-2, Magainin
2, MCD-P, PACAP ( 6-27), VIP (1-28), Indolicidin, Somatostatin 28, PACAP (6-38), Somatostatin, [D-Trp 7,9,10] -Substance P (1- ^{11) [D-Pro 2,} D-Trp 7,9,10] -Substance P (1-11), Substance P, pCNP (1-17), [D-Pro 4, D-Trp 7,9,10 ] -Substance P (1-11), eosinophil peroxidase peptide, Histatin 5, Compound 48/80, BAM (8-22), chaperonin 10 (1-20), catestatin, histamine releasing peptide, human HRP-1, Neurotensin An increase in intracellular free Ca concentration was observed. The results are shown in Table 2. The unit is μM. Compound
48/80 is expressed in μg / mL.

GTP binding test
Preparation of membrane fraction of Rec168 stably expressing 293 cells
Rec168 stably expressing 293 cells were cultured in a cell culture flask (culture area 175 cm ² ) in 50 mL of medium (Dulbecco's containing 10% fetal bovine serum, 50 units / mL penicillin, 0.05 mg / mL streptomycin.
Modified Eagle Medium (D-MEM; high
glucose) (manufactured by Nikken Biomedical Research Institute) at 37 ° C 5% CO ₂ . Cells that have become approximately 80% confluent after 2 days have passed are cooled with D-PBS (Phosphate buffered saline, pH 7.4; Ca ²⁺ free, Mg ²⁺ free) (Nikken Biomedical Research Institute) Washed). The collected cells were suspended in a cooled lysis buffer (15 mM Tris-HCl (pH 7.6), 2 mM MgCl ₂ , 0.3 mM EDTA, 1 mM EGTA, pH 7.4 to 7.5) and disrupted with a glass homogenizer. The cell lysate was centrifuged (50,000 × g, 30 minutes, 4 ° C.), and lysis buffer was added to the resulting precipitate. This was further centrifuged (50,000 × g, 30 minutes, 4 ° C), and the resulting precipitate was stored in a stock solution (75 mM Tris-HCl (pH 7.6), 12.5 mM MgCl ₂ , 0.3 mM).
EDTA, 1 mM EGTA, 250 mM
Sucrose, pH 7.5) was suspended to obtain a membrane fraction of Rec168 stably expressing 293 cells.

Binding test between Rec168 stably expressing 293 cells and GTP
The GTP binding experiment was performed using DELFIA GTP-binding kit AD0167 (PerkinElmer). A reaction buffer (GDP 0.03 μM, MgCl ₂ 1 mM, NaCl 10 mM, Saponin 300 μg / mL, expressed in final concentration) was added to a 96-well filter plate (AcroWell Filter Plate). To this, 20 μL of a test substance diluted to an appropriate concentration with 50 mM HEPES buffer was added per well. Furthermore, the membrane fraction prepared above was adjusted to 20 μL with 50 mM HEPES buffer and added to 3.858 μg per well. This was gently incubated on a plate shaker for 30 minutes at room temperature, and then europium-labeled GTP (GTP-Eu, 100 nM)
10 μL per well was added to initiate the reaction. After gentle incubation using a plate shaker at room temperature for 45 minutes, the reaction was stopped by filtering the filter plate (Multiscreen Vacuum Manifold, manufactured by Milipore). After filtration, the membrane was further washed twice with 300 μL of the cooled washing solution, and the amount of GTP-Eu bound to the membrane fraction on the filter was measured with a time-resolution fluorescence measuring apparatus.

Since the exchange reaction between GDP and GTP occurs even in the absence of the G protein-coupled receptor, the binding activity at the time of sample addition is the measured value (Stimulated signal) and the measured value in the absence of the sample (basal signal (Basal
signal)) and calculated as a percentage [% over basal = (Stimulated signal / Basal signal × 100) −100].

Nonspecific binding was measured in the presence of 5 μM (final concentration) GTP-γ-S per well. As a control, GTP binding activity was observed under the same conditions using a membrane fraction prepared in the same manner as above from 293 cells (parent strain) into which Rec168 had not been introduced. As test substances, MCD Peptide (Peptide Institute), Substance P (American Peptide), Human Arial Natriuretic Peptide (1-28) (Peptide Institute), Chaperonin10 (1-20), pCNP (1 -17) and Bovine Adrenal Medulla (8-22).

Results The results are shown in Figs.
As is clear from FIG. 4, an increase in the amount of GTP binding was observed depending on the concentration of Substance P.
As apparent from FIG. 5, an increase in the amount of GTP binding was observed depending on the concentration of Chaperonin 10 (1-20).
As is apparent from FIG. 6, an increase in the amount of GTP binding was observed depending on the concentration of pCNP (1-17).
As is clear from FIG. 7, an increase in the amount of GTP binding was observed depending on the concentration of the MCD peptide.
Based on this result, when the Rec168 stable expression 293 cell membrane fraction was used, Mast Cell Degranulating
Addition of Peptide, Substance P, Chaperonin 10 (1-20), and pCNP (1-17) increased the amount of GTP-Eu binding in a concentration-dependent manner. Among these, GTP binding was observed at the lowest concentration (30 nM or more) of MCD peptide, and then GTP binding was confirmed at 300 nM or more of Substance P and pCNP (1-17). Chaperonin 10 (1-20) was confirmed to have GTP binding activity at 1 μM or more. On the other hand, the test substance-dependent GTP binding was not observed in the membrane fraction prepared from 293 cells (parent strain).

Human cultured mast cell degranulation test

Obtaining umbilical cord blood 6 heparinized umbilical cord blood was obtained with informed consent from the Kyoto Monopoly Hospital. The acquisition dates for the six cases were February 23, February 26, March 2, April 12, April 21, and May 17, 2004. Donor 1, 2, 3, 4 , 5 and 6.

Isolation of hematopoietic stem cells <br/> 10-60 mL of umbilical cord blood collected from heparin with equal volume of Buffer (0.5% bovine serum albumin (BSA), 2 mM ethylenediamine
After dilution with tetra acetic acid / Phosphate Buffered Saline (-)), Ficoll-Paque
(Amersham Pharmacia Biotech) was layered (Ficoll-Paque: Blood (1: 2)), and the mononuclear cell fraction was obtained by centrifugation at 400 × g (1350 rpm) at 4 ° C for 30 minutes . Wash 3 times with Buffer (1500 rpm,
4 ° C, 5 min), count the number of cells, and 0.1 mL of Reagent A1 (Fc blocking, CD34) per 1 × 10 ⁸ cells
Progenitor Cell Isolation Kit, Miltenyi Biotec, 467-01) was added. After stirring, 0.1 mL of Reagent A2 (CD34
antibody-hapten) (final volume 0.5 mL / 1 × 10 ⁸
cell), and further incubated at 9 ° C for 15 minutes after stirring. Wash 3 times with Buffer (1500
After resuspending in Buffer (0.4 mL), add 0.1 mL of Reagent B (anti-hapten antibody-microbeads) and stir (final
volume 0.5 mL / 1 × 10 ⁸ cells), followed by incubation at 9 ° C for 15 minutes. After washing twice with Buffer (1500 rpm, 4 ° C, 5 minutes), resuspended in Buffer (0.5 mL), CS set in MACS (MAgnetic Cell Sorting System: Miltenyi Biotec, Daiichi Kagaku) Column (Miltenyi
Biotec) and washed with 30 mL of Buffer to remove CD34 ^- cells. The column was removed from the MACS and CD34 ⁺ cells bound with 30 mL of Buffer were eluted, and these were used as the hematopoietic stem cell population.

Differentiation into human mast cells by long-term culture of hematopoietic stem cells <br/> Isolated CD34 ⁺
recombinant human (rh) stem cell factor (SCF,
1 μg / mL, Peprotech), rh interleukin (IL) -6 (0.5 μg / mL, Peprotech),
rhIL-3 (10 ng / mL, Peprotech), Insulin-Transferrin (1%, Gibco),
1 × 10 ^{6 in} Iscove's Modified Dulbecco's Medium (IMDM, Gibco) medium containing 2-mercaptoethanol (2-ME, 5 × 10 ^-5 M, Gibco), BSA (0.1%, Sigma)
The cells were resuspended at a concentration of cells / mL and seeded on a 24-well culture plate at 0.1 mL / well. IMDM with 0.9% methylcellulose (Methocult, SFBIT,
StemCell technology) was added at 0.9 mL and culture was started. One week later, the above medium except 0.9% methylcellulose was added at 100 μL / well, and then the medium except IL-3 was added at 100 μL / well once a week. Dilute the cells while observing the degree of growth to approximately 10 ⁵
While maintaining the cells / mL / well level, the cells were cultured for about 8 weeks or more to differentiate into human cultured mast cells.

Fetal during culture
Examination of the effect of calf serum (FCS)
Cells from Donor 4 and Donor 6 were used. The cells on the 57th and 27th days from the start of the culture were divided in the presence and absence of 10% FCS, and then cultured until the 92nd and 57th days.

Degranulation test <br/> All of the following degranulation tests are 1%
Insulin-Transferrin, 5 × 10 ⁻⁵ M 2-ME, 0.1% BSA-containing IMDM medium was used. The obtained human cultured mast cells were washed with a medium, and then 6 × 10 ³
Cells / 180 μL / well were seeded on a 96 well U bottom culture plate. Compound 48/80 and various peptides were added at 20 μL to a concentration of 0.1-10 μM and stimulated at 37 ° C for 30 minutes. As a control, A-23187 (Sigma) dissolved in DMSO was added at 0.1-10 μM (final DMSO concentration).
20 μL was added to achieve 0.1%), and stimulation was performed at 37 ° C for 30 minutes. After stimulation, centrifugation (2000 rpm, 4 ° C, 5 minutes) was performed, and the supernatant was collected at 50 μL / well, and the activity of β-hexosaminidase was measured. P-nitrophenyl, N-acetyl, β, D-glucosaminide (1 mM, β-hexosaminidase substrate) is added to 50 μL of the culture supernatant.
0.1% Triton X-100) is added and reacted at 37 ° C for 2 hours. Carbonate buffer (0.1 M, pH 10)
100 μL was added to stop the reaction. Absorbance was measured using a Plate Reader (Molecular Devices) at a wavelength of 405 nm. The degranulation rate in each sample was calculated assuming that the value when the untreated cells were crushed with water (maximum release of intracellular granules; maximum release) was 100%.

The degranulation reaction of human cultured mast cells (Donor 3, 4, 5) by Compound 48/80 and 15 peptides was compared with that of A-23187. Reporter assay, Ca assay, reacted to Rec168, Compound
48/80 and Chaperonin 10 (1-20),
pCNP (1-17), Tropomyosin (1-12), Mast
cell degranulating peptide, Cortistatin 14, Substance P, Somatostatin, Angiopeptin, Indolicidin, [D-Trp 7,9,10] -Substance P (1-11), Pituitary adenylate cyclase activating polypeptide (PACAP) (6-27), Vasoactive The 12 peptides of intestinal peptide (VIP) all induce a concentration-dependent degranulation reaction at 0.1-10 μM, and the maximum reaction at 10 μm is the maximum release of intracellular granules. 44%, comparable to 29-30% at 10 μM for A-23187 (see Figure 8). The concentration at which they induce 25% of the maximum release of intracellular granules (EC25) is 0.2
-3.7 μM (see Table 3).

A report that the properties change in the presence of FCS in the mast cell culture and that it does not respond to peptides that induce degranulation (Moon TC, Lee E, Baek SH, Murakami M, Kudo I, Kim NS, Lee JM, Min HK ,
Kambe N, Chang HW. Degranulation and cytokine expression in human cord
blood-derived mast cells cultured in serum-free medium with recombinant human
stem cell factor. Mol Cells. 16: 154-160 (2003).) The human umbilical cord blood-derived mast cells obtained in this study were cultured in the presence or absence of FCS, and Substance P, Compound 48/80 and The degranulation reaction by A-23187 was compared. As a result, degranulation by Substance P and Compound 48/80 was not observed at all up to 10 μM in Donor 4 mast cells cultured in the presence of FCS from day 57 to day 92 of cord blood collection. Cells in the absence of FCS showed about 20-25% degranulation at 10 μM (see FIG. 9). On the other hand, A-23187 showed about 45-55% degranulation at 10 μM regardless of the presence or absence of FCS. Furthermore, as a result of the same examination with Donor 6, the examination using A-23187 was not conducted, but as with Donor 4, the degranulation reaction by Substance P and Compound 48/80 was performed in cells cultured in the presence of FCS. It was not recognized.

MRNA analysis of Rec168
The tissue distribution of Rec168 was analyzed by quantitative PCR (TaqMan method) using real-time monitoring. The TaqMan method uses a PCR-amplified specific PCR strand in real time according to the fluorescence intensity of a fluorescent probe called TaqMan probe.
It is based on the principle of detection and quantification by Sequence Detection System (Applied Biosystems). TaqMan probes and primers specifically recognizing Rec168 are Assays-on-Demand (registered trademark) Gene Expression Products (Assay ID:
Hs00365019 s1) was used.

The template for TaqMan PCR reaction is dorsal
cDNA synthesized from total RNA derived from root ganglion and 41 types of human tissue-derived poly (A) ⁺ RNA (CLONTECH) was used as a template. cDNA synthesis is 6.5 μg total RNA or poly (A) ⁺
From 1 μg of RNA, SuperScript II was used as a reverse transcriptase, random hexamer as a primer, and the procedure was performed at 42 ° C according to the attached instruction manual. The composition of the reaction solution is 2 × TaqMan Universal PCR Master, using 30 ng of cDNA synthesized from total RNA as the template cDNA and 4 ng of cDNA synthesized from poly (A) ⁺ RNA in terms of RNA. Mix (Applied
(Biosystems) 5 μL, Assays-on-Demand (registered trademark) Gene Expression Products 0.5 μL, TaqMan GAPDH Control Reagents (Applied Biosystems) 0.5 μL were added to make the total volume 10 μL. Rec168 tissue distribution was compared by measuring GAPDH expression level simultaneously with Rec168 expression level and normalizing. As standard DNA, pME-Rec168 was prepared to 2.3 × 101−2.4 × 108 copies / 4 μL and used. The PCR reaction was performed at 95 ° С for 10 minutes, followed by 40 cycles of 95 ° С for 15 seconds and 60 ° С for 1 minute. The results are shown in FIG.
As is clear from FIG. 10, high expression of Rec168 was observed in skin and adipose tissue.

Expression analysis in human blood mast cells derived from cord blood
Expression analysis of Rec168 and related receptors in cord blood-derived cultured human mast cells was performed by quantitative PCR (TaqMan method) with real-time monitoring. As a TaqMan probe and primer that specifically recognizes Rec168 and related receptors, Assays-on-Demand (registered trademark) Gene Expression Products was used.

The reaction composition of TaqMan PCR reaction consists of total RNA derived from umbilical cord blood-derived human cultured mast cells (Donor 4, Donor 5) and total RNA derived from umbilical cord blood-derived human cultured mast cells that were induced to differentiate in cultures with or without serum. Using SuperScript II as a reverse transcriptase and random hexamer as a primer from (FCS +, FCS-), total cDNA synthesized at 42 ° C according to the attached instruction manual
Use as RNA 5 ng template, 2 ×
TaqMan Universal PCR Master Mix (Applied Biosystems) 5 μL, Assays-on-Demand (registered trademark) Gene Expression Products 0.5 μL, TaqMan GAPDH Control Reagents (Applied Biosystems) 0.5 μL were added to make a total volume of 10 μL. The expression levels of Rec168 and related receptors in cord blood-derived human mast cells were compared by measuring GAPDH expression level simultaneously with Rec168 expression level or related receptor expression level. As a standard DNA, a vector holding each gene cDNA fragment was prepared to 2.3 × 10 ¹ −8.9 × 10 ⁶ copies / 4 μL. The PCR reaction was performed at 95 ° С for 10 minutes, followed by 40 cycles of 95 ° С for 15 seconds and 60 ° С for 1 minute. The results (copy number / 5 ng total RNA) are shown in Table 4.

Screening for Rec168 inhibitors
Rec168 inhibitors were screened using suppression of increase in intracellular free Ca concentration by Substance P in Rec168 stably expressing 293 cells as an index. Rec168 stably expressing 293 cells cultured in D-MEM medium (Nikken Biomedical Research Institute CM4402) supplemented with 200 μg / mL hygromycin B (Invitrogen 10687-010) and 10% fetal calf serum, 2 μM Fura- Ca ²⁺ assay buffer (20 mM HEPES (Nacalai Code 17514-15), 115 mM NaCl (Sigma Cat. S5150), 5.4 mM KCl (Nacalai Code 285-14), 0.8, containing 2 / AM (Dojindo Laboratories) mM MgCl ₂ (Nacalai Code 209-09), 1.8 mM CaCl ₂ (Nacalai Code 067-29), 13.8 mM Glucose (Nacalai Code 168-06), 0.2% BSA (Sigma Cat. A8806), 2.5 mM Probenicid (Sigma Cat P8761), adjusted to pH 7.4 with KOH) and incubated at 37 ° C for 1 hour. After washing twice with Ca ²⁺ assay buffer, Ca ²⁺ assay buffer 1 × 10 ⁶
It was suspended so that it might become cells / mL. The cell suspension was dispensed into a 384-well black plate at 20 μL / well, and 4 μL of a test substance diluted to 100 μM with Ca ²⁺ assay buffer was added. After 5 minutes, 16 μL of 25 μM Substance P (Peptide Laboratories, Inc.) was added with FDSS3000 (Hamamatsu Photonics Co., Ltd.), and changes in fluorescence intensity at excitation light wavelengths of 340 nm and 380 nm were measured. From the measured fluorescence intensity, calculate the fluorescence intensity ratio (fluorescence intensity at excitation light wavelength 340 nm / fluorescence intensity at excitation light wavelength 380 nm; Ratio value), and add Substance P from the maximum ratio value after adding Substance P. The amount of change in the Ratio value (ΔRatio value) obtained by subtracting the previous Ratio value was calculated. The inhibition rate of intracellular Ca ²⁺ release by the test substance was determined by the following formula.
In the formula, A and B represent the following meanings.
A: ΔRatio value when test substance and Substance P are added
B: ΔRatio value when both test substance and Substance P are not added
C: ΔRatio value inhibition rate when only Substance P is added [%] = [1− (A−B) / (C−B) × 100
The following three compounds were obtained as Rec168 inhibitory active compounds.

Inhibition of increase in intracellular free Ca2 ⁺ concentration in Rec168 stably expressing 293 cells by Rec168 inhibitor compound
2 μM of Rec168 stably expressing 293 cells cultured in D-MEM medium (Nikken Biomedical Research Institute CM4402) supplemented with 200 μg / mL hygromycin B (Invitrogen 10687-010) and 10% fetal calf serum
Ca ²⁺ assay buffer (20 mM HEPES (Nacalai Code 17514-15), 115 mM NaCl (Sigma Cat. S5150), 5.4 mM KCl (Nacalai Code 285-14) containing Fura-2 / AM (Dojindo Laboratories) , 0.8 mM MgCl ₂ (Nacalai Code
209-09), 1.8 mM CaCl ₂ (Nacalai Code 067-29), 13.8 mM Glucose (Nacalai Code 168-06), 0.2% BSA (Sigma Cat. A8806), 2.5 mM Probenicid (Sigma Cat. P8761), KOH (Adjusted to pH 7.4) and incubated at 37 ° C for 1 hour. After washing twice with Ca ²⁺ assay buffer, Ca ²⁺ assay buffer 1 × 10 ⁶
It was suspended so that it might become cells / mL. The cell suspension was dispensed into a 384-well black plate at 20 μL / well, and 4 μL of a test substance diluted to 30 μM to 100 μM with Ca ²⁺ assay buffer was added. After 5 minutes, 25 μM Substance P (Peptide Institute, Inc.), 2.5 μM as a stimulant with FDSS3000 (Hamamatsu Photonics)
PACAP (6-27) (synthetic product), 7.5 μM
^{[D-Pro 4, D-} Trp 7,9,10] -Substance P (1-11) ( synthetic), and 7.5 [mu] M Ionomycin the (BIOMOL CA-201) was added 16 [mu] L, and the pumping light wavelength 340 nm The change in fluorescence intensity at 380 nm was measured. From the measured fluorescence intensity, calculate the fluorescence intensity ratio (fluorescence intensity at excitation light wavelength 340 nm / fluorescence intensity at excitation light wavelength 380 nm; Ratio value), and add stimulant from the maximum ratio value after addition of stimulant The amount of change in the Ratio value (ΔRatio value) obtained by subtracting the previous Ratio value was calculated.

Substance P, the intracellular free Ca ²⁺ concentration increases in ^{[D-Pro 4, D-} Trp 7,9,10] -Substance P (1-11) and PACAP (6-27) Rec168 stably expressing 293 cells by stimulating Compound 1, compound 2 and compound 3 all inhibited in a concentration-dependent manner. Substance P Compound 1 on stimulation, inhibition rate of compound 2 and compound 3 (IC ₅₀ value), respectively 4.5,4.0 and ^{5.6 μM, [D-Pro 4} , D-Trp 7,9,10] -Substance P (1- 11) IC ₅₀ values for stimulation were 3.2, 3.8 and 5.9 μM, respectively, and IC ₅₀ values for PACAP (6-27) stimulation were 3.7, 4.2 and 5.8 μM, respectively. On the other hand, none of the compounds inhibited the increase in intracellular free Ca ²⁺ concentration in Rec168 stably expressing 293 cells in response to Ionomycin stimulation (see FIG. 11).

Examination of inhibitory activity of Rec168 inhibitory compound against mast cell degranulation The following materials were used. Recombinant human SCF (PeproTech, cat.300-07), Recombinant human (PeproTech, cat.200-06), Recombinant human IL-3 (PeproTech, cat.200-03), 55 mM 2-ME (GIBCO , Cat.21985-023), Iscove's Modified Dulbecco's
Medium (IMDM, GIBCO, cat.12440-053), Insulin-Transferrin (GIBCO, cat.41400-045), 35% BSA (SIGMA, cat.A-7409), MethocultSF ^BIT (StemCell
technology company, cat.04236), PBS (-) ( Nikka Kensha, cat.CM6201), Substance P (peptide Institute Inc., cat.4013-v), [D -Pro 4, D-Trp 7,9,10 ] -Substance P (1-11) (synthetic product), PACAP (6-27) (synthetic product), A23187 (SIGMA, cat.C-7522), p-Nitrophenyl-N-acetyl-β-D-glucosamine (PN-GlcNAc, SIGMA, cat.N9376), DMSO (SIGMA, cat.D-2660)

1 μg / mL recombinant human SCF, 100 ng / mL recombinant human IL-3 and 500 ng / mL recombinant human IL-6, 0.1% BSA, 1% insulin-transferrin, 1 mL of umbilical cord blood-derived CD34 positive cells ( 1 × 10 ⁵ cells) was suspended, 10 times the amount of MethocultSF ^BIT was added, and culture was started in a CO ₂ incubator. About 1x10 ⁵ cells / mL using basic medium containing 100 ng / mL recombinant human SCF, 50 ng / mL recombinant human IL-6, 1 ng / mL recombinant human IL-3 at intervals of once a week When the cells were diluted as described above and cell growth was no longer observed, half of the cells were exchanged in the same medium. Human cultured mast cells were obtained by maintaining the culture for 8 weeks or longer.

The obtained human cultured mast cells were collected, washed twice with 0.1% BSA, 1% Insulin-Transferrin, and IMDM medium, and then a 96-well U-shaped culture plate at about 2 × 10 ⁴ cells / well 160 μL each was seeded. 3, 10, 30, and 100 μM of Compound 1, Compound 2 and Compound 3 (1% DMSO, 0.1% BSA, 1% Insulin-Transferrin, IMDM medium solution) were added to each cell in 20 μL, and 10 μM Substance P, 10 μM
^{[D-Pro 4, D-} Trp 7,9,10] -Substance P (1-11), after addition to mix the 10 μM PACAP (6-27) and 3 [mu] M A23187 by 20 [mu] L, the CO ₂ incubator And incubated at 37 &ordm; C for 30 minutes. After centrifugation (4 ° C, 3,000 rpm, 5 minutes), 50 μL of the supernatant was collected. The amount of degranulation was measured using the β-hexosamidase activity contained in granules released in the culture supernatant as an index. An equal volume of 4 mM pN-GlcNAc (0.1 M citrate buffer, pH 4.5) was added to 50 μL of the culture supernatant and incubated at 37 ° C. for 2 hours. The reaction was stopped by adding an equal amount of 0.1 M carbonate buffer (pH 10), and the absorbance at a wavelength of 405 nm was measured with a plate reader. The degranulation rate by each stimulus was calculated with β-Hexosamidase activity obtained by disrupting cells with water as 100%. Further, Substance P in degranulation inhibitory activity in the presence 0.1% DMSO with ^{compound, [D-Pro 4, D} -Trp 7,9,10] -Substance P (1-11), PACAP (6-27) and A23187 The degranulation rate after stimulation was calculated as 100%.

^{Substance P, [D-Pro 4} , D-Trp 7,9,10] -Substance P (1-11) and PACAP (6-27) degranulation inducing compounds of the human cultured mast cells by stimulation 1, compound 2 and compound All 3 inhibited in a concentration-dependent manner (see Table 5). Substance Compound 1 against P stimulation, inhibition rate of compound 2 and compound 3 (IC ₅₀ value), respectively 1.5,0.6 and ^{1.0μM, [D-Pro 4,} D-Trp 7,9,10] -Substance P (1- 11) The IC ₅₀ values for stimulation were 3.0, 1.4 and 4.4 μM, respectively, and the IC ₅₀ values for PACAP (6-27) stimulation were 1.2, 0.4 and 1.0 μM, respectively. On the other hand, none of the compounds inhibited the degranulation induction of mast cells in response to A23187 stimulation.

The results of PC12h / zif reporter assay (1); Chaperonin 10 (1-20), 2 ', 3'-cyclic nucleotide 3' phosphodiesterase (1-17), and Tropomyosin (1-12) are shown. A shows the results with Rec168-expressing cells, and B shows the results with Mock. The results of PC12h / zif reporter assay (2); MCD-Peptide, Somatostatin 28, Substance P are shown. The results of PC12h / zif reporter assay (3); Compound 48/80 are shown. Substance P concentration-dependent GTP-Eu binding to membrane fractions; Substance P concentration-dependent increase in GTP binding amount. GTP-Eu binding to Chaperonin10 (1-20) concentration-dependent membrane fraction; shows concentration-dependent increase in GTP binding amount of Chaperonin10 (1-20). GTP-Eu binding to membrane fraction dependent on pCNP (1-17) concentration; GTP binding amount dependent on concentration of pCNP (1-17) is shown. GTP-Eu binding to MCD peptide concentration-dependent membrane fraction; shows the concentration-dependent increase in MTP peptide concentration. Fig. 2 shows degranulation of human cultured mast cells by ligand; concentration-dependent degranulation of ligand substance. A23187 was used as a positive subject. Effect of FCS during culture on degranulation reaction; A23187 was used as a positive target. Comparison of Rec168 mRNA expression levels by tissue; high expression of Rec168 in skin and adipose tissue. Inhibitory activity of Rec168 inhibitory compounds against mast cell degranulation

Claims (11)

Characterized in that it comprises a step of measuring a signal transduction ability for a ligand having a Rec168 agonistic action in the presence and absence of a test substance using a cell expressing Rec168, and a step of comparing both measured values. A method for screening for a substance that suppresses degranulation of mast cells via Rec168. The screening method according to claim 1, further comprising measuring and comparing the degranulation reaction of mast cells via Rec168 in the absence and presence of the test substance using mast cells. Characterized in that it comprises a step of measuring a signal transduction ability for a ligand having a Rec168 agonistic action in the presence and absence of a test substance using a cell expressing Rec168, and a step of comparing both measured values. A method for identifying a substance that inhibits Rec168-mediated mast cell degranulation. 4. The identification method according to claim 3, further comprising measuring and comparing the degranulation reaction of mast cells via Rec168 in the absence and coexistence of the test substance using mast cells. A degranulation inhibitor for mast cells comprising the degranulation inhibitor identified by the method according to claim 3 or 4 as an active ingredient. The degranulation inhibitor according to claim 5, comprising at least one compound selected from the following as an active ingredient.
1- (10H-phenazin-5-yl) -ethanone,
2-azepan-1-yl-7-benzyl-1,7-dihydro-purin-6-one, and 3- (pyridin-2-ylmethyl) -2-thioxo-2,3-dihydro-1H-quinazoline -4-one. The therapeutic agent of the inflammatory disease in which the mast cell which contains the degranulation inhibitor identified by the method of Claim 3 or Claim 4 as an active ingredient is concerned. The therapeutic agent for an inflammatory disease involving mast cells according to claim 7, comprising at least one compound selected from the following as an active ingredient.
1- (10H-phenazin-5-yl) -ethanone,
2-azepan-1-yl-7-benzyl-1,7-dihydro-purin-6-one, and 3- (pyridin-2-ylmethyl) -2-thioxo-2,3-dihydro-1H-quinazoline -4-one. A mast cell degranulation inhibitor comprising a substance having a Rec168 antagonistic action as an active ingredient. A therapeutic agent for inflammatory diseases involving mast cells comprising a substance having Rec168 antagonistic action as an active ingredient. A diagnostic agent for inflammatory diseases involving mast cells, comprising as a reagent a Rec168 protein or a partial peptide thereof and a salt thereof, a polynucleotide encoding Rec168, an antibody against Rec168 or an antibody against Rec168 ligand.