WO2001019985A1 - Novel receptors and genes encoding the same - Google Patents

Abstract

Novel proteins; novel genes encoding the same; plasmids or transformants containing these genes; a process for producing the above proteins; cells containing the above novel proteins or cell membrane fractions of the same; a method and a kit for screening agonists or antagonists to the above novel proteins; agonists or antagonists to the above novel proteins; a method of treating or preventing diseases caused by the deficiency or excess in leukotriene B4, hydroxyeicosatetraenoic acids or hydroxyperoxyeicosatetraenoic acids; utilization of the above agonists or antagonists; and antibodies against the above novel proteins or fragments of the same. These novel proteins involve novel receptors of human leukotriene B4, hydroxyeicosatetraenoic acids or hydroxyperoxyeicosatetraenoic acids and functionally equivalent modifications thereof.

Description

 Description Novel receptor and gene encoding the same Technical field

The present invention relates to a novel protein (a novel receptor), a gene encoding the same, a plasmid or a transformant containing the gene, a method for producing the novel protein, a cell containing the novel protein or a cell membrane thereof. min, wherein Agonisu Bok or antagonists Bok screening methods and screening kit Bok for new proteins, the Agonisuto and antagonists Bok for new proteins, leuco triethyl down B _4, hydroxy eicosatetraenoic E emissions acids or hydroperoxides O carboxymethyl Eiko satay Bok The present invention relates to a method for treating or preventing a disease caused by deficiency or excess of raenoic acids, use of the agonist or antagonist, and an antibody against the novel protein or a fragment thereof. Background art

Leukotriene day ₄ is an unsaturated fatty acid that is biosynthesized from arachidonic acid by various enzymes, is the most potent leukocyte chemotactic substance among naturally occurring substances, and is a leukocyte activator. is there. Leuco Toryen B ₄ is made of white blood cells, seeds' s condition (e.g., infection, immunodeficiency, ulcerative colitis, Crohn's disease, necrotizing expansion after myocardial infarction reperfusion, psoriasis, atopic dermatitis, bronchial asthma, It is thought to be involved in the formation of acute respiratory distress syndrome, delayed neuronal death, vasospasm after subarachnoid hemorrhage, or perfusion injury after organ transplantation.

Leuco Bok Rie> B ₄ is, G protein-coupled receptor (G- pr 0 te I ncoupledreceptor; GPCR) are believed to engage in a variety of cellular functions through the already first receptor that is isolated The structure has been determined [Yokomizo et al., Nature, 387, pp. 62-62 (19997), while hydroxyeicosatetraenoic acid (HETE) and Hydropel quiche Icosatetraenoic acid (HPETE) is also an unsaturated fatty acid that is biosynthesized from arachidonic acid by various enzymes. Fig. 9 shows the structures and biosynthetic pathways of leukotrienes, HETEs and HPETEs. Enzymes involved in the biosynthesis of HETEs and HPETEs have been isolated and analyzed from various animal species, and their primary structures and enzymatic properties are well preserved. It is presumed to play an important role, but there are still many unknowns, including its mechanism of action and the presence of receptors. In addition to suggesting the existence of specific cell membrane receptors, experimental facts that bind to the nuclear receptor PPAR (peroxisomeprolifera tor-activated receptors) and regulate the transcription of various genes are announced Have been.

 The experimental facts reported so far for HETEs and HPETEs include, for 12 (S) -HPETE, the inducing activity of c-fos and c-jun (Rao, GN et al., J. Biol. Chem., 271, 27770-2-77664, 1996), AP-1 activating action (Rao, G, N. et al., J. Biol. em., 271, 2776 0-2 7764, 1996), and the contractile activity of vascular smooth muscle (Nishiyama, M. et al., Eur. J. Pharmacol., 3). 4 1, 5 7 — 6 3, 1 998) and 12 (S)-HETE, tumor cell adhesion enhancing activity (Η ο π,. V. et al., Prostaglandins, 4 4 , 13-4 29, 1992 and Grossi, I. M. et al., Caneer Res., 49, 109-103, 18989), The biological activity of 15 (S) -HPETE and 15 (S) -HETE is unclear.

In addition, among the enzymes that produce 12 (S) -HPE, 12-lipoxygenase (leukocyte type) and 12-lipoxygenase (platelet type) mice lacking these were created and analyzed. I have. The phenotypes that have been identified so far include 12-lipoxygenase (leukocyte) -deficient mice (BI eich, D. et al., J. Clin. Invest., 103, 143). 1-13 36, 1999 and Sun, D. et al., J. Biol. Chem., 271, 24055—240062, 1996) , Increased production of leukotriene, decreased LDL (low density lipoprotein) cholesterol oxidizing capacity, and type 1 diabetes 耐性 2-lipoxygenase (platelet type) deficient mice (Johnson, E.N. et al., Proc. Natl. Acad. Sc in USA, 95 , 3100—3105, 1998 and Johnson, E.N., et al., J. Invest. Dermatol., 111,861—865, 1999. ), There is an increase in platelet aggregation by ADP, an increase in mortality in a thrombosis model by ADP administration, and a decrease in water retention in the skin basement membrane.

Various conventional leuco Bok Lien B ₄ receptor antagonists have been developed, these are mainly were those directed to the first receptor. However, it has been known that these antagonists alone cannot provide a sufficient therapeutic effect, and a stronger therapeutic agent or treatment method has been demanded.

In view of such a situation, the present inventor has found and found a completely novel receptor, determined the structure thereof, and found that this is a low-affinity second receptor of leukotriene day _4. Was found. The discovery of this second receptor is expected to lead to the development of new antagonists that are more potent (eg, inhibit both the first and second receptors), or to develop therapeutics using antibodies and the like. You.

Moreover, the present inventors, the present inventors the novel leuco which has found Toryen B ₄ second receptor, as well as leuco Bok Lien B _4, a plurality of hydroxy Eiko Sate Bok Lae phosphate (HETE) acids and Hidoroperu It has been found that the receptor has a binding ability to human xyeicosatetraenoic acids (HPETEs) and an ability to transmit intracellular signals by these ligands. No receptor that binds to HETEs or HPETEs has been reported in the past, and the present inventors have found its existence for the first time.

 The present invention is based on such findings. Disclosure of the invention

The present invention is, (1) protein comprising the amino acid sequence represented by SEQ ID NO: 2 (hereinafter, sometimes referred to as "LT 8 ₄ receptor protein">, (2) the functionally equivalent modified (Hereinafter sometimes simply referred to as “functionally equivalent variant”), or (3) a protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. Ie, LT 8 ₄ consisting of an amino acid sequence having 6 0% or more homologous sex respect to the amino acid sequence of the receptor protein) protein (hereinafter, Rukoto referred to as "homologous protein") relates.

Hereinafter, said L T已₄ receptor protein, variation functionally equivalent, and also to homologous proteins, referred to as "novel protein according to the present invention".

Further, the present invention, the novel proteins (i.e., to Ding 8 ₄ receptor protein, variation functionally equivalent, and the homologous protein) according to the present invention relates to a gene encoding a.

 The present invention also relates to a plasmid containing the gene.

 The present invention also relates to a transformant containing the gene.

 The present invention also relates to the method for producing a novel protein according to the present invention, which comprises culturing the transformant.

 The present invention also relates to a cell containing the novel protein according to the present invention or a cell membrane fraction thereof.

 The present invention also provides a novel protein according to the present invention, characterized by using the novel protein according to the present invention, a cell membrane fraction containing the same, or a cell containing the novel protein according to the present invention. The present invention relates to a method for screening an agonist or an antagonist for a protein and a screening kit. The present invention also relates to an agonist and an antagonist for the novel protein according to the present invention.

Further, the present invention provides a method for treating or preventing a disease caused by a deficiency of leucotriene- ₄ , hydroxyeicosatetraenoic acids, or hydroperoxyeicosatetraenoic acids by using the agonist according to the present invention. And administering to the subject in need thereof an effective amount.

Further, the present invention is that the antagonistic Bok by the of the present invention, leuco Toryen beta _4, hydroxy Eiko satay Bok Raen acids, or Hidoroperu old Kishieikosate Bok Raen excessively subject in need of treatment or prevention of caused by diseases of acids, The present invention relates to a method for treating or preventing the disease, which comprises administering an effective amount.

Further, the present invention relates to the use of Agonisu Bok by the of the present invention, leuco Bok Lien beta _4, human The present invention relates to a use for producing an agent for treating or preventing a disease caused by a deficiency of droxyeicosatetraenoic acids or hydroperoxyeicosatetraenoic acids.

Further, the present invention provides a method for producing a therapeutic or prophylactic agent for a disease caused by an excess of leukotriene B ₄ , hydroxyeicosatetraenoic acid, or hydropersylxieicosatetraenoic acid in the antagonist according to the present invention. Regarding the use of Furthermore, the present invention relates to an antibody or a fragment thereof, which specifically reacts with the novel protein according to the present invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing ^[3 H] and the concentration of LTB _4, the relationship between ^[3 H] binding amount of LTB ₄ to the cell membrane fraction.

 FIG. 2 is a graph showing the results of a scout yard analysis performed based on the results shown in FIG.

3, when using the C HO- FL AG BLT 1 cells is a graph showing the binding of ^[3 H] LTB ₄ to the cell membrane fraction.

FIG. 4 is a graph showing the amount of [ ³ H] LTB ₄ bound to a cell membrane fraction when CHO-HABLT 2 cells were used.

Figure 5 is based on the data shown in FIG. 3 and FIG. 4 is a graph showing the relative binding inhibition activity when formed into a 1 0 0% binding inhibition by LTB _4.

FIG. 6 is a graph showing the degree of increase in intracellular calcium ion (Ca ²⁺ ) concentration when CHO-FLAG BLT1 cells were used.

FIG. 7 is a graph showing the degree of increase in intracellular calcium ion ( ^{Ca2 +} ) concentration when CHO-HABLT2 cells were used.

Figure 8 is a repo one data one gene plus Mi de p CRE-L uc DNA and human Bok Roy co Bok Rie> B ₄ second receptor expression plasmid (ph BLT 2) and Bok lance Fuekushiyon simultaneously with HEK 2 9 is a graph showing the expression level of luciferase in 93 cells.

FIG. 9 is an explanatory diagram showing the structures and biosynthetic pathways of leukotrienes, HETEs and HPETEs. BEST MODE FOR CARRYING OUT THE INVENTION

A novel protein according to the present invention, (1) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 (i.e., LT 8 ₄ receptor protein), (2) functionally equivalent modifications of their body (i.e., variation functionally equivalent), and (3) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 (i.e., LTB ₄ receptor protein) 6 0% or more homology with respect to the amino acid sequence of (Ie, a homologous protein) comprising an amino acid sequence having the formula: The novel protein according to the invention, proteins consisting Amino acid sequence shown in SEQ ID NO: 2 (i.e., human Bok leuco Bok Rie> B ₄ second receptor), its functional equivalence variants, Alternatively, protein comprising the amino acid sequence represented by SEQ ID NO: 2 (i.e., human Bok 'leuco Toryen B ₄ second receptor) has a function to 6 0% or more homology to the amino acid sequence of Proteins consisting of an amino acid sequence are preferred. In the present specification, “protein” includes a salt of a protein, and further includes both a protein having no sugar chain and a protein having a sugar chain.

Human Bok leuco Bok Rie> B ₄ second receptor which is one of the novel proteins of the present invention, 3 5 2 amino acids the amino acid sequence of a known human Bok leuco Toryen B ₄ first receptor full length whereas consisting of residues, 3 5 consists of 8 amino acid residues, known prior Kihi Bok. leuco Bok Lien已₄ 4 amino acid sequence and the full length of the first receptor 5.2% of § Mino Has acid homology. Further, the Amino acid sequence of human Bok 'leuco Bok Rie> B ₄ second receptor according to the invention, so far plurality retain the amino acid common to a number The reported G protein-coupled receptor (GPCR) Therefore, it is presumed that it is a GPCR. The amino acid sequence of human Bok 'leuco Bok Rie> B ₄ second receptor according to the invention, the sequence is not registered in DDB JZEMB and / G Θ nbank database, an international gene database, heretofore It is a new receptor that was unknown.

Also, human Bok 'leuco Bok Lien已₄ second receptor according to the invention not only leuco Bok Lien B _4, a plurality of hydroxycarboxylic Eiko satay Bok Raen acid (HETE) acids and human mud pel O carboxymethyl Eiko satay The ability to bind to traenoic acid (HPETE) These receptors have intracellular signal transduction ability by these ligands.

 The HETEs and HPETEs are unsaturated fatty acids biosynthesized from arachidonic acid by various enzymes. HETEs include, for example, 1 2 (R) 1 HETE, 1 2 (S) — HETE, 15 (S) — HETE, 5 (S) — HETE, or 8 (S) — HETE; Classes include, for example, 15 (S) -HPETE, 5 (S) -HPETE, 8 (S) -HPETE, 12 (S) -HPETE, or 15 (R) -HPETE.

Thus, in the present specification, the "protein consisting of the amino acid sequence represented by SEQ ID NO: 2 of Sequence Listing," but referred to as "human Bok 'leuco Bok Rie> B ₄ second receptor" for convenience a term, this receptor, leuco Toryen B ₄ as well, HETE such multiple [e.g., 1 2 (R) -HETE, 1 2 (S) - HETE, or 1 5 (S) -HETE ] And HPETEs [eg, 15 (S) — HPETE].

As used herein, the term “functionally equivalent variant” means that the amino acid sequence has a deletion, substitution, or addition of one or more (particularly, one or several) amino acids in the amino acid sequence of the original protein. and an amino acid sequence, moreover, human Bok 'leuco tri E emissions B ₄ second receptor substantially the same activity (e.g., binding ability to the leuco Bok Lien B _4, HETE acids, and Z or HPETE acids, And the resulting various cell stimulating activities). The functional variant includes salts of the functional variant, and further includes both those having no sugar chain and those having a sugar chain.

Therefore, as long as these conditions are satisfied, the origin of the functionally equivalent variant is not limited to human. For example, human Bok 'leuco Bok Lien B ₄ second receptor variants in human Bok (e.g., human Bok' by alleles of the leuco Toryen B ₄ second receptor gene co - proteins de) only contains not, human Bok other organisms [eg ', baboon Bok mammal (e.g., mouse, rat Bok, hamster, or I j)] contained ₄ second receptor derived from leuco Toryen B or its variants. Furthermore, those of the native protein (i.e., mutants derived from human Bok, or human leuco Bok Rie from organisms other than preparative> B ₄ second receptor or a variant thereof) or human leuco Bok Lien B ₄ second Second reception Proteins artificially modified by genetic engineering based on the condition are included. As used herein, the term “variant” refers to “var I at I 0 n”, that is, an individual difference observed in the same protein within the same species, or a difference observed in a homologous protein between several species. means.

Human leuco Bok Rie> B ₄ second receptor variants in human Bok, or leuco derived from organisms other than human Bok Toryen B ₄ second receptor or variants thereof, those skilled in the art, human Bok leuco Bok Rie> B ₄ nucleotide sequence of the second receptor gene (i.e., the nucleotide sequence represented by SEQ ID NO: 1 of the sequence Listing) based on information, can be acquired. For example, based on information of the human leuco Triester> B ₄ nucleotide sequence of the second receptor gene to design appropriate primers one or probe, said primer one or probes, organisms and purpose [e.g., mammals Animals (eg, humans, mice, rats, hamsters, or dogs)] (eg, total RNA or mRNA fraction, cDNA library, or phage library). The desired protein can be prepared by obtaining the gene of the target protein by performing the PCR method or the hybridization method, and expressing the gene using an appropriate expression system.

 In addition, the above-mentioned protein which has been artificially modified by genetic engineering can be prepared by a conventional method, for example, by a site-directed mutagenesis method (site-specificmutagenews).

 Examples of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing include, for example, a fusion protein of a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and a fusion partner. it can. In the fusion protein, the fusion partner can be bound to the N-terminal and Z- or C-terminal of the protein having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.

Examples of the fusion partner include a protein for purification [eg, 'all or a part of glutathione S-transferase (GST)], a protein for detection [eg, hemagglutinin or /?-Galactosidase peptide] (All or part of LacZa)] or an expression protein (eg, ', signal sequence). Further, in the fusion protein, a proteolytic enzyme that undergoes limited degradation (for example, thrombin or factor) is interposed between the protein having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and the fusion partner. An amino acid sequence that can be cleaved at X a) can be introduced as appropriate.

Homologous protein of the present invention, as long as the amino acid sequence having 6 0% or more homology with respect to amino acid sequence of LT 8 ₄ receptor protein, is not particularly limited, the amino acid sequence of the LT beta receptor proteins With respect to amino acid sequences having a homology of preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more. Can be. Also, homologous proteins of the present invention, LTB ₄ receptor protein 6 0% or more with respect to the amino acid sequence of (preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably It is composed of an amino acid sequence having a homology of 95% or more, particularly preferably 98% or more, and has substantially the same activity as that of the human _4th receptor (for example, leuco). Toryen B _4, HETE compound, and or binding ability to HPETE acids, and is preferably thereby a protein with various cell-stimulating activity) occurring.

Novel proteins by these invention (i.e., LTB ₄ receptor protein, variation functionally equivalent, and the homologous protein), is possible to get by a variety of known methods, for example, by using a gene according to the invention known It can be prepared by the genetic engineering technique described above. More specifically, a transformant according to the present invention described later (that is, a transformant containing a gene encoding a novel protein according to the present invention) is used under conditions that allow expression of the novel protein according to the present invention. It can be prepared by culturing under the following conditions, and separating and purifying the target protein from the culture by a method generally used for separating and purifying proteins. The separation and purification methods include, for example, ammonium sulfate salting out, ion exchange column chromatography using ion exchange cellulose, molecular sieve column chromatography using molecular sieve gel, affinity column chromatography using protein A binding polysaccharide. Dialysis, lyophilization or the like.

The gene according to the invention is, in particular, so long as it encodes the novel protein according to the invention. It is not limited, and examples thereof include a gene consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing. The term “gene” in the present specification includes:

Includes both DNA and RNA. Nucleotide sequence or Ranaru the gene represented by SEQ ID NO: 1 is human • leuco Bok Lien 8 ₄ second receptor co one de consisting of the amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing .

 The plasmid according to the present invention is not particularly limited as long as it contains the gene according to the present invention.For example, the plasmid according to the present invention may be added to a known expression vector appropriately selected depending on the host cell used. Plasmids obtained by insertion can be mentioned.

 The transformant of the present invention is not particularly limited as long as it contains the gene of the present invention. For example, the transformant of the present invention is a transformant in which the gene of the present invention has been integrated into a chromosome of a host cell. Alternatively, it may be a transformant containing the gene according to the present invention in the form of a plasmid. Further, the transformant may be a transformant expressing the protein according to the present invention, or may be a transformant not expressing the protein according to the present invention. The transformant of the present invention can be obtained, for example, by transforming a desired host cell with the plasmid of the present invention or with the gene itself of the present invention.

Examples of the host cell include, for example, commonly used known microorganisms, for example, Escherichia coli or yeast (Saccharomyces cerevisiae) or known cultured cells, for example, animal cells (for example, CHO cells, HEK- 293 cells or COS cells) or insect cells (eg, BmN4 cells) Examples of the known expression vectors include, for example, p UC, p TV, pGEX, pKK, or pTrcHis; for yeast, pEMBL 丫 or pYES2; for CH0 cells, pcDNA3 or pMAM neo; HEK— For 293 cells, pcDNA3; for COS cells, pcDNA3; for BmN4 cells, the polyhedrin promoter of silkworm nuclear polyhedrosis virus (BmNPV). Vector (eg, pBK2283) can be mentioned. Novel protein (i.e., LT snake ₄ receptor protein, variation functionally equivalent, and the homologous protein) according to the invention the cells of the invention containing the novel proteins that by the present invention as long as they are expressed on the cell membrane surface Although not particularly limited, for example, the transformant according to the present invention (that is, a cell transformed with a plasmid containing a gene encoding a novel protein according to the present invention) may be used in accordance with the present invention. Or by injecting mRNA encoding the novel protein of the present invention into appropriate cells, and then culturing the novel protein according to the present invention. It can also be obtained by culturing under conditions that allow the expression of E.

 In addition, the cell membrane fraction according to the present invention containing the novel protein according to the present invention can be obtained, for example, by crushing the cells according to the present invention and separating a fraction containing a large amount of cell membranes. Examples of the method for crushing cells include a method of crushing cells with a homogenizer (for example, a Potter-E levehjem type homogenizer), a method of crushing with a single ring blender or a polytron (Kinematica), and a method using ultrasonic waves. Crushing, or crushing by ejecting cells from a fine nozzle while applying pressure with a French press or the like can be mentioned. Examples of the cell membrane fractionation method include, for example, a fractionation method using centrifugal force, for example, a fractionation centrifugation method or a density gradient centrifugation method.

For new protein according to the present invention (particularly human Bok leuco Bok Rienyo ₄ second receptor), the Agonisu Bok or antagonists Bok of subscription-learning process according to the present invention, a novel protein (i.e., LT 8 ₄ receptor according to the invention body protein, variation functionally equivalent, and the homologous protein), or cell membrane fraction according to the invention [ie, a novel protein (i.e. according to the invention, LT 8 ₄ receptor protein, functionally equivalent variants, or homologous protein) cell membrane fraction containing, or cells according to the present invention [i.e., a novel protein (i.e. according to the present invention, containing 1_ Ding 8 ₄ receptor protein, variation functionally equivalent, or homologous protein) Cells]. In the screening method of the present invention, when a cell containing the novel protein of the present invention or a cell membrane fraction thereof is used, a cell containing no leukotrien day ₄ first receptor or a cell membrane fraction thereof can be used. . Further, in the screening method according to the present invention, it is determined whether or not the test compound specifically binds to the novel protein according to the present invention, and if necessary, a natural ligand (ie, leuco) is added to the novel protein according to the present invention. Toryen B _4, HETE such, or cell stimulating activity caused by HPETE acids) are attached, for example, an increase in intracellular calcium concentration, activity inhibition of adenyl two Le cyclase, promotion of cell migration activity, My Bokujiko It utilizes activation of kinase activated kinase, c-fos and c-jun inducing activity, AP-1 activating effect, vascular smooth muscle contraction activity, or tumor cell adhesion enhancing activity.

In the screening method according to the present invention, for example, the novel protein according to the present invention or the cell membrane fraction according to the present invention or the cell according to the present invention is brought into contact with a test compound, and the novel protein according to the present invention or the cell membrane fraction according to the present invention is contacted. or a cell according to the invention, by a test compound to analyze whether binding, present invention Agonisu Bok or against the novel protein (particularly human Bok leuco Bok Rie> B ₄ second receptor) by The screening can be performed without distinguishing the antagonist.

Specifically, under each condition in the absence and presence of a test compound, a novel protein according to the present invention or a cell membrane fraction according to the present invention or a cell according to the present invention, and a labeled natural ligand (ie, leukotriene) B ₄ , HETEs or HPETEs), and the specific binding of the natural ligand to the novel protein according to the present invention, the cell membrane fraction according to the present invention, or the cell according to the present invention under the above conditions. by comparing the amount, novel protein according to the present invention (particularly a human 'Roikotoryen B ₄ second receptor) can be screened without distinction Agonisu Bok or antagomir- varnish Bok against. That is, when the test compound is an agonist or an antagonist against the novel protein according to the present invention, a natural protein against the novel protein according to the present invention, the cell membrane fraction according to the present invention, or the cell according to the present invention in the absence of the test compound. The specific binding amount in the presence of the test compound is lower than the specific binding amount of the ligand.

In the screening method of the present invention, when comparing the specific binding amount of the natural ligand to the novel protein or cell membrane fraction or cell according to the present invention, As the natural ligand, a labeled natural ligand can be used. As the label, for example, a radioisotope, for example, [ ³ H] or [′ ⁴ C] can be used.

As described above, in the screening method of the present invention, a compound that binds to the novel protein according to the present invention, the cell membrane fraction according to the present invention, or the cell according to the present invention, and inhibits the binding of these to the natural ligand, (the Japanese human Bok 'leuco Bok Rie> B ₄ second receptor) novel proteins by can be chosen indifferently as Agonisu preparative or Antagoni scan Bok against.

In another embodiment of the screening method according to the present invention, a cell according to the present invention and a labeled natural ligand (ie, leukotriene B or HETE) are used in the absence and presence of a test compound. , Or HPETEs), comparing the specific binding amount of the natural ligand to the cells according to the present invention under the above conditions, and further measuring the specific cell stimulating activity of the natural ligand under the above conditions. by comparison, the new protein according to the invention (in particular human Bok 'leuco Bok Rie> B ₄ second receptor) can be screened to distinguish Agonisu Bok or antagonistic Bok against.

In the embodiment, bound to the cell according to the present invention, a compound having a cell stimulating activity via the receptor contained in the cells, the novel protein (especially according to the invention inhibit Bok 'leuco Bok Rie> B ₄ second Receptor) can be selected as an agonist.

On the other hand, in the embodiment, although you inhibit the binding of the cells with the natural ligand of the present invention, a compound having no cell-stimulating activity, novel protein (in Japanese human Bok 'leuco Bok Rie> B ₄ first according to the invention (Two receptors).

 The screening method of the present invention can be carried out by utilizing, for example, the inhibition of the activity of adenylate cyclase as the cell stimulating activity.

In the screening method of this embodiment, for example, a novel protein according to the present invention expressed on the cell membrane (preferably, by introducing human Bok leuco Bok Lien已₄ second receptor including expression base Kuta scratch excess And the cyclic AMP response By using cells containing a luciferase gene (hereinafter referred to as screening cells) in which the system lj (CRE) is located 5 'upstream, novel proteins (particularly, human leuco triae) according to the present invention can be obtained. > B ₄ second receptor) to distinguish Agonisu Bok or § Ntago two be sampled for can be screened. This aspect, intracellular signal transduction caused by the natural ligand binds to the leuco Bok Lien B ₄ second receptor, i.e., the active suppression of adenylate cyclase, which is one of the cell-stimulating activity of the leuco Toryen B ₄ To use. Specifically, when the leuco Bok Lien B 4 natural ligand to the second receptor is bound, leuco Toryen B ₄ second receptor which is one of the G protein family one that coupled to G, the family, Suppresses adenylate cyclase and reduces the amount of cyclic AMP (generated from ATP by adenylate cyclase) in cells, resulting in the reduction of CRE introduced into the screening cells. It utilizes the fact that the transcription of the luciferase gene in the promoter region is suppressed.

Hereinafter, by the embodiment, the procedure novel protein (especially the human leuco Triester> B ₄ second receptor) is screened to distinguish Agonisu bets or antagonists Bok for the present invention will be described more specifically.

 That is, the CRE introduced into the screening cell is present in the transcriptional regulatory region of a group of genes (cyclic AMP inducible genes) whose expression is enhanced when the concentration of cyclic AMP in the cell is increased. Base sequence. Therefore, adding an activator of adenylate cyclase [eg, forskolin (FSK)] to screening cells increases the concentration of intracellular cyclic AMP, and consequently downstream of CRE. The expression level of the luciferase gene located at the site is increased. The luciferase expression level can be easily measured by measuring the luminescence derived therefrom.

Further, when adding the adenylyl two Le acid Siclà Ichize activator, (especially human Bok. Leuco Bok Rie> B ₄ second receptor) new protein according to the present invention the addition of natural ligand simultaneously, In addition to the promotion of the adenylate cyclase activity caused by the adenylate cyclase activator, the suppression of the activity of adenylate cyclase generated by the action of the natural ligand on the novel protein of the present invention. To happen, as a result, adenylic acid The luciferase expression level is reduced as compared to the case where the cyclase activator is administered alone.

Simultaneous addition of the adenylate cyclase activator, the natural ligand of the novel protein according to the present invention (particularly the human / leukotrienyo ₄ second receptor), and the test compound to the cells for screening When the test compound has an action as an antagonist, the same luciferase as when only the adenylate cyclase-active agent is administered alone (in the absence of the natural ligand and in the absence of the test compound). -Expression level is observed [ie, it antagonizes the action of the natural ligand on the novel protein (receptor) of the present invention]. On the other hand, when the test compound has an action as an agonist, the luciferase was compared with the case where only the adenylate cyclase activator was administered alone (in the absence of the natural ligand and the absence of the test compound). The expression level is reduced [that is, the novel protein (receptor) according to the present invention has the same action as a natural ligand].

It is, simply when the Agonisu Bok only Sukurini make one ring is Ade Nirusanshi class Ichize activator, novel protein according to the invention (in particular human leuco Bok Lien B ₄ second receptor) Instead of simultaneously adding the natural ligand and the test compound to the cells for screening, only the adenylate cyclase activator and the test compound are simultaneously added to the cells for screening. In this case, when the test compound has an action as an agonist, a natural ligand (ie, an adenylate cyclase activator; or a natural ligand is added to cells for screening) Similar luciferase expression levels are seen.

In this case, it can be easily confirmed that the effect of the test compound is an effect of binding to the novel protein according to the present invention. For example, in parallel with the above test using a screening cell (that is, a cell expressing the novel protein of the present invention on the cell membrane and further containing a luciferase gene located 5 ′ upstream of the CRE), A similar test is performed using control cells (for example, cells in which the CRE contains the luciferase gene located 5 ′ upstream but does not express the novel protein according to the present invention on the cell membrane). As a result, the action of the test compound is caused by the binding to the novel protein according to the present invention. In the case of no effect, the same phenomenon regarding the luciferase expression level is observed in the screening cells and the control cells, whereas the effect of the test compound is the effect of the binding to the novel protein according to the present invention. In some cases, different phenotypes of luciferase expression are observed between screening cells and control cells.

Note that the luciferase used in the above embodiment is an enzyme used when fireflies and the like emit light, and mammals do not have this. Therefore, the use of mammalian cells is preferable because the background is low. Luciferase is one of the reporter enzymes such as chloramphenicylase tranferase and /?-Galactosidase, but has excellent quantitative properties and high detection sensitivity. Because the measurement method is simple, it is widely used. Emission mechanisms are luciferin is a group substance _{(C, 3 H, 2 N} 2 S 2 0 3) is oxidized by Lucifera Ichize in the presence ATP, luminescent substance due to be produced. Luciferase has been applied to various Atsay methods (eg, promoter atsay, two hybrid assay, or screening for agonist or antagonist). Test compounds that can be used in the screening method of the present invention are not particularly limited, and include, for example, natural or synthetic compounds (eg, lipids, proteins, or peptides), fermentation products, cell extraction Liquid, plant extract, or animal tissue extract.

Screening kit Bok of the present invention, novel protein according to the invention (i.e., 1_ Chomi ₄ receptor protein, variation functionally equivalent, and the homologous protein), Moshiku cell membrane fraction according to the present invention (i.e., LT beta receptor protein, variation functionally equivalent, and cell membrane fractions containing homologous protein), or cells according to the present invention (i.e., L Ding 8 ₄ receptor protein, variation functionally equivalent, and containing homologous protein cells ) including. The screening kit I of the present invention, if desired, various reagents, for example ', labeled leuco Bok Rie> B ₄ or HETE acids or HPETE acids were unlabeled leuco Toryen B ₄ or HETE acids or HPETE acids, binding reaction And Z or a wash buffer. Specifically, the screening kit of the present invention comprises a novel protein according to the present invention. 1 includes a full or cell by cell membrane fraction or the present invention, optionally, labeled natural Li ligand (i.e., labeled leuco Bok Lien B ₄ or HETE acids or HPETE, in particular the ^[3 H] labeled leuco Bok Rie B ₄ or HETEs or HPETEs), unlabeled natural ligand (ie, unlabeled leukotriene day ₄ or HETEs or HPETEs), and / or a binding reaction buffer.

Another embodiment of the screening kit Bok of the present invention, express a novel protein on the cell membrane according to the present invention (preferably, over-expression by introducing the expression vector one containing human Bok leuco Toryen B ₄ second receptor) And a cell containing a luciferase gene having a cyclic AMP response element (CRE) located 5 ′ upstream, and optionally a luciferase substrate, an adenylate cyclase activator (eg, FSK) , A natural ligand, and / or a buffer for a binding reaction.

A screening kit according to yet another embodiment of the present invention is a kit for expressing the novel protein of the present invention on a cell membrane (preferably, by introducing an expression vector containing a human / leukotriene day ₄ second receptor). Overexpressed) and cyclic AMP response element

(CRE) contains the luciferase gene located at the 5 'upstream and the CRE contains the luciferase gene located at the 5' upstream, but expresses the novel protein of the present invention on the cell membrane. If desired, a luciferase substrate, an adenylate cyclase activator (eg, FSK), and a buffer for Z or binding reaction can be included.

Agonisu Bok for new protein according to the present invention (particularly human Bok leuco Bok Rie> B ₄ second receptors), for example, diseases caused by lack of leuco Toryen B _4, if example embodiment, such as infection or immunodeficiency It is useful as a therapeutic or prophylactic agent, and also as a therapeutic or prophylactic agent for diseases caused by deficiency of HETEs or HPETEs. The agonist of the present invention is administered alone or, preferably, together with a usual pharmaceutically acceptable carrier or diluent to a subject in need of treatment or prevention of the above-mentioned diseases in an effective amount. Can be. In addition, the agonist of the present invention can be used for producing an agent for treating or preventing the above-mentioned diseases. The agonist of the present invention can be obtained, for example, by the agonist screening method of the present invention. it can.

- How, antagonist to a novel protein (especially human Bok 'leuco Bok Lien 8 ₄ second receptor) according to the invention, for example, diseases you excessively due leuco Bok Lien B _4, for example, infectious diseases, immunodeficiency Ulcerative colitis, Crohn's disease, necrosis after myocardial infarction reperfusion, psoriasis, atopic dermatitis, bronchial asthma, acute respiratory distress syndrome, delayed neuronal cell death, vasospasm after subarachnoid hemorrhage, or organ It is useful as a therapeutic or preventive agent for post-transfusion perfusion injury, etc., and diseases caused by excess of HETEs or HPETEs, such as psoriasis vulgaris, arteriosclerosis, diabetes, Parkinson's disease, Alheimer's disease, cancer, Or it is useful as an agent for treating or preventing vasospasm after subarachnoid hemorrhage. The antagonist of the present invention is administered alone or, preferably, together with a usual pharmaceutically acceptable carrier or diluent to a subject in need of treatment or prevention of the above-mentioned diseases in an effective amount. can do. Further, the antagonist of the present invention can be used for producing an agent for treating or preventing the above-mentioned disease. Furthermore, antagonists Bok of the present invention are also useful in inhibiting the binding of human Bok 'leuco Bok Rie> B ₄ natural ligand against the second receptor. The antagonist of the present invention can be obtained, for example, by the antagonist screening method of the present invention.

 Antibodies according to the present invention include monoclonal and polyclonal antibodies.

Monochromator port one monoclonal antibody according to the present invention, as a an antigen for immunization and subscription-learning for antigen, a novel protein (particularly human Bok 'leuco Bok Rie> B ₄ second receptor) according to the invention or their partial fragment It can be obtained by a means known per se except for using.

For example, a mouse is immunized with the immunizing antigen, and spleen cells obtained from the mouse and mouse myeloma cells are isolated from each other by the method described in Nature, 256, 495 (1975). Cell fusion method, or cell fusion using the electric cell fusion method described in Imm unol. Method, 100, 181-189 (19897), and the screening antigen is used. By performing screening using E. coli, a hybridoma producing the monoclonal antibody of the present invention can be obtained. The medium for culturing the Hypri-doma may be any medium suitable for culturing Hypri-doma, and is preferably Dulbecco's modified Eagle's minimum essential medium (Du I beccos modified E eag I esminimumessent I a medium).培 地 A medium containing fetal serum, L-glutamine, L-pyruvic acid, and antibiotics (penicillin G and streptomycin) is used.

Culture of the High Priestess dormer, when carried out in a medium can be carried out in under conditions of 5% C_〇 ₂ concentration and 3 7 ° C for about 3 days. When culturing in the abdominal cavity of a mouse, it can be performed in about 14 days.

 The monoclonal antibody can be separated and purified from the thus obtained culture solution or mouse ascites by a method generally used for protein separation and purification.

 Such methods include, for example, ammonium sulfate precipitation, ion exchange column chromatography using ion exchange cellulose, molecular sieve column chromatography using molecular sieve gel-1, affinity column chromatography using protein A-binding polysaccharides, dialysis, Or lyophilization and the like can be mentioned.

Further, polyclonal antibody of the present invention also, as an antigen for immunization and antigen for screening (especially human Bok leuco Bok Rie> B ₄ second receptor) novel protein according to the invention but using or their partial fragment It can be prepared by a method known per se, for example, the method shown below. That is, a physiological saline solution containing an antigen is mixed with an equal amount of Freund's complete adjuvant or incomplete adjuvant, or an equivalent thereof, for example, Hunter's Titer Max ™ (Funakoshi; Cat. No. YT. It is emulsified and mixed with 001-000, Tokyo, Japan) and administered subcutaneously, intraperitoneally, or intramuscularly to mammals (especially egrets or goats) (first immunization). Thereafter, the same operation is performed at intervals of 2 to 4 weeks, and immunization is performed several times. Blood can be collected from the carotid artery or heart of a mammal one to two weeks after the final immunization, and the serum can be prepared by salting out with ammonium sulfate.

The antibody fragment according to the present invention is a partial fragment of the antibody of the present invention (including a monoclonal antibody and a polyclonal antibody) and has the same reaction characteristics as the original antibody. It is not particularly limited as long as it has isomerism. Examples of the antibody fragment according to the present invention include Fab, Fab ', F (ab') ₂ , or Fv. The antibody fragment of the present invention can be obtained, for example, by digesting the polyclonal antibody or the monoclonal antibody of the present invention with a proteolytic enzyme by a conventional method, and subsequently by a conventional method of protein separation and purification. be able to. Example

 Hereinafter, the present invention will be described specifically with reference to Examples, but these do not limit the scope of the present invention. In each of the following examples, unless otherwise specified, the genetic manipulation method using E. coli is described in "Molecular Cloning" [Maniatis et al., Old Spring Harbor Laboratory, (1989)]. According to the method described in

Example 1: Cloning of DNA encoding the human leukotriene B4 second receptor

After infecting Escherichia coli (XL-1 Blue MR F strain) with phage from a commercially available human genome library (Human Lymphocyte Genomic Library! Approximately 5 × 10 ⁴ plaques were spread on the top and incubated at 37 ° C. overnight to form plaques. After transferring the plaque onto the top of 20 nylon filters, denaturation solution (0.5 mo I ZL sodium hydroxide and 1.5 mo I ZL sodium chloride), neutralization solution [0.5 mo I / lot Lis monohydrochloric acid (pH 8.0), and 1.5 mo I ZL sodium chloride] and washing solution [0.2 mo IZL Tris monohydrochloric acid (pH 7.5), 2XS SC (20XS SC = 3 mo I ZL sodium chloride, ◦. 3 mo I sodium citrate)], air-dry, bake at 80 ° C for 2 hours, and filter the phage DNA with nylon filter. Fixed on top.

Meanwhile, for use as a probe, known human leuco Bok Rie> B ₄ first receptor c DNA [Y ok om izo et al, N ature, 3 8 7, 6, page 20 - 6 24 pp (1 997 )], A DNA fragment corresponding to the protein translation region is excised from the expression vector pLTBR1 with restriction enzymes EcoRI and BamHI. Purified using Ruth beads. This DNA fragment was labeled by a random priming method using random primer, [ ³² P] -dCTP, and Klenow (KIenow) enzyme.

 The filter on which the phage DNA was immobilized was mixed with a hybridization buffer (6 XSSC, lOXD enhardt's solution, 0.5% SDS, and 100 gZm L heat-denatured salmon) containing the labeled probe. Hybridization was performed at 65 ° C in sperm DNA). After the filter was finally washed at 65 ° C in 0.1 XSSC and 0.1% SDS solution, the obtained radiogram was searched for a black that hybridized with the probe.

 After extracting phage DNA from the phage clone (/ INOK) obtained by this method, the DNA fragment was cut out by digestion with restriction enzymes SaII and BamHI, and plasmid pBIuescript II was extracted. Was inserted into the S aII—BamHI site to obtain the plasmid p NOK2-2.

The nucleotide sequence of the inserted DNA fragment was determined by a usual method, and a homology search was performed using the international gene database DDBJ / EMB LZG enbank database. it has been found that contains the new DNA having the human leuco Bok Lien B ₄ first receptor homology. As shown in Example 4 described below, novel proteins encoded by the novel DNA is because it has a property of binding with leuco Bok Rie> B ₄ were confirmed, "human Bok leuco Bok Rie> B ₄ Second receptor ".

Example 2: Expression of human leukotriene B4 second receptor and its fusion protein Construction of plasmid

From plasmid p N_〇 K 2-2 containing a DNA fragment encoding the human 'leuco Bok Rie obtained in Example 1> B ₄ second receptor, by the following procedure, two kinds of outgoing current plasmid de, i.e., human leuco Toryen B ₄ second receptor expression plasmid ph and BLT 2, to厶Aguruchinintagu (HA tag) and N was added to the distal HA- human Bok 'leuco Bok Lien B ₄ second The expression plasmid pHA-hBLT2 of the receptor fusion protein was generated.

First, as a sense primer arsenide Bok 'leuco Bok Lien B ₄ for the second receptor, distribution Nucleotide sequence represented by SEQ ID NO: 3 in column list:

5 '-CGGGATCCCGCCATGTCGGTCTGCTACCGT-3'

The primer one consisting synthesized, hereinafter, similarly, HA-human 'as a sense primer for leuco Bok Lien已₄ second receptor fusion protein, nucleotide sequence shown in SEQ ID NO: 4 in Sequence Listing:

 A primer consisting of 5′-CGGGATCCCGCCATGTACCCCTACGACGTGCCCGACTACGCCTCGGTCTGCTACCGTCC-3 ′ was used as a dual-purpose antisense primer, and the nucleotide sequence represented by SEQ ID NO: 5 in the sequence listing:

 5 '-GGAATTCAAAGGTCCCATTCCGG-3'

Were synthesized respectively.

Using these primers, the plasmid pNOK22 obtained in Example 1 was subjected to a PCR reaction with the 錶 -type. The amplified fragment was digested with restriction enzymes EcoRI and BamHI, electrophoresed in 1% agarose gel, cut out from agarose gel, and purified using glass beads. By introducing this DNA fragment into a plasmid pcDNA3 (Invitrogen) cleaved with restriction enzymes Ec0RI and BamHI, two types of expression plasmids, namely, human 'leukotrie> B ₄ was obtained second receptor expression plasmid ph BLT 2, and HA- expression plasmid p HA- h BLT 2 arsenide Bok-mouth I co Toryen B ₄ second receptor fusion protein. The nucleotide sequence of the receptor translation region in the plasmid after the introduction was determined by an ordinary method, and it was confirmed that there was no inadvertent mismatch by PCR.

Example 3 Expression of HA—Human Leuco Trien B4 Second Receptor Fusion Protein in CHO-1 Cells

CH 0-K 1 cells (5 × 10 ⁵ cells) were placed in a Ham F F2 medium containing 10% fetal calf serum at 37 ° C. using a 10 cm diameter dish. and incubated for 24 hours at 95% air (air) / 5% C 0 2 conditions.

To the cells, Ripofuekushiyon method using the obtained in Example 2 HA- human Bok leuco Bok Rie> B ₄ expression of the second receptor fusion protein plasmid (p HA- h BLT 2)〗 5 / zg 3 days after the transfusion was performed, the selected drug, G418 (1 g / L), and 10% The medium was changed to a Ham F12 medium containing baby serum, and 18 clones of cells in which the expression plasmid had been integrated into the chromosome were selected. Colonies of selected cells were cloned from single cells by limiting dilution. The cells were stained using an anti-HA antibody, and three highly expressing cell clones were selected using a flow cytometer. Thereafter, the cells were subcultured in Ham's F12 medium containing G418 (0.3 gZL) and 10% fetal calf serum.

 One of the three clones was named "CHO-HABLT2J" and was used in Examples 5 and 6 described below.

Example 4: Expression and binding of human leukotriene B4 second receptor in HEK-293 cells

HEX-293 cells were seeded on a 15 cm plate, and 37% and 95% air was added using a modified Dalbecco's Eagle's medium (DMEM medium) containing 10% fetal serum. No. The cells were cultured for 24 hours under 5% C% ₂ conditions.

This cell was subjected to Bok run Sufuekushiyon in Ripofuekushiyon method using the expression of Example human leuco Toryen B ₄ obtained in 2 second receptor plasmid (ph BLT 2) 2 0 g . Three days after the transfection, the cells were collected, disrupted by sonication, and the cell membrane fraction (microsome fraction) was collected for binding experiments.

^[3 H] binding experiments for leuco Toryen B ₄ (LTB ₄₎ is as follows lines Natsuta. Binding assay buffer [5 0 mm ol ZL- T ris- HCI (p H 7. 4), 1 0 mm o I ZL- M g CI 2, 1 0 mmo IZL- N a C and and 0 - 0 5 % © shea serum albumin (BSA)] 0. in 1 m L, 2 0 μ g equivalent with ^[3 H] LTB ₄ at various concentrations (0. 2 5 O nmol ZL) , as the cell membrane fraction (protein ) and after the 1 hour of binding at 2 5 ° C, and adsorbed by passing through a quick glass fiber one filter one, and attached with its cell membrane fraction ^[3 H] LTB ₄ to Gurasufa I bar filter one Was. After washing 10 times with 0.2 mL of the binding measurement buffer, the glass fiber filter was dried at 50 ° C. The radioactivity was measured by adding a liquid scintillator to a glass fiber filter. In addition, the above procedure was repeated except that a 1000-fold concentration of unlabeled LTB was added during the binding reaction. The obtained value was defined as the amount of non-specific binding.

 The results are shown in FIGS. 1 and 2.

Figure 1 is a graph showing ^[3 H] and the concentration of LTB _4, the relationship between ^[3 H] binding amount of LTB ₄ to the cell membrane fraction. In FIG. 1, “open squares” represent the total amount of binding, “open circles” represent the amount of non-specific binding, and “black circles” represent the amount of specific binding.

FIG. 2 is a graph showing the results of Scatchard analysis performed based on the results shown in FIG. In FIG. 2, “8” means the amount of bound (B 0 und) [ ³ H] LTB ₄ and “BZF” means the amount of bound (B 0 und) [ ³ H] LTB ₄ ; unbound (F ree) ^[3 H] refers to the ratio between the concentration of LTB _4. As a result, _Kd was 22.7 nmol ZL and Bma iS IS fmol Zmg protein.

Example 5: HA-human Bok leuco Bok Lien B ₄ second receptor you express a fusion protein C HO- K 1 cell binding experiments using

Crushing the Example 3 HA-human Bok leuco Bok Rie created in> B ₄ second receptor fusion protein stably expressing CH 0 cells (i.e., C HO- HAB LT 2) ultrasound After that, the cell membrane fraction (microsome fraction) was collected and a binding experiment was performed.

^[3 H] binding experiments for leuco Bok Rie> B ₄ (LTB _4), except that was used in 5 nmo IL the ^[3 H] LTB _4, and the actual applied in accordance with the procedures described in Example 4. In addition, the above procedure was repeated except that a 1000-fold concentration (ie, 5 / mo I / L) of various unlabeled eicosanoids (see FIGS. 3 to 5 described later) was added during the binding reaction. Repeatedly, the obtained value was compared with the value when only [ ³ H] LTB ₄ was added. In addition, these eicosanoids used were Cayman Chemical Noka ISA.

Further, as a comparative example, in place of the C HO-HAB LT 2 cells, stably Furaggupe peptide (F LAG) a F LAG- human Bok leuco added to the N-terminal side Toryen B ₄ first receptor fusion protein The above procedure was repeated, except that specifically expressed CHO cells (hereinafter referred to as CHO-FL AG BLT1 cells) were used.

The results are shown in FIGS. FIG. 3 is a graph showing the amount of [ ³ H] LTB ₄ bound to a cell membrane fraction when CHO-FLAG BLT1 cells were used. FIG. 4 is a graph showing the amount of [ ³ H] LTB ₄ bound to the cell membrane fraction when CHO—HA BLT 2 cells were used. Figure 5 is based on the data shown in FIG. 3 and FIG. 4 is a graph showing the relative binding inhibition activity when formed into a 1 0 0% binding inhibition by LTB _4. In FIG. 5, "black bars" show the results when CHO-FLAG BLT1 cells were used, and "open bars" show the results when CHO-HABLT2 cells were used.

3 to 5, "ETE" means eicosatetraenoic acid, and similarly, "X" means lipoxin, "HETE" means hydroxyeicosatetraenoic acid, “HPETE” means hydroperic acid xyeicosatetraenoic acid, respectively. Further, "N 0 ne" as unlabeled Eikosanoi earth without addition means (ie, the case of adding only ^[3 H] LTB _4).

3 or as shown in FIG. 5, the binding of ^[3 H] LTB ₄ for FL AG- human leuco Toryen B 4 first receptor fusion _{protein, LTB 4, 2 0- OH- LTB} 4, or 1 against 2 the only was inhibited by epi -LTB _4, the binding of FIG. 4 or, as shown in FIG. 5, HA-human Bok leuco Toryen B ₄ ^[3 H] LTB ₄ with respect to the second receptor fusion protein Is not only LTB ₄ , 20 — OH — LTB ₄ , or 12-θ pi-LTB _4, but also 1 2 (R)-HETE, 1 2 (S)-HETE, 1 5 (S)-HETE, Or 15 (S) — also inhibited by HPETE. Example 6: HA-human leuco Bok Lien B ₄ second receptor fusion protein calcium rise response in C HO- K 1 cells express

The CHO-HABLT 2 cells prepared in Example 3 were mixed with 3 mol mol of ZL-Fura-IIAM (acetoxymethyl ester of Fura-2; Doujin Chemical) and 0.01% cremophor. HEPES—Tyrode-BSA buffer [25 mmol ZL— HEPES—NaOH (pH 7.4), 1 OmmoΙ / LNaCI, 2.7 mmol / L -CI, 1.0 mm o I / L — Ca CI ₂ , 12 mm o I / L Na HCO ₃ , 5.6 mm o I / L— Dg I ucose, 0.37 mm ol / _{L- N a H; P 0 4} , 0. 4 9 mmo I / L- M g CI 2, 0. 1% (w / v) fatty acid free (fattyacid- free) BSA] at 37 ° C for 1 hour.

After washing twice the cells in the HEPES- Tairodo BSA buffer, to a concentration of 1 X 1 0 ⁶ cells I, were suspended in HEPES- Tyrode one BSA buffer. Of the cell suspension, 0. 5 ml calcium concentration measuring apparatus; was applied to (CAF system manufactured by JASCO Corporation), various concentrations of various Eikosanoi earth _{[LTB 4, 1 2 (S} ) - HETE, or 5 (S) -HETE] in ethanol was added and the intracellular calcium elevation reaction was measured.

 Further, as a comparative example, the above operation was repeated except that CHO-FLAGBLT1 cells were used instead of the CHO-HABLT2 cells.

 The results are shown in FIGS.

FIG. 6 is a graph showing the degree of increase in intracellular calcium ion (Ca ²⁺ ) concentration when CHO-FLAG BLT1 cells were used. Figure 7 shows C HO—HAB

5 is a graph showing the degree of increase in intracellular calcium ion (C a ²⁺ ) concentration when LT 2 cells are used. 6 and 7, the symbol "white squares" indicates the results of LTB _4, below, similarly, the symbol "black circle" is the result of 1 2 (S) -HETE, symbol

“X” indicates the result of 15 (S) —HETE, respectively.

As shown in FIG. 6, FL AG- human leuco Bok Lien B 4 first receptor fusion protein reacts only LTB _4, whereas the increased intracellular calcium, as shown in FIG. 7 to, HA-human Bok 'leuco Toryen B ₄ second receptor fusion proteins not only _{LTB 4, 1 2 (S)} - HETE or 1 5 (S) also reacts with one HETE, increased intracellular calcium I let it.

From this example and the results of Example 5, human Bok leuco Bok Lien 8 ₄ second receptor, not LT 8 ₄ only, the ability to bind to multiple HETE acids and HPETE acids, cells with these ligands It has been clarified that the receptor has internal signaling ability. No receptor that binds to HETEs or HPETEs has been reported in the past, and this discovery is the first in the world.

Example 7: Screening of agonist or antagonist using human 'leukotrien B4 second receptor

To HE K-2 9 3 cells 6 cm cell culture dish, about 8 X 1 0 ⁵ cells / de We spread so that it could be made. As a medium for cell culture, a DMEM medium containing 10% fetal calf serum was used unless otherwise specified. After culturing at 37 ° C for one day, the reporter gene plasmid p CRE-Luc DNA (Stratagene) and the human leuco trienium ₄ second receptor expression plasmid obtained in Example 2 above were obtained. (Ph BLT 2) was simultaneously transfected into the cells. C The plasmid p CRE-Luc DNA was a plasmid containing a luciferase gene having a CRE (cyclic AMP response element) 5 ′ upstream. Is. The CRE is a nucleotide sequence that is commonly present in the transcriptional regulatory region of a group of genes (cyclic AMP-inducible genes) whose expression increases when the concentration of intracellular cyclic AMP increases.

The transfection was carried out using a commercially available transfection kit (Fu GENE 6 Transfectionreagent; Boehringer Mannheim) according to the attached instructions. After said transformer Fuekushon After incubation day at 3 7 ° C, poly one L- lysine to co one Bok was 9 6 Uerupure Bok, were seeded cells to approximately 2 XI 0 ⁴ cells Noueru. After further cultivation, 0.1% BSA, 0.1 mmo I / L—3-isobutyl-1- 1-methylxanthine (IBMX), 1 moi ZL fuirin ruskolin (forsko I i π; FS) ), and 0. 1 mol / L leuco Bok Rie> B ₄ (DM EM medium containing LTB ₄₎ (hereinafter, in referred to as the incubation medium), the medium was exchanged such that 1 0 0 mu 1_ Noueru Incubation was performed at 37 ° C for 4 hours. The FSK is an inducer that increases the expression level of a gene (ie, luciferase) located downstream of the CRE. After removing the medium, each of the cells was mixed with a luminescent substrate solution (LucLite; Packard) containing luciferin and ATP and a DMEM medium (excluding phenol red): One mixed solution (50 / L) was applied [I], and the generated luminescence was measured with a luminometer.

Moreover, the negative controls (i.e., the case of ₄ absence FSK absence and LTB) as, except using medium minus FSK and LTB ₄ from the incubation medium, was repeated the operation. Further, the positive control (ie, in the presence of FSK and in the absence of LTB ₄ ) may be Except using medium minus LTB ₄ from Yubeshiyon medium, it was repeated the operation.

 Fig. 8 shows the results. The unit of the measurement value “RLU” in FIG. 8 is a relative luminescence unit (RelatativeLightUnits).

As shown in FIG. 8, an increase in the amount of luciferase was observed by acting FSK alone. In addition, when FSK and LTB ₄ act simultaneously, the increase in the amount of luciferase caused by FSK is suppressed as compared with the case where FSK acts alone (that is, when LTB ₄ does not act). did. These results, by the screening method of the present invention, it is possible to perform Agonisu bets or antagonists Bok screening against the human Bok 'leuco Bok Rie> B ₄ second receptor was confirmed. Industrial applicability

According to the novel protein of the present invention, or the cell membrane fraction of the present invention containing the same, or the cell of the present invention containing the novel protein of the present invention, the novel protein of the present invention (particularly human 'leukotrie > B ₄ the Agonisu Bok or § Ntagonisu Bok for the second receptor) can be efficiently screened. According to the screening, for example, the development of powerful new antagonists are expected than known mouth I co Bok Lien B ₄ receptor antagonist to target human Bok-Roikotoryen B ₄ first receptor. Sequence listing free text

 Each nucleotide sequence described in SEQ ID NO: 3 or 4 in the sequence listing is a sense primer, and the nucleotide sequence described in SEQ ID NO: 5 in the sequence listing is an antisense primer. As described above, the present invention has been described according to the specific embodiments. However, modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.

Claims

The scope of the claims 1. Regarding the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the amino acid sequence of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, 60 A protein consisting of an amino acid sequence having a homology of at least%.  2. Regarding the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the amino acid sequence of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, 60 A gene encoding a protein consisting of an amino acid sequence having homology of at least%.  3. The gene c according to claim 1, which comprises the nucleotide sequence represented by SEQ ID NO:〗 in the sequence listing. 4. A plasmid containing the gene according to claim 2 or 3.  5. A transformant containing the gene according to claim 2 or 3.  6. A protein comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, a functionally equivalent variant thereof, or a sequence listing, wherein the transformant according to claim 5 is cultured. A method for producing a protein comprising an amino acid sequence having 60% or more homology with respect to the amino acid sequence of a protein comprising the amino acid sequence represented by SEQ ID NO: 2.  7. Regarding the amino acid sequence of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, 60 A cell containing a protein consisting of an amino acid sequence having homology of at least% or a cell membrane fraction thereof. 8. 60% of the amino acid sequence of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing A protein comprising the amino acid sequence having the above homology, or the cell membrane fraction according to claim 7, or the cell according to claim 7, which is represented by SEQ ID NO: 2 in the sequence listing. 60% or more homology with the amino acid sequence of a protein containing the amino acid sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, or a protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. A method for screening an agonist or an antagonist for a protein having an amino acid sequence having the same.  9. More than 60% of the amino acid sequence of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing 9. The screening method according to claim 8, wherein cells containing a protein comprising a homologous amino acid sequence and further containing a luciferase gene having a cyclic AMP response element 5 ′ upstream are used.  10. Regarding the amino acid sequence of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, 60 A protein comprising an amino acid sequence having homology of at least% or a cell membrane fraction according to claim 7, or a cell according to claim 7, characterized by comprising SEQ ID NO: 2 in the sequence listing. Amino acid sequence having amino acid sequence of 60% or more with respect to the amino acid sequence of the protein containing the amino acid sequence to be identified, its functional equivalent variant, or the protein including the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. A kit for screening an agonist or an antagonist for a different protein.  11 1. Regarding the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the amino acid sequence of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing 6 An agonist for a protein consisting of an amino acid sequence having 0% or more homology.  1 2. Regarding the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the protein amino acid sequence containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing 6 Antagonists for proteins consisting of amino acid sequences having 0% or more homology. 1 3. The Agonisuto of claim 1 1, leuco Bok Lien B _4, requires treatment or prevention of hydroxy rays Kosate Bok Raen acids or hydroperoxides O carboxymethyl diseases attributable to a lack of Eiko Sate Bok Raen acids A method for treating or preventing the disease, comprising administering to a subject an effective amount. 14. The subject in need of treatment or prevention of a disease caused by an excess of leukotriene B ₄ , hydroxyeicosatetraenoic acid, or hydroperoxycytic eicosatetraenoic acid according to claim 12. A method for treating or preventing the disease, which comprises administering an effective amount of the disease. 1 5. Of Agonisu Bok according to claim 1 1, leuco Bok Lien B _4, hydroxy rays Kosate Bok Raen acids, or produce a therapeutic or prophylactic agent for diseases caused for attributable to lack of Hidoroperu old carboxymethyl eicosatetraenoic E emissions acids Use to do. 1 6. Of antagonists Bok according to claim 1 2, leuco Bok Lien B _4, to produce a therapeutic or prophylactic agent for hydroxy eicosatetraenoic E emissions acids, or Hidoroperu old alkoxy diseases caused by the excessive eicosatetraenoic E emissions acids Use for.  1 7. Regarding the amino acid sequence of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, its functional equivalent variant, or the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, 6 An antibody or a fragment thereof characterized by specifically reacting with a protein having an amino acid sequence having 0% or more homology