WO1999033876A1

WO1999033876A1 - Novel seven-pass transmembrane receptor protein

Info

Publication number: WO1999033876A1
Application number: PCT/JP1998/005886
Authority: WO
Inventors: Takeshi Ohno; Takehiro Koshio; Hiroshi Ishimaru
Original assignee: Asahi Kasei Kogyo Kabushiki Kaisha
Priority date: 1997-12-24
Filing date: 1998-12-24
Publication date: 1999-07-08
Also published as: JP2002241399A; AU1688899A

Abstract

A seven-pass transmembrane receptor protein originating in humans which has the amino acid sequence represented by SEQ ID NO:2 and a DNA encoding the same. Use of this protein and the DNA encoding the same makes it possible to screen substances for treating or preventing autoimmune diseases, etc., to diagnose diseases and to produce diagnostics. An expression vector of the above protein; transformed microorganisms or cells; the above protein produced thereby; a method for screening a ligand to the above protein or a substance inhibiting the binding of the ligand to the protein; and an antibody against the protein. A fragment of a seven-pass transmembrane receptor protein originating in mouse having the amino acid sequence represented by SEQ ID NO:4; and a DNA encoding the same. This protein fragment and the DNA encoding the same are useful in identifying and isolating a novel full-length mouse seven-pass transmembrane receptor protein and a DNA encoding the same.

Description

Description New seven-transmembrane receptor protein technology

The present invention relates to a novel seven-transmembrane receptor protein present on leukocytes and a DNA encoding the same. More specifically, the present invention relates to a human-derived seven-transmembrane receptor protein comprising the amino acid sequence of SEQ ID NO: 2, and a DNA encoding the same. The use of the seven-transmembrane receptor protein and the DNA encoding the protein of the present invention makes it possible to screen for substances useful for treating or preventing diseases involving leukocyte functions such as autoimmune diseases. And diagnostic methods and agents for such diseases. Also, the present invention provides a replicable recombinant DNA obtained by incorporating the above DNA into a replicable expression vector; a microorganism or a cell transformed with the above replicable recombinant vector; A seven-transmembrane receptor protein produced on the cell membrane surface of the transformant; a ligand for the above-mentioned seven-transmembrane protein and a substance that inhibits the binding of the ligand to the seven-transmembrane protein A screening method; and an antibody capable of binding to the seven-transmembrane receptor protein. Furthermore, the present invention relates to a mouse-derived seven-transmembrane receptor protein fragment comprising the amino acid sequence of SEQ ID NO: 4, and a DNA encoding the same. Conventional technology

Leukocytes are a kind of blood cells and are cells that control various functions such as immunity and inflammation. In particular, during infection, leukocytes protect the body through beneficial immune and inflammatory response mechanisms (translated by Yoshio Aso, at-a-glance immunology, published by Metigull 'Science' International, 4 8-61, 1993 (Japan)]. However, it also causes unwanted immunity and inflammatory effects such as autoimmunity [translated by Yoshio Aso, immunology at a glance, published by Medical Sciences International, 62-73, 199 3 years (japan)]. Finding ways to control the function of leukocytes could help cure infections and tumors with a beneficial immune response, or treat autoimmune diseases with a diminished harmful immune response. Have been.

The functions of leukocytes, that is, their proliferation, differentiation, activation, chemotaxis, etc., are controlled by various receptor proteins expressed on leukocytes. Receptors are extracellular events that occur on the cell surface and bind with high affinity to specific substances (signal molecules) present on the surface of other cells or in body fluids. Is a protein that converts cells into intracellular signals and triggers cell responses [Keiko Nakamura and Kenichi Matsubara, Molecular Biology of Cells (2nd ed.), Kyoikusha, p. 936, 1990 (Japan) )] Substances that bind to receptors are generally called ligands. Alters receptor function Substances that stimulate the cell by binding to the receptor, those that block the stimulation of other ligands by binding to the receptor, or that are stimulated by other ligands inside the cell If a substance that inhibits transmission could be obtained, the obtained substance would positively or negatively regulate the function of leukocytes, and would be useful for treating diseases caused by insufficient or excessive functions of leukocytes. Is done.

There are various leukocyte receptors such as the family of cytokine receptors, the family of EGF (Epiderma 1 Growth Factor) receptors, and the family of seven transmembrane receptors. The Leukocyte Antigen Facts Book, Academic Press Inc., 38-49 (1993)], has a wide variety of functions.

The seven-transmembrane receptor protein family is one of such receptor families, G-protein-coupled receptor, rhodopsin-type receptor. It is also called the body. Studies of seven transmembrane receptor proteins in leukocytes have been relatively recently started, and it is believed that there are still many unknown seven transmembrane receptor proteins.

Receptors identified to date as seven-transmembrane receptor proteins present on leukocytes include receptors that bind to anaphylatoxin, receptors that bind to chemokines, and PAF (platelet activation Factors) and other receptors.

For example, the receptor for anaphylatoxin is neutrophils and macrophages. It is involved in the function of phage, for example, the production of reactive oxygen species, the chemotaxis, and the activation of cell adhesion [Bouley, F. et al., Biochemistry 30, 2993-2999, (1991)]. In addition, administration of an inflammation-inducing substance intraperitoneally to mice deficient in IL-8 (interleukin 8) receptor homolog, one of the receptors that bind to chemokines, caused neutrophil infiltration. At the same time, neutrophilia and an increase in granulocytes and plasma cells in the bone marrow and lymph nodes were observed [Hisashi Iizasa, Tsunaharu Matsushima, Clinical Immunity, 28, 731-173, 1 996 years (Japan)]. As is clear from the above, these seven transmembrane receptor proteins regulate leukocyte proliferation, differentiation, activation, chemotaxis and the like.

Among the substances that act on such receptors, those that may be considered as therapeutic agents for diseases include IL-8 and MCP-1 (Monocyte Chemotactic Protein 1). There are those that bind cells to stimulate cells and those that bind to receptors and block the stimulation by ligands, such as IL-18 mutants (Howard, OMZ et al. TIBTECH, 14, 46-51, (1996)).

In seven transmembrane receptors, the receptor often binds to multiple signal molecules, and the signal molecule also binds to multiple receptors. Therefore, knowing the signal molecule is not enough when considering the treatment of a disease. For example, in the case of serotonin, for a single signal molecule called serotonin, in addition to a seven-transmembrane receptor, an ion channel receptor A total of 14 types of receptors, including receptors for completely different signal transduction pathways, are known, and compounds that specifically bind to individual receptors are also known [1996]. Scientifically, Recept or lon, Hannell Nomenclature supplement, Trends Pharmacol. Sc, (1996)], and the application of each receptor to treatment of different diseases is also considered. In the case of chemokines, a single signal molecule (a kind of chemokine) reacts with many receptors, while a single receptor becomes a large number of signal molecules (multiple kinds of chemokines). Many examples are known [A. Power et al., Trends Pharmaco 1. Sc i. 17, 209-213, (1996)].

As is clear from the above, even if a single signal molecule is the cause of the disease, there are multiple receptors that differ depending on the cell type. In order to specifically control the function of a specific cell group that causes a disease, the receptor expressed in the cell is specified rather than the signal molecule that acts on the cell. This is important. For example, taking the chemokine group, one of the signal molecules, as an example, the signal molecule RANT ILS (Regulated on Activation, Norma 1 T cell ex pressed and secreted) acting on leukocytes Because of the variety of leukocytes that react, it is not possible to identify leukocytes that are reactive with RANTES. However, eosinophils, a type of leukocyte, specifically express CCR3, one of the chemokine receptors, so using the receptor CCR3 specifically regulates eosinophils. You It is also possible to search for methods that use this method [Howard, OMZ et al., T1BTECH, 14, 46-51, (1996)].

It is also known that receptors in humans and other species of organisms respond differently to the same compound [for example, Marleau S. et al., J. Immun. 157, 4141-4146 (1996)]. It is known that a substance that activates human receptors acts as a substance that inhibits the activation of other types of receptors. Therefore, it is important to use human-derived receptors to develop drugs for humans.

In addition, some receptors are known to act as receptors during viral infection [see, for example, Choe H. et al., Ce 1185, 1135-1148, (1996), It is also known that molecules that bind to such receptors prevent virus infection (eg, B 1 eu 1

Nature, 382, 829-833, (1996)]. In these cases, it is also important to know the receptors that are expressed on the virus-infected cells. It is also known that certain viruses can be infected by binding to receptors for certain species (SPecies). Therefore, human-derived receptors are important when developing pharmaceuticals to prevent human infection with humans. To date, signal molecules acting on leukocytes, such as the known chemokines PF4 and HCC1, have been presumed to be seven-transmembrane receptor proteins. Although no receptor protein has been identified [Premack Β, Α. Et al., Nature Medicine 2, 1174-1178, (1996); Loetscher M. et al., J. Exp. Med. 84, 963-969, (1996)]. In particular, it is estimated that there are more new chemokines in the chemokine group [Howard, OMZ et al., TIBTECH, 14, 46-51, (1996)]. The receptor is expected to be present. As described above, not all receptors for molecules acting on leukocytes have been understood, and it is considered that leukocytes have more seven-transmembrane receptor proteins. It is thought that identifying a substance (signal molecule) that alters the action of such a receptor will provide a method for controlling the function of leukocytes and, consequently, a disease.

For example, autoimmune diseases such as rheumatoid arthritis and multiple sclerosis are characterized by refractory chronic inflammation that is systemic or organ-specific. At present, it has been pointed out that there are various inadequacies such as the low therapeutic effect of the drug used for the treatment, or the strong side effect even if the therapeutic effect is high. In order to solve these problems, drugs that clarify the pathogenesis of autoimmune diseases and that specifically inhibit only those mechanisms are needed. The pathogenesis of autoimmune diseases is thought to involve genetic factors and infection in addition to acquired immune abnormalities, and the details have not yet been elucidated. However, animal models have recently shown that cellular immunity is involved in the induction and exacerbation of chronic inflammation in autoimmune diseases. It became clear. The reaction between a type of leukocyte, a self-reactive T cell, and an autoantigen on an antigen-presenting cell triggers the secretion of cytokins and chemokines, the migration of cells to it, and other reactions. The cascade of reactions begins to move, creating inflammation with the infiltration of more self-reactive T cells and another leukocyte, a mononuclear cell, which is thought to trigger an autoimmune disease I have. Therefore, suppressing the activation of autoreactive T cells is thought to suppress various subsequent reactions and lead to the treatment of autoimmune diseases.

Multiple sclerosis is an autoimmune disease of the central nervous system, and its pathological model is experimental allergic encephalomyelitis of the mouse (EA £, experiment al al ergic encepha l omye litis). Is a pathological model whose analysis of the pathogenesis is advanced. This disease model is based on the myelin basic protein found in the nerve myelin.

(MBP, myelin basic protein) and autoreactive T cells that respond to proteolipid apoproteins are known to be induced, and cloned T cells are also available. It has been established at the research institute of Tsutsuji [eg, J. Neuro immuno 1.58, 167-176 (1995)]. However, no receptor that controls the function of these cells, especially the seven-transmembrane receptor protein, has been reported so far. Thus, not all molecules and their receptors that act on leukocytes, especially on autoreactive T cells, have been understood, nor have they been known on autoreactive T cells. The presence of a transmembrane receptor protein is expected. By identifying substances that alter the actions of these various novel receptors, it is necessary to control the function of autoreactive T cells and, consequently, establish a method for controlling autoimmune diseases. Be expected.

As a causative substance acting on the seven-transmembrane receptor protein, various substances are known for various receptors. For example, glutamate-dopamine, which is a physiological amide, binds to glutamate receptors and dopamine receptors, respectively. In addition, the neural peptide Yendocerin, which is a peptide, binds to the neuropeptide Y receptor group and the endocrine receptor group, respectively. And Steve Arkinstall, The G-protein 1 in kedrefactors Book, Academic Press Inc., (1994)). These include substances known to act on leukocytes, such as the chemokine group and PAF, and substances that do not act on leukocytes.

The substance that activates the seven-transmembrane receptor protein as described above, whether natural or non-natural, is the substance, the seven-transmembrane receptor protein itself, or a cell that expresses the receptor. Depending on the status of each of the three, various intracellular signal molecules fluctuate. Fluctuations in the signal molecule include, for example, reactions such as an increase or decrease in intracellular cAMP concentration, an increase in inositol phosphate concentration, and an increase in intracellular calcium concentration (Watson, S. and St. eve Ar ki ns tall 荖, The G-pro teinli nke dre cep tor Factors Book, Academic Press Inc., (1994)], and a method for measuring each of them has been developed. Therefore, by measuring the reaction as described above, it is possible to determine whether a specific substance activates a specific seven-transmembrane receptor protein or inhibits its activation. You can do it. Also, a method for observing physiological phenomena such as cell proliferation, fluctuations in gene expression, and chemical migration caused by binding of such a substance to the transmembrane receptor protein seven times is also known. Similarly, a substance can be used as an indicator to determine whether a substance activates a specific seven-transmembrane receptor protein or prevents it from activating. it can.

As described above, there are various methods for identifying a substance that acts on the seven-transmembrane receptor protein, but in order to obtain a substance useful as a human drug by using these methods, First, it is important to obtain seven transmembrane receptors from humans.

Based on these considerations, in order to control the function of leukocytes, especially autoreactive T cells, and to control diseases, it is necessary to obtain a previously unknown seven-transmembrane receptor. In particular, obtaining a seven-transmembrane receptor derived from human is a major issue in order to obtain a substance useful as a human drug.

As described above, a method for controlling the function of leukocytes, particularly autoreactive T cells, and controlling disease has not yet been completed. The biggest cause is controlling the function of leukocytes associated with the disease Is that the receptor protein, especially the seven-transmembrane receptor protein, has not been identified. That is, an object of the present invention is to find a novel seven-transmembrane receptor protein present in leukocytes and a cDNA encoding the protein, and to use the same for searching for a drug that controls the function of leukocytes. To provide Summary of the Invention

The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, thought that the seven-transmembrane receptor protein present in leukocytes would be useful for searching for a drug that regulates the function of leukocytes. First, we established EAE-causing autoreactive mouse T cells 4R312 strain as research material and used that RNA to express a known seven-transmembrane receptor expressed on leukocytes. A mouse cDNA fragment similar to the protein gene sequence was obtained. Furthermore, two types of mouse cDNA fragments having homology to the obtained cDNA fragments were found on a publicly known database, and based on these sequences, a novel seven-transmembrane receptor protein ( Hereinafter, the gene fragment of ET240 is often successfully linked to the cDNA library of the mouse 4R312 strain described above. Next, the present inventors searched a known database using the ET240 cDNA fragment obtained from the mouse T cell 4R312 strain and found that the homologous to the mouse ET240 described above. CDNA fragments derived from two independent humans were discovered. The two arrays obtained from the database are Based on the full-length cDNA coding region of a novel seven-transmembrane receptor protein derived from human, which has a high homology (84.4%) with a mouse-type ET240 fragment. Was successfully completed. In addition, the cloned mouse and human cDNAs were translated into amino acid sequences to obtain novel mouse ET240 protein fragments and full-length human ET240 proteins. . Furthermore, the present invention was completed by establishing an expression system for human ET240 and producing an antibody capable of specifically binding to human ET240. .

Therefore, one main object of the present invention is to provide a human-derived seven-transmembrane receptor protein useful for the search for a drug that controls the function of leukocytes.

Another object of the present invention is to provide a DNA encoding the above seven-transmembrane receptor protein, a recombinant DNA obtained by incorporating this DNA into an expression vector, and this recombinant DNA A microorganism or cell transformed by the method described above.

Another object of the present invention is to provide a method for screening a ligand that binds to a seven-transmembrane receptor protein, and a seven-transmembrane receptor protein and a ligand for the protein. It is intended to provide a method for screening a substance that inhibits the binding to a metal.

Another object of the present invention is to provide an antibody that can bind to a seven-transmembrane receptor protein. Another object of the present invention is to provide a mouse seven-transmembrane receptor protein fragment and a DNA encoding the same.

The above and other objects, features and advantages of the present invention will become apparent from the following detailed description and the appended claims, taken in conjunction with the accompanying Sequence Listing. Free text of sequence listing

The sequence of SEQ ID NO: 5 is a primer for cloning a mouse ET240 fragment based on the sequence of a known seven-transmembrane receptor protein expressed in leukocytes.

The sequence of SEQ ID NO: 6 is a primer for cloning a mouse ET240 fragment based on the sequence of a known seven-transmembrane receptor protein expressed in leukocytes.

SEQ ID NO: 7 is a primer for subcloning human ET240 based on GenBank entry H67224. SEQ ID NO: 8 is a primer for subcloning of human ET240 based on GenBank entry AA2155707.

SEQ ID NO: 9 is a primer for subcloning of human ET240 based on the sequence of a known vector ATrip1Ex arm.

SEQ ID NO: 10 is a primer for subcloning of human ET240 based on the trapping of GenBank entry H67224.

SEQ ID NO: 11 is the phase of the GenBank entry H67224. Primer for subcloning of human ET240 based on the complementary chain.

SEQ ID NO: 12 is a primer for subcloning of human ET240 based on the sequence of a known vector Trip1Ex.

SEQ ID NO: 13 is a primer for subcloning of human ET240 based on GenBank entry AA2155707.

SEQ ID NO: 14 is a primer for subcloning of human ET240 based on the GenBank entry AA2155707.

SEQ ID NO: 15 is a primer for the analysis of mouse ET240 based on SEQ ID NO: 3.

SEQ ID NO: 16 is a primer for the analysis of mouse ET240 based on SEQ ID NO: 3.

SEQ ID NO: 17 is a primer for subcloning human ET240 based on SEQ ID NO: 1.

SEQ ID NO: 18 is a primer for subcloning human ET240 based on SEQ ID NO: 1.

SEQ ID NO: 19 is a partial sequence of a guinea pig-derived myelin basic protein used to induce EAE (experimental allergic encephalomyelitis) in mice.

Detailed description of the invention According to one aspect of the present invention, there is provided a substantially pure human-derived seven-transmembrane receptor protein having the amino acid sequence of SEQ ID NO: 2. . Next, in order to facilitate understanding of the present invention, first, basic features and preferred embodiments of the present invention will be listed.

1. A substantially pure human-derived seven-transmembrane receptor protein having the amino acid sequence of SEQ ID NO: 2.

2. A substantially pure peptide characterized by being a partial sequence consisting of at least five amino acids of the amino acid sequence of SEQ ID NO: 2.

3. An isolated DNA encoding the seven-transmembrane receptor protein described in 1 above.

4. The isolated DNA according to item 3, which has the nucleotide sequence of SEQ ID NO: 1.

5. A DNA or derivative thereof comprising at least 12 bases in the base sequence according to claim 3 or 4. 6. A DNA consisting of at least 12 bases in a DNA complementary to the base sequence according to claim 3 or 4, or a derivative thereof.

7. An RNA consisting of at least 12 bases in the RNA complementary to the base sequence according to claim 3 or 4, or a derivative thereof.

8. A replicable recombinant DNA comprising the DNA according to any one of the above items 3 to 7 incorporated into a replicable expression vector.

9. A microorganism or cell transformed with the replicable recombinant DNA according to item 8 above.

10. (A) The DNA described in 3 or 4 above is bound to a replicable expression vector and a replicable recombinant DNA comprising the DNA and the replicable expression vector is obtained. Get

(b) transforming a microorganism or a cell with the replicable recombinant DNA to form a transformant,

(c) selecting the transformant from the parent cell of the microorganism or cell,

(d) culturing the transformant to cause the transformant to express the DNA; A seven-transmembrane receptor protein produced on the cell membrane surface of the transformant by a method including the above.

11.7 The method of screening for a ligand that binds to a 1.7-transmembrane receptor protein includes the use of the protein described in 1 or 10 above or the peptide described in 2 above. A protein that is brought into contact with the sample material and binds to the transmembrane receptor protein seven times, using as an index the change that occurs in response to the binding of the protein or the peptide to the ligand. A method that involves detecting a gand.

12.7 The method of screening a substance that inhibits the binding of a seven-transmembrane receptor protein to a ligand to the protein, the protein described in the above item 1 or 10, or the protein described in the above item 2 The peptide of the present invention and the ligand are brought into contact with the sample material, and the change caused by the binding of the protein or the peptide to the ligand is used as an index, and the membrane is repeated seven times. A method comprising detecting a substance that inhibits the binding between a penetrating receptor protein and a ligand.

13. An antibody that binds to the seven-transmembrane receptor protein described in 1 above.

14. A substantially pure seven-transmembrane receptor protein derived from a mouse, having the amino acid sequence of SEQ ID NO: 4. A piece of quality.

15. An isolated DNA encoding the fragment of the seven-transmembrane receptor protein described in 14 above.

16. The isolated DNA according to the above item 15, which has the nucleotide sequence of SEQ ID NO: 3. Hereinafter, the present invention will be described specifically.

The left and right ends of the amino acid sequence described in the sequence listing are the amino terminal (hereinafter referred to as N terminal) and the carboxyl terminal (hereinafter referred to as C terminal), respectively. The left and right ends of the nucleotide sequence are the 5 'end and the 3' end, respectively.

In the present invention, A in the DNA base sequence indicates adenine, C indicates cytosine, G indicates guanine, and T indicates thymine.

In the present invention, in the three-letter display, A1a in the amino acid sequence is alanine, Arg is arginine, A sn is asnolagin, Asp is asanoraginate, Cys Is cystine, G1n is glutamine, G1u is glutamic acid, G1y is glycine, His is histidine, I1e is isoleucine, and Leu is royin. Syn, Lys is lysine, Met is methionine, Phe is phenylalanine, Pro is proline, Ser is serine, Thr is threonine, Trp is tolane. Rip fan, T yr is Ciro Shin, V a1 is valine.

The term “seven-transmembrane receptor protein” in the present invention is a seven-transmembrane receptor present on leukocytes, particularly autoreactive T cells, and was first discovered by the present inventors. Protein. The protein of the present invention is a protein having a specific ligand-binding activity for a receptor and a signal transmission activity present downstream in a signal transduction pathway.

One such protein is human ET240 having the amino acid sequence of SEQ ID NO: 2, but the protein of the present invention is limited to the amino acid sequence of SEQ ID NO: 2. It is not. If the polypeptide has the above-mentioned properties as a receptor, a variant (i.e., one or more mutations) caused by a mutation, such as intra-species mutation or allelic mutation, which is known to occur in nature. The present invention also includes amino acid sequences in which several amino acids have been deleted, substituted, or added (amino acid sequences).

Further, the protein of the present invention may be modified after translation. In the amino acid sequence of SEQ ID NO: 2, there is a portion expected to add a sugar chain. For example, the sixth Asn (Asn-Gln-Ser) of SEQ ID NO: 2 is considered to be Asn of N-glycosidic consensus sequence Asn-X-Ser / Thr, and N-g It may have been modified by the risk. In addition, a portion where serine or threonine residues frequently appear may be considered as a portion for estimating the 0-glycosidic bond of N-acetyl-D-galactosamine. These sugar chains are added It is considered that the obtained protein is generally more stable to degradation in vivo than the protein itself to which no sugar chain is added, and has a stronger physiological activity. Therefore, the protein of the present invention contains N-daricoside or N-acetyl, such as N-acetyl-D-gnorecosamine or N-acetyl-D-galactosamine, in the amino acid sequence of SEQ ID NO: 2. It also includes a protein containing a sugar chain having a dalicoside bond.

Further, the protein of the present invention may have a known tag sequence such as an antigen epitope. For example, tags such as FLAG (DY DDDDK) T 7 (MASMTGGQQMG), HSV (SQP EL AP EDP ED), S (ETAAAKFERQH DS), Myc (EQKLISEEDL), His (HHHHHHHH), HA (YP YDVPDYA), etc. It may have a system ij (all sequences in kazuko are one-letter notation of amino acid). Since the tag sequence is present at the C-terminus or N-terminus of the ET240 protein, the protein of the present invention can be obtained by a method such as flow cytometry or Western blotting. And can be easily detected.

In addition, a natural cDNA sequence derived from human of the present invention, which encodes a protein consisting of the amino acid sequence of SEQ ID NO: 2, is described in SEQ ID NO: 1 together with the amino acid sequence. did. In chromosomal DNA or cDNA isolated from the natural world, the base sequence of DNA is often mutated without changing the amino acid sequence encoded by the DNA due to the degeneracy of the genetic code. Is recognized. In fact, in Example 6, the human natural cDNA library was used. And the amino acid at position A, which is the 678th base of SEQ ID NO: 1, has become G. Due to this mutation, the codon changes from ACA to ACG, and both codify for amino acid as Thr (threonine). The resulting amino acid sequence does not change. A DNA sequence containing such a mutation is also included in the DNA of the present invention. Further, since the 5 'untranslated region and the 3' untranslated region are not involved in the definition of the amino acid sequence of the protein, the nucleotide sequence of the untranslated region is easily mutated. The nucleotide sequence obtained by such mutation or degeneracy of the genetic code is also included in the DNA of the present invention.

In the present invention, “a fragment of a seven-transmembrane receptor protein derived from a mouse” refers to a seven-transmembrane receptor of a mouse corresponding to the seven-transmembrane receptor derived from human. It is a fragment of the body. Specifically, it is a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 from the second transmembrane region of the receptor to the C-terminal. However, similarly to the seven-transmembrane receptor protein described above, the fragment of the seven-transmembrane receptor protein is not limited to the amino acid sequence shown in the sequence listing, but may be mutated. Thus, the resulting variants are also included in the present invention. Further, it may have a known tag sequence such as post-translational modification or antigen epitope.

In addition, the natural cDNA sequence derived from the mouse of the present invention encoding a seven-transmembrane receptor fragment consisting of the amino acid sequence of SEQ ID NO: 4 has amino acid sequence of SEQ ID NO: 3 Described along with the sequence Was. As with the human-derived cDNA of the present invention described above, the mouse cDNA also has a nucleotide sequence obtained by mutation of the nucleotide sequence without a change in amino acid after translation and degeneracy of the genetic code. Included in the DNA of the invention.

In addition, production of cDNA necessary for the genetic manipulation described in the present invention, examination of expression by Northern plot, screening by hybridization, production of recombinant DNA, nucleotide sequence of DNA A series of molecular biology techniques such as determination of DNA library and preparation of a cDNA library can be performed by a method described in an ordinary experimental book. More specifically, Molecular cloning, Almanatory manual, (1989), Sambrook, J., Fritsch, E.F., and Maniatis, T. Eds., Cold Spr. ing Harbor Laorator Press, etc. can be referred to.

Next, in order to further clarify the basic features of the present invention, the technical features included in the present invention will be described while following the process leading to the completion of the present invention.

A primer (sequence) designed based on the nucleotide sequence of a known seven-transmembrane receptor protein known to be expressed in leukocytes for the purpose of closing a novel seven-transmembrane receptor protein Using Nos. 5 and 6), a gene fragment was amplified from a mouse EAE-causing T cell 4R312 strain cDNA library. When the amplified DNA sequence was searched on the mouse EST database, two EST fragments were found. Using the mouse DNA sequence cloned by the present inventors and the EST fragment obtained from the database, a novel seven-transmembrane mouse derived from the mouse described in SEQ ID NOs: 3 and 4 in the sequence listing A fragment of the type receptor protein was obtained. This novel protein was named ΤΤ240 because of its ability to be the first receptor protein found in EAE-causing T cells (abbreviated as “ET”) (hereinafter often referred to as mouse-derived T cells). The 7-times transmembrane receptor protein is called “mouse type Ε Ε 240”. The fact that the mouse-type Τ240 fragment was cloned into Ε Ε pathogenetic mouse は cells means that Τ-240 mRNA was expressed in EAE-onset mouse T cells. It suggests and. In subsequent experiments, native mRNA for mouse ET240 was detected in mouse lung, heart, and liver tissues, as well as in mouse tongue and intestinal lymphocytes. Therefore, a new seven-time membrane It was revealed that the penetrating receptor exists not only in mouse EAE-causing mouse T cells but also in various mouse tissues. Furthermore, the present inventors used the mouse-type cDNA fragment obtained as described above to derive a mouse-type E-frog from a cDNA library derived from human lung. We succeeded in cloning the full-length cDNA of a novel seven-transmembrane receptor protein corresponding to 0 (this "human-derived seven-transmembrane receptor protein" is often referred to as " Human ET240 ”). Later studies found that native mRNA encoding ET240 was strongly expressed in the human small intestine and heart.

From the above experimental results, it can be seen that the natural mRNA of the mouse and human seven-transmembrane receptor protein ET240 of the present invention is expressed systemically, especially lymphocytes of the mucosal system. It was presumed to be expressed in leukocytes. Because mRNA is translated into proteins by cellular mechanisms, detection of ET240 native mRNA in these tissues is considered equivalent to the presence of ET240 protein. Can be

The seven-transmembrane receptor protein ET240 of the present invention comprises a full-length human ET240 represented by SEQ ID NO: 2 in the sequence listing and a mouse ET240 represented by SEQ ID NO: 4. This is a fragment peptide. The present inventors used Gene X-Mac / DB Ver. 37.0 (Software Development Co., Ltd.) and prepared the amino acid sequences of SEQ ID NOS: 2 and 4 and the database (GenBank CDS ( Re 1.100 Apr il 1 997; a sub-database of Primates, Rodent, MammaIs, Vertebrate, and Patent)). The results are shown in Tables 1 and 2.

Mouse type ET240 GenBank cds Search result Rank Entry name Comment Similarity

1 S63848 ij-protein coupled receptor 84.0% type B {clone PPRl}.

2 HSU45982 Human Gpr ₀ te in-coup 1 ed 37.4% receptor GPR-9-6 gene, complete cds.

3 HUMGPCRA Human Epstein-Barr virus 36.4% induced G-protein coupled

receptor m NA.

4 HUMEBI 1CDN Human G protein-coupled 36.4% receptor (EB I 1) mRNA, com-p 1 e t cds.

5 Awake EBI103 Human G prot ein ~ coup 1 ed 36.4% receptor (EB I 1) gene exon 3, com 1 e t e cds.

6 USIL8GR0A Mouse IL8 / gro-a lpha receptor 33.8% gene, comp 1 e t e cds.

7 U31207 Mus mu s c u 1 u s i n t e r 1 e u k i n-8 33.8% receptor gene, complete cds.

8 USIL8RB Mouse inter leukin-8 receptor 33.8% type B (I 18rb) gene, complete cds.

9 MUSITLK8 Mouse mRNA for i n t e r 1 euk Ί n-8 33.8% receptor (IL-8R), comp 1 e t e

cds.

10 RNIL8R R. norveg icus mRNA for inter- 3.1%

1 euk i n-8 rece tor. |

1 Table 2 Results of GenBank cds search for human ET240s Rank Ranking Entry name Comment Similarity

1 S63848 υ-protein coupled receptor 86.0% type B {clone PPR1}.

2 Application EBIICDN Mouse Gprote in-coup 1 ed 39.4% receptor (EB I 1) mRNA, complete cds.

3 HU EBI103 Human Gprote in-cou 1 ed 39.2% receptor (EB I 1) gene exon

3, com 1 e t e cds.

4 HUMGPCRA Human Epst e inn-Barr virus 38.9% induced G-protein cou led

receptor mRNA.

5 HUMEBI1CDN Human G protein-coupled 39.2% receptor (EB I 1) mRNA, complete cds.

6 HSU20350 Human G protein-coupled 35.5% receptor V28 mRNA, complete cds.

7 So-called 28934 Human beta chemok i ne rece 35.5% tor-like 1 mRNA, com lete

cds.

8 HSU45982 Human G protein-coupled 36.8% receptor GPR-9-6 gene, complete cds.

9 FCU63558 Felis c atus CXCR-4 homo Iog 35.1% mRNA, complete cds.

10 HSNPYRLA H. sapiens mRNA for neuro34.6% ί peptide Y-like rece tor.

1 Table 1 shows that among the amino acid sequences registered in GenBank CDS, the top 10 proteins with high homology to the mouse ET240 fragment (SEQ ID NO: 4) of the present invention and their homology showed that. The proteins determined to be highly homologous in comparison with the mouse ET240 fragment of the present invention were all seven-transmembrane receptor proteins. No sequences were found to be identical. Therefore, the present inventors confirmed that mouse-type ET240, a part of which was cloned, had a novel sequence.

Table 2 shows that among the amino acid sequences registered in GenBank CDS, the top 10 proteins with high homology to human ET240 (SEQ ID NO: 2) of the present invention and their homology Showed sex. The protein determined to have high homology as compared with the human ET240 of the present invention was a transmembrane receptor protein having a seven-fold translocation, but the human ET240 of the present invention was the same. No sequence that could be judged to be the same as 0 was found. Therefore, the present inventors confirmed that human-type ET240 whose full-length had been cloned was a novel sequence.

The sequence having the highest homology with the mouse type ET240 fragment (SEQ ID NO: 4) and the human type ET240 (SEQ ID NO: 2) of the present invention is a seven-transmembrane receptor protein PPR derived from Escherichia coli. The homology with mouse ET240 and human ET240 was 84.0% and 86.0%, respectively. PPR 1 is from tongue The possibility of a neuropeptide receptor has been discussed, and strong expression in the lung has been reported (Biochem. Biophys. Res. Comm. 194, 504-511 (1993). However, nothing is known about the expression of PPR1 in leukocytes and its role in autoimmune diseases, especially in multiple sclerosis and its model, EAE. Thus, there is no known possibility that PPR1 regulates the function of leukocytes, particularly autoreactive T cells, and thus regulates disease.

Furthermore, the amino acid sequence of the human ET240 of SEQ ID NO: 2 and the amino acid sequence of the mouse ET240 fragment of SEQ ID NO: 4 were respectively changed to the Swiss Prot [Release 34.0]. , October, (1996)) and the amino acid sequence registered in the patent database (DGENE Derwent Information Lt d .; 971130 UP)}. It was confirmed that the sequence was correct.

In addition, according to the results of the search using the above-mentioned database, since the proteins having high homology with ET240 of the present invention are all seven transmembrane receptors, the mouse and human types of the present invention were not identified. It was suggested that ET240 is a protein belonging to the seven-transmembrane receptor.

Further, the present inventors have made the hydrophobic part and the hydrophilic part of the amino acid sequence of SEQ ID NO: 2 in accordance with the method of Kyte-Doo 1 itt 1 e UM.B'ioL 157: 105, (1982)]. Analyzed. As a result, the present invention It has been clarified that ET240, a seven-transmembrane receptor protein derived from G, is expressed on the cell surface as a cell membrane protein having seven cell membrane translocating parts. With respect to the amino acid sequence of SEQ ID NO: 4, analysis of the hydrophobic portion and the hydrophilic portion was carried out in the same manner as described above, according to the method of Kyte-Doolyle U. Mo and Bio, 157: 105, (1982)]. In other words, the sequence of SEQ ID NO: 4 consists of a hydrophobic portion and a hydrophilic portion (ie, a C-terminal portion from the middle of the second transmembrane portion) almost corresponding to the latter half of the sequence of SEQ ID NO: 2. And was predicted to be a fragment constituting a part of a cell membrane protein having seven cell membrane transit portions.

As analyzed by the amino acid sequences of the human ET240 and mouse ET240 fragments, the present inventors encoded the human ET240 described in SEQ ID NO: 1. The nucleotide sequence of the DNA to be encoded and the nucleotide sequence of the DNA encoding the mouse ET240 fragment described in SEQ ID NO: 3 were analyzed. The present inventors used Genetyx-Mac / DB Ver. (Re: 100 April 1997; a sub-database of Primates, Rodent, Mammals, Vertebrate, and Patent)]. The results are shown in Tables 3 and 4. Table 3 Results of GenBank search for human ET240 cDNA

Rank Entry One Comment Comment Similarity

1 S63848 ϋ- r o t e in cou led receptor 82.0% type B {clone PPR1}.

2 HUMGPCRA Human Epst ein-Barr virus 54.2% induced G-protein coupled

rece tor mRNA.

3 HUMEBllCDN Human G p ro t e inn-cou p 1 e d 54.0% receptor (EB I 1) mRNA, complete cds.

4 HSDNABLR2 H. sapiens BLR2 gene.54.0%

5 HU EBI103 Human G protein-coupled 54.0% receptor (EBI 1) gene exon

3, c om 1 e t e c d s.

6 USIL8GR0A Mouse I L8 / gr ₀ -a 1 pha recep-50.6% tor gene, complete c ds.

7 MUSIL8RB Mouse inter 1 euk i n ~ 8 recep50.6% tor type B (I 18rb) gene,

c omp 1 e t e c d s.

8 strokes 31207 Mus mus c u 1 u s int e r 1 euk i n ~ 8 50.6% receptor gene, c omp 1 e t e

cds.

9 USITLK8 Mouse mRNA for interleukin- 50.6%

8 receptor (IL-8R), complete cds.

10 i RABIL8REC Or y c t o 1 a gu s c un i cu 1 u s inter-50.6%

1 euk i n ~ 8 (IL-8) receptor

mRNA, complete cds. Table 4 Results of GenBank search for mouse ET240c DNA

Table 3 shows the top 10 cDNAs with high homology to the gene encoding the mouse ET240 fragment of the present invention among the cDNA sequences registered in GenBank. Indicated. The cDNAs with high homology compared to the gene encoding the mouse ET240 fragment of the present invention were all cDNAs of the seven-transmembrane receptor protein. None of the nucleotide sequences determined to be the same as the mouse type ET240 of the present invention was found. Therefore, mouse ET240 was confirmed to be a novel sequence.

Table 4 shows that among the cDNA sequences registered in GenBank, the top 10 cDNAs with high homology to the gene encoding the full length human ET240 of the present invention and their homology showed that. The cDNAs that were found to have a high degree of homology with the gene encoding human ET240 of the present invention were all cDNAs of the seven-transmembrane receptor protein. None of the nucleotide sequences determined to be the same as human ET240 of the present invention was found. Therefore, human ET240 was confirmed to be a novel sequence. Similar to the amino acid sequence search results, the nucleotide sequence having the highest homology with the gene of the seven-transmembrane receptor protein ET240 of the present invention is a seven-transmembrane type protein derived from Escherichia coli. The homology between the cDNA of the receptor protein PPR1 and the mouse and human ET240 was 82.0% and 88.4%, respectively. And then As described above, PPR1 has a completely different sequence from the protein and DNA of the present invention.

In addition, the results of the search using the above database show that all the cDNAs having high homology to ET240 of the present invention are seven-transmembrane receptors, indicating that the mouse type and the human type of the present invention are used. It has been suggested that G-type ET240 is a protein belonging to the seven-transmembrane receptor.

The present invention relates to a fragment peptide comprising a human full-length ET240 protein and a partial sequence thereof.

The method for obtaining the protein of the present invention is not particularly limited, but specifically, a method for preparing a synthetic peptide based on the amino acid sequence information, or a method for encoding a peptide. A method of synthesizing a peptide by introducing it into a host cell may be mentioned. A method for synthesizing a peptide by introducing a gene encoding the peptide into a host cell is described in "Krieg 1 er, Gene Transferana Expression-A Laboratory Manual, Stockton Press. , (1990); and a number of methods known by Yokota et al., Biomanual Series 4, Gene Transfer and Expression 'Analysis, Yodosha, 1994]. it can.

Human ET240 and its fragments are useful for the production of antibodies for diagnostic purposes and for the search of drugs for therapeutic purposes. Since mouse-type ET240 is expressed in EAE-causing T cells in mice, for example, diagnosis of autoimmune diseases including multiple sclerosis and autoimmunity including multiple sclerosis It is particularly useful for searching for drugs intended for the treatment of sexually transmitted diseases. In addition, since it is expressed on mucosal lymphocyte leukocytes, it is particularly useful for searching for drugs for the purpose of diagnosing and treating autoimmune gastrointestinal diseases. Like the full-length protein, the peptide consisting of a partial sequence of the human ET240 protein can be used for antibody production and ligand screening. It is useful in searching for a substance that binds to ET240 and regulates the response of white blood cells to treat a disease. As a peptide used for preparing an antibody, that is, as an antigen, for example, a peptide of 5 to 8 amino acid residues at a site corresponding to an extracellular region or a cytoplasmic region is appropriate. Examples of partial peptides used for screening of ligands and the like include, for example, Ν240, which is considered to be a ligand-binding site, Ν-terminal extracellular region (1st to 29th of SEQ ID NO: 2) Amino acid residue) and the first extracellular loop portion (amino acid residues 97 to 108 of SEQ ID NO: 2) or the second extracellular loop portion (SEQ ID NO: 1 Peptides containing (amino acid residues at positions 78 to 195) can be used.

Further, the present invention is a D type which codes the above-mentioned human type {240}.

In order to obtain DNA encoding human ET240 of the present invention, the ability to extract from a tissue in which the expression of ET240 has been confirmed, such as the small intestine and heart of a human, can be obtained by the method described in the present specification. It may be synthesized based on the nucleotide sequence described in SEQ ID NO: 1. In addition, in order to obtain DNA encoding human ET240 described in SEQ ID NO: 1, cDNA encoding the entire amino acid sequence of human ET240 is included. Transformant into which plasmid PET 240 H has been introduced E. coli: INV a F'-p ET 240 H [Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology 0 4 6 1-3-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan) Contract number: F on February 22, 1997 ERM BP—deposited as 6213).

A DNA encoding a polypeptide substantially equivalent to the amino acid sequence represented by SEQ ID NO: 2, for example, a DNA consisting of the base sequence represented by SEQ ID NO: 1 is derived from the human of the present invention. It is useful when examining the detailed function of the ET240, a seven-transmembrane receptor protein of the above.

Specifically, the nucleotide sequence of SEQ ID NO: 1, DNA or RNA complementary to the nucleotide sequence, at least 12 or more, preferably 16 or more, and more preferably If a DNA or RNA consisting of 18 or more bases or a derivative of these nucleic acids is used, cDNA, cDNA clone, genomic DNA, and the like of the seven-transmembrane receptor protein ET240 of the present invention can be used. Genome gene clones can be detected. The length of the required nucleic acid depends on the specificity of the sequence and the stability of binding to the nucleic acid to be detected, but the target gene is determined by PCR using DNA (polymerase chain reaction). When Tm is detected, it is desirable that Tm (duplex dissociation temperature) be 45 ° C or more. When DNAs bind to each other as in PCR, one GC bond is set at 4 ° C and one AT bond is set at 2 ° C, and the Tm can be estimated. Accordingly, a nucleic acid of 12 bases is required when the GC content is high, and a nucleic acid of 16 bases is required in a region having a general GC content of about 50%. Also, nucleic acid induction with stable binding to DNA When using a body, it is possible to detect the target gene using a shorter nucleic acid. For example, in order to examine the expression of these genes for diagnostic purposes, a nucleic acid which can be complementary to the nucleotide sequence of SEQ ID NO: 1, or at least 12 or more, preferably 16 or more, Preferably, the nucleic acid consists of at least 18 bases, i.e., antisense DNA or RNA, or antisense nucleic acid is methylated, methylphosphated, deaminated, or thiophosphorylated. Hybridization, primer extension, nuclease / protection / assay, reverse transcription gene amplification (RT-PCR), etc., using the phosphorylated derivatives Can be recruited. In Example 4 of the present specification, detection of natural mRNA by hybridization was performed.

Furthermore, a method of genetic diagnosis using ET240 includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, that is, DNA or RNA, or at least 12 nucleotides thereof. As described above, a nucleic acid consisting of preferably 16 or more bases, more preferably 18 or more bases, may be methylated, methylphosphated, deaminated, Or genomic southern hybridization using a chitophosphated derivative thereof. In a similar manner, it is possible to detect homologs of the gene of the present invention in other organisms such as rats, and to close such genes. Furthermore, genetics on genomics including humans and mice Cloning of the child is possible as well. Therefore, by using these genes thus cloned, the more detailed function of the seven-transmembrane receptor protein ET240 of the present invention can be clarified.

For the purpose of clarifying the more detailed function of the seven-transmembrane receptor protein ET240 of the present invention, administration of antisense nucleic acid to cells or living organisms is also conceivable. In addition, if an abnormality of the gene of the present invention is found in human genome, the discovered abnormality can be applied to gene diagnosis and gene therapy. For example, it is possible to treat a disease in which an excessive response of ET240 is a condition by suppressing gene expression using these antisense nucleic acids. It is also possible to use a recombinant nucleic acid obtained by incorporating an antisense nucleic acid into an appropriate vector. For examples of the preparation of such antisense nucleic acids ′, see Murray, A.H.

ANTISENSE RNA AND DNA, Wiley-Liss, Inc., (1992).

As described above, the DNA of SEQ ID NO: 1, its complementary nucleic acid, and derivatives thereof are useful for diagnosis and therapy. Since the seven-transmembrane receptor protein ET240 gene of the present invention was first found in mouse EAE-causing T cells, it has been shown that autoimmune diseases including multiple sclerosis can be It is particularly useful for searching for drugs for diagnostic or therapeutic purposes. Also, the ET240 gene Because it is expressed on mucosal and lymphocyte leukocytes (see Example 4), it is considered to be particularly useful for drug discovery for the purpose of diagnosing and treating autoimmune gastrointestinal diseases. Can be

Furthermore, the present invention is a recombinant DNA, comprising any one of the above-mentioned DNAs of the present invention.

The vector used for preparing the recombinant DNA of the present invention is not particularly limited, and any commonly used vector can be used. Specifically, PBR32, PUC8, pUC19, pUC18, and PUC119 derived from Escherichia coli (all manufactured by Takara Shuzo Co., Ltd., Japan), Bacillus subtilis-derived plasmid, and yeast-derived Plasmid vectors such as Plasmid, etc. Bacteriophage vectors such as gt10 and λgt11 (both manufactured by Stratagene, USA), and animal violales such as Retro Winorescunia senior Winores. And the like, but any other substances can be used as long as they can be propagated in the host. As a specific example of the recombinant DNA of the present invention, cDNA encoding the entire amino acid sequence of human ET240 is inserted into the vector pCR3.1. The obtained plasmid PET240H (see Example 6) is mentioned. E. coli: INV aF '— 4 Ε 4 240 Η (Deposit number: FE RM BP — 62) 13) The Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry of Japan (〒 30 5 — 0 4 6) No. 3 on February 22, 1997).

Further, it is preferable to introduce the recombinant DNA of the present invention into a known host. That is, the present invention is a microorganism or cell transformed with the recombinant DNA.

The host into which the recombinant DNA of the present invention is introduced is not particularly limited, but is a microorganism or a cell capable of expressing the recombinant DNA of the present invention. Specifically, prokaryotic cells such as Escherichia (Escherichia) genus (Escherichia coli) and Bacillus (Bacillus subtilis) are assembled using the calcium chloride method or the like. Recombinant DNA can be introduced. Examples of the above Escherichia bacteria include Escherichia coli K12, HB101, MC1061, LE392, JM109, and INVaF '. No. Bacillus subtilis Ml114 is mentioned as an example of the Bacillus genus. The phage vector was introduced into the grown E. coli using, for example, the in vitro packaging method (Pr0c.Nat 1.Acad.Sci. 74: 3259-3263, 1977). can do. Eukaryotic cells such as animal cells and insect cells can also be used as hosts.

Further, the present invention relates to a seven-transmembrane receptor protein produced on the cell membrane surface by a transformant. Specifically, (a) a DNA encoding ET240 is bound to a replicable expression vector, and the replicable expression vector is integrated with the DNA and the replicable expression vector. To obtain a recombinant DNA, (b) transforming a microorganism or cell with the replicable recombinant DNA to form a transformant,

(c) selecting the transformant from the parent cell of the microorganism or cell, and (d) culturing the transformant to cause the transformant to express the nucleic acid,

It is ET240 produced on the cell membrane surface of the transformant by a method including the above.

The recombinant vector used to produce ET240 on the cell membrane surface of the transformant is a translation initiation codon at the 5 'end of the ET240-encoding DNA inserted into the vector. It may have a translation termination codon at its 3 'end. A translation initiation codon and a translation termination codon can also be added using an appropriate synthetic nucleic acid adapter. Further, in order to express the target DNA, it is preferable to connect a promoter upstream of the DNA. The promoter used in the present invention is not particularly limited as long as it is a promoter corresponding to a host used for gene expression. If the host is a bacterium belonging to the genus Escherichia, a tac promoter, a tr ρ promoter, a 1 ac promoter, etc. are preferred. New When the host is a prokaryotic cell, the recombinant DNA to be introduced preferably has a ribosome binding site together with a promoter. If the host is yeast, PGK promoter, GAP promoter If the host is an animal cell, such as an SV40-derived promoter, a retrovirus promoter, a metalthione promoter, a heat shock promoter, etc. Is available.

The DNA used for producing ET240 of the present invention is not particularly limited as long as it encodes ET240 substantially equivalent to the amino acid sequence of SEQ ID NO: 2. . Specifically, the nucleotide sequence of SEQ ID NO: 1 can be used. In addition, in order to produce ET240 with a special function D, a known nucleotide sequence can be bound to DNA encoding ET240. For example, to ensure expression on the membrane surface, DNA encoding a signal peptide may be added to the 5 'end (the N-terminus of the peptide) or to facilitate detection of the produced protein. In addition, DNA encoding the antigen epitope can be added thereto. For an example of such a technique, reference can be made to Choe, H. et al., Cels. 85, 1135-1148, 1996.

A transformant for producing ET240 can be obtained by introducing the recombinant DNA constructed as described above into a host cell capable of expressing a vector. As the cells used as the host, the above-mentioned Escherichia bacteria, Bacillus bacteria, yeast, animal cells, and the like can be used. Specifically, animal cells are preferred, and monkey cells such as C〇S—7, Ver0 cells, Chinese hamster cells CHO, and silkworm cells SF9 are examples. Can be

As described in Examples 7 and 9 of the present specification, a transformant can be produced by introducing the above-described recombinant DNA into CHO cells or 2993 cells. By culturing the transformant, ET240 can be produced on the cell membrane surface of the transformant. The production of ET240 by the cultured transformant can be confirmed by the Western blot method or FACS (Fluorescence Actuated Cell Source) used in Example 7; it can.

The seven-transmembrane receptor protein of the present invention can be used to screen a ligand for the seven-transmembrane receptor protein. Specifically, the present invention provides substantially pure human ET240, a peptide comprising a partial sequence thereof, or human ET240 produced on the cell membrane surface of a transformant. Contact the sample material and bind to the transmembrane receptor protein seven times, using as an index the change that occurs in response to the binding of ET240 or its peptide to the ligand. It is a method that includes detecting the ligand of the target.

E-240 used in the screening method of the present invention may be a purified protein or an unpurified protein, but must have the same ligand binding activity as in ViV0. In particular, it is preferable to use the ET240 transformant of the present invention (that is, ET240 expressed on the cell membrane) for screening. For example, as was performed in Example 8, 293 transfected with a recombinant vector containing DNA encoding the full length of the human seven-transmembrane receptor protein ET240. Cells can be used for screening.

The sample material which is considered to include the ligand used in the screening method of the present invention is not particularly limited. For example, a tissue or a cell derived from a living body which is considered to include a physiological ligand Extracts or culture supernatants derived from the culture, and culture supernatants of synthetic compounds and microorganisms can be used. In particular, since the receptor of the present invention belongs to the seven-transmembrane receptor protein group, candidate ligands belong to the chemokine group, which is a ligand of the seven-transmembrane receptor group. Substance is conceivable.

The method for detecting the ligand for ET240 is not particularly limited, but, for example, the sample material is brought into contact with ET240, and the resulting ET240 and ligand are detected. Examples include a method for measuring the amount of the complex and / or the amount of unbound sample material, and a method for measuring a reaction caused by the binding of the sample material to ET240. Methods for measuring the amount of complex and / or the amount of unbound sample material include, for example, labeling the sample material with a radioactive compound, dye, etc., and then contacting the sample material with ET240. The ET240-.ligand complex is then separated from unbound sample material and the amount of complex and Z or unbound sample material are determined using the label. Can be measured. As an example, in Example 8, the amount of a complex formed between the radiolabeled candidate ligand compound and the receptor was measured. If a substance that binds to the receptor can be identified, the substance can be labeled, and the binding of the sample material can be measured based on whether the sample material competes with the labeled substance. Specific examples of these methods are given in Mikito Asanuma et al., Experimental Medicine, 11, 22-29, 1993 (Japan). Other than that, like SPA (Scintill at ion Proximi tity As say), the ligand complex can be separated without separating the ET240-ligand complex from unbound sample material. There is also a method of measuring the amount of binding.

As a method for measuring the reaction caused by the binding between the sample material and ET240, various methods using a signaling system to which ET240 is conjugated can be considered. Examples of such a method include a method for measuring intracellular calcium concentration, for example, as described in Experimental Medicine 7, pp. 26-109, 1989 (Japan), edited by Hideaki Karaki et al. Samson, M., et al., Biochem. 35, pp. 3362-3367, (1996), a method using a micrometer, a method for measuring the amount of intracellular cAMP, and the like. As one example, in Example 9, the chemotaxis of human type ET240 transformed cells using LPS-administered rat serum was observed.

Further, as a method for determining the ligand using a fragment peptide consisting of a partial sequence of the seven-transmembrane receptor protein of the present invention, Examples include a method using Bia Core and a method using purification using a resin column. BiaC0re is a device that detects the association of proteins with proteins using surface plasmon resonance (Protein Nucleic Acid Enzyme Vol. 37: 2997 7-29884,, 1992) . In this case, the purified peptide of the present invention, preferably the N-terminal extracellular region, is immobilized on a Bia Core sensor chip, and a sample material that is a ligand candidate is added thereto. The binding between the partial peptide and the sample material (that is, the ability of the sample material to be a ligand for the seven-transmembrane receptor protein of the present invention is examined. The fragment peptide of the present invention is also examined. By fixing it to a resin for column chromatography and preparing an affinity column, for example, the ligand for human ET240 present in cell culture supernatants is removed. The ligand purified in this way can be isolated and identified.

The substance obtained by the above-described screening method using the full-length protein and its fragment peptide of the present invention is a substance that binds to ET240, controls a reaction of white blood cells, and treats a disease. Is useful.

Furthermore, when a substance acting on the seven-transmembrane receptor protein ET240 of the present invention, that is, a ligand for the protein of the present invention, is discovered, ET240 and the ligand are compared. A substance that changes the action, that is, activates a reaction caused by binding, or conversely It is also possible to search for substances that inhibit activation. Specifically, substantially pure human ET240, a peptide comprising a partial distribution thereof, or human ET240 produced on the cell membrane surface of a transformant, and the like. The ligand for the protein or peptide is brought into contact with the sample material, and the change that occurs in response to the binding of the protein or peptide to the ligand is used as an index to determine ET240. This method involves detecting a substance that inhibits binding to a ligand.

As a specific screening method, for example, the chemical migration test of the transformant performed in Example 10 can be employed. In addition, like the ligand for ET240, a substance that inhibits the binding of the ligand to the protein or peptide of the present invention binds to the seven-transmembrane receptor protein and binds to leukocyte cells. It may be useful as a substance that controls reactions and treats diseases. Further, as a method for screening a substance that inhibits the binding between a peptide and a ligand, using a fragment peptide consisting of a partial sequence of the seven-transmembrane receptor protein of the present invention. In the same manner as the method for determining the ligand, a method using BiaC0re can be adopted. Further, the present invention is an antibody capable of binding to a seven-transmembrane receptor protein derived from human.

Antigens used to produce antibodies specifically recognizing the human seven-transmembrane receptor protein include cells expressing human ET240 or full-length ET240. 0 The protein or its fragment peptide can be used in a purified or unpurified state. When cells are used as an antigen, the cells are not particularly limited as long as they do not cause an immune reaction in the recipient individual to whom the cells are administered. For example, as in Example 6, a vector having a DNA encoding human ET240 was introduced into cells derived from a specific mouse individual, and the obtained transformant was used as a transformant. By returning the host cell to its original individual, an antibody that specifically recognizes human ET240 can be produced by the same method as in Example 11.

When a protein is used as an antigen, the protein is not particularly limited as long as it is capable of inducing an antibody that specifically recognizes human ET240. It is preferable that the full-length amino acid sequence of type ET240 is fused with GST (daltathione S-transferase) or the like. This protein is cross-linked with KLH (keyhole-l-etetemoemocyanin) or BSA (bovine serum alumin) or carrier protein, and then inoculated with an adjuvant, if necessary, to the animal. Recognition of human ET240 by collecting serum An antiserum containing antibodies (polyclonal antibodies) is obtained. Absorbing the obtained antiserum to the Pacific PPR1 protein makes it possible to produce an antiserum that does not react with the Pacific PPR1 protein and specifically recognizes human ET240. You.

Furthermore, a fragment peptide consisting of a partial sequence of the amino acid sequence shown in SEQ ID NO: 2 can also be used as an antigen for producing an antibody. The fragment peptide as an antigen is a partial sequence consisting of 5 or more, preferably 8 or more contiguous amino acids, the extracellular region of human ET240 full-length protein or Preferably, the peptide is obtained from a site corresponding to the intracytoplasmic region. The peptide of the present invention, similarly to the above-described protein of the present invention, is cross-linked with a carrier protein such as KLH or BSA, and then, if necessary, is brought into contact with an animal together with an adjuvant to recover the serum. By doing so, an antiserum containing an antibody that recognizes the ET240 protein can be obtained. In particular, if a peptide consisting of a partial sequence of the N-terminal extracellular region important for binding to the ligand is used as the antigen, the binding of the ligand to the seven-transmembrane receptor protein of the present invention will be inhibited. Such antibodies, or conversely, monoclonal antibodies that substitute for the action of ligands, can be expected to be obtained.

Further, it is also possible to purify the antibody from the antiserum and use it. In addition, a monoclonal antibody can be prepared based on a known method for preparing a hybridoma cell. As an animal to be inoculated with the antigen, a sheep, a goat, a goat, a rabbit, a mouse, a rat, and the like can be used. And egrets are preferred, and mice are preferred for production of monoclonal antibodies.

Furthermore, the antibody of the present invention can be prepared by various methods and gene cloning methods shown in a compendium (Antibodies al aboratory manua l, E. Harlow et al., Cold Spring Harbor Laborator). Using the isolated immunoglobulin gene, it can also be produced as a recombinant antibody expressed in cells. The antibody produced by such a method can also be used for purification of human ET240 of the present invention.

Utilizing an antibody capable of binding to the human seven-time transmembrane receptor protein ET240 of the present invention (for example, the antibody prepared in Example 11), the human ET240 is produced. Detection and measurement can be performed. Therefore, the antibody of the present invention can also be used as a diagnostic agent for diseases associated with abnormal cell differentiation, such as malignant tumors, viral infections, and autoimmune diseases. As a method for measuring and detecting the human ET240, Western Blotting, FACS Luo rescence Ac tivat ed Ce11 as described in Example 7, and the like. Sorter) can be adopted. For example, in Antibodiesl abo ratory manua l, E. Harlow eta, Cold Sirng Harbor Labo ra tory, pp. 471—510 (in addition, Wes tern Blo tt ing nob 1 otti ng) for the detection and measurement of protein peptides.For immunoprecipitation and immunoassay, see PP.421-470 and pp.553- Details are given in 612 respectively. In addition, edited by Tenjin Mio et al., Flow cytometry handbook, Science Forum, Japan, Part 4 of 1998 (japan). Application of FACS provides an example of clinical diagnosis using FACS. For details on the staining of cells during FACS, see Seiji Takatsu and Shinsuke Taki, Basic Techniques for Immunological Research, Yodosha, 1995, pp. 16-61. The details are shown in the book “Flow Site Metrics Handbook, Science Forum, Inc., edited by Mio Tenjin et al., 1998, Πap an).

As described above, an antibody against human ET240 that is characterized by containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 The present invention is useful for identifying cells expressing ET240.

Furthermore, the present invention is a fragment of a mouse-type ET240 protein. That is, a substantially pure mouse-derived seven-transmembrane receptor protein fragment having the amino acid sequence of SEQ ID NO: 4.

Further, the present invention is a DNA encoding the above-mentioned mouse ET240 fragment. Specifically, a mouse-derived seven-transmembrane receptor characterized by having the nucleotide sequence of SEQ ID NO: 3 An isolated DNA that encodes a protein fragment.

The mouse-derived seven-transmembrane receptor protein fragment of the present invention is a large fragment from the second transmembrane region to the C-terminus of mouse ET240. Furthermore, the mouse cells were the first to find a novel seven-transmembrane receptor protein, and the results of Northern hybridization in Example 3 showed that mouse-type ET240 Expression was confirmed in the lungs and hearts of mice, the use of this mouse-type ET240 fragment and the DNA encoding the same enabled the expression of the mouse-type ET240 full-length protein and its protein. It is easy to isolate the coding DNA. Due to the development of various genetic engineering techniques in mice, transgenic mice, gene targeting mice, and double knockers that inactivate another gene related to the gene of the present invention. It is also possible to clarify more detailed functions of the seven-transmembrane receptor protein ET240 of the present invention by using a technique such as a mouse. Therefore, the mouse ET240 full-length protein and the gene encoding the same have the same function as the human ET240 full-length protein of the present invention and the DNA encoding the same. Screening of substances useful for the treatment or prevention of the diseases involved, and methods for diagnosing such diseases and diagnostic agents can also be applied. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to Examples and Comparative Examples, but these do not limit the scope of the present invention. Example 1 Obtaining a novel seven-transmembrane receptor protein fragment derived from mouse

(Establishment of 4R312 shares)

Solid phase method using MBP (myel in basic rotein) peptide (amino acid sequence of SEQ ID NO: 19 in the Sequence Listing) using a 43 OA peptide synthesizer manufactured by Applied Biosystems, USA And a 16 mg / ml MBP peptide solution was prepared. Next, tuberculosis-killed bacteria (Aoyama B strain) (obtained from Nippon BSI, Japan, National Institute for Preventive Health) was added to complete Freund's adjuvant (manufactured by Difco, USA) at a final concentration of 4 mg / day. ml, and the solution was made into an adjuvant solution. The MBP peptide solution 900 μl and the adjuvant solution 900 μl are mixed well in a glass syringe, and the MBP peptide concentration is 8 mg Zm 1 and the MBP peptide emulsification solution is mixed. And

This MBP peptide emulsion solution was subcutaneously injected into both hind limbs L of SJL / J mouse (9 weeks old, female; obtained from Japan, Japan, Japan) with 25 μl each. did. Further, 1 μg of islet activating protein (manufactured by Kaken Pharmaceutical Co., Ltd., Japan) solution (5 g / mi) was added to the tail vein immediately after immunization and 2 days later. A 200 μl saline solution was injected.

Based on visual observation, mice were selected to develop clinical symptoms of EAE (experimentalallergic encephalomye litis) and selected, and on day 63 after immunization, axillary, inguinal and popliteal lymphocytes were selected. The lymph node was cut into small pieces with a female thread, rubbed with a silicone stopper using a mesh No. 150 to obtain a single cell suspension. The solution was added to a medium containing 10 μg / m 1 MBP peptide [Click's EHAA medium (US, irv, containing 5% FCS (petal fetal serum; obtained from Bioser)). The cells were sub-cultured in a Ca strain from Ine S cienti Πc Co. No.9582) In each culture, the cell count was adjusted to 4 × 10 ⁶ cells / ml, and a 25 cm ² (Corning, USA) with 10 ml flask and the culture was started with the flask standing.

The following factors were added to the subculture medium. 1 0 mu g / 1 of co Nkanapa Li down A (US, S i gm a Inc.) and RPMI 1 6 4 0 medium containing 1% FCS (US, GIBCO BRL Co.) 1 0 ⁶ cells / m 1 After stimulating the spleen cells of an SD rat (Chart River) for 48 hours, the supernatant was collected and the final concentration was 20 mg Zm1 (1-1111611 ^ 1). —0— 11131111 ₀ 5; (Add 16 and add RGF (Rat Growth Factor). Also, lng / ml phorbo noremyristate acetate (Sigma, USA) D including) - MEM (GI BCO- BR L manufactured;. in containing 5% FCS) 1 0 ⁶ mice EL 4 was prepared in a cell Z ml IL - 2 cells (ATCC Re-available. After stimulating ATCCTIB-181) for 24 hours, the culture supernatant was collected to obtain EL4sup as a source of IL-12, a growth factor of T cells.

On the 6th day of culture, the above-mentioned lymph node cell culture medium was subcultured (C1ick's EHAA medium, 5% FCS, 15% RGF, EL4sup).

1%), adjust to 4 × 10 ⁶ cells / m 1 in the same medium, dispense into a 24-well plate with 1.2 ml no-well, and add 5% The cells were cultured at 37 ° C in a carbon atmosphere.

After about 1 week of culture, 1 g / ml of MB P peptide Dooyo beauty about 2 X 1 0 ⁶ X-ray irradiated cells / m 1 of antigen-presenting cells (SJLZJ mouse splenocytes with 3 0 0 0 R The mouse single cells described above were cultured at 37 ° C in a 5% carbon dioxide atmosphere (antigen stimulation) in the presence of), and after about one week, the medium was replaced with a subculture medium. The cells were cultured at 37 ° C for about 1 week in a 5% carbon dioxide atmosphere. This was defined as one cycle of culture, and the culture cycle was repeated every two weeks. The number of cells to be planted during the passage was gradually reduced, and subculture was finally performed at 2.5 to 5 × 10 ⁵ cells / ml.

After repeating this culture for 3 cycles, each clone was divided into individual clones by ultra-dilution. Select clones that respond to the MBP antigen peptide in vitro, and then inject the clones into mice to develop EAE (tail loss of tail tone and hind limb paralysis). The target clone was selected based on this. That way One clone was named 4R312 strain. The 4R312 strain was also subjected to expanded culture by repeating the same culture cycle as described above, and used in subsequent experiments.

(Confirmation of AE onset of 4R312 strain)

4 R 3 1 culture expanded the two strains, 4 R 3 1 2 strain cells 5 X 1 0 ⁶ pieces of 2 mice SJLZJ mice (8 weeks of age 3 days after antigen stimulation, main scan; Chiya one pulse Li server Was injected into the tail vein. EAE onset was observed on day 5 in one animal and on day 6 in the other animal by visual observation.

(Acquisition of cDNA fragment of novel seven-transmembrane receptor protein derived from mouse)

Ma c scan 4 R 3 1 culture expanded 2 strain was used as a material by culturing about 1 X 1 0 ⁷ cells in total. After suspending the cells by pipetting, centrifuge at 100 rpm for 15 minutes (KS-8300, manufactured by Kubota Seisakusho, Japan; RS300 / 6/6) ), Aspirate the supernatant, discard the supernatant, and apply PBS (Phosphate Buffered Saline) (Dainippon Pharmaceutical Co., Ltd., Japan; Ca. No. 28-103-05) 30 m After adding 1 suspension, it was centrifuged again under the same conditions. Hereafter, the manufacturer's protocol (Rev.4. XV—025—00—) was used using Quick Prep mRNA Purifi cat on Kit (Spain, manufactured by Pharmacia Biotech). 1 1 to 1 page 4 of 07) ί this therefore _m R NA was extracted (however, s econd columum purification was not extracted). After ethanol precipitation, Moecullar Cloning, Alaborator manua l, (1989), Sambrook, J., Fr its ch, EF, and Maniatis, T. Eds., RNA was quantified according to E5, Spec tro pho to ome tric e ti n a tio n Amount of DNA or RNA of Cold Spirng Harbor Labo r espresses. Approximately 2 μg of the obtained RNA was used as a suspension, and 5 XF irststr and bufffer Ο, μ Ι, 0 Su Im MDTT 5 ^ 1. Add 5 μl of RNa sin (Promega, USA) and 1 μl of Rasefree DNase (Boehr inger, Germany) and sterilize. The total volume was adjusted to 50 μl with water and left at room temperature for 5 minutes. After that, phenol-chloroform extraction and ethanol precipitation were performed, and cDNA synthesis was performed as follows.

CDNA synthesis was performed using Supe r sc r ip pt Cho ic Sys tem fo rc DNA Snt he s ys (manufactured by Uie Technolog ies, USA). A double-stranded (ds) DNA was synthesized using oligo (dT) primer in accordance with pages 11 to 17 (Protocol 1 and 2) of P. Tokonore. Then, after phenol / chloroform extraction and ethanol precipitation, the synthesized cDNA was dissolved in 40 μl of sterilized water (this is referred to as a cDNA sample).

PCR (Polymerase cha in reaction) was performed using 41 of the cDNA samples. Raze (Code R001A, manufactured by Takara Shuzo, Japan) was used. 5 μl of the buffer attached to the enzyme, 4 μl of the dNTP mixture attached to the enzyme and the synthetic oligonucleotide Α shown in SEQ ID NO: 5 in the Sequence Listing, and SEQ ID NO in the Sequence Listing Each of the synthetic oligonucleotides B shown in Fig. 6 was subjected to a force of 200 pmo1, and the final volume was set to 50 µl.

The mixture was subjected to 95 ° C for 1 minute, 40 ° C for 2 minutes, and 72 ° C for 3 minutes for 5 cycles using TaKaRa PCR thermal Cycler 480 (Takara Shuzo, Japan). This was performed for 25 minutes at 95 ° C for 1 minute, at 50 ° C for 2 minutes, and at 72 ° C for 3 minutes. A part of the obtained PCR product was subjected to electrophoresis in a 1.5% agarose gel, stained with ethidium bromide (manufactured by Nippon Gene, Japan), and observed under ultraviolet light. It was confirmed that bp cDNA was amplified. The bands the gel force ^ et al switching Li out to Sup 〖ec01 (Japan, Takara Shuzo Co., Ltd.) after purification by, TA c loning kit (Ola down da country, I n V itf made ₀ gen Co., Ltd.) Crowjung was used.

Specifically, the vector used was pCRIIVector (Invitrogen, The Netherlands; hereinafter, referred to as pCRII), and the vector and the preceding DNA had a molar ratio of 1: 3. The DNA was mixed into the vector, and the DNA was incorporated into the vector using T4 DNA ligase (Invitrogen, The Netherlands). The vector with the DNA incorporated, pCRII, was transformed into Escherichia coli One Shot Competent Cells 1NV F '(manufactured by Inviogen, The Netherlands). L-Broth (Takara Shuzo Co., Ltd., Japan) containing 50 μg Z ml of ampicillin (Sigma, USA) was seeded on a plate of semi-solid medium. It was left at 37 ° C for about an hour. The colonies that appeared were randomly selected, inoculated in 2 ml of L-Br0th liquid medium containing the same concentration of ampicillin, and cultured with shaking at 37 ° C for about 8 hours. Next, the cells were collected and plasmid was separated using Withomi Dopply (Promega, USA) according to the attached instructions. Digestion of this plasmid with the restriction enzyme Ec0RI revealed that approximately 700 bp of DNA was cut out, confirming that the PCR product had been incorporated. The nucleotide sequence of the incorporated cDNA was determined for the confirmed clone.

The nucleotide sequence of the inserted cDNA fragment was determined using a fluorescent sequencer manufactured by Applied Biosystems, USA. Sequence samples were prepared using PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems, USA). In a 0.5 ml microtube, 9.5 μl of reaction stock solution, 4.5 μμΟ of 0.8 ρmoI /; Primer (manufactured by Applied Biosystems, Inc., USA) and 6.5 μl of 0.16 μg / μ1 sequence type DNA for sequencing are added and mixed, and 1 μm of mineral is added. After lamination of Larnoinore, PCR amplification reaction is performed at 96 ° C for 30 seconds and 55 ° C for 15 seconds and at 60 ° C for 4 minutes. Was performed for 25 cycles and kept at 4 ° C for 5 minutes. After the reaction, 80 μl of sterilized purified water was added and stirred, and after centrifugation, the aqueous layer was subjected to three times phenol / close-mouth extraction. To the 100 μl aqueous layer, add 10 μl of sodium triacetate (ρρ5.2) and 300 μl of ethanol, stir, and then add room temperature to room temperature. The precipitate was collected by centrifugation at 0 rpm for 15 minutes. After the precipitate was washed with 75% ethanol, it was allowed to dry under vacuum for 2 minutes to obtain a sequence sample. The sequence sample was dissolved in formamide containing 4 μl of lOmM EDTA, denatured at 90 ° C. for 2 minutes, cooled on ice, and subjected to a sequence.

When the DNA sequence was determined for 75 clones, two clones had a sequence corresponding to the 6th to 627th C of the DNA sequence of SEQ ID NO: 3 ( It does not include the sequences of the primers at both ends). A search for GenBank release 100.0, April, 1997, showed that this sequence was similar to the seven transmembrane receptor group. Mouse-type E-clone (derived from EAE-causing T cells) 240 fragment].

(Acquisition of a sequence containing the C-terminal region of a mouse-derived novel seven-transmembrane receptor protein ET240 fragment)

The mouse-derived ET240 fragment cDNA sequence obtained above was We originally searched the mouse's EST (Expressed Sequence Tag) database (GenBank release 100, 0, April, 1997). As a result, two types of EST fragments, mouse AA 0143733 and AA0550273, which are considered to encode almost the same gene, were used. From 13.5 dpc and 14.5 dpc embryos). By using these sequences, the cDNA sequence of the DNA sequence U of SEQ ID NO: 3 from the 501st base G onward was obtained.

The sequence of the mouse type ET240 obtained above and the sequence of the EST fragment were connected, and the DNA sequence shown in SEQ ID NO: 3, that is, a novel mouse-derived seven-transmembrane receptor protein ET240 was obtained. A portion of the cDNA sequence was obtained. In addition, the sequence was translated into amino acid to obtain a part of the amino acid sequence of the novel seven-transmembrane receptor protein ET240 derived from mouse shown in SEQ ID NO: 4.

In addition, in each of the two entries of the above-mentioned EST database, the residue corresponding to G at the 598th base in the DNA sequence of SEQ ID NO: 3 becomes C, and the sequence shown in SEQ ID NO: 3 Was different. Example 2 Acquisition of human seven-transmembrane receptor protein

(Acquisition of a cDNA fragment encoding a novel human seven-transmembrane receptor protein)

CDNA sequence of mouse ET240 fragment obtained in Example 1 Based on the columns, we searched the human EST (Epressed Sequence Tag) database (GenBank release 100.0, April, 1997). As a result, two types of EST fragments, AA 2 1 5 5 7 7 (tonsl 11 arcells enr iched for germi nalc ent er B ce lls by flow sorti ng (CD20, IgD—) And H67224 (derived from the library of Weismann Olfactory Epielium).

Based on the sequences of these two fragments, the oligonucleotide F1 (system IJ number 7: 5 'GCTGTAGCAG ATTTACTCCT TCTATTCAC 3'; a part of the sequence of H67224; 1: 259th G force to 287th base C to C) and oligonucleotide R1 (system iJ number 8: 5 'GCCGATGTCC ATGCGTTTGC TCATGTC 3'; SEQ ID NO: 1 8 35 Corresponding to the complementary strand of the C base of the 86th base, from the G base at the 5th base, but since a part of the complementary strand of the sequence of AA2155577 was used, the C base of the 860th base was used. Base (corresponding to the second base of SEQ ID NO: 8), instead of complementary G, is now C), and human-derived genomic DNA (Clontech, USA) No. 6550-1) was used as a 铸 type to amplify the gene by PCR (Polymerase), and used for gene sequence analysis.

Taq polymerase (Takara Shuzo, Japan) was used for PCR. c Mix 1 μl of DNA library solution and 38.5 μl of deionized water and heat at 95 ° C for 7 minutes. aq Polymerase 5 l attached to polymerase, 2.5 μM dNTP mixture (Takara Shuzo Co., Ltd., Japan) 4 μl, and oligonucleotides F 1 and R 1 After adding 20 pm 01 each, heating at 95 ° C. for 3 minutes, 0.5 μl of 39 polymerase was added to make a final volume of 50 μl.

Using a TaKaRa PCR template 480, this mixture was subjected to 94 ° C for 1 minute, 65 ° C for 2 minutes, and 72 ° C for 3 minutes to 5 cycles, 94 ° C for 1 minute, 6 ° C. 3 ° C 2 min, 72 ° C 3 min 5 cycles, 94 ° C 1 min, 60 ° C 2 min, 72 ° C 3 min 5 cycles, then 95 ° C 1 Min, 50 ° C for 2 minutes, and 72 ° C for 3 minutes for 25 cycles, and finally, 72 ° C for 7 minutes for 1 cycle. The PCR product was subjected to electrophoresis in 1% agarose gel, stained with ethidium Muvuchi Mide (manufactured by Japan Gene, Japan), and observed under ultraviolet light. A PCR product of about 600 bp was cut out from the gel, and the DNA was purified using TaKaRa SuperECOl. This purified DNA was incorporated into a pCR2.1 vector using a TA cloning kit (InVit0rogen) according to the attached protocol. The pCR2.1 vector into which the DNA was incorporated was transfected into E. coli INV cF 'Competent Cells (manufactured by Invitrogen). Bacterium containing 50 μg Zm1 of Ampicillin (Sigma, USA) and Broth (Takara Shuzo, Japan) Transfected E. coli on a plate of semi-solid medium. 1 Approximately 2 hours Leave the colony at 37 ° C and select it randomly. The cells were inoculated on 2 ml of L-BrQth liquid medium containing silicon. The cells are recovered by shaking culture at 37 ° C for about 18 hours, and the plasmid is separated using Wizard Dominipple (Promega, USA) according to the attached instructions. did. The base sequence of the separated plasmid was determined. The DNA sequence of the three clones was analyzed using a DNA sequencer, and the obtained consensus sequence was used as a cDNA sequence. And the nucleotide sequence from the C to the 834th C was determined (excluding the primer sequences at both ends). This sequence was strongly similar to the cDNA sequence of mouse-type seven-transmembrane receptor protein ET240 obtained in Example 1.

The DNA fragment obtained in this manner was designated as a human ET240 fragment.

In the known entry H67224, a partial force corresponding to CCCATTTTCCCCC at the 564th and subsequent nucleotides of the DNA sequence of SEQ ID NO: 1; NCCATTTTTCCCCC, which is shown in SEQ ID NO: 3 It was different from the rooster. In addition, in the known entry AA21δ5777, an extra A is inserted after the base corresponding to the base C at the 744th base of the DΝII sequence of SEQ ID NO: 3. The nucleotides corresponding to the bases 777, 787, 807, and 807 become N (that is, A, G, C or T), and the sequence shown in SEQ ID NO: 3 Was different. (Determination of human full-length transmembrane receptor protein ET240 full-length gene sequence)

Using a human lung-derived cDNA library (CL0NTECH, CatK HL 5030 t) as a type III gene, the gene was amplified by PCR (po1 ymerase cha in reaction), and the gene sequence was analyzed. It was subjected to analysis.

The following method was used to determine the 5 'end of the human transmembrane receptor protein ET240 gene.

Taq polymerase (Takara Shuzo, Japan) was used for PCR. Mix the cDNA library solution 11 with 38.5 μl of deionized water, heat at 95 ° C for 7 minutes, and add the buffer attached to the Taq polymerase 5 μ 1 and 2.5 mM d NTP mixture (Takara Shuzo Co., Ltd., Japan) 4 μ 1 and the oligonucleotide LD using the vector arm sequence of this cDNA library — 5: 5 'CTC GGG AAG CGC GCC ATT GTG TTG GT 3' (SEQ ID NO: 9), and oligonucleotide R3: 5 'GTG TGT ACA AGG CTG AAG TTA TTT TGC AC 3' (distribution system) 1J No. 10; the nucleotide sequence of the nucleotide sequence from 342nd G to 370th C of SEQ ID No. 1) was added at 20 pmol each, and the mixture was heated at 95 ° C for 3 minutes. 0.5 μl of polymerase was added to make a final volume of 50 μl (this reaction solution is referred to as reaction solution R3). At the same time, instead of the oligonucleotide R3, the oligonucleotide R2: 5 'GCA TTA ACA GCC CAA AAA GGC AGA GTG 3' (SEQ ID NO: 11; SEQ ID NO: 1) 2 8 5th C Capra 3 1 1 A reaction solution using the G sequence (antisense sequence of the base sequence of G) was also prepared (this reaction solution is referred to as reaction solution R 2).

Each of these two mixtures was subjected to TaKaRa PCR thermal Cycler 480 for 1 minute at 94 ° C, 4 minutes at 72 ° C for 5 cycles, 1 minute at 94 ° C, and 2 minutes at 68 ° C. 5 minutes at 72 ° C for 3 minutes, 1 minute at 94 ° C, 2 minutes at 65 ° C, 5 cycles at 72 ° C for 3 minutes, then 1 minute at 95 ° C and 55 ° C C 2 minutes, 72 ° C 3 minutes were performed for 20 cycles, and finally 72 ° C 7 minutes were performed for 1 cycle. This PCR product was subjected to electrophoresis in 1.5% agarose gel, stained with ethidium bromide (manufactured by Nippon Gene, Japan), and observed under ultraviolet light. The band detected as a somewhat clear band is compared between the reaction solutions R 3 and R 2, and about 450 bP for the reaction solution R 2 and about 450 bP for the reaction solution R 3. A band of about 500 bp which was 60 bases long was found, the PCR product was cut out from the gel, and DNA was purified using Ta KaRa SuprecOl. Each of the obtained purified DNAs was incorporated into a pCRII vector according to the attached protocol using a TA cloning kit (Invitrogen, The Netherlands). Each of the two pCRII vectors containing the DNA was transfected into E. coli INV aF 'Competent Ce 1 Is (1 nvirogen, The Netherlands). B [o th (Takara Shuzo, Japan) containing 50 g / m 1 of ampicillin (Sigma, USA). And leave it at 37 ° C for about 12 hours. Was. Two colonies that appeared each were randomly selected, inoculated in 2 ml of L-Br0th liquid medium containing the same concentration of ampicillin, and shaken at 37 ° C for about 18 hours. After culturing, the cells were collected. Plasmid was separated using Wizard Dominiprep (Promega, USA) according to the attached instructions, and the nucleotide sequence of DNA was determined. The clone DNA sequence was analyzed using a DNA sequencer in the same manner as in Example 1.

As a result, only one clone derived from the reaction solution R3 had, in addition to the primer portion, a sequence common to the sequence of the human ET240 fragment obtained above, which is shown in SEQ ID NO: 1. It was a DNA sequence from the first A to the 34th T of the DNA sequence.

In addition, the determination of the 3 'end of the human seven-transmembrane receptor protein ET240 gene was determined using the same method as that for obtaining the 5' end as follows.

In order to obtain the 5 'end of primer LD-5, use oligonucleotide vector H using ATACGACTCACTATAGGGCGAATTGGC (§self ij number 12), using the arm sequence of the vector of the cDNA library. , And oligonucleotides R 3 and R 2, respectively, instead of oligonucleotides F 3: GCTGCGACATGAGCAAACGCATGG AC (SEQ ID NO: 13; 8th G protein in SEQ ID NO: 1) And the nucleotide sequence corresponding to the 8th and 5th C) and the oligonucleotide F2: GTCAGTTATAGTTTTCATTGTCACTCAACTG (No. 14; No. 1; No. 1) No. 1 7 4 2nd G-para 7 1 This is the sequence corresponding to the 1st G, but since the sequence of GenBank entry AA 2 1 5 5 7 7 was used, SEQ ID No. 14 4 4 (Excess A is included at the base). After the electrophoresis, an approximately 400 bp fragment derived from the reaction using the oligonucleotide F 3 and an oligonucleotide F 2 that is approximately 110 bP longer were obtained. A fragment of about 5100 bP derived from the reaction solution used was cut out from the gel. The obtained fragments were each cloned in the same manner as in the acquisition of the 5'-end, and the colonies that appeared in each case were randomly selected two by two, and the DNA sequence was determined. As a result, only one clone derived from the reaction solution using the oligonucleotide F2 had a sequence common to the above-obtained sequence in addition to the primer portion. It was the nucleotide sequence from the 772nd base to the 115th base A of a certain DNA sequence. In the entry AA2155557, the base sequence from the 859th base G to the 870th base C of the DNA sequence in SEQ ID NO: 1 becomes the IGG force ^ GGCNATCCAAGTN. This was different from the sequence of SEQ ID NO: 1.

By joining these sequences and the above-obtained sequence, the DNA sequence of SEQ ID NO: 1 was obtained, which was further translated into amino acid to obtain the amino acid sequence of SEQ ID NO: 2. The acid sequence was obtained.

Example 3 Analysis of Mouse Tissue Expressing ET240 The expression of the mouse-type seven transmembrane receptor protein ET240 derived from the 4R312 cell line obtained in Example 1 in each organ was analyzed by Northern hybridization. Using a 1 p 1 e Tissue Northern (MTN) Blot filter (manufactured by CLONTECH, code: 762-1) for Mu 1, a 5-fold concentration of SSPE solution (a 1-fold concentration of SSPE solution is 0.1%). 5 MNaCl, 10 mM NaHPO 1 mM EDTA, pH 7.4), Denhardt's solution at 10 times concentration (Wako Pure Chemical Industries, Japan), 2% SDS (dodecyl sulfate) Sodium, Wako Pure Chemical Industries, Japan), final concentration 50% formamide (Wako Pure Chemical Industries, Japan), and 100 μg Zm1 boiling water bath The cells were immersed in a hybridization solution containing denatured salmon sperm DNA (manufactured by Sigma, USA) and shaken at 42 ° C for 2 hours.

Mouse-derived ET 2 4 0 fragment probe labeled with radioisotope ³² P was prepared in the Hare good follows. The vector containing the mouse ET240 fragment was pCRII, and the inserted fragment was excised from the vector with the restriction enzyme EcoRI and electrophoresed in a 0.8% agarose gel. Was done. The gel after electrophoresis was stained with ethidium bromide (manufactured by Nippon Gene, Japan), observed under ultraviolet light, and a band of about 600 bp was cut out from the gel to remove GENECLEANM I Kit (Funakosi, Japan). The obtained DNA fragment was used as a DNA labeling kit (Megaprime DNA labeling system; Amersham, UK, code RPN). 1607). That is, add 10 μl of the primer solution, 5 μl of the reaction buffer solution and 20 μl of the reaction buffer solution to 100 ng of DNA, make the total volume 86 μl, and perform a boiling water bath for 5 minutes. Then, add α-32 ^2- d CTP (manufactured by Amersham, code AA00005) (10 μl) and Klenow enzyme solution (4 μl), and add at 37 ° C. After water bath for 0 minutes, radiolabeled mouse ET240 fragment was synthesized. After that, the product was purified using a Sephadex column (Quick Spin Co 1um Sephadex G-50; manufactured by Boehringer Mannheim, Germany), boiled in a water bath for 5 minutes, and then cooled for 2 minutes on ice. .

The prepared ³² P-labeled mouse ET240 gene fragment probe was added to the hybridization solution, and the mixture was further shaken at 42 ° C for 16 hours, followed by hybridization. Was done.

Next, the plate was immersed in a double concentration SSC solution containing 0.1% SDS, washed three times at room temperature, and further washed with the same solution at room temperature for 15 minutes. Furthermore, washing was performed at room temperature for 15 minutes using a 0.2% or twice concentration SSC solution containing 0.1% SDS. After the washing, the filters were subjected to autoradiography at 185 ° C using an intensifying screen. As a result, bands around 1.5 to 2.2 kb were observed in all lanes, and strong bands were observed particularly in the lungs, heart, and liver. Example 4 Analysis of Human Tissue Expressing ET240 The gene expression of the human seven-time transmembrane receptor protein ET240 obtained in Example 2 in each organ was analyzed by Northern hybridization.

Using a Multiple Tis sue ort hern (MTN) B 1 ot filter (CLONT ECH, code # 7760-1), use a 5x SSPE solution (1x SSPE solution is 0%). . 1 5 MN a C 1, 1 0 m MN a H 2 PO lm MEDTA, p H 7. 4), 1 0 -fold concentration Denharu preparative liquid (Japan, manufactured by Wako Pure Chemical Industries, Ltd.), 2% SDS (dodecyl Sodium sulfate, manufactured by Wako Pure Chemical Industries, Japan), final concentration 50% formamide (manufactured by Wako Pure Chemical Industries, Japan), and a boiling water bath of 100 μg / m 1. denatured salmon sperm DNA (US, S i gma Ltd.) soaked in hybridization Dizeshiyo down solution containing, after cormorants preparative vibration for 2 hours at 4 2 ° C, is ³² P-labeled by the same method as in example 3 The resulting human ET240 gene fragment probe was prepared, added to a hybridization solution, and shaken at 42 ° C for 16 hours to perform hybridization.

Next, the plate was immersed in a double concentration SSC solution containing 0.1% SDS, washed three times at room temperature, and further washed with the same solution at room temperature for 15 minutes. Further, washing was performed at room temperature for 15 minutes using a 0.2-fold concentration SSC solution containing 0.1% SDS. After the washing, the finalizer was subjected to autoradiography at 185 ° C using a sensitizing screen. As a result, bands around 1.5 to 2.2 kb were observed in all lanes except the prostate. A strong band was observed in the heart and small intestine. Example 5 Analysis of Mouse Tissue Expressing ET240 by PCR

The mouse ET240 gene expression in the intestine and tongue was examined by PCR.

Preparation of intestinal lymphocytes was in accordance with the Current Protocols in Immunology, Supplement 15, 3 / 19.1 to 3.19 / 4.

Specifically, two C3HZH e mice (male, 12 weeks old) were sacrificed by cervical dislocation under ether anesthesia. After washing, the obtained small intestine was cut into a length of about 1.5 cm and collected in a triangular flask. RPMI 164 medium (manufactured by GIBCO BRL, USA) supplemented with 45% FCS (manufactured by GIBCO BRL, USA) in a flask at 37 ° C Stirred for 30 minutes. Thereafter, the filtrate was recovered using a tea strainer. The residue remaining in the tea strainer was placed in a 50 ml centrifuge tube, and 15 ml of RPMI 1640 medium was shaken, shaken vigorously for 15 seconds, and the filtrate was collected using a tea strainer. . This was repeated three times. These filtrates were combined, passed through a glass wool column, centrifuged to precipitate the cells, and the cells in the pellet were collected and resuspended in 15 ml RPMI 1640 medium. The suspension was returned to the flask again, and the procedure of starting stirring in the flask was repeated twice to collect the cells. The cell recovered solution is centrifuged to collect the cells again to form one stock, and twice more with PBS (Phosphate Buffered Saline) (Dainippon Pharmaceutical Co., Ltd., Japan). , Ca o. 28-103-05). After suspending the cells in 8 ml of 40% Perc 011 solution (in RPMI 1640 medium), halve 2 ml of 75%

It was overlaid on a Percoll solution (in RPMI 1640 medium) and centrifuged at 800 X g for 20 minutes at room temperature. The cell fraction at the interface between the 40% solution and the 75% solution was collected as IEL (Integral Lymphocy tes).

The obtained 5 × 10 ⁶ IELs were recovered, and total RNA was purified using a QuickPrep Total RNA extraction kit (Pharmacia, Sweden) according to the manufacturer's protocol. .

For the tongue, the entire tongue was cut off after the craniotomy, cut into small pieces with surgical scissors, and manufactured using the QuickPrep Total RNA extraction kit (Pharmacia, Sweden). Total RNA was purified according to the manufacturer's protocol.

CDNA was synthesized using a kit of these RNA force, Superscript Preampli i'i cation (GIBCO BR, USA, Cat. No. 18089-011).

The mouse DNA genome was purchased from C10ntech in the United States (Ca. 6650-1).

The above cDNA 3 / l (genomic DNA was diluted to 100 μg equivalent to 3 μl. The same amount was used for negative control) Primers d NTP mixture 7 / l in water was added), 4 mu 1 of 1 0 X buffer, 2 mu 1 of ₂ 5 m MM g C l 2 solution, by ll of (2 0 pmo by 1) m240F (SEQ ID NO: 15: 5 'GCTCATCAAG ATGCCCAACA TTAAAAAGTC TC 3', corresponding to the 4th 17th G protein from the 4th sequence of the base sequence of J number 3) Limer m 2 4 OR (Robot system ij number 16: 5 'TTAAATGGTAAAAG AACT GGTTGGCTCTGTAG3', Robot system IJ No. 3 base sequence 7 U 1 C or Ί 9 9 T and st (Corresponding to the complement of the codon TAA) to obtain a PCR mixture. This mixture is

Using PCR eCycler 480, 30 cycles of 1 minute at 94 ° C, 2 minutes at 55 ° C, and 3 minutes at 72 were performed, followed by a reaction at 72 ° C for 7 minutes. Was. This PCR product was subjected to electrophoresis in a 0.8% agarose gel, stained with ethidium mouth mold (manufactured by Nippon Gene, Japan), and observed under ultraviolet light. As a result, a band with the same mobility of about 400 bp was observed in the three samples excluding the negative control. The size of this band closely matched the size of the mouse fragment (375 bp) obtained in Example 1, and was considered to be the same gene. Example 6 Preparation of 7 Transmembrane Receptor Protein ET240 Expression Vector

Human lung-derived cDNA library (CLONTECH, Cat I HL 5030 type III, PCR of full-length ET240 gene (po 1 yme rase cha in reaction). 0 1 This was used (11'1811 Fidility Taq polymerase (Beringer Mannheim, Germany). 2 μl of this phage library solution and deionized water 36 Mix 1 μl, heat at 95 ° C for 7 minutes, add 1 μl of Taq polymerase, and 5 μl of buffer attached to Taq polymerase. 5 μM dNTP mixture (Takara Shuzo Co., Ltd., Japan) 4 μ 1 and Origo Nucleotide F 4 (SEQ ID NO: 17: 5 'ACTACAAAAGCTTGGAGCCATGGCTTTGGAAC 3'; AAG (: plus a spacer sequence, ACTACAA) to introduce a recognition site for the restriction enzyme HindIII on the 5 'side of the 21 bases from T force to the 2nd C, and oligo Nucleotide R 4 (system ij No. 18: 5 'AAAAGCTCGAGCAGTTTTACCTTTAA ATGCTAAAAG 3'; SEQ ID NO: 1 C base of the 10th ninth C to 107th C base CTC for introducing the recognition site of the restriction enzyme XbaI on the 5 'side of the antisense sequence and AAAAG as the spacer sequence []) were added to the psense of 20 pmo 1, respectively. In addition, the final volume was 50 μl.

This mixture was subjected to 95 ° C for 1 minute, 65 ° C for 2 minutes, and 72 ° C for 3 minutes using TaKaRa PCR thermal Cycler 480, and then to 95C for 1 minute. 6 2 ° C for 2 minutes, 72 ° C for 3 minutes, 5 cycles, and 95 ° C for 1 minute, 59 ° C for 2 minutes, 72 ° C for 3 minutes, 5 cycles Then 95. C 1 minute, 50 ° C. 2 minutes, 72 ° C. 3 minutes were performed for 20 cycles, and finally a reaction was performed at 72 ° C. for 7 minutes. This PCR product Perform electrophoresis in 8% agarose gel, stain with ethidium bromide (manufactured by Nippon Gene, Japan), and observe under ultraviolet light. It was confirmed that the DNA was amplified. The target gene product was cut out from the gel, and DNA was purified using SuprecOl (Takara Shuzo, Japan). This purified DNA was purified using Euka Ryotic TACl on Ing Kit (manufactured by InVitrogen, The Netherlands) according to the attached protocol and the pCR3.1 vector. Incorporated into one. The DNA-incorporated pCR3.1 was transfected into E. coli INV aF's Competent Cells (manufactured by 1nVi10rogen, The Netherlands), and ampicillin (US Sigma) (50 g / ml) was plated on a plate of Broth (Takara Shuzo Co., Ltd.) semi-solid medium and left at 37 ° C. for about 12 hours. The colonies that appeared were randomly selected, and the selected colonies were inoculated in 2 ml of L-Br0th liquid medium containing the same concentration of ampicillin and left for about 18 hours. After culturing with shaking in C, the cells were collected. The recovered cells were separated from plasmid using Wizard Dominiprep (Promega, USA) according to the attached instructions. By cutting with the restriction enzyme HindI1I, one having the fragment inserted in the correct direction was selected, and the nucleotide sequence of the DNA was determined.

The determination of the DNA base sequence was performed in the same manner as in Example 1 using a fluorescent sequencer manufactured by Applied Biosystems, USA. The sequence primer is Euka ryotic TA C l on inng K it (O The primers attached to the Netherlands (1 nviogen) and the F1 and R1 primers described in Example 2 were used. When the nucleotide sequences of the seven clones were determined, the consensus sequence was identical to the sequence of the cDNA shown in SEQ ID NO: 1, and the expression plasmid having the consensus sequence was subjected to PET. It was named 240H. In addition, out of 7 clones, 3 clones had the base A at position 678 of the DNA sequence of SEQ ID NO: 1 changed to G. Since this change was frequent, it was determined to be an alias mutation.

The strain E. coli in which this plasmid PET 240H was introduced into E. coli INV aF ': Deposited with the National Institute of Advanced Industrial Science and Technology under the accession number: FE RM BP — 6 2 13

Example 7 Transfection of Expression Vector into Cells and Its Expression The expression vector prepared in Example 6 was obtained from 293 cells (available from Dainippon Pharmaceutical Co., Ltd., Japan, original ATCC No. CRL— The gene was transfected into 1 5 7 3). For cell passage before transfection, 10% horses were collected in MEM Wis Ear One Salts (MEM Earl Liquid Medium, Japan, Dainippon Pharmaceutical Co., Ltd. C. No. 12-102-54CN). Serum (manufactured by ICN Biomedicals Inc., USA, Ca No. 2921149 heat-treated at 56 ° C for 20 minutes and inactivated), 1 volume of Penicill in- Septoniycin solution (Dainippon Pharmaceutical Co., Ltd., Japan; Ca No. 16-70D-49DN) was used. Cells planted in the medium at a concentration of 5 X 1 0 ⁴ cells / m 1 Kakara 5 X 1 0 ⁵ cells Z m 1, and incubated at 3 7 ° C, 5% carbon dioxide, humidity 1 0 0% The cells were cultured until they became confluent. The culture medium was discarded and discarded, and the cells were removed from the bottom of the plate by treating with EDTA trypsin solution (Cosmo Bio Co., Ltd., Japan). The reaction was stopped by adding the above MEM R liquid medium (including serum). Thereafter, the cells are uniformly suspended by pipetting, centrifuged (1000 rpm, 15 minutes) (KS-8300, Kubota Manufacturing Co., Ltd., Japan; RS300 / Collected, resuspended in fresh medium and subcultured. The gene transfer was carried out by the calcium phosphate coprecipitation method using a kit (Ca-No. IV2780-1) from 1 nV Itrogen in the Netherlands (protocol 6 Page) 7 times transmembrane of the present invention Transformants were prepared which harbor DNAs encoding the type receptor protein ET240. For the DNA, 5 μg of a plate having a diameter of 35 mm was used.

Next, the membrane fraction was prepared as follows. Cells transfected with PET240 and cells transfected with pcDNA3 plasmid as a control were cultured for 24 to 48 hours. After that, the medium was aspirated with aspirator and discarded. The plate was added to PBS, and the plate was peeled off using Cell Scrape r-L (manufactured by Sumitomo Beilite Co., Ltd., Japan, Ca No. MS-93300). Thereafter, 1 ml of PBS was added to each of the three plates, and the plate was washed and added to the collected cells. This cell suspension is crushed with PQ tr0n (PT10-SK, manufactured by KI Corporation, Switzerland), and a microcentrifuge for microtubes (MRX-150, Tomi Issei, Japan) ) And centrifuged at 1300 Xg for 15 minutes at 4 ° C. The supernatant was discarded, the cells were suspended twice by adding PBS twice, and centrifuged under the same conditions. The protein was quantified using a protein assay kit from Bio-Rad Laboratories, USA, and PBS was added so that the protein content would be 1 mg Zml. This suspension was used as a membrane fraction preparation. Expression of ET240, a seven-transmembrane receptor protein, by Western blotting using the membrane fraction preparation thus obtained It was confirmed. Membrane fractions were analyzed using ADS Japan SDS-PAGE electrophoresis tank and SDS-PAGE polyacrylamide gel (gradient gel 5 to 15%) according to the attached instruction manual. SDS-PAGE was performed. The sample was treated with 2-menolecaptoethanol (2-ME) and reduced by heating in a boiling water bath for 5 minutes. As a marker, use Rainbow Marker 1 (for high molecular weight) manufactured by Amersham, UK, and use the attached instruction manual for each sample buffer and electrophoresis buffer. It was produced according to. After completion of SDS-PAGE, Acrynorea midgel was transferred to a PVDF membrane separator (Bio-Rad Laboratories, USA) using a mini trans blot cell.

The filter prepared in this manner was treated with TBS-T [20 mM Tris · HC1, 1337 mM NaC1 (pH 7.0) containing Block Ace (Dainippon Pharmaceutical, Japan). 6), in 0.1% Tween 20] at 4 ° C with shaking. Using the anti-ET240 antiserum described in Example 11 as the primary antibody according to the instructions attached to the ECL Western blotting detection system (Amersham, UK), A peroxidase-labeled anti-mouse Ig donkey antibody (manufactured by Amersham, UK) was reacted as an antibody.

The reaction time of each antibody is 1 hour at room temperature. Repeated. After the last wash, the finolators are immersed in the reaction solution of ECL Western Blotting Detection System (Amersham, UK) for 5 minutes, wrapped in polyvinylidene wrap, and X-ray finol Exposed to light.

As a result of comparison with the molecular weight marker, a band of about 38 to 42 kD was obtained only for the pET240H transfected gene, but not observed for the pcDNA3 transfected cells. . Example 8 Screening of ligands

The membrane fraction of the 293 cells into which the gene had not been introduced and the ET 240 transformant 293 cells was prepared in the same manner as in Example 7. 50 μl of membrane fraction preparation solution or PBS [Ca-No. 28-103-05, manufactured by Dainippon Pharmaceutical Co., Ltd., Japan], radiolabeled ligand candidate compound CGS 2 16 50 (manufactured by Dupont NEN, USA, Cat No. NET-1021) 50 μI (final concentration Ι Ο η η Μ) and 50 μl of PBS were added to make the total volume 150 μl. After mixing and bringing the candidate compound into contact with the transmembrane receptor protein 7 Τ 240 seven times, keeping the mixture at 37 ° C for 30 minutes, centrifuge for microtubes (Tomi Issei, Japan) Centrifugation at 1500 g for 15 minutes at room temperature at room temperature (MRX-150 type) to separate those not bound to the candidate compound bound to the ET240 protein. The supernatant was added to 1 and 10 ml of a liquid scintillator capsule (EC0M0FL-J0R-2, manufactured by Dupont NEN, USA) and mixed. Then B The radioactivity was counted using an ec kman LS6000LL scintillation counter (manufactured by Bee kman, USA), and the radioactivity obtained with and without the introduction of the ET240 gene was compared.

As a result, there was no difference in the obtained radioactivity with or without the introduction of the ET240 gene. Example 9 Screening of ligands

The expression vector prepared in Example 6 was transfected into CHO cells [original ATC No. CCL-61] [available from Dainippon Pharmaceutical Co., Ltd., Japan].

No. 11765-047, F-12 Nutrient Mixture (HAM medium, GIBCO BRL Cat.No. 11765-047) was used for the passage of the cells before the gene transfer. No. 10099-141, manufactured by GIBCO BRL, USA; heat-treated at 56 ° C. for 20 minutes to render it inactive), 100 parts of Penicilli n-S A trept omy cin solution [Ca. Nippon Dainippon Pharma Co., Ltd. NQ. 16-70D-49DN] was added. Cells are seeded in medium at a concentration of ⁵ × 10 ⁵ cells / m 1, ⁵ × 10 ⁴ cells m 1, and cultured at 37 ° C., 5% carbon dioxide, 100% humidity. And cultured until confluent. The medium was aspirated, discarded, and treated with EDTA trypsin solution (Cosmo Bio Co., Ltd., Japan) to remove cells from the bottom of the plate. The reaction was stopped by adding the above medium (including serum). The cells are then evenly distributed by pipetting. The suspension was suspended and centrifuged (1000 rpm for 15 minutes; KS-8300, Kubota Seisakusho, Japan, RS3000 Z6). The cells were resuspended in a new medium and subcultured.

Gene transfer was performed by electroporation using GenePu 1 ser from Bio-Rad Laboratories, USA. C Η Ο cells are treated with trypsin as described above, peeled off from the bottom of the plate, and electroporation buffer (272 mM Suerose, 1 mM MgCl _2, washed with 7 mM-phosphate buffer), 5 X 1 0 ⁶ were suspended in the cell / 5 0 0 cormorants by becomes mu 1 same elect Roporeshiyo down buffer, the GenePu l se r Cuvet te Dispensed. As a gene to be introduced, the plasmid DNA pET240H (5 μg) prepared in Example 6 was added to CuveUe. The resulting mixture was ice-cooled for 5 minutes, placed in a GenePu1 ser cell chamber, and pulsed twice under the conditions of 3F and 550V. After cooling again on ice for 5 minutes, add 10 ml of the medium kept at 37 ° C to Cuvet te, and then transfer the mixture in Cuvet te to a 10 cm diameter cell culture dish. Was cultured. After about 24 hours, the medium was replaced with a new one and cultured for about 24 hours. The cells were replaced with a medium containing neomycin (Genetic iii) (181B-023, GIBCO BRL, USA) at various cell concentrations at a concentration of 400 μg / ml. Thereafter, the cells grown and cultured for about two weeks were designated as human ET240 protein-expressing cells. Human seven-time transmembrane receptor protein E prepared as described above Using T240-expressing cells, the measurement of chemical migration was performed. LPS (lipopolysaccharide) -administered rat serum prepared as follows was used as a candidate ligand. A 7-week-old Wistar rat was purchased from Japan Biological Materials Co., Ltd. in Japan. LPS (Sigma, USA) derived from Salmonella Minnesota RE595 was suspended in physiological saline in the Japanese Pharmacopoeia to a final concentration of lmg / m1. The suspension was sonicated with a sonicator (Branson, Japan) to obtain a clear liquid. This was diluted 10-fold with physiological saline in the Japanese Pharmacopoeia, and 400 μl was administered via the tail vein. Rats approximately 22 hours after administration were laparotomized after ether anesthesia and blood was collected from the heart. Centrifuge in a microtube centrifuge (MRX-150, manufactured by Tomi Issei Co., Ltd., Japan) at 4 ° C for 15 minutes at 300 xg and centrifuge the supernatant. Stored at 0 ° C. This was used as the test substance solution.

9 6-hole microplate chamber [Funacosi, Japan

Cat. No. FE—2 292—96] manufactured by KK Co., Ltd. with a 96-hole microplate [Funakoshi KK, Japan, Cat No. FE-2300-02] ] And 15 µg / m 1 of fibronectin (manufactured by Sigma, USA; dissolved in PBS (Dainihon Pharmaceutical Co., Ltd., Japan, Cat No. 28-103-05)). A treated 8 μm pore size frame finoletor [Cat. FE-2340-08, manufactured by Funakoshi Co., Ltd., Japan] was installed. 0.15% B S A in lower chamber

RPMI 1640 (Sigma, U.S.A.) The test substance solution was diluted 10-fold with Ca BRL Co., Ltd., Ca No. 22400-071) and added. To the upper chamber, ET240 expressing cells, a seven-transmembrane receptor protein suspended in RPMI 164 containing 0.15% BSA, are added, and the test substance is brought into contact with the ET240 protein. Was. The 96-well microplate chamber in this state was kept at 37 ° C for 5 hours at 5% carbon dioxide. The filter was fixed and stained and observed under a microscope. As a result, chemi-migrating cells were observed. Example 10 Screening of a substance antagonistic to ligand

Using the E.sub.T240 transformed C.sub.HO cells, the transformant was subjected to a chemical migration test. (I) The seven-transmembrane receptor protein ET240 ′ expressed in the ET240 transformed CHO cells prepared in Example 9 and the LPS-treated rat serum shown in Example 9 were religated. As in Example 9, chemi-migrating cells were observed in the same manner as in Example 9. In addition, () N-ethylcarboxymide adenosine (manufactured by Sigma, USA) having a final concentration of 10 μm was added to the solutions contained in the upper and lower chambers in the experiment of Example 9. As in Example 9, chemi-migrating cells were observed. After that, the results of (i) and (ii) were compared, but there was no difference between them. Example 1 Preparation of Antibody Recognizing 1E T240 Protein

The expression vector prepared in Example 6 was replaced with BALB / c3T3 The gene was transfected into cells [available from Dainippon Pharmaceutical Co., Ltd., Japan, original ATCC No. CCL-163].

Cells were passaged before gene transfer using Dulbecco's modified MEM medium (D-MEM; GIBC0-BRL, USA) at a final concentration of 10% FBS (Fetal Bovine Serum) (US, GIBCO BRL, Ca No. 10099-141; heat-treated at 56 ° C for 20 minutes and inactivated), a 1/100 amount of Penicci 11 in-St reptomycin solution [Japan Country, Dainippon Pharmaceutical Co., Ltd., Ca No. 16-70D-49 DN] was prepared. Cells planted in the medium at a concentration of 5 X 1 0 ⁴ cells / m 1 Power et 5 X 1 0 ⁵ cells Z m 1, incubated at 3 7 ° C, 5% diacid carbon, humidity 1 0 0% And cultured until confluent. The medium was aspirated and discarded, and treated with EDTA trypsin solution (Cosmo Bio Co., Ltd., Japan) to remove cells from the bottom of the plate. The reaction was stopped by adding the above medium (including serum). Thereafter, the cells are uniformly suspended by pipetting, and centrifuged (l OOO rpm 15 minutes) (KS-830, Kubota, Japan; RS300 / 6 rotor). Use) The cells were collected. The cells were resuspended in a new medium and subcultured.

Gene transfer was performed by electroporation using GenePulser manufactured by Bio-Rad Laboratories, USA. BALB / c3T3 cells are treated with trypsin as described above, the bottom of the plate is peeled off, and a buffer for electroboration (2 7 2 m M Sucrose, 1 m MM g C 1 2, 7 m M Li washed with phosphate buffer), 5 X 1 0 ⁶ cells / 5 0 0 cormorants by becomes 1 the same d Lek collected by filtration Pollet one to The suspension was suspended in a buffer solution for use, and dispensed into Gene Puserservet. As a gene to be introduced, 5 g of the DNA of the ET240 expression vector prepared in Example 6 was added to Cuvette. The resulting mixture was ice-cooled for 5 minutes, placed in a Gene Pulser cell chamber, and pulsed twice under the conditions of 3 F and 550 V. After ice-cooling again for 5 minutes, add 10 ml of the medium kept at 37 ° C to Cuvet te, and then transfer the mixture in Cuvet te to a 10-cm-diameter cell culture dish, and transfer the cells. Cultured. After about 24 hours, the medium was replaced with a new one, and the cells were further cultured for about 24 hours. The further cultured cells were replaced with a medium containing neomycin (Gene cin) (GIBCO BRL, 1811-023) at a concentration of 400 μg / m 1 at various cell concentrations. . Thereafter, the cells grown and cultured for about two weeks were designated as human ET240 protein-expressing cells.

The cells were immunized into a BALBZc mouse to prepare a human-derived ET240 protein antibody of the present invention. BALB / c mice (Chiya one pulse Li server one by Li purchased, main scan, 7 weeks old) 1 X 1 0 ⁷ cells, prepared cormorants good in the intraperitoneally administered, every two weeks The administration was repeated 5 times. The last immunization administered the cells intravenously. Seven days later, whole blood was collected, and the serum obtained therefrom was used as anti-human ET240 protein antiserum. Industrial applicability

Use of the novel seven-transmembrane receptor protein derived from human of the present invention and a DNA encoding the protein are useful for treating or preventing diseases associated with leukocyte functions, such as autoimmune diseases. It is possible to screen substances and to create diagnostic methods and diagnostic agents for such diseases. Furthermore, the novel seven-transmembrane receptor protein fragment derived from the mouse of the present invention and the DNA encoding the same encode the novel full-length mouse transmembrane receptor protein and encode the same. Useful for identification and isolation of DNA.

Claims

The scope of the claims

3. An isolated DNA encoding the seven-transmembrane receptor protein of claim 1.

4. The isolated DNA of claim 3, which has the nucleotide sequence of SEQ ID NO: 1.

5. DNA or a derivative thereof comprising at least 12 bases in the base sequence according to claim 3 or 4.

6. A DNA consisting of at least 12 bases in a DNA complementary to the base sequence according to claim 3 or 4, or a derivative thereof.

7. An RNA complementary to the nucleotide sequence according to claim 3 or 4 RNA consisting of at least 12 bases or its derivative.

8. A replicable recombinant DNA comprising the DNA according to any one of claims 3 to 7 incorporated into a replicable expression vector.

9. A microorganism or cell transformed with the replicable recombinant DNA of claim 8.

10. (a) A replicable set comprising the DNA according to claim 3 or 4 linked to a replicable expression vector and incorporating the DNA and the replicable expression vector. Get the recombinant DNA,

(d) culturing the transformant to cause the transformant to express the DNA;

A seven-transmembrane receptor protein produced on the cell membrane surface of the transformant by a method including the above.

11. The method according to claim 1 or 10, wherein the method for screening for a ligand that binds to a 1.7-transmembrane receptor protein is used. White matter or the peptide according to claim 2 is brought into contact with the sample material, and transmembrane seven times using the change occurring in response to the binding of the protein or the peptide to the ligand as an index. A method comprising detecting a ligand capable of binding to a type receptor protein.

12. The protein according to claim 1 or 10, wherein said method is a method for screening a substance that inhibits binding of a 2.7 transmembrane receptor protein to a ligand for said protein. The peptide of 2 and the ligand are brought into contact with a sample material, and the change occurring in response to the binding of the protein or the peptide to the ligand is used as an index, A method comprising detecting a substance that inhibits binding between a seven-transmembrane receptor protein and a ligand.

13. An antibody that binds to the seven-transmembrane receptor protein according to claim 1.

14. A substantially pure mouse-derived seven-transmembrane receptor protein fragment having the amino acid sequence of SEQ ID NO: 4.

15. An isolated DNA encoding the seven-transmembrane receptor protein fragment according to claim 14.

16. The isolated DNA of claim 15, which has the nucleotide sequence of SEQ ID NO: 3.