WO2003089643A1 - Novel proteins and dnas encoding the same - Google Patents

Abstract

The base sequences of cDNA clones involved in a catalogued full-length cDNA library are analyzed. Concerning cDNA clones having novel sequences from among those analyzed above, the physiological activities (functions) of proteins encoded thereby are specified. Based on these physiological activities (functions), a method of utilizing the proteins and DNAs encoding the same is proposed. Namely, the following proteins (a) and (b), DNAs encoding the same and a method of utilizing the same are provided: (a) a protein comprising an amino acid sequence represented by any of SEQ ID NOS:17 to 32; and (b) a protein comprising an amino acid sequence derived from an amino sequence represented by any of SEQ ID NOS: 17 to 32 by deletion, substitution and/or addition of one to several amino acids and having a G protein-coupled receptor activity.

Description

 Description Novel proteins and their technical fields

 The present invention has introduced a novel protein, a DNA encoding the protein, a full-length cDNA encoding the protein, a recombinant vector having the DNA, an oligonucleotide comprising a partial sequence of the DNA, and the DNA. The present invention relates to a transgenic cell, an antibody specifically binding to the protein, and the like. Background art

 Obtaining cDNA and analyzing its base sequence are essential for analyzing the physiological activity of a protein expressed in a living body and developing a method of using the protein based on the activity. In addition, creating a library that catalogs full-length cDNAs for all genotypes is one of the important tasks of the Human Genome Project. The cataloged library means that there is no duplication in the cDNAs contained in the library, and is a library containing one kind of each cDNA. The full-length cDNA cloning method is described in JP-A-9-1248187 and JP-A-10-127291. In this method, a tag molecule is bound to a diol structure present at the 5 ′ cap site of the mRNA. The mRNA bound to the tag molecule is type II, and RNA-DNA complex is formed by reverse transcription using oligo dT as a primer. A method comprising preparing a body, and separating a complex having a DNA corresponding to the full-length mRNA from the complex using the function of a tag molecule.

Further, as an efficient reverse transfer method, a method for performing the method at a high temperature such that the type III does not form a higher-order structure has been developed (Japanese Patent Application Laid-Open No. H10-84961). Furthermore, regarding the DNA fragments contained in the synthesized full-length cDNA library, Regardless, a cloning vector that can be cloned uniformly has been developed (Japanese Patent Application Laid-Open No. 11-9273).

 The full-length cDNA library produced by such a technique does not include all the elements that are different evenly as individual elements of the library. Some clones do not exist. Since a clone present in only a trace amount is likely to be novel, a subtraction method and a normalization method for enriching such clones have also been developed (Japanese Patent Application Laid-Open No. 2000-325080; Carninci, P. et al., Genomics, 37, 327-336 (1996)).

 When the nucleotide sequence of each clone of the cataloged full-length cDNA library thus obtained is analyzed by a known method, the nucleotide sequence is identified, but the physiological activity of the protein encoded by the cDNA is It remains unknown. Disclosure of the invention

 The present invention analyzes the nucleotide sequence of a cDNA clone contained in a cataloged full-length cDNA library, and among those having a novel sequence, identifies the physiological activity of the protein encoded by the nucleotide sequence. The purpose of the present invention is to propose a method of using a protein based on activity and a DNA encoding the protein.

 The present inventors analyzed the nucleotide sequence of a cDNA clone in a mouse full-length cDNA library and searched a database based on the homology of the sequence, and found that a protein having a specific function was found in the sequence. Was found. The expression levels of these cDNAs in each tissue were analyzed. The present invention has been achieved based on these findings.

 That is, according to the present invention, the following inventions (1) to (15) are provided.

 (1) The following protein (a) or (b):

 (&) A protein comprising the amino acid sequence of any one of SEQ ID NOs: 17 to 32.

(b) in the amino acid sequence of any one of SEQ ID NOs: 17 to 32, A protein comprising an amino acid sequence in which several amino acids have been deleted, substituted and / or added, and having G protein-coupled receptor activity.

 (2) DNA encoding the protein of (1) above.

 (3) A full-length cDNA encoding the protein described in (1) above.

 (4) Any one of the following DNAs (a), (b) or (c):

 (a) DNA having the nucleotide sequence of any one of SEQ ID NOs: 1 to 16.

 (b) in the nucleotide sequence of any one of SEQ ID NOs: 1 to 16, the nucleotide sequence has one or several nucleotides deleted, substituted and Z or added, and has G protein-coupled receptor activity. DNA encoding a protein having

 (c) a G protein-coupled receptor having a nucleotide sequence capable of hybridizing under stringent conditions to DNA having the nucleotide sequence of any of SEQ ID NOs: 1 to 16 or a sequence complementary thereto, and DNA encoding an active protein.

 (5) A recombinant vector containing the DNA according to any one of (2) to (4).

(6) A transgenic cell into which the DNA according to any of (2) to (4) or the recombinant vector according to (5) is introduced, or an individual comprising the cell.

 (7) The protein according to (1), which is produced by the cell according to (6).

 (8) A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA according to any of (2) to (4) above, and having a sequence complementary to the sense oligonucleotide. An oligonucleotide selected from the group consisting of an antisense oligonucleotide and an oligonucleotide derivative of the sense or antisense oligonucleotide.

 (9) An antibody or a partial fragment thereof that specifically binds to the protein of (1) or (7).

 (10) The antibody according to the above (9), wherein the antibody is a monoclonal antibody.

(1 1) The monoclonal / antibody is a protein according to (1) or (7) above. The antibody according to the above (10), which has an action of neutralizing the G protein-coupled receptor activity of the above.

 (12) contacting the protein according to (1) or (7) with a test substance, and measuring a change in the activity of the protein caused by the test substance; Screening method for modulators.

 (13) contacting the gene-transfected cell according to (6) with a test substance, and detecting a change in the expression level of the DNA introduced into the cell; Screening method.

 (14) At least one or more amino acid sequence information selected from the amino acid sequence of the protein described in (1) above, and Z or the DNA base sequence described in any of (2) to (4) above. A computer-readable recording medium storing at least one or more selected base sequence information.

 (15) A carrier to which the protein according to (1) and / or the DNA according to any of (2) to (4) are bound. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in more detail.

 (1) Acquisition of full-length cDNA and analysis of nucleotide sequence

 The DNA of the present invention may be a protein or an amino acid sequence comprising the amino acid sequence of SEQ ID NOs: 17 to 32, or one or several (the number is not particularly limited; And preferably 15 or less, more preferably 10 or less, and even more preferably 5 or less) amino acid residues containing substitutions, deletions, insertions, additions, or inversions, and G Any protein can be used as long as it can encode a protein having protein-coupled receptor activity. Specifically, it may be only the translation region encoding the amino acid sequence, or may include the full length of its cDNA.

Specifically, the DNA containing the full-length cDNA includes, for example, SEQ ID NOs: 1 to 1 And DNA comprising the nucleotide sequence according to any one of the above (6). Also, as the translation area,

A sequence represented by base numbers 107 to 1858 of SEQ ID NO: 1,

A sequence represented by base numbers 507 to 1874 of SEQ ID NO: 2,

A sequence represented by base numbers 9 to 1169 of SEQ ID NO: 3,

A sequence represented by base numbers 474 to 1841 of SEQ ID NO: 4,

A sequence represented by base numbers 281 to 1267 of SEQ ID NO: 5,

A sequence represented by base numbers 173 to 1456 of SEQ ID NO: 6,

A sequence represented by base numbers 53 to 1354 of SEQ ID NO: 7,

A sequence represented by base numbers 64 to 1161 of SEQ ID NO: 8,

A sequence represented by base numbers 88 to 1185 of SEQ ID NO: 9,

A sequence represented by base numbers 160 to 1236 of SEQ ID NO: 10,

A sequence represented by base numbers 92 to 1207 of SEQ ID NO: 11,

A sequence represented by base numbers 661 to 2196 of SEQ ID NO: 12,

A sequence represented by base numbers 108 to 1184 of SEQ ID NO: 13,

A sequence represented by base numbers 218 to 970 of SEQ ID NO: 14,

A sequence represented by base numbers 1557 to 2489 of SEQ ID NO: 15,

A sequence represented by base numbers 311-1210 of SEQ ID NO: 16,

Can be mentioned.

 Furthermore, even if not the full-length of the above-mentioned cDNA, those containing the above-mentioned translation region and a part adjacent to the 3, and Z or 5 'end thereof, which contains the minimum necessary for expression of the translation region, etc., are also included in the DNA of the present invention. included.

The DNA of the present invention may be obtained by any method as long as it can be obtained. Specifically, it can be obtained by the following method, for example. First, niRNA is prepared from a suitable animal, preferably a mammalian tissue or the like, by a method known per se and generally used. Next, using this mRNA as type I cDNA Although synthesize, 5 to synthesize c DNA of the full-length this time, a molecule consisting the tag-specific diol structures in the cap ^(7Me G _PPP N) site is chemically bonded, the m RNA as铸型After reverse transcription using oligod T as a primer, it is possible to use a method of separating only full-length cDNA using the function of a tag molecule (JP-A-9-1248187; JP-A-10_127291). preferable. In addition, in the case of reverse transcription, in order to prevent the type III from forming a higher-order structure and reducing the efficiency of reverse transcription, the temperature is increased by using a thermostable reverse transcriptase in the presence of trehalose or the like. It is preferable to use a method of performing reverse transcription below (JP-A-10-84961). Here, high temperature means 40 to 80 ° C.

 The cDNA thus obtained is inserted into an appropriate cloning vector for cloning. The vector used here has a recombinase recognition sequence at both ends of the cloning site that can uniformly clone DNA of various chain lengths, and is a linear vector that can be inserted into the host by a method other than infection. The vector (JP-A-119-1273) is preferably used. In the thus obtained cDNA library, not all clones exist uniformly (hereinafter, this may be referred to as “cataloged”). A clone that exists only in a plant has a high probability of being new. Therefore, a subtraction method for concentrating such clones and a normalization method (Japanese Unexamined Patent Publication No. 2000-325080; Carninci, P. et al., Genomics, 37, 327-336 (1996)) have been proposed. Preferably, it is used.

 The nucleotide sequence of the cataloged cDNA library is analyzed by a commonly used method known per se. In the case of the DNA of the present invention, in the case of full-length cDNA, the base sequence obtained from the sequence based on the terminal 100 is obtained by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/; National Center). of Biotechnology Information) and searched databases such as NCBI Genbank, EMBL, DDB J, and PDB.

0% or less and shows the highest homology with respect to the entire length of the translated region of the DNA Sequences with a homology of 40% or less were newly submitted to the following analysis. Examples of a DNA having such a full-length cDNA base sequence and its translation region include those described above.

 The novel nucleotide sequence obtained in this way is subjected to homology search (homology search) by BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990)). ) And HMME R (sequence analysis method using hidden Markov model; Eddy, SR, Bioinformatics 14, 755-763 (1998)). : // pfam. Wustl. Edu), etc., to estimate the function of the protein encoded by the nucleotide sequence.

In the homology search by BLAST, the function of the clone to be analyzed can be estimated from various annotation information associated with the hit sequence in which the homology obtained as a result of the search is sufficiently significant. Here, a sufficiently significant hit sequence means that the identity between the catalytic domain portion of the registered sequence and the corresponding portion of the DNA of the present invention is less than 10 ^-4 as e-value, or 30 % Or more.

 For example, if many of the catalytic domain sequences hit at the top are confirmed to function as G protein-coupled receptors, clones to be analyzed that are sequence-similar to those will also have the same function. The expectation holds.

 In HMMPFAM, analysis is performed by a method for identifying whether or not a base sequence to be analyzed has the characteristics of the base sequence of an entry in a database in which a protein profile called Pfam is accumulated. Profiles are extracted from a series of proteins with the same characteristics, and cannot be clarified by comparing one-to-one sequences over their entire length. And function prediction.

A specific example of the prediction of the function of a protein thus performed will be described below. The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 is a BLAST search. Has a 99% homology with G protein coupled receptor affecting testicular descent (Great) and e-value: 0.0, 579 amino acid residues.

 In addition, a protein feature search using the HMM PFAM for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 showed that the low-density lipoprotein receptor domain class A had a special feature (P fam: “ldl-recept — A sequence that shows the characteristics of Leucine Rich Repeat over 10 amino acids in the amino acid sequence encoded by nucleotides 467-1186 (entry as “LRR” in Pfa) Base sequence).

 From these facts, it can be inferred that the protein encoded by the nucleotide sequence of SEQ ID NO: 1 has G protein-coupled receptor activity including Leucine Rich Repeat, and testis development based on the function of homologous proteins. It is inferred that it has the function of maintaining the function.

More PROBABLE G PROTEIN-COUPLED RECEPTOR GPR72 PRECURSOR amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 2 in BLAST searches (GLUCOCORTICOID - INDUCED RECEPTOR) and e- value is ^{5 X 1 0- 136, 2 4} 0 Amino acid residue has a 1 0 0% homology with groups, also the orphan G- protein coupled receptor GPR72, e- va 1 ue: in 1 X 1 0- ^84, with 4 5% degree of coincidence over the 301 amino acid residues a further glucocorticoid- induced receptor, e- value: in 3 X 1 0 one ^84, has a 4 4% homology over 3 0 1 § amino acid residues.

 In addition, according to the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2, a sequence showing the characteristics of the G protein-coupled receptor according to the protein feature search by HMM PFAM (the nucleotide sequence "7tm_lj From these results, it is presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 2 is a G protein-coupled receptor that may be induced by dalcocorticoid. Highly homologous proteins are upregulated when antipsychotics are stopped.

The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3 was More PROBABLE G- PROTEIN - COUPLED RECEPTOR GPR72 PRECURSOR (GLUCOCORTICOID- INDUCED RECEPTOR and by ¾ 100% homology with 5 X 10- ⁹⁶ of e- value, 1 73 Amino acid residues, Si was orphan G - protein coupled receptor and GPR72, e- va 1 ue: with 2 X 10- ^84, it has a 45% homology over 301 amino acid residues, even more, glucocorticoid - and induced receptor, e- va 1 ue: 4X 10 -84 And has 4'4% homology over 301 amino acid residues.

 A protein characteristic search using HMMP FAM was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 3. A sequence showing the characteristics of the G protein-coupled receptor (base sequence that is entered as 7tm_lJ in P f am) It can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 3 has a G-protein-coupled receptor activity that can be induced by dalcocotide. Higher proteins are upregulated when antipsychotics are stopped.

Sequence amino acid sequence encoded by the nucleotide sequence set forth in ID NO: 4 B LAS T More search PROBABLE G PROTEIN-COUPLED RECEPTOR GPR72 PRECURSOR (GLUCOCORTICOID- INDUCED RECEPTOR) and e- value is 5 X 10- ^136, 240 Amino acid residues in a 10 0% homology, also the orphan G- protein coupled receptor GPR72, e - va 1 ue: at 1 X 10 one ^84, 301 Amino acid residue! : 45% degree of coincidence is, further glucocorticoid - and induced receptor, e- value: with 3 X 10- ^84, has a 44% homology over 301 § amino acid residues.

In addition, according to the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4, a sequence showing the characteristics of a G protein-coupled receptor according to HMM PFAM protein search (“7tm-1” was entered in P f am Base sequence). From these facts, it is presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 4 has a G protein-coupled receptor activity that may be induced by dalcocorticoid. In addition, the expression of highly homologous proteins increases when antipsychotic drugs are stopped. SEQ ID NO: G protein-coupled receptor (GPR82) More Amino acid sequence BLAST search base sequence is co one de described in 5 gene and e- va 1 ue force 5X 10- ^159,

A sequence that is similar to 82% at 328 amino acid residues and shows characteristics of a G protein-coupled receptor according to a protein feature search by HMMP FAM (base sequence entered as “7tm_l” in P f am) ) Is found.

 From these facts, it is presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 5 has G protein-coupled receptor activity. In addition, proteins with high homology are frequently expressed in the colon.

According the Amino acid sequence nucleotide sequence is co one de set forth in SEQ ID NO: 6 in B LAS T Search TGR21 G- protein coupled receptor (GPCR) and e-value power 5 X 10 one ^176,

A protein characteristic search by HMMP FAM, which is similar to 442 amino acid residues at 73%, shows a characteristic of G protein-coupled receptor (base sequence entered as “7tm 1” in P f am) ) Is found.

 From these facts, it can be inferred that the protein encoded by the nucleotide sequence of SEQ ID NO: 6 has G protein-coupled receptor activity.

 According to the BLAST search, the amino acid sequence encoded by the nucleotide sequence described in SEQ ID NO: と is 6% with G protein-coupled receptor (GPR103): 0.0, 432. With homology. According to a protein feature search using HMMP FAM, a sequence (a base sequence that is entered as “7tm-1” in P f am) is found that shows the characteristics of a G protein-coupled receptor.

 From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 7 has G protein-coupled receptor activity. In addition, proteins with high homology have high homology between the neuropeptide FF2, neuropeptide Y2, and galanin GalRl receptor and the B transmembrane region. .

If the Amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 8 good in B LAST search G protein- coupled receptor (GPR103) and e- value force 5 X 10 ^-157, similar 73% 377 Amino acid residues And HMM PF AM for protein characterization When the cable was searched, a sequence (a base sequence entered as “7tm1” in P f am) showing characteristics of the G protein-coupled receptor was found.

 From these results, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 8 is a G protein-coupled receptor. Highly homologous proteins have high homology between the neuropeptide FF2, neuropeptide Y2, and the galanin GalRl receptor and the transmembrane domain.

Amino acid sequences are G protein-coupled receptor (GPR103) and e- value force 5X 10 one ¹⁶⁰ according to the B LAS T search to nucleotide sequence encoding a SEQ ID NO: 9,

It shows 73% homology at 386 amino acid residues. According to a protein feature search using HMMPFAM, a sequence (a base sequence that is entered as “7tm-1” in P f am) showing characteristics of a G protein-coupled receptor is found.

 From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 9 is a G protein-coupled receptor. Highly homologous proteins have high homology between the neuropeptide FF2, the neuropeptide Y2, and the galanin GalRl receptor and the transmembrane domain.

According the Amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 10 in the BLAST search G protein - coupled receptor and (GPR103) e- value: 5X 10- 172,

A protein characteristic search using HMMPFAM with 73% homology at 409 amino acid residues showed a characteristic of G protein-coupled receptor (base sequence entered as “7tm-1” in P f am) Is found.

 From these facts, it can be assumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 10 is a G protein-coupled receptor. Highly homologous proteins have high homology between the neuropeptide FF2, neuropeptide Y2, and the galanin GalRl receptor and the transmembrane domain.

According the Amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 1 to BLAST search, a novel human g protein coupled receptor and e- va 1 ue: 5 X 1 0 ^179, 348 87% with an amino acid residue Shows homology. Also shown in SEQ ID NO: 11 When the amino acid sequence encoded by the nucleotide sequence was searched for protein characteristics using HMMPFAM, a sequence (a nucleotide sequence entered as “7tm-1” in P f am) showing characteristics of a G protein-coupled receptor was found.

 From these facts, it can be assumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 11 is a G protein-coupled receptor.

 According to the BLAST search, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 12 is gpcrxll as a functional receptor validated by angiopeptin and useful for screening of agonists and antagonists and e-value: 0.0, 507 amino acids remaining Characteristic search by HMMPFAM with a homology of 72% in the amino acid group revealed a sequence (base sequence that is entered as “7tm-1J” in P f am) indicating the characteristics of a G protein-coupled receptor .

 From these facts, it can be assumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 12 is a G protein-coupled receptor. In addition, highly homologous proteins are activated by the somatostatin analog angiopeptin, which suppresses atherosclerosis.

According to the BLAST search, the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 13 and G protein-coupled receptor C5L2 GPR77 are e-value: 5 X 10 1 ¹¹⁴ , 61% homology at 331 amino acid residues, 61% homology In addition, it shows 44% homology with Oryctolagus cuniculus anaphylatoxin C5a receptor gene with e-value: 5 X 10 ^_60 and 329 amino acid residues. In addition, HMMPFAM was used to perform a protein feature search on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 13, and the sequence showing the characteristics of the G protein-coupled receptor (“7tm-1” in P f am) Base sequence to be entered).

 From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 13 is a G protein-coupled receptor. Highly homologous proteins are involved in C5L2 expressed in granulocytes and undifferentiated dendritic cells and chemotaxis.

According to the BLAST search, the nucleotide sequence of SEQ ID NO: 14 is Bos taurus latrophilin 3 splice variant abbf and e- value: 3 X 10 shows one ^51, 302 § amino acids 37% homology at residues indicates the character of the G protein-coupled receptor proteins, wherein at the retrieval was performed by HMMPFAM The sequence (base sequence entered as “7tm 2” in P f am) is found.

 From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 14 is a G protein-coupled receptor. A highly homologous protein is brain-specific Latrophilin, a splicing variant of the exogenous toxin alpha-latrotoxin receptor Latrophilin.

SEQ ID NO: 1 5 nucleotide sequence according to the serpentine receptor (Cyt28) according to BLAST search e- value: 5 X 10 one ^56, 271 38% ho th same or live was not in Amino acid residue, also , G protein-coupled receptor 56 and ^{e_v alue: 8 X 10- 51,} 281 36% of homology with Amino acid residue, further G-protein - is coupled receptor (GPR56), e- va 1 ue: 1 X At ^10-50, it shows 36% homology over 281 amino acid residues. When the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 15 was searched for protein characteristics using HMMPFAM, a sequence exhibiting the characteristics of a G protein-coupled receptor (“7tm-2” in P f _am) Entry, and Latrophilin / CL-1-likeGPSdomain (base sequence entered as “GPS” in P fam).

 From these facts, it can be assumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 15 is a G protein-coupled receptor. CDNA encoding a similar protein was cloned from mouse hematopoietic stem cell mRNA.

According the nucleotide sequence of SEQ ID NO: 16 in the B LAS T Sa '- over switch, G- protein coupled receptor GPR34 (GPR34 ) and ^{e- value: 1 X 10- 19,} 294 Amino acid residues in the 27% homology, further G protein-coupled receptor Gpr34 (Gpr34 ) gene and, e- value: in 5X 10- ^18, has a 38% homology over 294 Amino acid residues. A program tmHMM (S. Moller, MDR) that predicts a transmembrane helix for the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 16 Croning, R. Apweier 丄 er. Evaluation of methods for the prediction of membrane spanning regions.Bioinformatics, 17 (7): 646-653, 2001). With the amino acid number of SEQ ID NO: 32, transmembrane sites were predicted at 30 to 52, 65 to 87, 107 to 129, 141 to 163, 197 to 219, 239 to 261 and 276 to 298).

 From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 16 is a G protein-coupled receptor. The gene encoding a similar protein is on the X chromosome, is abundantly expressed in various organs, and has weak homology to the PAF receptor.

 The DNA of the present invention may be obtained in a state in which a base sequence has been deleted or inserted in the translated sequence. As a result of the above-described homology search and protein feature search, the DNA If a deletion or insertion in the nucleotide sequence is suspected, use a method generally used by those skilled in the art, such as library screening, PCR cloning, etc., to delete the full length without deletion or insertion of bases. cDNA can be obtained. The protein of the present invention is expressed using the full-length cDNA thus obtained, and can be used for functional analysis.

The DNA of the present invention thus obtained, whose nucleotide sequence is determined, and whose function is presumed, has the nucleotide sequence shown in SEQ ID NOS: 1 to 16 or the nucleotide sequence shown above as its translation region. In addition, in these base sequences, one or several (the number is not particularly limited, but is, for example, 60 or less, preferably 30 or less, more preferably 20 or less, and still more preferably Means 10 or less, particularly preferably 5 or less). A protein having a base sequence in which bases are deleted, substituted and / or added, and having G protein-coupled receptor activity. And a DNA that hybridizes with these under stringent conditions and encodes a protein having G protein-coupled receptor activity. As described above, in these DNAs, one or several amino acid sequences in the amino acid sequences described in SEQ ID NOs: 17 to 32 are deleted, substituted and / or substituted. Includes those encoding a protein consisting of an added amino acid sequence and having G protein-coupled receptor activity.

 Here, DNA that hybridizes under stringent conditions refers to a base sequence represented by SEQ ID NOS: 1 to 16 and a BLAST analysis of 80% or more, preferably 90% or more, and more preferably 95% or more. DNAs containing a base sequence having homology are exemplified. Hybridization under stringent conditions refers to a reaction in a normal hybridization buffer at a temperature of 40 to 70 ° C, preferably 60 to 65 ° C, and the like. Washing can be performed according to a method of washing in a washing solution having a salt concentration of 15 mM to 30 O mM, preferably 15 mM to 6 O mM.

 Further, the DNA of the present invention may be obtained by the above-described method or may be synthesized. The DNA base sequence can be easily replaced with a sales kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene). it can.

 The nucleotide sequence of SEQ ID NOs: 1 to 16 is derived from a mouse.1S A human cDNA library was prepared according to the above-described method for preparing a cDNA library. By performing hybridization using a DNA fragment having the nucleotide sequence of SEQ ID NOS: 1 to 16 as a probe, a human homolog protein of the protein encoded by the nucleotide sequence of SEQ ID NOs: 1 to 16 (Hereinafter, this may be referred to as "human homolog DNA"). Such a DNA homologous to DNA that hybridizes under stringent conditions to DNA having the nucleotide sequence of SEQ ID NOS: 1 to 16 or a sequence complementary thereto according to the present invention is also included.

 Further, it is also possible to predict the base sequence S of the human homolog DNA by using informatics, and obtain the human homolog DNA from the above human cDNA library or the like based on the nucleotide sequence. .

Generally, homologues of the target protein using informatics Methods for predicting the nucleotide sequence encoding a protein include, for example, (i) a cDNA database of a human or the like (a cDNA database predicted by informatics) using a query as a base sequence of a target cDNA; And (ii) homology search using BLAST etc. against EST databases such as humans using the base sequence of the target cDNA as a query. Then, a method is used in which the sequence of the hit EST is linked with reference to the base sequence of the target cDNA, and (iii) the base sequence of the target cDNA is used as a query against the genome database of humans or the like. A homology search is performed using LAST, etc., to identify the location on the genome where the cDNA gene of interest is located, and Genscan (http: // genes, mit.edu/ GENSC A. html), Sim4 (Genome Res., 8: 976-74 (1998)), and the like, and a method of predicting the nucleotide sequence of the gene portion in the genome.

 When predicting the nucleotide sequence of human homolog DNA of mouse-derived cDNA, any of the above methods can be used, but any of the cDNAs having the nucleotide sequences of SEQ ID NOs: 1 to 16 of the present invention is novel. Since it is considered that the method (i) cannot obtain the nucleotide sequence of the human homologous DNA, the method described in (ii) or (iii) is preferably used.

 Based on the predicted nucleotide sequence of the human homolog DNA, a DNA encoding the human homolog protein of the protein encoded by the nucleotide sequence of SEQ ID NOS: 1 to 16 was obtained from the above human cDNA library. You can also. As a specific acquisition method, for example, the above-mentioned human cDNA library is prepared by using a primer having a nucleotide sequence complementary to the nucleotide sequence at the 5, 5, and 3 'end of the predicted human homolog DNA. Examples include a method of performing PCR as a template, and a method of performing hybridization on the human cDNA library using a partial sequence of the predicted human homolog DNA as a probe.

In general, a similar gene having a nucleotide sequence having a high homology to the nucleotide sequence of the target gene is called a “homolog”, and the above-mentioned method also requires the acquisition of a human homolog. Although it is intended, it is important in gene function analysis not only to confirm that the nucleotide sequences are similar, but also to confirm that the gene obtained as a homolog is a family member of the target gene. Genes acquired as “homologs” between two species are identical genes that evolved from a common ancestral gene

It may be an "ortholog" or a "paralog" which is a different gene created by duplication from a common ancestral gene.

 In other words, in order for the human-derived DNA obtained as a homologue described above to have the same function as the protein of the present invention, the function of the protein encoded by the human-derived DNA must be In order to estimate and verify the function of the protein of the present invention in mice, it is preferable to confirm that the human homolog is an ortholog of a closely related species of the mouse gene of the present invention.

 For example, the following method is used as a method for confirming the ortholog.

(1) First, the homology between the nucleotide sequence of the cDNA of the present invention and the nucleotide sequence of the obtained human homolog DNA is analyzed. Next, using the nucleotide sequence of the cDNA of the present invention as a query, homology search was performed on international nucleotide sequence databases such as DDB J, EMBL, Gen Bank, and human nucleotide sequences contained in patent databases. Confirm that the degree of matching between the obtained human homolog DNA and the base sequence of the query is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query. Further, (2) homology is analyzed for the nucleotide sequence of the obtained human homolog DNA and the corresponding nucleotide sequence of the cDNA of the present invention. Next, using the obtained nucleotide sequence of the human homolog DNA as a query, homology search was performed on the international nucleotide sequence database such as DDB J, EMBL, GenBank, and the mouse nucleotide sequence contained in the patent database. Confirm that the degree of matching between the cDNA of the invention and the base sequence of the query is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query. By confirming the above (1) and (2), the obtained human homolog can be identified as a human ortholog corresponding to the cDNA of the present invention. The homology analysis described in (1) and (2) above is based on the amino acid sequence ratio. Comparison may be used, and a molecular evolutionary phylogenetic tree may be drawn and examined. In addition, it is preferable that the degree of matching by the homology analysis described in (1) and (2) above be analyzed as the degree of matching over the entire length of the query.

 Estimating and confirming the function of the protein encoded by the base sequence of the human homolog DNA or ortholog DNA thus obtained by performing homology search by BLAST or protein characteristic search by HMMPFAM, etc. Can be.

 The DNA that hybridizes under stringent conditions with the DNA having the nucleotide sequence of SEQ ID NOS: 1 to 16 or the complementary sequence thereof includes DNA encoding such a human homolog or ortholog protein. It is.

(2) Novel protein encoded by cDNA

 The translation region of the protein encoded by the DNA of the present invention is, for example, the nucleotide sequence of the DNA, which is converted into amino acids by three reading frames, and the range encoding the longest polypeptide is translated by the present invention. The amino acid sequence of the region can be determined. Such amino acid sequences include, for example, those described in SEQ ID NOs: 17 to 32. Further, the protein of the present invention is not limited to the above-described amino acid sequence, but comprises an amino acid sequence in which one or several amino acids have been substituted, deleted, and / or added in the amino acid sequence. And those having G protein-coupled receptor activity.

 As a method for obtaining the protein of the present invention, the method of transcription / translation of the DNA of the present invention described in the above (1) by an appropriate method is preferably used. Specifically, a recombinant vector inserted into a suitable expression vector or a suitable vector together with a suitable promoter is prepared, and this recombinant vector is used to transform a suitable host microorganism, or to a suitable cultured cell. The expression can be obtained by introducing the DNA into a plasmid, and it can be obtained by purifying the protein.

When the protein thus obtained is obtained in a free form, a known method or Can be converted into a salt by a method analogous thereto, and conversely, when obtained as a salt, it can be converted into a free form or another salt. Such salts of the protein of the present invention are also included in the protein of the present invention. In addition, the protein produced by the above transformant may be modified before or after purification with an appropriate protein modifying enzyme to optionally modify the protein or partially remove the polypeptide to modify the protein. Quality. These modified proteins are also included in the scope of the present invention as long as they have G protein-coupled receptor activity.

 When producing the protein of the present invention, the vector used for the production of the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant. And phage vectors. Of these, usually, a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for a host into which the DNA is introduced has already been inserted is used. Specific examples of such a protein expression vector include pET3 and pET11 (manufactured by Stratagene) and GEX (manufactured by Amersham Armacia Biotech) when the host is Escherichia coli. In the case of (1), p ESP-I expression vector (manufactured by Stratagene) and the like can be mentioned. In the case of insect cells, Bac PAK6 (manufactured by Clontech) can be used. When the host is an animal cell, examples include ZAP Express (Stratagene), SVK3 (Amersham Pharmacia Biotech) and the like.

When a vector without an expression control region is used, it is necessary to insert at least a promoter as the expression control region. The promoter used herein is not limited to a promoter that can be used by a host microorganism or a promoter possessed by a cultured cell. Specifically, for example, when the host is Escherichia coli, T3, T7, tac , 1 ac promoter and the like, and in the case of yeast, nmt1 promoter, Ga11 promoter and the like can be used. When the host is an animal cell, SV40 promoter, CMV promoter and the like are preferably used. When a host capable of functioning as a mammalian promoter is used, a promoter specific to the gene of the present invention can also be used. Insertion of the DNA of the present invention into these vectors may be performed by linking the DNA or a DNA fragment containing the DNA to the amino acid sequence of the protein encoded by the gene DNA downstream of the open motor in the vector.

 The recombinant vector thus prepared can be transformed into a host described below by a method known per se to prepare a DNA-introduced body. As a method for introducing the vector into a host, specifically, a heat shock method (J. Mol. Biol., 53, 154, (1970)) and a calcium phosphate method (Science, 221, 551, (1983)) ), DEAE dextran method (Science, 215, 166, (1982)), in vitro packaging method (Proc. Natl. Acad. Sci. USA, 72, 581, (1975)), virus vector method (Cell, 37, 1053, (1984)), and the electric pulse method (Chu. Et al., Nuc. Acids Res., 15, 1331 (1987)).

 The host for preparing the DNA transfectant is not particularly limited as long as the DNA of the present invention is expressed in the body. Examples of the host include Escherichia coli, yeast, baculovirus (arthropod polyhedrovirus), insect cells, Alternatively, animal cells and the like can be mentioned. Specifically, BL21 and XL-2B1ue (manufactured by Stratagene) for E. coli, SP—Q01 (manufactured by Stratagene) for yeast, and AcNPV for baculovirus.

(J. Biol. Chem., 263, 7406, (1988)) and its host, Sf-9 (J. Biol. Cem., 263, 7406, (1988)). Examples of animal cells include mouse fibroblast C 127 (J. Viol., 26, 291, (1978)) and Chinese hamster ovary cell CHO cells

(Proc. Natl. Acad. Sci. USA, 77, 4216, (1980)), and the like. Preferably, expression level か ら cos-7 derived from African green monkey kidney is preferred because of simple screening.

(ATCC CRL1651: American type Karyuchi collection collection preserved cells) Used.

In addition to the expression method using a protein expression vector as described above, a homologous recombination technique for directly inserting a promoter-ligated DNA fragment of the present invention into the chromosome of a host microorganism (AA Vertes et al., Biosci. Biotechnol. Biochem., 57, 2036, (1993)), Alternatively, a DNA transfectant can be prepared using a transposon, an insertion sequence (AA Vertes et al., Molecular Microbiol., 11, 739, (1994)) and the like. In the obtained culture, cells or cells are collected by a method such as centrifugation, suspended in a suitable buffer, and then collected by a suitable method known per se such as ultrasonication, lysozyme, and / or freeze-thawing. After disruption by the method, a crude protein solution is obtained by centrifugation, filtration, or the like, and further purified by a combination of appropriate purification methods. Thus, the protein of the present invention is obtained. In addition to the above-described expression method using a protein expression recombinant vector, protein expression is induced by subjecting the DNA of the present invention obtained in the above (1) to a cell-free transcription / translation system, and the protein of the present invention Can be obtained. The cell-free transcription / translation system used in the present invention is a system containing all elements necessary for transcription from DNA to mRNA and translation of mRNA to protein, and by adding DNA thereto. It refers to any system by which the protein encoded by the DNA is synthesized. Specific examples of the cell-free transcription / translation system include a transcription / translation system prepared based on an eukaryotic cell, a bacterial cell, or an extract from a part thereof, and a particularly preferred example is Egret A transcription / translation system prepared based on extracts from reticulocytes, wheat germ, and Escherichia coli (Escherichia coli S30 extract) may be mentioned.

 Separation and purification of the protein of the present invention from the obtained transcription-translation product of the cell-free transcription / translation system can be carried out by a commonly used method known per se. Specifically, for example, a DNA region encoding an epitope peptide, a polyhistidine peptide, a glutathione-S-transferase (GST), a maltose binding protein, and the like is introduced into the DNA to be transcribed and translated, and And purified using the affinity of the protein with a substance having affinity.

The expression of the target protein is separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (manufactured by Sigma), or an antibody that specifically binds to the protein of the present invention described below. Detection method, etc. Can be confirmed. It is generally known that the expressed protein is cleaved (processed) by a proteolytic enzyme present in the living body. The protein of the present invention is naturally included in the protein of the present invention, even if it is a fragment of the amino acid sequence that has been cleaved, as long as it has G protein-coupled receptor activity.

 By analyzing the interaction of other proteins and nucleic acids, etc., the proteins obtained by the effort can be used to find out the multifaceted functions in vivo. As a method for analyzing the interaction, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, liposome method One example is the multiple display method.

 (3) Preparation of oligonucleotide

 Using the DNA of the present invention or a fragment thereof obtained by the method described in (1) above, antisense oligonucleotides having a partial sequence of the DNA of the present invention, Oligonucleotides such as oligonucleotides can be prepared.

 Examples of the oligonucleotide include a DNA having the same sequence as the continuous 5 to 100 bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA. As a specific example, DNA having the same sequence as 5 to 100 consecutive nucleotides in the base sequence represented by any of SEQ ID NOS: 1 to 16 or DNA having a sequence complementary to the DNA Can be mentioned. When used as a sense primer or antisense primer, the above-mentioned oligonucleotides whose melting temperature (Tm) and number of bases do not extremely change are preferable. The length of the sequence is generally 5 to: L00 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.

In addition, derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention. Examples of the oligonucleotide derivative include an oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond. Nucleotide derivative, an oligonucleotide derivative in which the phosphodiester bond in the oligonucleotide is converted to an N3,1 P5'phosphoamidate bond, and a report and a phosphodiester bond in the oligonucleotide that are converted to a peptide nucleic acid bond Oligonucleotide derivatives, Oligonucleotide derivatives in which peracyl in oligonucleotides are substituted with C-5 propynylperacyl, Oligonucleotide derivatives in which peracyl in oligonucleotides are substituted with C-15 thiazole peracyl, Oligonucleotides Oligonucleotide derivatives in which cytosine in nucleotides has been substituted with C-15-propynylcytosine; cytosine in oligonucleotides has been substituted with phenoxazine-modified cytosine; oligonucleotide derivatives, oligonucleotides Oligonucleotide derivatives in which the report in the oligonucleotide is substituted with 2'-O-propylribose, or oligonucleotide derivatives in which the ribose in the oligonucleotide is substituted with 2, -methoxyethoxyribose, etc. .

 In addition, the oligonucleotide of the present invention is prepared as a double-stranded RNA, introduced into a transfectant, and inhibits the expression of a target gene by an RNA interference method (hereinafter referred to as “RNAi method”). ) May be used. As the RNA interference method, for example, the method described in (Elbashir, S., et al., Nature, 411, 494-498 (2001)) can be used. Also, not all of the above-mentioned double-stranded RNAs need to be RNAs. For example, those described in WO 02/13734 can be used.

Here, the target gene may be any as long as it is the DNA of the present invention. A double-stranded RNA consisting of a sequence substantially identical to at least a part of the base sequence of these DNAs (hereinafter sometimes referred to as “double-stranded polynucleotide”) is referred to as a base sequence of a target gene. It is composed of a sequence substantially identical to a sequence of 15 bp or more, which may be any part of the sequence. Here, “substantially identical” means that the sequence has 80% or more homology with the sequence of the target gene. The length of nucleotides ranges from 15 bp to the full length of the open reading frame (ORF) of the target gene. Any length may be used, but a length of about 15 to 50 Obp is preferably used. However, it is known that mammalian-derived cells have a signal transduction system that activates in response to long double-stranded RNA of 3 O bp or more. This is called an interfering reaction (Mareus, PI, et al., Interferon, 5, 115-180 (1983)), and when the double-stranded RNA enters the cell, PKR (dsRNA-responsive) protein kinase: The translation initiation of many genes is non-specifically inhibited via Bass, BL, Nature, 411, 428-429 (2001)), and at the same time, 2, 5, oligoadenylate synthetase

(Bass, B 丄, Nature, 411, 428-429 (2001)), activation of RNaseL occurs, and nonspecific degradation of intracellular; RNA is caused. These non-specific reactions mask the specific response of the target gene. Therefore, when a mammal, or a cell or tissue derived from the animal is used as a transfectant, a double-stranded polynucleotide of 15 to 30 bp, preferably 19 to 22 bp, more preferably 21 bp It is preferable to use The double-stranded polynucleotide does not need to be entirely double-stranded, and includes those with a partially protruding 5 'or 3' end, but preferably those having two or three bases protruding.

 The double-stranded polynucleotide means a double-stranded polynucleotide having complementarity, but may be a self-annealed single-stranded polynucleotide having self-complementarity. Examples of the single-stranded polynucleotide having self-complementarity include those having an inverted repeat sequence.

The method for preparing the double-stranded polynucleotide is not particularly limited, but it is preferable to use a chemical synthesis method known per se. In chemical synthesis, a single-stranded polynucleotide having complementarity can be separately synthesized, and can be converted into a double-stranded strand by associating them by an appropriate method. Specific examples of the method of association include a method in which the synthesized single-stranded polynucleotide is mixed, heated to a temperature at which the double-strand is dissociated, and then gradually cooled. The associated double-stranded polynucleotide is confirmed using an agarose gel or the like, and the remaining single-stranded polynucleotide is removed by, for example, decomposing with a suitable enzyme. The transfectant into which the double-stranded polynucleotide prepared in this way is introduced may be any as long as the target gene can be transcribed into RA or translated into protein in the cell. Specific examples include those belonging to plant, animal, protozoan, virus, bacterial, or fungal species. The plant can be a monocotyledonous, dicotyledonous or gymnosperm, and the animal can be a vertebrate or invertebrate. Preferred microorganisms are those used in agriculture or industry, and are pathogenic to plants or animals. Fungi include organisms in both power and yeast forms. Examples of vertebrates include mammals, including fish, sea lions, goats, pigs, sheep, hamsters, mice, rats, and humans, and invertebrates include nematodes and other insects. Includes creatures, Drosophila, and other insects. Preferably, the cells are vertebrate cells.

 The transductant means a cell, tissue, or individual. Here, the cells may be from germ line or somatic, totipotent or pluripotent, divided or undivided, parenchymal or epithelial, immortalized or transformed, and the like. The cells may be gametes or embryos, in the case of embryos, single cell embryos or constitutive cells, or cells from multicellular embryos, including fetal tissue. Further, it may be an undifferentiated cell such as a stem cell, or a differentiated cell such as a cell of an organ or tissue including fetal tissue, or any other cell present in an organism. Differentiating cell types include fat cells, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, neurons, glial, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, and eosinophils Basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, liver cells and cells of the endocrine or exocrine glands.

As a method for introducing the double-stranded polynucleotide into the recipient, when the recipient is a cell or tissue, calcium phosphate method, electroporation method, lipofection method, virus infection, two For example, immersion in a strand polynucleotide solution or a transformation method is used. Examples of the method for introduction into an embryo include microinjection, electoral poration, and virus infection. Can be When the recipient is a plant, a method of injecting or perfusing the plant into the body cavity or stromal cells, or spraying is used. In the case of an individual animal, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular, and intravenous administration), vaginal, rectal, nasal, ophthalmic, and intraperitoneal administration Method, electoral poration method, virus infection, etc. are used. For methods for oral introduction, the double-stranded polynucleotide can be mixed directly with the food of the organism. Furthermore, when introduced into an individual, it can be administered, for example, by administration as an implanted long-term release preparation or the like, or by ingesting an introduced body into which a double-stranded polynucleotide has been introduced. The amount of the double-stranded polynucleotide to be introduced can be appropriately selected depending on the introduced substance and the target gene, but it is preferable to introduce an amount sufficient to introduce at least one copy per cell. Specifically, for example, when the transfectant is a cultured human cell and the double-stranded polynucleotide is introduced by the calcium phosphate method, 0.1 to 100 OnM is preferred.

 By suppressing the expression of the gene of the present invention in the transfectant by RNA interference, the function of the protein encoded by the gene of the present invention can be confirmed, or a new function can be analyzed.

 (4) an antibody that specifically binds to the protein of the present invention

 As a method for preparing an antibody that specifically binds to the protein of the present invention, a commonly used known method can be used. Even polypeptides used as antigens have high antigenicity according to known methods, A sequence suitable as (antigenic determinant) can be selected and used. As a method of selecting the epitope, for example, commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used.

As the polypeptide used as the above antigen, a synthetic peptide synthesized according to a known method, or the protein itself of the present invention can be used. A polypeptide serving as an antigen may be prepared in an appropriate solution or the like according to a known method, and immunization may be performed on a mammal, for example, a rabbit, a mouse, a rat, or the like. In order to increase the antibody titer, it is preferable to use the antigen peptide as a conjugate with an appropriate carrier protein, or to carry out immunization by adding an adjuvant or the like.

 The route of administration of the antigen upon immunization is not particularly limited, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular route may be used. Specifically, for example, a method of inoculating a BALB / c mouse several times every several days to several weeks with an antigen polypeptide is used. The antigen intake is preferably about 0.3 to 0.5 mg / time when the antigen is a polypeptide, but is appropriately adjusted depending on the type of the polypeptide and the animal species to be immunized.

 After immunization, test blood is collected as appropriate, and an increase in antibody titer is confirmed by enzyme-linked immunosorbent assay (hereinafter sometimes referred to as “ELISA”) or Western blotting. Blood is collected from animals with elevated antibody titers. A polyclonal antibody can be obtained by subjecting this to an appropriate treatment used for antibody preparation. Specifically, for example, a method of obtaining a purified antibody obtained by purifying an antibody component from serum according to a known method, and the like can be mentioned. The antibody component can be purified by a method such as iontophoresis, ion exchange chromatography, and affinity chromatography.

 Alternatively, a monoclonal antibody can be produced by using a hybridoma fused with spleen cells and myeloma cells of the animal according to a known method (Milstein, et al., Nature, 256, 495 (1975)). it can. A monoclonal antibody can be obtained, for example, by the following method.

First, antibody-producing cells are obtained from an animal whose antibody titer has been raised by immunization with the above-mentioned antigen. The antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any of the individuals, but is preferably obtained from spleen, lymph nodes, peripheral blood and the like. The myeloma to be fused with these cells is generally a cell line obtained from a mouse, for example, P3X63-Ag8.6653, an 8-azaguanine-resistant mouse (derived from BALB, etc.) myeloma cell line (ATCC: CRL -1580), P3-NSl / lAg4.1 (RIKEN cell bank: RCB0095) and the like are preferably used. For cell fusion, antibody-producing cells and myeloma cells are mixed at an appropriate ratio, and an appropriate cell fusion medium such as RPMI 1640 is used. And Iscov's Modified Dulbecco's Medium (IMDM), Certain, can be performed by using 50% polyethylene dalicol (PEG) dissolved in Dulbecco's Modified Eagle's Medium (DMEM) or the like. It can also be performed by the electrofusion method (U. Zimmermann. Et al., Naturwissenschaften, 68, 577 (1981)).

Hypridoma was prepared using 5% CO _{2 in a} normal medium (HAT medium) containing an appropriate amount of hypoxanthine 'aminopterin' thymidine (HAT) solution, utilizing the fact that the myeloma cell line used was an 8-azaguanine resistant strain. , At 37 ° C for an appropriate time. This selection method can be appropriately selected and used depending on the myeoma cell line used. The antibody titer of the antibody produced by the selected hybridoma is analyzed by the method described above, the hybridoma producing the antibody with a high antibody titer is separated by a limiting dilution method, etc., and the separated fused cells are cultured in an appropriate medium. A monoclonal antibody can be obtained from the culture supernatant obtained by the purification by an appropriate method such as ammonium sulfate fractionation and affinity chromatography. A commercially available monoclonal antibody purification kit can also be used for purification. Furthermore, ascites containing a large amount of the monoclonal antibody of the present invention can also be obtained by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or a nude mouse. .

 When a human-derived protein is obtained as the protein of the present invention, the above-described polypeptide or a partial peptide thereof is used as an antigen in Severe combined immune deficiency (SCID) mice transplanted with human peripheral blood lymphocytes. Human antibodies can also be prepared by immunization in the same manner as described above and preparing a hybridoma between antibody-producing cells of the immunized animal and human myeloma cells (Mosier, DE, et al. Nature , 335, 256-259 (1988); Duchosal, MA, et al., Nature, 355, 258-262 (1992)).

In addition, RA is extracted from the obtained hybridoma producing the human antibody, the gene encoding the desired human antibody is cloned, this gene is inserted into an appropriate vector, and the gene is introduced into an appropriate host. To express even more First, a humanized antibody can be prepared. Here, an antibody having a low binding property to an antigen can be obtained as an antibody having a higher binding property by using an evolutionary engineering technique known per se. A partial fragment such as a monovalent antibody can be prepared by, for example, cleaving the Fab portion and the Fc portion using papain or the like, and collecting the Fab portion using an affinity column or the like.

 The antibody that specifically binds to the protein of the present invention thus obtained can also be used as a neutralizing antibody that specifically binds to the protein of the present invention and thereby inhibits the activity of the protein. Although the activity of the protein is inhibited, the selection method is not particularly limited. For example, the function of the target protein in the introduced product is inhibited by bringing the antibody into contact with the DNA-introduced product prepared in (2) above. There is a method of analyzing whether or not this is the case.

 Such a neutralizing antibody can be used alone for the clinical application, but can also be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, or syrups for oral administration, or injections, drops, ribosomes, Parenteral administration using suppositories and the like can be mentioned. The dose can be appropriately selected depending on the condition, age, body weight, and the like.

 (5) Confirmation and analysis of the activity of the protein of the present invention

 The protein of the present invention is prepared as a recombinant protein as described in the above (2), and the protein is analyzed by chromatography to confirm that it has the activity estimated in the above (1). . Furthermore, analysis can also be performed by combination with an antibody or the like prepared as described in (4) above.

Whether the protein of the present invention has G protein-coupled receptor activity can be assayed, for example, as follows. Ligand sources for activity evaluation include transmitters, honolemon, cytokines, and G protein-coupled receptors. It contains routine agonists, biologically active peptides, cell culture supernatants, tissue extracts, and compounds not found in nature but that activate G protein-coupled receptors. Using this ligand source, a functional assay or a binding test is performed using a ligand, a reporter gene, cAMP, a microphysiometer, or the like as described below, and a ligand for a G protein-coupled receptor is performed. Can be identified and the activity can be evaluated.

 The following functions of the G protein-coupled receptor can be used as a method of functional access. For example, G protein-coupled receptors are linked to specific intracellular second messengers in response to G proteins coupled to them. Gq-type G protein-coupled receptors are functionally linked to the increase in intracellular calcium concentration through activation of PLC, and Gs-type Gi-type G protein-coupled receptors are It has been shown to increase or decrease cAMP levels through activation or inhibition of rate cyclase.

 As an example of such a functional assay, a calcium assay will be described below. HEK293 cells, CHO cells, cos cells, etc. expressing the recombinant protein of the present invention are loaded with calcium fluorescent dye fura2 or F1uo3, etc. at l-5 mM, and then the above-mentioned ligand source is added to the medium. After culturing, the transient increase in calcium concentration induced by agonists is evaluated using an intracellular calcium concentration measurement device (for example, CAF-100: Nihon Kohden or FD SS 6000: Hamamatsu Photonics, etc.). can do.

 Further, cAMP assays include a reporter gene assay system using a cAMP response element (CRE) or a standard cAMP assay. Specifically, for example, after the above-mentioned ligand source is added and cultured in a medium such as HEK293 cells, CHO cells, and cos cells expressing the recombinant protein of the present invention, stimulation or inhibition of cAMP production is performed. This can be done by evaluating

Due to the above-mentioned atssay, transient increase in calcium concentration or change in cAMP concentration Agonists that are active are tested in mottainfection cells in which only the vector has been introduced into similar cells to determine if the response is specific to the transformed cell expressing the receptor. Can be.

 On the other hand, the activation of a wide range of second messenger systems secretes hydrogen ions out of cells. This change in the extracellular hydrogen ion concentration can be detected by a site sensor, such as a microphysiometer (CYTO SENSOR micropy s i ome t er) (Molec cu l ar de vic e s L t d s, Men l o P ark, C soil). Therefore, the site sensor can detect receptor activation linked to an intracellular signaling pathway that uses energy, such as the G protein-coupled receptor of the present invention.

 The binding of the above-mentioned ligand to the protein of the present invention can be examined by a normal binding experiment using a labeled ligand or a test using surface plasmon resonance.

 The above functional assay system can also be used to evaluate agonists and antagonists of proteins having G protein-coupled receptor activity of the present invention. Note that confirmation of the activity of the protein of the present invention is not limited to these methods. Further, these functional assay systems can also be used for screening for a function activator or a function inhibitor of the protein of the present invention and a screening for a protein expression regulator of the present invention, which will be described later.

 As a method for analyzing the function of the protein of the present invention, generally, for example, (i) a method for comparing and analyzing the expression state in each tissue, disease or developmental stage, and (ii) a method for analyzing the expression of other proteins and DNA (Iii) a method for introducing into appropriate cells or individuals and analyzing changes in phenotype; (iv) a method for inhibiting the expression of the protein in appropriate cells or individuals, and And a method of analyzing the change in Further, according to such a method, the activity specific to the target protein can be analyzed from many aspects.

In the method (i), the expression of the protein of the present invention is determined at the mRNA level or Can be analyzed at the protein level. When the expression level is analyzed at the mRNA level, for example, the in situ hybridization method (In situ hybridization: Application to Developmental Biology & Medicine., nd. by Harris, N. and Wilkinson, DG, Cambridge University) Press (1990)), a hybridization method using a DNA chip, a quantitative PCR method, and the like. In the case of analyzing at the protein level, a tissue staining method using an antibody that specifically binds to the protein of the present invention described later, an ELISA, a Western plotting method, and the like can be mentioned. If a known variant exists in the protein to be analyzed, use a probe that exists only in the cDNA encoding the protein to be analyzed and does not hybridize with the cDNA that encodes the known variant. Is preferred. In the case of the quantitative PCR method, the method is performed by selecting primers that can generate amplified fragments of different lengths between the target cDNA and the known variant (Wong, Y., Neuroscience Let., 320: 141-145 (2002)). And the like. Also, when analyzing at the protein level, it is preferable to use an antibody that reacts only with the target protein and does not react with a known variant.

 In the method (ii), the function of the protein of the present invention can be analyzed by examining the presence or absence of interaction between the protein of the present invention and a known protein. As a method of analyzing interaction, the ability to use a conventional method known per se, specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosome A multiple display method and the like can be mentioned. In this method, if a known variant exists in the protein to be analyzed, the known variant also analyzes the interacting substance in the same manner and identifies a substance that interacts specifically with the target protein. Is preferred.

In the method (iii), the cell into which the cDNA of the present invention is introduced is not particularly limited, but human cultured cells and the like are particularly preferably used. Methods for introducing DNA into cells include those described in (2) above. In addition, the phenotype of the transfected cells includes cell viability, cell growth rate, cell differentiation, neurite outgrowth if the cells are neurons, This includes those that can be observed with a microscope, such as the localization and translocation of intracellular proteins, and those that can be analyzed by biochemical experiments, such as changes in the expression of specific proteins in cells. When a known variant of the target protein exists, these phenotypes can be similarly introduced into cells, and the phenotype associated with the target protein can be identified by comparative analysis. . Further, since it is known that the protein of the present invention has a G protein-coupled receptor activity, it is also possible to analyze the protein by focusing on the phenotype and the like found in diseases associated with the G protein-coupled receptor. preferable.

 In the method (iv), the method can be efficiently performed by a method using an oligonucleotide described later or an RNA interference method. In this method, if a known variant is present in the target protein to be analyzed, the same analysis is performed for the known variant and other variants, and the target protein-specific protein is obtained by comparative analysis. Function can be identified.

(6) Screening for a molecule that regulates the activity of the protein of the present invention Screening for a substance that specifically binds to the protein of the present invention and has an effect of inhibiting, antagonizing or enhancing the function (activity) of the protein of the present invention By doing so, a function modulator of the protein of the present invention (hereinafter, this may be referred to as “modulator”) can be obtained.

 This method of screening for a regulatory substance may be any method as long as it can obtain a substance that specifically binds to the protein of the present invention and has an activity of inhibiting, antagonizing, or enhancing the activity of the protein. . For example, a method is used in which a protein of the present invention is first brought into contact with a test substance, selection is performed using the binding property of the protein as an index, and then a change in the activity of the protein of the present invention is used as an index. be able to.

The test substance may be any substance as long as it interacts with the protein of the present invention and may affect the activity possessed by the protein. , Peptide, protein, non-peptide compound, small molecule compound Products, synthetic compounds, fermentation products, cell extracts, animal tissue extracts, and the like. These substances may be novel substances or known substances. As a method for analyzing the interaction between the test substance and the protein of the present invention, a conventional method known per se can be used. Specifically, for example, the yeast two-hybrid method, the fluorescence depolarization method, the surface The plasmon method, the phage display method, the ribosomal display method, or the competition analysis method with the antibody described in the above (4) can be used. By such a method, the substance found to bind to the protein of the present invention is then analyzed by analyzing how the activity of the protein of the present invention is affected in the presence of the substance. Whether it is used as a modulator or not is identified. Here, for the purpose of screening for a pharmaceutically active ingredient, it is preferable to use the above-described human homologous protein or orthologous protein for the DNA or recombinant protein of the present invention to be used. Further, the substance screened by the above method may be selected as a drug trap by screening in vivo.

As a specific analysis method, for example, when analyzing a substance that regulates G protein-coupled receptor activity, a protein serving as a substrate is introduced into the DNA transfectant described in (2) by the same method. I do. The dephosphorylation of the protein serving as a substrate in the presence / absence of the substance selected for the transductant is analyzed by a commonly used method known per se. Specifically, it can be performed using the method described in (5) above. If the calcium concentration, c AMP concentration, etc., are increased as compared to the absence of the substance, the substance may function as a G protein-coupled receptor activator, or may decrease, or When inhibited, the substance can be identified as possibly functioning as a G protein-coupled receptor inhibitor. Here, for the purpose of screening for a pharmaceutically active ingredient, it is preferable to use the above-mentioned human homologous protein or orthologous protein as the DNA or recombinant protein of the present invention to be used. Furthermore, substances screened by the above method can be used as drug candidates by screening in vivo. You may make a selection.

 The G protein-coupled receptor having the G protein-coupled receptor activity of the protein of the present invention is a superfamily of essential membrane proteins that transmit extracellular signals. GPCRs include receptors for living amines such as dopamine, epinephrine, histamine, glutamic acid (a metabolic effect), acetylcholine (a musculinic effect), and serotonin, as well as prostaglandins and platelet activation. Receptors for factors, lipid mediators of inflammation such as leukotriene, and receptors for calcitonin, C5a anaphylatoxin, follicle-stimulating hormone, gonadotropin-releasing hormone, neurokinin, oxytocin, thrombin, etc. And receptors for sensory signaling media such as retinal photopigments and olfactory stimulants.

 Therefore, compounds that can be identified by this screening method include hypertension, angina, myocardial infarction, atherosclerosis, ulcer, asthma, allergy, psychosis, Alhaima's disease, chorea, schizophrenia, depression, and migraine , Ischemic brain disease, anxiety, vomiting, benign prostatic hypertrophy, Parkinson's disease, acute heart failure, diabetes, diabetic retinopathy, ophthalmic disease, endocrine disease, immune disease, inflammatory disease, respiratory disease, hypotension, urine It can be used as a remedy for closure, infertility, contraception, liver disease, and osteoporosis. Such modulators can be used alone as the above active ingredients when applied to clinical applications, but can also be used as pharmaceutical compositions by blending with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, or syrups for oral administration, or injections, drops, ribosomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, weight, and the like.

 (7) Screening of the DNA expression regulator of the present invention

Screening methods include the use of the protein of the present invention in the presence of a test substance. Or a method of analyzing the expression level of mRNA encoding the same. Specifically, for example, cells expressing the protein of the present invention described in (2) above are cultured in an appropriate medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined by ELISA. Or by analyzing the amount of mRNA encoding the protein of the present invention in the cells by quantitative reverse transcription PCR, Northern blotting, or the like. Can be.

 As the test substance, those described in the above (6) can be used. As a result of this angular dissolution, if the amount of the protein or mRNA expressed in the cells cultured in the absence of the test substance is increased as compared with the amount of the test substance, the substance promotes the expression of the DNA of the present invention. When the substance may function as a substance, and conversely decreases, it can be determined that the substance can be used as the DNA expression inhibitor of the present invention.

 The above-mentioned active ingredient can be used alone for clinical application, but it can also be used as a pharmaceutical composition by blending it with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. The drug can be administered in various forms. Examples of the dosage form include tablet, capsule, granule, powder, syrup, and the like, oral administration, injection, drip, ribosome And parenteral administration with suppositories and the like. In addition, the dose can be appropriately selected depending on symptoms, age, weight, and the like.

 (8) The DNA-introduced animal of the present invention

The transfected DNA containing the DNA of the present invention described in the above (1) is constructed, introduced into a fertilized egg of a mammal other than a human, and transplanted into a female individual uterus to generate the present DNA. A non-human mammal into which the DNA of the present invention has been introduced can be produced. More specifically, for example, a female individual is superovulated by hormone administration, then mated with a male, a fertilized egg is excised from the oviduct on the first day after mating, and the DNA is introduced into the fertilized egg with a micro DNA. It is introduced by a method such as injection. After culturing by an appropriate method, the surviving fertilized eggs are transplanted into the uterus of a pseudopregnant female individual (foster parent) and allowed to give birth. You. Whether or not the desired DNA has been introduced into the newborn can be identified by Southern blot analysis of DNA extracted from cells of the individual. Non-human mammals include, for example, mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, and the like.

 The DNA-introduced animal of the present invention thus obtained is bred with this individual, and its offspring are bred in a normal breeding environment while confirming that the introduced DNA is stably maintained. Obtainable. Also, by repeating in vitro fertilization, the offspring can be obtained and the strain can be maintained.

 The non-human mammal into which the DNA of the present invention has been introduced can be used as an analysis of the function of the DNA of the present invention in a living body, or as a screening system for a substance regulating the function.

 (9) Other uses of the protein of the present invention and the DNA containing the nucleotide sequence encoding the same

 The protein of the present invention can be used as a carrier having it bound on a substrate. In addition, a nucleotide sequence encoding the protein of the present invention, for example, a DNA having the nucleotide sequence of any one of SEQ ID NOs: 1 to 16 and a partial fragment thereof can be used as a carrier obtained by binding them to a substrate. . Hereinafter, these may be referred to as “protein chips”, “DNA chips” or “DNA arrays” (DNA microarrays and DNA macroarrays). These protein chips or DNA chips or arrays may contain other proteins ゃ DNA in addition to the protein and DNA of the present invention.

Here, a resin substrate such as a nylon film or a polypropylene film, a nitrocellulose film, a glass plate, a silicon plate, or the like is used as a substrate for binding proteins and DNA, but the detection of hybridization is not performed by non-RI. Specifically, for example, in the case of using a fluorescent substance or the like, a glass plate or a silicon plate containing no fluorescent substance is preferably used. The binding of the protein or DNA to the substrate can be easily carried out by a commonly used method known per se. These protein chips, DNA chips, or DNA arrays are also included in the scope of the present invention.

 Further, the amino acid sequence of the protein of the present invention and the nucleotide sequence of DNA can also be used as sequence information. Here, the nucleotide sequence of the DNA of the present invention includes the nucleotide sequence of the corresponding RNA. That is, a database of amino acid sequences and nucleotide sequences can be constructed by storing the obtained amino acid sequences and nucleotide sequences in an appropriate recording medium in a computer-readable predetermined format. This database may contain the nucleotide sequences of other types of proteins and the DNA encoding them. Further, in the present invention, the database also means a computer system that writes the above-mentioned sequence on an appropriate recording medium and performs a search according to a predetermined program. Suitable recording media include, for example, magnetic media such as flexible disks, hard disks, and magnetic tapes; optical disks such as CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RW; and semiconductors. Examples include memory. Example

 Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited by these Examples.

Example 1 Preparation of cDNA Library

 (1) Preparation of mRNA

 mRNA-prepared mouse (C57BL / 6) 0.5 to lg of each organ or tissue is homogenized with a 10 ml suspension, and the same amount of fuminol / clonal form as lm 1 of 2M sodium acetate at pH 4.0 (Volume ratio 5: 1) The mixture was added and extracted. When the same amount of isopropanol was added to the aqueous layer after the extraction, RNA separated and precipitated from the aqueous phase.

After incubating the sample on ice for 1 hour, the precipitate was collected in a refrigerated centrifuge at 4,000 rpm for 15 minutes. Wash this sample with 70% ethanol, dissolve in 8 ml of water, and add 2 ml of 5 M Na C 1, 1% C TAB. RNA was precipitated by adding 16 ml of an aqueous solution of pH 7.0 containing (cetyltrimethy-1 ammonium bromide), 4 M urea, and 50 mM Tris to remove polysaccharide (CTAB precipitation).

 The RNA was then centrifuged at room temperature at 4,000 rpm for 15 minutes to dissolve the RNA in 4 ml of 7 M guanidine-C1. After adding twice the volume of ethanol, the mixture was incubated on ice for 1 hour, centrifuged at 4,000 rpm for 15 minutes, and the resulting precipitate was washed with 70% ethanol to recover RNA. And the RNA purity was determined by reading the OD ratios 260Z280 (> 1.8) and 230Z260 «0.45).

 (2) Preparation of first strand cDNA

 Using 5-μg of the mRNA prepared in (1) above, 5-methyl-dCTP, dATP, dTTP, and dGTP were reacted with 3,000 units of reverse transcriptase in a final reaction volume of 165 μl. 0.54 mM, 0.6 M trehalose, 50 mM Tris-HCl (l8.3), 75 mM KC1, 3 mM MgC12, 10 mM DTT, 52 ng / μ1 BSA respectively The reverse transcription reaction was performed under the condition of 5 units of RNase inhibitor. Oligonucleotide containing recognition sequence of restriction enzyme Xho I (SEQ ID NO: 33) (wherein, V indicates A, G, or C, and N indicates A, G, C, or T) 12.6 μ1 was used as a primer.

At the beginning of this reaction, the 1Z4 of the reaction solution was taken and thereto 1. 5 zl in the shed one ³² P] one dGTP (300 0 C i / mm o 1, 1 0 μ C i 1; manufactured Am Ersam Co.) Thus, the efficiency of the synthesis of the first strand cDNA was measured. 0.5 μl of the RI-labeled reaction solution was spotted on DE-81 paper, and the RI activity before and after washing three times with 0.5Μ sodium phosphate buffer (ρΗ7.0) was measured and calculated. did. Then, mix the RI-labeled reaction solution with the unlabeled reaction solution, add 0.5Μ ED ^ 8 ^ 1, 10% SDS 2 zl, and proteinase (Proteinase) K 20 / g, and add the mixture at 45 ° C. Heated for 15 minutes. After extraction with phenol Z-form and ethanol precipitation, the precipitate was treated with RNAse-free water (hereinafter referred to as (Sometimes referred to as "RNase-free water").

 (3) 5 'cap structure and 3.

 Biotinylation of RN Α diol Two steps to bind biotin to the diol site of RN ((present at both the 5 'end with the Cap structure and the 3' end report with the poly A chain) The reaction was performed. These are the oxidation of the diol group followed by the coupling reaction of biotin hydrazide with the oxidized RNA. First, 15 μg of the RNA-first strand cDNA complex obtained by the reverse transcription reaction was purified using 6.6 mM sodium acetate buffer (PH4.5) and sodium periodate as an oxidizing agent. Ο μ

Treated in reaction 1 This oxidation reaction was performed on ice for 45 minutes under light-shielded conditions. Then add 11 μl of 5Μ sodium chloride, 0.5 μl of 10% SDS and 0.5 μl of the same amount of isopropanol, leave on ice for 60 minutes, and then at 4 ° C for 15 minutes.

The precipitate was obtained by centrifugation at 15,000 rpm. The precipitate was washed with 70% ethanol and redissolved in 50 μl of RNase-free water. To the sample, add 5 μl of sodium acetate (ρ 6.1), 5 μl of 10% SDS, 10 μm of biotin hydrazide (Sigma) 150 ^ 1, and add it to room temperature (22 to 26 ° C ) Overnight. Finally, add 5 μl of 5 M NaCl, 1 M sodium acetate (pH 6.1), 751, and 2.5 volumes of ethanol, cool on ice for 1 hour, and centrifuge at 4 ° C for 15 minutes And biotinylated. After the reaction, the reaction solution was centrifuged for 15 minutes to precipitate the RNA-DNA complex again. The precipitate was washed once with 70% ethanol and once with 80% ethanol, and dissolved in RNase free water 701.

 (4) Selection of full-length c DN A by RNa s e I

When the biotinylated RNA-DNA complex obtained in (3) above was treated with RNase I that digests single-stranded RNA, complete cDNA extension was not obtained during the reverse transcription reaction. mRNA and the biotin residue labeled at the 3 'end of the mRNA were removed. Specifically, 10 X RNase I buffer (10 OmM Tris-HC1 (pH 7.5), 5 OmM EDTA, 2 M NaOAc) was added to 70 μl of the sample obtained in (3) above. μ1, RNase I (RNa s eOne ™; manufactured by Promega) was added, and the single-stranded RNA was digested at 37 ° C for 15 minutes.

 (5) Collection of full-length cDNA

 To prevent nonspecific adsorption of cDNA to streptavidin-coated magnetic beads, 100 μg of yeast tRNA (treated with DNase I) was added to 5 mg (500 μl) of magnetic beads (matrix). In addition to gneticporousglass (MPG) particles coated withst reptavidin (CPG, NJ)), the mixture was left on ice for 1 hour, and then washed with a solution of 50 mM EDTA and 2 M NaC1.

The beads were suspended in 500 μl of a solution of 50 mM EDTA and 2 M NaCl, and the RNase I-treated cDNA obtained in (4) was added. By stirring at room temperature for 30 minutes, the magnetic beads and the full-length cDNA were bound. The beads that captured the full-length cDNA were washed four times with a solution of 5 OmM EDTA and 2 M NaC1, 0.4% SDS, once with 50 μg ZiU1 yeast tRNA, and OmM NaC1, 0. 2 mM EDTA, 1 OmMT ris—HC 1 (pH 7.5), once with 20% glycerol, once with 50 μg / μ1 yeast tRNA aqueous solution, RNase H buffer (20 mM Tris_HCl ( p H 7. 5), 10 mM Mg C 1 2, 2 OmM KC 1, 0. 1 mM EDTA, washed once with 0. 1 mM dithiothreitol one Honoré (D TT), RNa se H buffer 100 mu Suspend in 1 and add RNase H 3 unit and heat for 30 minutes at 37 ° C. Then, add 10 μl of 10% SDS and 2 μl of 0.5Μ EDTA, and add 10 μl of 65 ° C for 10 minutes. C and the supernatant was collected.

The single-stranded full-length cDNA recovered in this manner was extracted with phenol / chloroform, and the volume of the solution was reduced to 100 μl or less using a speed bag, and then subjected to G25ZGIOOS ephadex chromatography. Fractions having RI activity were collected in a silicon-treated microtube, 2 μg of dalycogen was added, and the precipitate obtained by ethanol precipitation was dissolved in 30 μl of ultrapure water. (6) Add oligo dG to single-stranded cDNA

30 μl of the single-stranded cDNA recovered in the above (5) was used in a reaction solution having a final volume of 50 μl in a reaction solution of 20 OmM sodium codylate (ρΗ6.9), 1 mM MgCl ₂ , ImM _{C o C l 2, 1 mM} 2- mercaptoethanol, l OO ^ original uM dGTP conditions, terminal de o carboxymethyl nucleotidyl transferase (T a K a R a, Inc.) 3 3 7 ° with 2 Unit C. The mixture was subjected to an oligo dG addition reaction for 30 minutes. At the end of the reaction, EDTA was added to 5 OmM, and it was dissolved in 31 μl of ultrapure water through a series of extraction with phenol / chloroform and ethanol precipitation.

(7) Second strand cDNA synthesis

The synthesis of the second-chain cDNA in which the first-chain cDNA was changed into a 铸 -type was performed as follows. In a reaction system with a final volume of 60 μl, use a second strand low buffer (20 OmM Tris-HC1 (pH 8.75), 10 OmM KC1, 100 mM (NH ₄ ) ₂ SO ₄ , 2 OmM Mg SO ₄ , 1% Triton X—100, lmg / 1 BSA) 3/1, 2nd strand high buffer (20 OmM Tris-HCl (pH9.2) 60 OmM KCl, 2 OmM Mg C 1 ₂ ) 3 1, d CTP, d ATP, d TTP, d GTP each 0.25 mM, β-NADH 6 μ1, first strand cDNA A3 with oligo dG, 1 μ1, Add 600 ng of a two-strand primer-one adapter (SEQ ID NO: 34), add 15 units of ExTaq DNA polymerase (TaKaRaExTaq; manufactured by TaKaa), 15 units, and thermophilic DNA ligase ( Second strand cDNA was synthesized using 150 units of Amp 1 igase (E picentre) and 3 units of heat-resistant R Nase H (Hybridase; E picentre).

 Stop the reaction by adding 1 μl of 0.5 M EDTA, and further dissolve the protein components by 45 ° in the presence of 1 μl of 10% SDS and 10 μg of proteinase K. The mixture was heated at C for 15 minutes, and finally, a double-stranded full-length cDNA was purified by extraction with phenol-noroform form and ethanol precipitation.

(8) Library preparation The full-length double-stranded cDNA obtained by the above method was inserted into a ΙII vector and recovered as a library. The λ ZAP III vector is obtained by modifying SEQ ID NO: 35, which is a partial sequence of the multiple cloning site of λ ZAP II (manufactured by STRATAGENE) vector, to SEQ ID NO: 36, and newly introducing two Sfi I sites. .

 Further, LPS (RIKEN) vector was prepared and cDNA was inserted. PS

(RI KEN) (named λ-FLC-1 (FLC stands for FULL-LENGTH cDNA)) is a modified version of the LPS vector from Mo Bitec (Germany) for cDNA. is there. That is, BamHI and Sa1I, which are convenient for cDNA insertion, were introduced into the cloning sites on both sides of lO kbpstuffer, and cDNA from 0.5 kb to about 13 kb was cloned. A 6 kb DNA fragment inserted into the Xba I site

(Japanese Patent Laid-Open No. 2000-325080). Using this λ-FLC-1, for example, in the case of a lung cDNA library, the average insert length is 2.57 kb, and actually inserts from 0.5 kb to 12 kb are cloned. I was able to do it. In the case of the conventional method; in the case of LZAP, the average length of the insert was 0.97 kb, so the use of L-FLC-1 enables large-sized cDNA to be more efficient than LZAP. It turns out that it can be cloned.

Example 2 Normalization of the full-length cDNA library No subtraction

 (1) Preparation of driver

The mRNA prepared in Example 1 (1) (hereinafter, this may be referred to as “(a) RNA driver”) and the RNA prepared by the in vitro transcription reaction were used as drivers. The latter RNA is further divided into two types (hereinafter referred to as “(b) RNA driver” and “(c) RNA driver”). In the first, cDNA was recovered from RNA-cDNA that had been removed by normalization and cloned into a phage vector. One starting material after infection with E. coli One thousand (2,000) plaques are mixed together to form one library (mini-library), which is converted to plasmid DNA by a conventional method. (The phage is again infected with Escherichia coli together with a helper phage, converted into a phagemid, and further infected again with a plasmid.) Get DNA).

 The obtained DNA was subjected to an in vitro transcription reaction (using T3 RNA polymerase or T7 RNA polymerase), and treated with DNase I (RQ1-R Nasefree; manufactured by Promega) and Proteinase K, followed by phenol / clonal form. After extraction, an RNA (b) RNA driver was obtained. In this case, as a starting material, mini-libraries are created from nine types of tissues (pancreas, liver, lung, kidney, brain, spleen, testes, small intestine, stomach), and mixed with nine types of mini-libraries. To obtain RNA. Another RNA is cultured from a library (about 20,000 clones) that is already stored as a non-overlapping clone, and the resulting DNA is subjected to in vitro transcription reaction in the same manner as the RNA driver (c). RN A driver.

 These three types of RNA were labeled with biotin using the Label_ITB iotin labeling kit (manufactured by Mirus Corporation), and then added to tester cDNA at a ratio of 1: 1: 1. Reaction at t 10

(42) was performed, and the second strand was synthesized from the supernatant collected by streptavidin bead (CPG) treatment.

Example 3 Nucleotide sequencing of full-length cDNA clone

, 1) rear ray of black ¹

One representative clone was selected from each cluster. Representative clones were selected using Q-bot (manufactured by G ENET IXLI MITED) and arrayed on a 384-well plate. At that time, E. coli was cultured in 50 µl of LB medium at 30 ° C for 18 to 24 hours. At this time, if the cDNA library has been introduced into the PS vector and Escherichia coli DH10B has been transformed, add 10 Omg / ml of ampicillin and 5 Omg / ml of kanamycin and introduce it into the Zap vector. Led to SOLR system If so, 10 Omg / ml of ampicillin and 25 m / ml of streptavidin were added.

 (2) Extraction of plasmid and InsSizinng

 Each clone cultured in (1) above is further cultured in 1.3 ml HT solution containing 10 OmgZm1 ampicillin, and after collecting cells by centrifugation, Q

Plasmid DNA was recovered and purified using I-Aperp 96 Turbo (manufactured by QIAGEN). In order to check the chain length of the cDNA inserted in the obtained plasmid, 1Z30 of the plasmid DNA obtained above was

After digestion with II, 1% agarose gel electrophoresis was performed.

 (3) Sequence determination

 Three types of sequencers were used for full-length nucleotide sequence analysis of the full-length cDNA inserted into the plasmid thus obtained. Plasmids were divided into two categories: those with insert sequences shorter than 2.5 kb and those with longer insert sequences. Of these clones, the clone having an insertion sequence shorter than 2.5 kb was analyzed for the nucleotide sequence from both ends. At this time, the plasmid was prepared using the primers described in SEQ ID NO: 37 (sense strand) when the vector was PS, and the primers described in SEQ ID NO: 39 (sense strand) when the vector was Zap. Using the primers described in Sections 40 and 40 (antisense strand) (Hermo Sequenase Primer Cycling Sequencing Kit, manufactured by Amersham Pharmacia Biotech). And analyzed using Licor DNA 4200 (longreadsequencer).

Gaps that could not be analyzed by the above nucleotide sequence analysis were determined by the primer walking method. At that time, AB IPris m377 and Z or AB IPri sm3700 (manufactured by Applied Biosystems Inc.), Big Dy ete rm inatorkit, and Cyclic Sequencing FS ready Reacti on Kit (Ap plied B iosysterns Inc.). In addition, sequencing of clones with inserted cDNAs longer than 2.5 kb was performed by the shotgun method. At that time, Shimadzu RISA384 and DYEnam1cEtterminatorcyclesequencingkit (manufactured by Amersham Pharma Pharmacia Biotech) were used. To generate a shotgun library, 48 DNA fragments grown in PCR from 48 independent representative clones were used. The ends of the amplified DNA fragments were blunt-ended with T4 DNA polymerase.

 This DNA fragment was inserted into a pUC18 vector, and Escherichia coli DH10B was transformed with the recombinant vector. A plasmid was prepared from this E. coli in the same manner as in (2) above.

 For those representative clones, the nucleotide sequence was determined by nucleotide sequence analysis from both ends, and the nucleotide sequences were ligated on a computer, followed by Double Stroke S hearing Device (Fiore Inc. Was performed. Nucleotide sequencing by the shotgun method was performed with duplication of 12 to 15 clones. The gap whose sequence could not be determined by the nucleotide sequence determination was determined by primer walking in the same manner as described above.

Example 4 Analysis of base sequence

 (1) d n a f o rm34147 (SEQ ID NO: 1, 17)

 dn a fo rm34147 is composed of 2386 bases, as shown in SEQ ID NO: 1, of which base numbers 107 to 1858 have an open reading frame.

(Including the stop codon). The amino acid sequence predicted from the open reading frame consists of 583 amino acid residues (SEQ ID NO: 17). When a homology search was performed using the BLAST for the amino acid sequence encoded by SEQ ID NO: 1, the SPTR protein database (SWI SS-PROT protein sequence database and the TrEMBL nucleic acid translation database were integrated). (I) Database registration symbol Mus musc 1 us AF 346501, G roteinco up ledreceptoraffectingt esticula rdescent (Great) force e— value: 0.0 hit with 579 amino acid residue with 99% coincidence.

 In addition, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 was searched for protein characteristics using HMM PFAM. As a result, the amino acid sequence encoded by nucleotide numbers 182—301 in SEQ ID NO: 1 was encoded by amino acid sequence [J ow—density 1 ipoproteinreceptordoma inclass A (J (base sequence sequenced as “1 d1—recept—a” in P f am)) was identified. In 10 amino acid sequences encoded by, a sequence exhibiting the characteristics of Leucine Rich Reeat (base sequence entered as “LR R” in P fam) was found.

 The above-mentioned Great is involved in the descent of the testis from the abdominal cavity to the scrotum based on the literature information in the database (Genesis 2001 May; 30 (1): 26-35), and stops when this gene is abnormal. It has been found to be testicular. From these facts, it is inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 is a G protein-coupled receptor including Leucine Rich Repeat, and also has a function to control testis development and reproductive function. Was presumed to have.

 (2) d n a f o rm37308 (SEQ ID NOs: 2, 18)

 As shown in SEQ ID NO: 2, dnaform37308 is composed of 2558 bases, of which base numbers 507 to 1874 are open reading frames.

(Including the stop codon). The amino acid sequence predicted from the open reading frame consists of 455 amino acid residues (SEQ ID NO: 18). When a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 2, the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database) (I) Database registration code AK018327, proteins im ilarto PRO BABLE G PROTE IN—COUP LED RECEPTOR GPR 72 PRECURSOR (GLUCOCORT I CO I D- I NDUCED RECEPTOR) force e- va 1 ue: 5 in X 1 CT ^136, with 100% degree of coincidence over the 240 amino acid residues, also have (ii) a database registration mark Q 9NYM4, orphan G- proteincouledreceptor R 72 , e- va 1 ue: 1 with X 1 CT ^84, 45% one致度over 301 amino acid residues, yet (iii) a database registration mark AY 029071, R attusnorvegicusglucoc orticoid- inducedreceptor force e- value: 3 X 1 (T 84 The hit was 44% over 301 amino acid residues.

 In addition, the amino acid sequence encoding the nucleotide sequence shown in SEQ ID NO: 2 was subjected to protein characteristic search using HMM PFAM. As a result, it was found that the amino acid sequence encoded by nucleotide numbers 840 to 1613 of SEQ ID NO: 2 had a G protein A sequence (a nucleotide sequence entered as “7 tm-1” in P f am) showing the characteristics of the conjugated receptor was found.

 The above-mentioned GPR72 is found to be an orphan GPCR similar to the Y-receptor based on the literature information in the database (Biochim Biophys Acta 2000 Apr 25; 1491 (1-3): 369-75). 7 The glucocorticoid of intestinal ci ci-in du cedrecepto is based on the literature information in the database (J Neurosci 2001 Nov 15; 21 (22): 9027-35). Therefore, it is shown that the expression is enhanced upon withdrawal after chronic amphetamine administration.Therefore, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 2 may be induced by darcocorticoid. It is a G protein-coupled receptor, and its ligand is peptide, and it was speculated that it is involved in neuroadaptation to stress and psychostimulants.

 (3) d n a f o rm37953 (SEQ ID NO: 3, 19)

As shown in SEQ ID NO: 3, dnafo rm37953 was composed of 3015 bases, of which base numbers 9 to 1169 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 386 amino acid residues (SEQ ID NO: 19). SEQ ID NO: 3 When a homology search was performed using BLAST for the amino acid sequence to be read, the S PTR protein database (a combination of the SWI SS—P ROT protein sequence database and the Tr EMB L nucleic acid translation database) contained ( i) database registration symbol AK018327, PROBABLE G-pROTE I N -COUPL ED RECEPTOR GPR 72 PRECURSOR (GLUCOCOR TI CO I D- I NDUCED RECEPTOR is, e- value: at 5 X l 0-96, i 7 ₃ amino acid residues 10 _0% degree of coincidence over the group, also (ii) data base over scan registration mark Q 9 NYM4, orhan G- proteincoupledrecepto r GPR 72 force e- value: with 2 X 10- ^84, 30 1 amino acid residue in 45 ° / ₀ degree of coincidence over, further (iii) a database registration mark AiT0290 ί '1 attusnorveg ι cusglucocorticoid- in du cedreceptor force ^s, e- value: with 4 X 10 one ^84, over a 301 Amino acid residue 44 Hits with% match.

In addition, a protein characteristic search using the HMM PFAM for the amino acid sequence encoding the nucleotide sequence shown in SEQ ID NO: 3 revealed that the G protein was conjugated to the amino acid sequence encoded by nucleotide numbers 135-908 of SEQ ID NO: 3. A sequence (a nucleotide sequence entered as “7 tm-1” in P i am) showing the characteristics of the type receptor was found. The above-mentioned GPR72 was found to be an orphan GPCR similar to the Y-receptor from literature information in the database (Biochim Biophys Acta 2000 Apr 25; 1491 (1-3): 369-75). 7 The glucocorticoid-induced receptor in §d above is expressed in the brain based on literature information in the database (J Neurosci 2001 Nov 15; 21 (22): 9027-35), and that the ligand is a peptide GPCR. However, it has been shown that the expression is enhanced at the time of drug withdrawal after chronic amphetamine administration. From these facts, the protein encoded by the nucleotide sequence shown in SEQ ID NO: 3 is an orphan G protein-coupled receptor that may be induced by glucocorticoid, the ligand is peptide, and the stress or psychostimulant It was speculated that it might be involved in the neural adaptation to the disease. (4) dn afo rm42851 (SEQ ID NOs: 4, 20)

 dnafor m42851 consists of 3138 bases as shown in SEQ ID NO: 4, of which base numbers 474 to 1841 have an open reading frame.

(Including the stop codon). The amino acid sequence predicted from the open reading frame consists of 455 amino acid residues (SEQ ID NO: 20). When a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 4, the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database) Inside, (i) Database registration code AK018327, ptoteins im ilarto PRO BABLE G—PROTE IN—COUP LED RECEPTOR GPR 72 PRECURSOR (GLUCOCORT I CO I D- I N DUCED RECEPTOR) I e-va 1 ue: 5X in 10 one ^136, also at 240 Amino acid residues 100% degree of coincidence over the group, also (ii) a database registration mark Q 9 NYM4, Homo sapiensorpnan Ur- proteincouledreceptor GPR 72 (GPR 72) power e- value: 2 X l 0 one ^84, 45% degree of coincidence over the 301 amino acid residues, yet (iii) database registration mark AY 029071, Ra ttusnorvegicusg 1 ucocorticoid- inducedreceptor force, e- va 1 ue: in 4 X 1 (gamma ⁸⁴ And a hit of 44% over 301 amino acid residues. For amino acid sequences encoded by the nucleotide sequence shown in 4, sequences showing characteristics of G protein-coupled receptor protein, characterized retrieval by HMM PF AM to the amino acid sequence nucleotide numbers 807- 1 580 of SEQ ID NO: 4 where having conducted encoded

(Base sequence entered as “7 tm_l” in P f am). The above-mentioned GPR72 is found to be an orphan GPCR similar to the Y-receptor based on the literature information in the database (Biochim Biophys Acta 2000 Apr 25; 1491 (1-3): 369-75). In addition, the glucocorticoid-in du cedreceptor described above is based on literature information in the database (J Neurosci 2001 Nov 15; 21 (22): 9027-35), which indicates that it is expressed in the brain, that the ligand is a peptide GPCR, and that expression is enhanced upon discontinuation of the drug after chronic amphetamine administration. The protein encoded by the nucleotide sequence shown in SEQ ID NO: 4 is an orphan G-protein-coupled receptor that may be induced by glucocorticoids, its ligand is peptide, and it has a neuronal response to stress and neurostimulants. It was presumed to be involved in adaptation.

 (5) dn a f o rm27636 (SEQ ID NOS: 5, 21)

As shown in SEQ ID NO: 5, dnafo rm27636 was composed of 2499 bases, of which nucleotides 281 to 1267 were an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 328 amino acid residues (SEQ ID NO: 21). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 5 using BLAST, and a SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEML nucleic acid translation database) Among them, (i) database registration code A F41 11 11 1, Homo saiens G protein-couledreceptor (GPR82) genej, e—va 1 ue: 5 X 10 " ¹⁵⁹ , and 82% over 328 amino acid residues The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 5 was subjected to a protein feature search using HMM PFAM, and the amino acid sequence encoded by nucleotides 374-1210 in SEQ ID NO: 5 was determined. A sequence exhibiting characteristics of a G protein-coupled receptor (a nucleotide sequence entered as “7 tm-1” at P f am) was found. From the literature information (Gene2001 275 (1): 83-91) in the database for the human ortholog of GPR82, it is shown that ESTs are obtained from human colon tissue. From these facts, it was inferred that the protein encoded by the nucleotide sequence of SEQ ID NO: 5 is a novel mouse orphan G protein-coupled receptor and is expressed in the colon.

(6) dnafo rm47734 (SEQ ID NOs: 6, 22) As shown in SEQ ID NO: 6, dnafo rm47734 is composed of 2146 bases, of which base numbers 173 to 1456 are open reading frames.

(Including the stop codon). The amino acid sequence predicted from the open reading frame consists of 427 amino acid residues (SEQ ID NO: 22). When a homology search was performed on the amino acid sequence encoded by SEQ ID NO: 6 using BLAST, the SPTR protein database (SWI SS—PROT protein sequence database integrated with the TrEMBL nucleic acid translation database) ), (I) Database registration code AX 37657 5, human TGR 21 G—protein coupled receptor (GPCR) force e—value: 5 × 10 1 ¹⁷⁶ , and 442 amino acid residues over 73 Hit with% match.

 In addition, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 6 was subjected to protein characteristic search using HMM PFAM, and the amino acid sequence encoded by nucleotide numbers 368-1 207 in SEQ ID NO: 6 was found to have a G protein-coupled receptor. An array indicating the features

(Base sequence entered as “7 tm—1” in P f am). From these facts, it was presumed that the protein encoded by the nucleotide sequence of SEQ ID NO: 6 had a G protein-coupled receptor activity similar to that of human TGR21.

 (7) d n a f o rm29894 (SEQ ID NOs: 7, 23)

As shown in SEQ ID NO: 7, dnafo rm29894 was composed of 1575 bases, of which nucleotides 53 to 1354 had an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 433 amino acid residues (SEQ ID NO: 23). A homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 7, and it was found in the SPTR protein database (integrated SWI SS—PROT protein sequence database and TrEMBL nucleic acid translation database). In addition, (i) database registration BG "^ A F41 11 l7, Homo saiens G protein—coup 1 edreceptor (GPR 103) power S, e—value: 0.0 And a hit of 74% over 432 amino acid residues.

 The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 7 was subjected to a protein feature search using HMM PFAM. As a result, it was found that the amino acid sequence encoded by nucleotide numbers 236-1048 of SEQ ID NO: 7 had a G protein-conjugated A sequence (a nucleotide sequence entered as “7 tm-1” in P f am) showing the characteristics of the receptor was found. The above-mentioned human GPR103 is based on the literature information in the database (Gene 2001 Sep 5; 275 (1): 83-91) based on the information of neuropeptide FF2, neuropeptide Y2 and galanin GalRl receptor and transmembrane region. The agreement is 34-38%, and Northern Hidden Reduction experiments show that mRNA is expressed in the cerebral cortex, pituitary gland, thalamus, hypothalamus, basal forebrain, midbrain, and pons. ing. From these facts, it was conjectured that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 7 was expressed in the brain, and that the ligand was a peptide G protein-coupled receptor and had GPCR activity. .

 (8) dn a f o rm53455 (SEQ ID NOS: 8, 24)

As shown in SEQ ID NO: 8 of 53455, dnafo rm was composed of 2864 bases, of which base numbers 64 to 1161 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 365 amino acid residues (SEQ ID NO: 24). A homology search was performed for the amino acid sequence encoded by SEQ ID NO: 8 using BLAST, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). , (i) No. database Noboru d a F41 1 1 17, Homo sapiens G: rotein- cou 1 edreceptor (GPR 103) power e- value: 5 X 1 0 - 157 the 3 77 Amino acid residues 73% of over Hits were made for the degree of coincidence.

In addition, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 8 was searched for protein characteristics using HMM PFAM. As a result, the amino acid sequence encoded by nucleotide numbers 91 to 903 in SEQ ID NO: 8 Characteristic array (P The nucleotide sequence entered as “7 tm—1” at f am.

 The above human GPR103 is based on the literature information in the database (Gene 2001 Sep 5; 275 (1): 83-91), and indicates the degree of coincidence between neuropeptide FF2, neuropeptide Y2 and galanin GalRl receptors and the transmembrane domain. Is 34-38%, and Northern Hidden Reduction experiments show that mRNA is expressed in the cerebral cortex, pituitary gland, thalamus, hypothalamus, basal forebrain, midbrain, and pons . From these facts, it was conjectured that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 8 was expressed in the brain, and that the ligand was a peptide G protein-coupled receptor and had GPCR activity. .

 (9) d n a f o rm53728 (SEQ ID NOS: 9, 25)

As shown in SEQ ID NO: 9, dnafo rm53728 was composed of 1642 bases, of which base numbers 88 to 1185 were an open reading frame (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 365 amino acid residues (SEQ ID NO: 25). A homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 9, and it was found in the SPTR protein database (integrated SWI S_PROT protein sequence database and TrEMBL nucleic acid translation database). to, (i) a database registration No. ΰ a F41 1 1 1 7, Homo saiens G protein- cou 1 edreceptor (GPR103) power e- value: 5 X 1 0 - over at ¹⁶ °, also 386 Amino acid residues Hits with 73% match.

 The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 9 was searched for protein characteristics using HMMP FAM. As a result, it was found that the amino acid sequence encoded by SEQ ID NO: 9 nucleotide number 115-927 showed the characteristics of G protein-coupled receptor. The sequence shown (base sequence entered as “7 tm-1” in P f am) was found.

The above-mentioned human GPR103 is based on the literature information in the database (Gene 2001 Sep 5; 275 (1): 8391), such as force, et al., Neuropeptide FF2, neuropeptide Y2 and galanin GalRl receptor and 34-38% match, plus Northern Yong experiments show that niRNA is expressed in the cerebral cortex, pituitary gland, thalamus, hypothalamus, basal forebrain, midbrain, and pons. From these facts, it was speculated that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 9 was expressed in the brain, and that the ligand was a peptide-type G protein-coupled receptor and had GPCR activity. .

 (10) d n a f o rm62766 (SEQ ID NO: 10, 26)

As shown in SEQ ID NO: 10, dnafo rm62766 was composed of 1698 bases, of which base numbers 160 to 1236 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 358 amino acid residues (SEQ ID NO: 26). A homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 10, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). (I) Database registration code AF 41 1 117, Homo saiens G protein—cou 1 edreceptor (GPR 103) force S, e—va 1 ue: 5 × 10 ” ¹⁷² and over 409 amino acid residues 73 The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 10 was subjected to a protein feature search using HMMP FAM, and as a result, the amino acid sequence encoded by nucleotide numbers 118 to 930 in SEQ ID NO: 10 was found. A sequence (a nucleotide sequence entered as “7 tm-1” in P f am) was found that exhibits the characteristics of a G protein-coupled receptor in the sequence. The above-mentioned human GPR103 is based on the literature information in the database (Gene 2001 Sep 5; 275 (1): 83-91), which indicates that neuropeptide FF2, neuropeptide Y2 and galanin GalRl receptor and transmembrane region The agreement is 34-38%, and the Northern Hidden Reduction experiments show that mRNA is expressed in the cerebral cortex, pituitary gland, thalamus, hypothalamus, basal forebrain, midbrain, and pons. ing. Therefore, the protein encoded by the nucleotide sequence of SEQ ID NO: 10 is expressed in the brain, and the ligand is a peptide G protein-coupled receptor and has GPCR activity. That was speculated.

 (11) d n a f o rm32567 (SEQ ID NOS: 11, 27)

As shown in SEQ ID NO: 11, dn afo rni325 & 7 consisted of 3792 bases, of which base numbers 92 to 1207 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 371 amino acid residues (SEQ ID NO: 27). A homology search was performed using BLAST on the amino acid sequence encoded by SEQ ID NO: 11, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). (I) The database registration symbol AX 339740, anovel huma ngroteincouled receptor force S, e—value: 5 X 10 to ¹⁷⁹ , and 87% of the hits over 348 amino acid residues were hit.

 The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 11 was subjected to a protein feature search using HMMP FAM. As a result, it was found that the amino acid sequence encoded by nucleotide numbers 287 to 1081 in SEQ ID NO: 11 had G protein-coupled A sequence (base sequence entered as “7 tm-1” in P f am) showing body characteristics was found. From these facts, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 11 has G protein-coupled receptor activity.

 (12) d n a f o rm33729 (SEQ ID NOS: 12, 28)

dn afo rm33729 consists of 3336 bases as shown in SEQ ID NO: 12, of which base numbers 661 to 2196 are open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 511 amino acid residues (SEQ ID NO: 28). When a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 12, the SPTR protein database (integrating the SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database) (I) Database registration code AX 375432 gpcrxllasafunctio na 1 receptorvalidatedbyan giopeptinandusefulfor screeningofagonistsand dantagonists power ^s , e-value: 0.0, 'and also hit 507 amino acid residues with a 72% match.

 In addition, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 12 was subjected to a protein feature search using HMMP FAM, and the G protein was conjugated to the amino acid sequence encoded by nucleotide numbers 796 to 1548 in SEQ ID NO: 12. A sequence (a nucleotide sequence entered as “7 tm-1” in P f am) showing the characteristics of the type receptor was found. AX3755332 described above is a human GPCR, which is a patent information (W0198330-A927-DEC-2001) in the database, and has been shown to be activated by angiopeptin. Angiopeptin is an analog of somatostatin and is shown to inhibit atherosclerosis in the coronary artery of the egret, based on literature information (Atherosclerosis 1989 Aug; 78 (2-3): 229-36) in the database. . From these facts, it is inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 12 is a G protein-coupled receptor for somatostatin-like ligand, has GPCR activity, and is associated with arterial sclerosis and cancer. Was.

 (1 3) d n a f o r m 4847 7 (SEQ ID NOS: 13, 29)

As shown in SEQ ID NO: 13, dnaform48477 was composed of 3379 bases, of which base numbers from 108 to 1184 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 358 amino acid residues (SEQ ID NO: 29). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 13 using BLAST, and it was found in the SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database). , (I) Database registration code AB 0 382 3 37, Homo saiens mRNA for G protein— coupled receptor C 5 L 2 ^ PR 77, e— va 1 ue: 5 × 10 1 ¹¹⁴ , and 3 3 1 1% over 1 amino acid residue In the matching degree, (ii) AF 068680, O ryctolaguscunicu 1 usanap hy latoxin C 5 areceptorgene force ^s, e- value: hit at 5 X 10- ⁶ °, and in 44% of the degree of coincidence over the 329 amino acid residues did.

 In addition, the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 13 was subjected to a protein feature search using HMMP FAM. (A nucleotide sequence entered as "7 tm-1" in P f am) was found.

 The above-mentioned C5L2 is expressed in granulocytes and undivided dendritic cells from literature information in the database (Mol Immunol 2000 Jun; 37 (8): 407-12), and C3a And homology with C5a chemoattractant receptor have been shown to be high. From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 13 had a G protein-coupled receptor activity related to chemotaxis.

 (14) d n a f o rm54572 (SEQ ID NOs: 14, 30)

 As shown in SEQ ID NO: 14, dnaform54572 consists of 3005 bases, of which base numbers 218 to 970 have an open reading frame.

(Including the stop codon). The amino acid sequence predicted from the open reading frame consists of 250 amino acid residues (SEQ ID NO: 30). A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 14 using BLAST, and a SPTR protein database (integrated SWI SS-PROT protein sequence database and TrEMBL nucleic acid translation database) during, (i) data base registration mark AF 1 1 1088, B ostaurus 1 atrophi 1 in 3 splicevariantabbf, e- value: hit with 3 X 10- ^51, also 302 Amino acids 37% degree of coincidence over the residues .

The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 14 was searched for protein characteristics using HM MPF AM. Array indicating (Base sequence entered as “7 tm—2” in P f am). From the literature information in the database (FEBS Lett 1999 Jan 29; 443 (3): 348-52), latrophilin 3 splice variant abbf indicates that Latrophilin is a receptor for alpha-latrotoxin, a potent prosynaptic toxin. Latrophilin-3 has been shown to be specifically expressed in the brain. From these facts, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 14 is an orphan G protein-coupled receptor and has activity in the brain and the like.

 (15) d n a f o rm29069 (SEQ ID NOs: 15, 31)

As shown in SEQ ID NO: 15, dnafo rm29069 consisted of 3034 bases, of which base numbers 1557 to 2489 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 310 amino acid residues (SEQ ID NO: 31). When a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 15, the S PTR protein database (SWI SS-PROT protein sequence database and Tr EMB L nucleic acid translation database were integrated). ) in, (i) database registration mark AF 1 66382, Mu s mu scu 1 usserentinereceptor (Cy t 28) force e - value: in 5X 10 one ^56, and in 38% of the degree of coincidence over the 271 amino acid residues and (ii) a database registration symbol _{BC 008770 N Homo saiens, G protein-} cou 1 edreceptor 56 force e- value: at 8 X 10 one ^51, 2 81 36% degree of coincidence over the amino acid residues, and ( iii) database registration § himself No. AF 106858 Homo saiens G- protein- cou 1 edreceptor (GPR56) force e_v alue: at l X 10 one ⁵⁰ was hit with 36% degree of coincidence over 28 1 Amino acid residues.

The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 15 was subjected to a protein feature search using HMMP FAM. Show body characteristics Sequence (base sequence entered as “7 tm—2” in P f am), and the amino acid sequence encoded by base Nos. 1647–1790 contains Latrophi 1 in / C L-1-like GPS doma in (Base sequence that is entered as “GP S” in P fa).

 According to the description in the database entry number AF 166382, the above Cyt28 was shown to be cloned from mouse hematopoietic stem cells. From the literature information in the database (Genomics 1999 Feb 1; 55 (3): 296-305), GPR 56 is strongly expressed in the thyroid gland, heart and brain, and is particularly strongly expressed in the hippocampus and thalamus in the brain, Its extracellular mucin-like structure has been suggested to be involved in cell-cell interactions. From these facts, it was presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1.5 was a G protein-coupled receptor and had an activity related to cell-cell interaction. +

 (16) d n a f o rm45616 (SEQ ID NO: 16, 32)

As shown in SEQ ID NO: 16, dnafo rm45616 was composed of 2774 bases, of which base numbers 311 to 1210 were open reading frames (including a stop codon). The amino acid sequence predicted from the open reading frame consists of 299 amino acid residues (SEQ ID NO: 32). When a homology search was performed using BLAST for the amino acid sequence encoded by SEQ ID NO: 16, the SPTR protein database (the SWI SS-PROT protein sequence database and the TrEMBL nucleic acid translation database were integrated) (i) the data base registration mark AF 039686, Homo saiens G- proteincouledreceptor GPR 34 (GPR3) force e- va 1 ue: at 1 X 10- ^19, and in 27% of the degree of coincidence over the 294 amino acid residues, and ( 1 ¾0 data base over to register symbol <391 11: 6,1 ^ 1 3 mu sculus G protein- co up ledreceptor Gp r ^ 4 (G r 34) gene force e- value: at 5 X 10 one ^18, 294 Amino The hit was achieved with 38% identity over the acid residues, and the amino acid encoded by the nucleotide sequence shown in SEQ ID NO: 16 A program that predicts transmembrane helices for acid sequences tm marauder (S. Moller, MDR Croning, R. Apweiler. Evaluation of methods for tne prediction of membrane spanning regions. Bioinformatics, 17 (7): 646-653, 2001) When the transmembrane site was predicted using, the seven transmembrane sites (30-52, 65-87, 107-129, 141-163, 197-219, 239- 261, 276-298).

 The above GPR 34 is analogous to the platelet-activating factor (PAF) receptor from literature information in the database (BiochimBiophys Acta 1999 Jul 7; 1446 (1-2): 57-70). It has been shown to be abundantly expressed in various human and mouse organs. Also, from the literature information in the database (um Genet 2000 Jul; 107 (1): 89-91), GPR34 is located near the causal locus of congenital night blindness (CSNB) on the X chromosome and is expressed in the eyes, It has been shown not to be the causative gene of CSNB. From these results, it was presumed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 32 had a novel G protein-coupled receptor activity. From these results, it was presumed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 32 had G protein-coupled receptor activity. In addition, genes encoding similar proteins are on the X chromosome, are abundantly expressed in various organs, and have weak homology to PAF receptors.

Example 5 Tissue expression analysis

 Tissue expression analysis was performed as described in Miki, R., et al., Proc. Natl. Acad. Sci. USA, 98, 2199-2204 (2001).

 (1) Creation of DN A microarray

Mouse cDNA clone containing the nucleotide sequence of SEQ ID NO: 1: dnafo rm 34147, mouse cDNA clone containing the nucleotide sequence of SEQ ID NO: 4: dnafo rm42851, mouse cDNA clone containing the nucleotide sequence of SEQ ID NO: 7 Mouth: dn afo rm29894, mouse cDNA clone containing the nucleotide sequence of SEQ ID NO: 11: dn afo rm32567, nucleotide sequence of SEQ ID NO: 14 Mouse cDNA clone containing: cDNA clone belonging to the same cluster as dnafo rm54572: FANTOM 9330 161N09

 (http: // fantom. gsc. riken. go. jp /), and a mouse cDNA clone containing the nucleotide sequence of SEQ ID NO: 15: cDNA clone belonging to the same cluster as dnafo rm29069: FAN TOM 3830420G05

 (http: //fantom.gsc.riken.go.jp/) After amplifying the DNA containing the nucleotide sequence of the mouse cDNA portion using Ml3 forward and reverse primers, the PCR product was amplified with isopropanol. Precipitated and dissolved in 3XS SC solution of 15β1. The obtained DNΑ solution was spotted on a glass slide coated with poly-L-lysine using a DNA arrayer of 16 chips (S MP d, 丄, ele! I Internatlona Sunny vale, CA), and a DNA microarray was prepared. It has been created and is listed in Manpo's Itoda fihttp: // cmgm. Stanford.edu/pbrown/mguide/index.htm. Mouse i3 actin and daricell aldehyde-3-phosphate dehydrogenase cDNA were used as a positive control, and Arabidopsis thaliana cDNA was used as a negative control.

 The detection sensitivity of this DNA microarray was 1 to 3 copies of mRNA per cell. The signal intensity of clones with approximately 80% match with the target sequence was one-tenth that of clones with perfect sequence match. The signal intensity of clones with less than 80% match with the target sequence was reduced at the background level.

 (2) Preparation of probe

49 tissues of fetal, neonatal and adult C57BL / 6J mice (kidney, brain, spleen, lung, liver, testis, kidney, stomach, small intestine, colon, cecum, placenta, heart, tongue, thymus, breast Glands (1st day of pregnancy), cerebellum, medulla oblongata, olfactory brain, epididymis, eyes, cortex, follicles, uterus, ovaries and ovary (1st day of pregnancy), bones, muscles, mammary glands (10th day of lactation) ), Whole 10-day-old fetus, 11-day-old fetal whole body, 13-day-old fetal whole body, 11-day-old fetal head, 12-day-old fetal head, 13-day-old fetal head, 15-day-old fetal head Part, 16 days old fetal head, 17 days old fetal head Part, 16-day-old fetal lung, 13-day-old fetal liver, 14-day-old fetal liver, 0-day-old newborn whole head, 6-day-old newborn whole head, 10-day-old newborn whole head, 10 Random prime reverse transcription reaction of mRNA lg extracted from day-old neonatal intestine, 0-day-old neonatal lung, 10-day-old neonatal cerebellum, 0-day-old neonatal skin, 10-day-old neonatal skin, SV40 infection Then, the fluorescent dye Cy 3 (Amersham Pharmacia) was incorporated. On the other hand, 1 μg of mRNA extracted from the whole body of a 17.5-day-old fetus was subjected to a random prime reverse transcription reaction, and the fluorescent dye Cy5 was taken in as a reference for expression analysis. The CyDye-labeled cDNA probe was purified using CyScribe GFX Purification Kit (Amersham Pharmacia), and eluted from the column with sterile water 171, and 3 μl was added thereto. 1 of 10 μg no μ 1 oligo (dA), 3 μ 1 of yeast tRNA 20 μg / μ 1, Ι μ 2 2

A CyDye-labeled cDNA probe was prepared by mixing a blocking solution consisting of 0 μl mouse Cοt1 DNA, 5.1 μl of 20 XSSC, and 0.91 of 10% SDS. .

 (3) DNA microarray hybridization

 A 30 μl solution of the cDNA analysis target tissue-derived cDNA probe (Cy3 label) and the reference 17.5-day-old fetal cDNA probe (Cy5 label) was mixed at 95 ° C. Heat treatment was carried out for 1 minute and cooled at room temperature. Add the above probe solution to the DNA microarray and cover with a force bar slip.Hybricacsette (Arraya

It was hybridized at 65 ° C- 晚 in 1 t company). Next, the DNA microarray was washed with 2 × SSC and 0.1% SDS, and then rinsed with 1 × SSC for 2 minutes and with 0.1 × SSC for 2 minutes. The microarray was scanned using a ScanA 500 000 confocal laser scanner and the images were analyzed on an I MAGENE (BioDiversive).

 (4) Data analysis

The mRNA level (Cy3 label) in each tissue is expressed as the ratio (Cy3 / Cy5) to the reference 17.5-day-old fetal whole-body mRNA level (Cy5 label) as a logarithm (1 og). ₂ ) in displayed. That is, if the expression level of mRNA corresponding to the full-length cDNA of each mouse to be analyzed is larger in each tissue than in the reference tissue, a positive value is used, if it is smaller, a negative value, and if it is equal, Indicated by 0. Experiments were performed twice independently to increase the accuracy of the data, and reproducible results were used. The results are shown in Table 1. In general, the results of expression analysis using a DNA microarray are considered to be experimental errors if the value is increased or decreased by a factor of about two. It was interpreted that the amount of mRNA to be analyzed in the tissue was more than twice as large as the mRNA amount of the whole fetal body at 17.5 days of age, and that the amount was significantly increased. Conversely, if the value of the expression analysis result in a certain tissue is less than or equal to -1, the amount of the mRNA to be analyzed in the tissue is one-half of that of the control 17.5-day-old fetus whole body. The following were interpreted as significantly reduced.

 When comparing the mRNA expression levels between tissues, when the difference between the values in each tissue is 1, the mRNA level is 2 times, and when the difference is 2, the mRNA level is 4 times. If the difference between the values is -1, the mRNA amount is 1Z 2 times, and if it is _2, the mRNA amount is 1/4 times.

Mouse cDNA clones belonging to the same cluster as the DNA spotted on the microarray and having a region having a nucleotide sequence identity of at least 80% with at least 200 bases are also shown in Table 1 And the numerical value of the measurement result of the DNA spotted on the microarray was used instead.

table 1 :

Analysis target Spot on cDN microarray Kidney Brain Spleen Lung Liver

A _ _ fcD A

dnaform34147 dnaform34147 0--1 0.604644 -0.918401 -0.394848 0.003062 dnaform42851 dnaform42851 -0.028749 0 -0.035681 0.263115 0.512224 dnaform29894 dnaform29894 0.211066 1.21558 0.525265 -0.218343 0.712455 dnaform32567 dnaform32567 0 1.52081 0.375na0 0.792 ANT017201F 072 0.297707 -0.817491 —0,694666 -0.009974 0.177833 Test spots spotted on the cDN microarray to be analyzed testis knee small intestine colon

A was DNA

dnaform34147 dnaform34l / 0.002757 0.389407 0.04 38 i 0.699333 -0.333782 dnaform42851 dnaform42851 -0.49458 1.16233 0.217832 -0.070995 0.658057 dnaform29894 dnaform29894 0.007426 1.79989 0.483078 0.310433 -0.213772 dnaform32567 dnaform32567 1.01920 1.596 0. 0.992702 0.549824 1 0.305854 -0.074891 -0.001997 Analyzed target spot on cDN microarray Placental heart Thymus

A was DNA

dnaform34l 47 dnaform3414 / ichi 0.283769 -0.372257-0.659996 -1.14156 0.361602 dnaform42851 dnaform42851 0.567144 0.188979 -0.661836 0.669685 0.139335 dnaform29894 dnaform29894 0.148378 0.049242 0.171353 -0.440989 -0.091025 dnafortn320.1 0.194930.10940.18 dnaform29069 FANTOM NO3830420G05 1.80195 -0.294741 -0.140147 -0.943278 -0.16034 Analysis target Spot on cDN microarray Bone muscle Muscle Dorsal kidney Derived from epididymis Visceral fat A fcDNA Original fat cell Adipocyte

dnaTorm34l47 dnaform34147 -0.3937 0.012674 - 0.332067 -0.358397 - 1.09285 dnaform42851 dnaform42851 0.200827 0.4848 0.241472 0 dnaform29894 dnaform29894 -0.100222 0.633098 0.54399 0.505093 0.086003 dnaform32567 dnaform32567 -0.91889 0.54258 0.635356 0.431214 0.150435 dnaform54572 FANTOM NO: 9330161 N09 0.320531 0.74831 1.17013 1.30375 0.912332 dnaform29069 FANTOM NO3830420G05 1.85781 0.161977 - 0.247306 -0.361052 -0.086625 Spot on the analysis target cDN microarray.

A DNA Liver cerebellum Child skin ―

dnaform34147 dnaform34147 -0.579103 -0.935412

dnaform42851 dnaform42851 0.246094 0.517231

dnaform29894 dnaform29894 0 0.55205

dnaform32567 dnaform32567 -0.1335 —0.124381

dnaform54572 FANTOM NO: 9330161 N09 1.12068 0.643064

dnaform29069 FANTOM NO3830420G05 -0.494198 -0.204861 As can be seen from Table 1, dnaform 3 4 1 4 7 has increased fluorescence in the spleen and uterus compared to the control (17.5 day fetal whole body) The expression was similar to that of the control in testis and kidney, but the expression level was reduced as a whole as compared to the control. The expression of dnaform 42851 increased in muscle and spleen, increased in cerebellum, colon, and placenta compared to controls, and was equivalent in brain. dnaform 2 9 8 9 4 is more strongly expressed in the spleen compared to controls Increased, and also increased in the brain. Compared to controls, dnafo rm32567 increased strongly in spleen and brain, increased in uterus and testis, and tended to increase in liver and colon. dn afo rm54572 is up-regulated in spleen, adipocytes, and 10-day-old neonatal cerebellum as compared to control, and is expressed to the same extent as control in brain, but is up-regulated overall as compared to control There was a tendency. dnaio rm29069 increased strongly in bone and placenta and tended to increase in spleen and muscle as compared to control.

Example 6 Tissue expression analysis using PCR method

 In order to examine the changes in tissue expression in normal and diseased mice of niRNA encoding the protein of the present invention, PCR was performed according to a standard method (Higuchi R, et al., Biotechnology, 11: 1026-30 (1993)). Was used to perform tissue expression analysis.

 (1) cDNA synthesis

 Total RA was extracted from 19 tissues of the following mice (Kazuo Moriwaki, 1st Edition, Molecular Medicine Separate Volume, Vol. 36 “Model Animals with Spontaneous Diseases”, Nakayama Shoten, 1999), and oligo dT was used as a primer. CDNA synthesis was performed using reverse transcriptase. (a) Tissue of normal mice and tissue of diabetic model mice

 ① Control mouse C57BL / KsJ-+ m / + m Jcl (female, 8 weeks old) whole brain, thalamus, lung, kidney, bone marrow, knee, adipocyte, liver, eye

 ② Diabetes model mouse CWBL / KsJ-db / db Jcl (female, 8 weeks old) spleen, fat cells, liver, eyes

 (b) Aging-promoting mouse tissue

 (1) Hippocampus and frontal cortex of normal aging mouse SAM Rl / TA Sic (13 weeks old)

② Senescence-accelerated mouse SAM P8 / Ta Sic (Oss, 15 weeks old) hippocampus, frontal cortex

(c) Tissue of cancer metastasis model mouse

 ① Normal colon of control mouse Balb / c (female, 5 weeks old)

② Colon metastasis of cancer metastasis model mouse Balb / c (female, 6 weeks old) (Colony 26 colon cancer cells are transplanted into the abdominal cavity of the mouse and colon cancer is removed 2 weeks later) (2) Quantification by PCR method

 Expression of the following four mRNAs encoding the protein of the present invention was attached to the product using a light cycler quantitative PCR device (Roche Diagnostics) and LightCycler-FastStart DNA Master SYBR Green I reagent. Quantification was performed according to the protocol provided. The synthetic DNA sequences used for quantitative PCR are shown below.

 (a) dn a f o rm33729

 5. Side primer: (AGCAAAGGGTCTCCATTGTC) (SEQ ID NO: 41)

 3. Side primer: (AAACCCAAGCTGAGGTAGCA) (SEQ ID NO: 42)

 (b) d n a i o rm456 16

 5, side primer: (TGATCATTGAACTTTGGGTCA) (SEQ ID NO: 43)

 3. Side primer: (CCCTGAAGTATGGGAGCAAA) (SEQ ID NO: 44)

 (c) d n a f o r m47734

 5, side primer: (CAAACAACTAAGCGCAAATCC) (SEQ ID NO: 45)

 3. Side primer: (ACCGCTTAAAAGGCCAAAGT) (SEQ ID NO: 46)

 (d) d n a f o r m48477

 5, side primer: (CTGGGGAACTATTCCAAGCA) (SEQ ID NO: 47)

 3. Side primer: (TCTGGGCATTTGTGTTGAAG) (SEQ ID NO: 48)

The quantification results were corrected using Glyceraldehyde 3-phosphate dehydrogenase (GAP DH) as the internal standard [5 standards]. That is, the expression level (copy number / μ1) of the target gene in each tissue is divided by the GAPDH expression level (copy number / 1), and the result is raised to a constant (1 × 10 ⁶ ) (Note: 10 to the sixth power). And displayed. The results are shown in Table 2. Table 2:

表 2から明らかなように、 dn a f o rm33729は眼での発現が高く、肺、 脂肪、 膝臓で発現が観察され、 結腸癌で発現が減少した。 d n a f ₀ rm456 16は肺での発現が高く、 勝臓、 脂肪、 結腸で発現が観察されたが、 糖尿病の膝 臓おょぴ結腸癌では発現が減少した。 d n a f o rm47734は脳組織特に前 頭葉皮質および海馬での発現が高く、記憶学習との関連が示唆され、膝臓や脂肪、 および骨髄でも発現が観察された。 d n a f o rm48477は骨髄、 肺で発現 が高く、 糖尿病の脂肪組織で発現が増加した。 上記クローンの cDNAおよぴ該 cDNAによってコードされるタンパク質は、 糖尿病や癌などの治療や診断に応 用できる。 また該 cDNAによってコードされるタンパク質は、 上記のような m RNA発現の変動が見られる組織あるいは mRNA発現量の多い組織に関わる疾 患に関与している可能性がある。 実施例 7 機能予測 (1) dn a f o rm34 147

As is evident from Table 2, expression of dnafo rm33729 was high in eyes, expression was observed in lung, fat and knee, and decreased in colon cancer. The expression of dnaf ₀ rm456 16 was high in the lung, and was observed in the viscera, fat, and colon, but decreased in diabetic knee and colon cancer. dnafo rm47734 was highly expressed in brain tissues, especially in the frontal cortex and hippocampus, suggesting a relationship with memory learning, and was also observed in knees, fat, and bone marrow. dnafo rm48477 was highly expressed in bone marrow and lung, and increased in diabetic adipose tissue. The cDNA of the above clone and the protein encoded by the cDNA can be applied to the treatment and diagnosis of diabetes and cancer. Further, the protein encoded by the cDNA may be involved in a disease relating to a tissue in which the mRNA expression is varied or a tissue having a high mRNA expression level as described above. Example 7 Function prediction (1) dn afo rm34 147

 From the results of Example 4 (1), it is known that the proteins encoded by the present cDNA are similar proteins involved in testis development, function maintenance, and fertility. In addition, from the results of Examples 5 and 6, the expression of the protein encoded by the cDNA tends to increase in the viscera and uterus as compared to the control (17.5-day-old fetal whole body), The expression was similar to that of the control in such as kidney.

 Based on these facts, the protein encoded by this cDNA has functions related to infertility, contraception, and diabetes, because it is involved in testis development and maintenance, function in fertility, and is expressed in the uterus. It was considered useful for the analysis of these therapeutic agents.

 (2) d n a f o rm37308

 From the results of Example 4 (2), Examples 5 and 6, the GPCR that the protein encoded by this cDNA is expressed in the brain, induced to be expressed by dalcocorticoid, and enhanced when the psychotropic drug is stopped And homology was higher.

 From these facts, it was predicted that the protein encoded by this cDNA is involved in neuroadaptation to stress and psychostimulants. Therefore, this protein was considered to be useful for developing drugs for withdrawing from drug dependence, schizophrenia, depression, anxiety, Alzheimer's disease, and Parkinson's disease.

 (3) dn a f o rm37953

 From the results of Example 4 (3), Examples 5 and 6, the protein encoded by this cDNA is expressed in the brain, is induced to be expressed by dalcocorticoid, and has a GPCR that enhances the expression when the psychotropic drug is stopped. The homology was high. For these reasons, it was predicted that the protein encoded by this cDNA would be involved in neural adaptation to stress and psychostimulants. Therefore, this protein was considered to be useful for the development of therapeutic drugs for withdrawal from drug dependence, schizophrenia, depression, anxiety, Alzheimer's disease, Parkinson's disease, and the like.

 (4) d n a f o rm42851

From the results of Example 4 (4), Examples 5 and 6, the protein encoded by the cDNA The protein was expressed in the brain, induced to be expressed by dalcocorticoids, and had a high homology with GPCRs, whose expression was enhanced when psychotropic drugs were stopped. In addition, the expression was increased in muscle and spleen compared to control, and increased in cerebellum, colon and placenta, and was similar in brain and the like.

 These results suggest that the protein encoded by this cDNA is involved in neuroadaptation to stress and psychostimulants. Therefore, this protein was considered to be useful for the development of a drug for withdrawal from drug dependence, schizophrenia, depression, anxiety, Alzheimer's disease, Parkinson's disease, and diabetes.

 (5) d n a f o rm27636

 From the results of Example 4 (5), and Examples 5 and 6, the protein encoded by the present cDNA was similar to GPR82 expressed in the colon. Based on these findings, it was considered that this protein has functions related to Crohn's disease, irritable colitis, gastrointestinal tract function regulation, cancer, etc., and is useful for the development of these therapeutic agents.

 (6) dn a f o rm. 47734

 From the results of Example 4 (6) and Examples 5 and 6, the protein encoded by the present cDNA is similar to human TGR21, and is also found in brain tissue, especially in the frontal cortex and hippocampus. High expression was observed in spleen and fat.

 The protein has functions related to Alzheimer's disease, Parkinson's disease, chorea, ischemic brain disease, schizophrenia, depression, anxiety, and diabetes. It was considered useful for development.

 (7) d n a f o rm29894

 From the results of Example 4 (7) and Examples 5 and 6, the protein encoded by this cDNA is a human GPR103 with high homology between neuropeptide FF2, neuropeptide Y2 and galanin GalRl receptor and the transmembrane domain. Was similar to It was also expressed in the cerebral cortex, pituitary gland, thalamus, hypothalamus, basal forebrain, midbrain, and lungs. Expression was strongly increased in the knee and increased in the brain.

Based on these strengths, this protein is useful for schizophrenia, depression, anxiety, parkin It has functions related to Sohn's disease, Alzheimer's disease, ischemic brain disease, diabetes, and endocrine diseases, and was considered to be useful as a therapeutic drug for these.

 (8) dn a f o rm53455

 From the results of Example 4 (8) and Examples 5 and 6, the protein encoded by this cDNA was converted to human GPR103, which has high homology between neuropeptide FF2, neuropeptide Y2 and the galanin GalRl receptor and the transmembrane region. Was similar. It was also expressed in the cerebral cortex, pituitary gland, thalamus, hypothalamus, basal forebrain, midbrain, and lung. From these facts, this protein has functions related to schizophrenia, depression, anxiety, Parkinson's disease, Alzheimer's disease, ischemic brain disease, diabetes, and endocrine disease, and is useful as a therapeutic drug for these. It was thought that there was.

 (9) dn a f o rm53728

 From the results of Example 4 (9) and Examples 5 and 6, the protein encoded by this cDNA is a human GPR103 with high homology between neuropeptide FF2, neuropeptide Y2 and galanin GalRl receptor and transmembrane domain. Was similar to It was also expressed in the cerebral cortex, pituitary gland, thalamus, hypothalamus, basal forebrain, midbrain, and lung. From these facts, this protein has functions related to schizophrenia, depression, anxiety, Parkinson's disease, Alzheimer's disease, ischemic brain disease, diabetes, and endocrine disease, and is useful as a therapeutic drug for these. It was thought that there was.

 (10) dn a f o rm62766

 From the results of Example 4 (10) and Examples 5 and 6, the protein encoded by the present cDNA is a human GPR having high homology with the neuropeptide FF2, neuropeptide Y2 and galanin GalRl receptor and the transmembrane region. It was similar to 103. It was also expressed in the cerebral cortex, pituitary gland, thalamus, hypothalamus, basal forebrain, midbrain, and pons. From these facts, this protein has functions related to schizophrenia, depression, anxiety, Parkinson's disease, Alzheimer's disease, ischemic brain disease, diabetes, and endocrine disease, and is useful as a therapeutic drug for these. It was thought that there was.

(1 1) dnafo rm32567 From the results of Example 4 (1 1) and Examples 5 and 6, the protein encoded by the present cDNA increased strongly in the kidney and brain, increased in the uterus and testis, and increased in the liver compared to the control. And the colon tended to increase.

 Based on these findings, this protein has functions related to diabetes, schizophrenia, depression, anxiety, Parkinson's disease, Alzheimer's disease, ischemic brain disease, infertility, contraception, and liver disease. It was considered as useful.

 (12) d n a f o rm33729

 From the results of Example 4 (1 2), Examples 5 and 6, the protein encoded by the present cDNA is similar to human AX 375432, and AX 375432 is activated by the somatostatin analog angiopeptin, Atherosclerosis was suppressed. In addition, the expression of this protein was high in the eyes, and was observed in lung, fat, and spleen, and decreased in colon cancer.

 Based on these facts, it was considered that the present protein has functions related to atherosclerosis, cancer, diabetes, glycemic retinopathy, and ophthalmic diseases, and is useful for the development of these therapeutic agents.

 (13) d n a f o r m48477

 From the results of Example 4 (13) and Examples 5 and 6, the protein encoded by this cDNA was found to be resistant to C5L2 and chemotaxis expressed in granulocytes and undifferentiated dendritic cells. Similar to the GP CR involved. The expression of this protein was high in bone marrow and lung, and increased in diabetic adipose tissue.

 From these facts, this protein has functions related to immune diseases, inflammatory diseases, allergic diseases, respiratory diseases such as lungs and trachea, and diabetes, and is useful for the development of these therapeutic agents. It was considered.

 (14) d n a f o rm54572

From the results of Example 4 (14), Examples 5 and 6, the protein encoded by this cDNA is similar to brain-specific latrophilin 3, which is a splicing variant of the exogenous toxin alpha-latrotoxin receptor Latrophilin. I was The protein Was increased in the knee, adipocytes, and 10-day-old neonatal cerebellum as compared to the control, and was expressed to the same extent in the brain and the like.

 Based on these facts, this protein has functions related to endocrine diseases such as schizophrenia, depression, anxiety, Parkinson's disease, Alzheimer's disease, chorea and diabetes, and is useful for the development of these therapeutics. It was thought that there was.

 (15) d n a f o rm29069

 From the results of Example 4 (15) and Examples 5 and 6, the protein encoded by the present cDNA was similar to Cyt 28 cloned from mouse hematopoietic stem cells. GPR56, a similar protein, is strongly expressed in the thyroid gland, heart, and brain, is particularly strongly expressed in the hippocampus and thalamus in the brain, and has an extracellular mucin-like structure. Has been suggested. In addition, the present protein increased strongly in bone and placenta, and tended to increase in knee and muscle compared to control.

 Based on these findings, this protein has functions related to endocrine diseases such as osteoporosis and diabetes, schizophrenia, depression, anxiety, Parkinson's disease, Alzheimer's disease, immune diseases, inflammatory diseases, and allergic diseases. However, it was considered useful for the development of these therapeutic agents.

 (16) d n a f o rm456 16

 From the results of Example 4 (16) and Examples 5 and 6, the GPR34 gene, which is similar in the protein encoded by this cDNA, is on the X chromosome, and is abundantly expressed in various organs. It was found that there was weak homology with the body.

In addition, expression of this protein was high in lung and was observed in kidney, fat and colon, but decreased in diabetic knee and colon cancer.

From these facts, it is considered that the present protein has functions related to respiratory diseases (such as asthma), diabetes, cancer, and the like, and is useful for the development of these therapeutic agents. Industrial applicability Since the protein of the present invention and the DNA encoding the same have the above-described G protein-coupled receptor activity, a substance that regulates the activity can be screened using the protein or the DNA encoding the protein. It can also be used for the development of a medicament capable of acting on diseases associated with the protein. In particular, G-protein coupled receptors (G-protein coupled receptors) are membrane protein receptors that transmit signals from various extracellular ligands via G proteins. It is involved in various signal transduction of cells and is important as a target for drug development, and many inhibitors and activators of G protein-coupled receptor-related signal transduction are listed as drugs.巿 has been used. Therefore, finding a novel G protein-coupled receptor is very useful in the pharmaceutical development industry. This application is for a Japanese patent application filed on April 19, 2012 (Japanese Patent Application No. 2002-111), and for a Japanese patent application filed on February 1, 2002 (Japanese Patent Application No. 2002-3502252), the contents of which are incorporated herein by reference. The contents of the documents cited in this specification are also incorporated herein by reference.

Claims

匚 = range of f  1. The following protein (a) or (b):  (a) a protein comprising the amino acid sequence of any one of SEQ ID NOs: 17 to 32; (b) one or more amino acids in the amino acid sequence of any one of SEQ ID NOs: 17 to 32 comprising deletion, substitution, Z or addition of an amino acid sequence, and G protein-coupled receptor activity A protein having  2. A DNA encoding the protein of claim 1.  3. A full-length cDNA encoding the protein of claim 1.  4. DNA of any of the following (a), (b) or (c):  (a) a DNA having the nucleotide sequence of any one of SEQ ID NOs: 1 to 16;  (b) in the nucleotide sequence of any one of SEQ ID NOs: 1 to 16, having one or several nucleotides deleted, substituted and / or added, and having a G protein-coupled receptor activity DNA encoding a protein having  (c) having a nucleotide sequence capable of hybridizing under stringent conditions to DNA having the nucleotide sequence of any one of SEQ ID NOs: 1 to 16 or a complementary sequence thereof, and a G protein-coupled type DNA encoding a protein having receptor activity.  5. A recombinant vector containing the DNA according to any one of claims 2 to 4.  6. A gene-transfected cell into which the DNA according to any one of claims 2 to 4 or the recombinant vector according to claim 5 has been introduced, or an individual comprising the cell.  7. The protein of claim 1, which is produced by the cell of claim 6. 8. A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the nucleotide sequence of DNA according to any one of claims 2 to 4, an antisense having a sequence complementary to the sense oligonucleotide. An oligonucleotide selected from the group consisting of an oligonucleotide and an oligonucleotide derivative of the sense or antisense oligonucleotide. 9. An antibody or a partial fragment thereof that specifically binds to the protein of claim 1 or 7.  10. The antibody according to claim 9, wherein the antibody is a monoclonal antibody.  11. The body according to claim 10, wherein the monoclonal antibody has an action of neutralizing the G protein-coupled receptor activity of the protein according to claim 1 or 7.  12. The method according to claim 1, wherein the protein according to claim 1 or 7 is brought into contact with a test substance, and a change in the activity of the protein caused by the test substance is measured. Screening method.  13. A method for regulating the expression of a DNA, comprising bringing the gene-introduced cell according to claim 6 into contact with a test substance and detecting a change in the expression level of the DNA introduced into the cell. A method for screening substances.  14. At least one or more amino acid sequence information selected from the amino acid sequence of the protein according to claim 1 or selected from the DNA base sequence according to any one of claims 2 to 4. A computer-readable recording medium that stores at least one or more base sequence information.  15. A carrier to which the protein according to claim 1 and / or the DNA according to any one of claims 2 to 4 are bound.