JP2003125780A - Novel GPCRs, genes encoding them and their uses - Google Patents

Abstract

(57) [Problem] To provide a novel GPCR, a gene encoding the same, and a new drug screening means using the same. Provision of a method for diagnosing a disease using them. SOLUTION: A novel GPCR, C-HEP15374 protein,
The fragment, the DNA encoding them, C-HEP1
5374 or a method for screening a ligand using the fragment thereof, a method for screening an agonist or antagonist of C-HEP15374 using the protein or a fragment thereof and the ligand, a ligand for C-HEP15374 using the protein and the G protein, A method for screening an agonist or antagonist, a method for diagnosing various diseases using DNA encoding C-HEP15374 or a fragment thereof, or an antibody against C-HEP15374.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel G protein-coupled receptor (GPCR), a DNA encoding the same, an antibody against the GPCR, and uses thereof. More specifically, the present invention relates to a novel GPCR which is highly expressed in the thyroid gland, testis, olfactory brain and the like of normal animals and which is significantly different from normal tissues in various disease tissues including cancer,
The present invention relates to a diagnostic agent for various diseases having a partial peptide, an antibody against them or a DNA encoding them as a main component, and a diagnostic method for various diseases using the same. On the other hand,
The present invention uses a screening reagent for the ligand containing the GPCR, a screening method for the ligand using the same, a screening kit for a substance that inhibits or promotes the activity of the GPCR containing the GPCR and the ligand, and the same. Screening Method, Ligand to GPCR Constituting Membrane System Containing G Protein and G Protein Coupled to It, or Cells Expressing Both, or G
The present invention relates to a screening system for a substance that inhibits or promotes PCR activity and a screening method for the substance using the same. The present invention also provides an antisense nucleic acid of the GPCR gene, a ribozyme capable of specifically cleaving a transcription product of the GPCR gene, and a ribozyme that may be effective for treating a disease caused by abnormal expression of the GPCR activity. Expression vector.

[0002]

2. Description of the Related Art Cells have several systems for receiving information from the outside world, and among them, cells are mediated by trimeric GTP-binding proteins (G proteins) by activation of receptors on the cell membrane. The mechanism for transmitting information within is the most typical. These types of receptors are collectively called G protein-coupled receptors (GPCRs) and have a common structure with seven transmembrane regions in the molecule,
"It is also called the 7-transmembrane receptor (7TMR).

According to a study, about half of existing medicines exert their actions through receptors on the cell membrane, and among them, GPCRs account for a particularly large proportion, which makes them most likely. Has attracted particular attention as a potential drug discovery target (eg, Science, 287, 1960-196).
4, 2000).

With the recent rapid progress in genomic analysis research, new GPCR genes have been found one after another, and it is estimated that the total number of human GPCR genes is 2000 or more. Since most of these GPCRs have unknown physiological ligands (that is, “orphans”), most of them could not be targets for drug discovery until now. However, due to the recent advances in combinatorial chemistry technology, it has become possible to use a substantial library of compounds, and due to the development of high throughput screening (HTS) through the introduction of robot technology etc., the time and effort required for ligand search have been reduced to the conventional level. It is being greatly reduced in comparison. Furthermore, with the progress of recombinant DNA technology, a screening system for GPCR agonists or antagonists that does not require a ligand has been established.

On the other hand, the fact that most of the pharmaceuticals express their pharmacological action via GPCR strongly suggests that GPCR can be used as a disease marker.
That is, by comparing the expression of a predetermined GPCR in a sample derived from a patient's specific tissue, body fluid, etc. with that in a normal person, the presence or absence of disease or the degree of disease progression can be determined,
Furthermore, disease typing can be performed, and it can be expected as a useful tool for so-called tailor-made medicine.

[0006] Examination of the expression of the orphan GPCR gene in various tissues is also important from the aspect of ligand search. To perform efficient ligand search,
It is more preferable to use an extract from a tissue containing a large amount of ligand as a source for screening, but it is expected that a large amount of the ligand will be present in a tissue where the receptor is highly expressed.

[0007]

DISCLOSURE OF THE INVENTION The present invention isolates a gene encoding a novel orphan GPCR,
It provides a means of producing recombinant cells engineered to express R or a fragment thereof, producing a recombinant protein or a fragment thereof, and using these to screen a ligand for the GPCR. The present invention further provides a means for screening an agonist or antagonist of the GPCR using the obtained ligand and the GPCR or a fragment thereof, and further provides a substance having a therapeutic activity against a disease associated with the GPCR. The purpose is to do. The present invention also utilizes the above-described GPCR-expressing recombinant cell or its cell membrane system to produce the GPC.
It is intended to provide a means for screening a ligand or agonist or antagonist for R. Further, another object of the present invention is to provide a DNA encoding the GPCR or a fragment thereof, or an antibody having a specific affinity for the GPCR or a fragment thereof, and using them, means for diagnosing various diseases. Is to provide. Still another object of the present invention is a nucleic acid having a base sequence complementary to the DNA encoding the GPCR (eg, antisense nucleic acid, ribozyme or double-stranded oligo RNA causing RNA interference), or expression encoding the same. It is to provide a vector.

[0008]

[Means for Solving the Problems] As a result of intensive studies to achieve the above object, the present inventors have found that seven transmembrane domains, which are considered to be transmembrane domains, in a cDNA library derived from a liver cancer cell line. We succeeded in isolating a cDNA clone, C-HEP15374, encoding a polypeptide having Next, the present inventors determined the nucleotide sequence of this cDNA clone and confirmed by homology search with a known gene sequence that the clone encodes a novel GPCR which has not been known so far. Furthermore, the present inventors constructed a primer pair capable of specifically amplifying a fragment of the DNA based on the obtained base sequence, and performed quantitative PCR using cDNA derived from organs of normal humans and patients with various diseases as templates. Was performed to compare the expression levels. As a result, the C-HEP15374 gene was highly expressed in the thyroid gland, testis, olfactory brain, etc. in normal individuals, and was significantly expressed in normal brain, thyroid, lung, stomach, colon, prostate, testis, uterus, etc. cancers. On the contrary, it was found that the expression was significantly increased in the frontal lobe and hippocampus in pancreatic cancer, renal cancer, cirrhosis, and Alzheimer's disease, compared with normal tissues. From these results, the present inventors, as a ligand source of C-HEP15374, thyroid, testis and olfactory brain tissue extract or secretions can be a preferred ligand source,
And confirmed that C-HEP15374 can be used as a disease marker for various cancers, liver cirrhosis, Alzheimer's disease, etc.,
The present invention has been completed.

Therefore, the present invention provides (a) SEQ ID NO: 2
A protein consisting of the amino acid sequence described above or (b) an amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids are deleted, substituted, inserted, added or modified, and Provide a protein that exhibits GPCR activity by coupling with the same G protein that the protein couples. The invention also provides a polypeptide or oligopeptide that is a fragment of the protein, which retains the ligand binding properties of the protein. Such a peptide can be used as an antigen for screening a ligand for C-HEP15374 and producing an anti-C-HEP15374 antibody.

The present invention is a DNA containing a nucleotide sequence encoding the above protein or peptide, preferably (a) a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or (b)
Provided is a DNA that hybridizes with the DNA of (a) under stringent conditions and that encodes a protein that exhibits GPCR activity by being coupled to the same G protein as that of the protein encoded by the DNA of (a). To do. D like this
NA can be used for recombinant production of the GPCR protein or a peptide fragment thereof by inserting it into an appropriate expression vector and introducing it into a host cell.
It can be used for diagnosis of various diseases as a probe or a primer for detecting gene expression.

The present invention also provides an antibody having a specific affinity for the above protein or peptide. Such an antibody can be used not only for detecting the expression of the GPCR, but also as a therapeutic neutralizing antibody for inhibiting the activity of the GPCR.

Another aspect of the present invention provides a method for diagnosing diseases such as cancer in various organs by detecting the expression of C-HEP15374 in a biological sample at the protein level or RNA level and comparing it with the expression in normal animals. provide. In this regard, the present invention also relates to (a) DNA encoding the above protein or peptide, and (b) DNA encoding the above protein.
There is provided a disease diagnostic agent comprising, as a main component, a primer pair capable of specifically amplifying all or a part of the above or (c) an antibody against the above-mentioned protein or peptide.

Still another aspect of the present invention provides a method for screening a ligand for C-HEP15374 by contacting a test sample with the above protein or peptide and selecting a substance that binds to the protein or peptide. In this regard, the present invention also provides a reagent for screening a ligand for C-HEP15374, which contains the above protein or peptide. Contacting the ligand obtained by such a method with the above-mentioned protein or peptide in the presence or absence of a test sample, and comparing the binding activity of the protein or peptide with the ligand under both conditions. Thus, a substance that inhibits or promotes C-HEP15374 activity can be screened. In this regard, the present invention also provides a kit for screening a substance that inhibits or promotes C-HEP15374 activity, which comprises the above protein or peptide and a ligand for C-HEP15374.

Screening for a substance that inhibits or promotes C-HEP15374 activity is carried out by contacting a cell expressing the above protein with a ligand for the protein in the presence or absence of a test sample, and It can also be done by comparing the change in cells due to binding under both conditions.

The present invention also provides a system in which the above protein and G protein or its α subunit (Gα) can act in a coupled manner, and optionally a system including an effector that further interacts with the G protein, A method for screening a ligand for C-HEP15374, or a substance that inhibits or promotes the activity of the receptor using them is provided.

In still another aspect of the present invention, (a) a nucleic acid having a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or (b) a nucleic acid having a base sequence shown in SEQ ID NO: 1 or the base obtained by post-transcriptional processing It consists of an initial transcription product giving a sequence and a nucleotide sequence that can hybridize under physiological conditions of mammalian cells, and can inhibit the translation of a protein encoded by the nucleotide sequence of SEQ ID NO: 1 in a hybridized state. Provided are nucleic acids, or expression vectors encoding those nucleic acids. Such nucleic acids are
Since it can inhibit the expression of C-HEP15374 gene,
It can be used for the treatment of various diseases related to the GPCR.

The above and other advantages of the present invention, as well as further features of the present invention, will be apparent in the following embodiments of the invention.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is a novel orphan GP.
C-HEP15374 protein that is CR is provided. C-HEP15374
Is a translation product (comprising the amino acid sequence set forth in SEQ ID NO: 2) of a cDNA clone originally isolated from a human hepatoma cell line, and is a protein having a 7-transmembrane domain belonging to the selectin family. In the present invention, the origin of C-HEP15374 is not particularly limited, in addition to those of human origin, it is derived from mammals such as cow, horse, monkey, pig, dog, cat, goat, sheep, mouse, rat, hamster, and guinea pig. The thing is also illustrated preferably. Further, the C-HEP15374 of the present invention is not limited to wild-type receptors derived from various mammals as described above, but activates the same G protein to which wild-type receptors are coupled to transduce a signal into cells. As long as possible, one or two or more amino acids in the amino acid sequence of the wild-type protein may be substituted, deleted, inserted, added or modified. Therefore, in the present invention, "C-HEP15374" means a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, and amino acids in which one or several amino acids have been substituted, deleted, inserted, added or modified in the amino acid sequence. A protein having a sequence and having the GPCR activity coupled to the same G protein as that to which the protein having the amino acid sequence of SEQ ID NO: 2 is coupled.

The C-HEP15374 protein of the present invention is a cell that expresses it, for example, mammals (for example, human, cow, horse, monkey, pig, dog, cat, goat, sheep, mouse,
Rat, hamster, guinea pig, etc. thyroid, colon,
It can be isolated from cell membrane-containing fractions derived from tissues such as testis, lung, stomach and brain (especially olfactory brain) by affinity chromatography using anti-C-HEP15374 antibody. Alternatively, from the cDNA library or genomic library derived from the tissue, a DNA clone isolated using the C-HEP15374 cDNA clone or a fragment thereof as a probe is cloned into an appropriate expression vector, and introduced into a host cell. Expressed from the membrane-containing fraction of the cell culture, anti-C-HEP15374 antibody or His-ta
It can also be purified by affinity chromatography using g, GST-tag, MBP-tag and the like. Alternatively, C-HEP15374 can also be obtained from the above expression vector by in vitro synthesis using a cell-free translation system derived from rabbit reticulocyte extract or wheat germ extract. Furthermore, C-HEP15374 can also be chemically synthesized based on the amino acid sequence of SEQ ID NO: 2 using a technique such as solid phase synthesis.

The C-HEP15374 of the present invention may be a mutant of C-HEP15374 isolated from nature, or C-HEP15374
It may be an artificial mutant obtained by treating a cell expressing HEP15374 with a physical or chemical mutagen. Alternatively, based on the C-HEP15374 cDNA clone,
A part of the mutation may be introduced by gene manipulation such as site-directed mutagenesis. However, it is desirable not to introduce mutations into such regions, as the ligand binding domain, preferably the extracellular loop and N-terminal chain to which an inverse agonist may bind, should be highly conserved. In addition, GDP in Gα
In order to retain the activity of promoting the GTP exchange reaction, it is preferable that the Gα activation domain existing on the intracellular third loop is also highly conserved. Conservative amino acid substitutions are well known, and those skilled in the art can use C- as long as the characteristics of the receptor are not changed.
An appropriate mutation can be introduced into HEP15374.

The present invention also provides a polypeptide or oligopeptide which is a fragment of C-HEP15374 (in the present specification, a peptide consisting of 100 amino acids or more is a "polypeptide" and a peptide consisting of 99 amino acids or less is a "polypeptide". For convenience, it is defined as "oligopeptide"). The peptide preferably contains at least the ligand binding domain of the receptor for use in a method for screening a ligand for C-HEP15374 or a substance that inhibits or promotes C-HEP15374 activity, which will be described later. The extracellular region of the receptor is preferably exemplified as the ligand binding domain. Specifically, the N-terminal chain or the first to third extracellular loops can be mentioned. These regions are, for example, the method of Kyte and Doolittle (J. Mol. Bio
l., 157, 105-132, 1982), and can be easily predicted from the hydropathy plot (see Example 2 and FIG. 1). Alternatively, the peptide is C-HEP1
When producing an antibody having a specific affinity for 5374, it can also be used as an antigen, in which case the peptide only needs to have an amino acid sequence of sufficient length to function as an epitope, For example, it is about 8 amino acids or more, preferably about 10 amino acids or more, more preferably about 15 amino acids or more.

The peptide fragment of C-HEP15374 is C-
It can be obtained by partially degrading the receptor protein isolated from cells expressing HEP15374 using an appropriate proteolytic enzyme or chemical substance. It can also be chemically synthesized by a known method such as solid phase synthesis based on the amino acid sequence shown in SEQ ID NO: 2. Alternatively, c-HEP15374 c obtained by the method as described above
DNA is digested with an appropriate restriction enzyme to prepare a cDNA fragment encoding a desired partial amino acid sequence, which is inserted into an appropriate expression vector and introduced into a host cell. The peptide fragment of can also be prepared by purification using affinity chromatography or the like.

The present invention also includes a D encoding C-HEP15374.
Provide NA. The DNA is a DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted, added or modified in the amino acid sequence, and the sequence There is no particular limitation as long as it is a DNA that encodes a protein that exhibits GPCR activity by being coupled with the same G protein that is coupled with the protein consisting of the amino acid sequence of No. 2. Preferably, the DNA of the present invention is a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, or is hybridized with the DNA under stringent conditions and is coupled with the same G protein as C-HEP15374 is coupled with. It is a DNA encoding a protein exhibiting GPCR activity. The "stringent condition" as used herein refers to C-HEP15374 protein having about 60% or more identity at the amino acid level, preferably about 80% or more identity, more preferably about 90% or more identity. The conditions under which DNA encoding a protein can hybridize are, for example, “1 × SSC, 0.1% SDS, 37
° C ", preferably" 0.5 x SSC, 0.1% SD "
S, 42 ° C. ”, more preferably“ 0.2 × SSC, 0.
1% SDS, 65 ° C. ”and the like, but those skilled in the art can understand the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction. The desired stringency can be easily adjusted by appropriately changing the salt concentration of the washing solution, the washing temperature, and the like.

The DNA encoding C-HEP15374 expresses the receptor in mammals (eg, human, cow, horse, pig, monkey, dog, cat, goat, sheep, mouse, rat, hamster, guinea pig, etc.). The cDNA or genomic library derived from cells can be isolated by colony or plaque hybridization using the cDNA having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof as a probe. Alternatively,
A DNA encoding C-HEP15374 can also be cloned from an cDNA derived from cells expressing the receptor or a genomic DNA library by using an anti-C-HEP15374 antibody by an antibody screening method. Further, as a method for obtaining a cDNA clone more quickly and easily,
The RACE method can be used. That is, oligonucleotides homologous to the partial base sequences of the sense strand and antisense strand of double-stranded DNA corresponding to a part of C-HEP15374 cDNA were synthesized, and each oligonucleotide was paired with an appropriate adapter primer. A full-length cDNA clone can be obtained by carrying out 5'and 3'RACE reactions as PCR primers and ligating each amplified fragment by a method such as using a restriction enzyme and ligase. Alternatively, the DNA encoding C-HEP15374 is SEQ ID NO: 1.
Fragments of the sense strand and antisense strand based on the described nucleotide sequence are chemically synthesized using a commercially available DNA automatic synthesizer in such a length that they overlap each other, and these fragments are hybridized, PC
The full length can also be produced by combining the R method and the like.

The nucleotide sequence of the DNA obtained as described above can be determined by using a known sequencing technique such as Maxam-Gilbert method or dideoxy termination method.

The present invention also provides a DNA encoding a polypeptide or oligopeptide which is a fragment of the C-HEP15374 protein. Such DNA is C-HE
To examine the expression of C-HEP15374 in a tissue as a probe or primer when cloning the DNA encoding P15374 from a cell that expresses it, and as a sequence primer when determining the nucleotide sequence of the cloned DNA. As a probe or primer for C-HEP1 by inserting it into an appropriate expression vector.
It can be used to produce fragments of the 5374 protein. Considering the above applications, it is desirable that the DNA comprises at least about 15 bases or more, preferably about 18 bases or more, more preferably about 20 bases or more.
The DNA is preferably a DNA encoding a partial amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2, more preferably a DNA having a partial base sequence of the base sequence shown in SEQ ID NO: 1. When the DNA is intended to be used as a probe or a primer, it may be a fragment encoding a fragment containing any region of the C-HEP15374 protein. However, the DNA
Is intended to produce a peptide to be used in a method for screening a ligand for C-HEP15374 or a substance that inhibits or promotes C-HEP15374 activity, it contains a region likely to be a ligand binding domain as described above. It is desirable to encode the peptide.

The DNA encoding the fragment of C-HEP15374 protein is C-HEP15374 isolated as described above.
It can also be obtained by digesting the DNA encoding the enzyme with an appropriate restriction enzyme or by physically shearing it. In addition, the DNA is, for example, a commercially available DNA based on the nucleotide sequence shown in SEQ ID NO: 1.
It can also be chemically synthesized by an automatic synthesizer.

The present invention also provides a recombinant vector containing either the above-mentioned DNA encoding C-HEP15374 or DNA encoding a peptide which is a fragment of the receptor. The recombinant vector of the present invention is not particularly limited as long as it can be replication-retained in various host cells of prokaryotic and / or eukaryotic cells, and includes a plasmid vector, a viral vector and the like. The recombinant vector is simply a known cloning vector or expression vector available in the art, in which a DNA encoding the above-mentioned C-HEP15374 or a fragment thereof is subjected to appropriate restriction enzymes and ligases, or if necessary. Furthermore, it can be prepared by ligating with a linker or adapter DNA. Examples of such a vector include plasmids derived from E. coli, such as pBR322 and pB
As yeast-derived plasmids such as R325, pUC18, pUC19,
Examples of plasmids derived from Bacillus subtilis such as pSH19 and pSH15 include pUB110, pTP5 and pC194. In addition, as viruses, bacteriophage such as λ phage, SV40, bovine papilloma virus (BPV)
Papovaviruses such as, and Moloney murine leukemia virus (MoMuLV) and other retroviruses, adenovirus (AdV), adeno-associated virus (AAV), vaccinia virus, baculovirus, and other animal and insect viruses.

In particular, the present invention provides an expression vector in which a DNA encoding C-HEP15374 or a fragment thereof is placed under the control of a promoter functional in the intended host cell. The vector used is a promoter region that functions in various host cells of prokaryotic and / or eukaryotic cells and can control the transcription of genes located downstream thereof (for example, when the host is Escherichia coli, the trp promoter, lac promoter, lecA promoter, etc. when the host is Bacillus subtilis, SPO1 promoter, SPO2 promoter, penP promoter, etc., when the host is yeast, PHO5 promoter, PGK promoter, GA
When the host is a mammalian cell such as P promoter and ADH promoter, SV40-derived early promoter, MoM
viral promoters such as uLV-derived long terminal repeats and adenovirus-derived early promoters),
A sequence containing a transcription termination signal of the gene, that is, a terminator region, wherein the promoter region and the terminator region include at least one restriction enzyme recognition site, preferably a unique restriction site that cuts the vector only at that position. It is not particularly limited as long as it is linked via the, but a selectable marker gene for transformant selection (a gene that imparts resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin). , A gene that complements an auxotrophic mutation, etc.) is preferably contained.
Furthermore, when the DNA encoding the C-HEP15374 or a fragment thereof to be inserted does not include a start codon and / or a stop codon, the start codon (ATG, or GTG in some cases) and / or the stop codon (TAG, T
Vectors containing GA and TAA) downstream of the promoter region and upstream of the terminator region are preferably used.

When bacteria are used as the host cells, the expression vector generally needs to contain a replicable unit capable of autonomously replicating in the host cells in addition to the above promoter region and terminator region. In addition, the promoter region contains an operator and Shine-Da near the promoter.
Includes the lgarno (SD) sequence.

When yeast, animal cells or insect cells are used as the host, the expression vector is an enhancer sequence, C-
HEP15374 mRNA 5'side and 3'side untranslated regions,
It is preferable to further include a polyadenylation site and the like.

When the DNA encoding C-HEP15374 of the present invention is isolated from genomic DNA and obtained in a form containing the original promoter and terminator regions,
The expression vector of the present invention can be prepared by inserting the DNA into an appropriate site of a known cloning vector that can be replication-maintained in the target host cell.

The present invention also provides a transformant obtained by transforming a host cell with the above C-HEP15374 expression vector. The host cell used in the present invention is not particularly limited as long as it is compatible with the above expression vector and can be transformed, and a natural cell or an artificially established natural cell usually used in the technical field of the present invention. Various cells such as mutant cells or recombinant cells (for example, bacteria (E. coli, Bacillus subtilis, lactic acid bacteria, etc.), yeast (Saccharomyces,
Pichia, Kliberomyces, etc.), animal cells, insect cells, etc.) are exemplified. Preferably, the host cell is preferably a mammalian cell, particularly a cell of human, monkey, mouse, rat, hamster, or the like, especially a human-derived cell. Specifically, COP, L, C127, Sp2 / 0,
Mouse-derived cells such as NS-1 and NIH3T3, rat-derived cells, hamster-derived cells such as BHK and CHO, CO
Examples include monkey-derived cells such as S1, COS3, COS7, CV1, Vero, and HeLa, diploid fibroblast-derived cells, myeloma cells, human-derived cells such as Namalwa, and the like.

The expression vector can be introduced into the host cell by a conventionally known method. For example, in the case of bacteria, the method of Cohen et al. [Proc. Natl. Acad. Sci. USA.,
69,2110 (1972)], protoplast method [Mol. Gen. Gene
t., 168, 111 (1979)], competent method [J. Mol. Bio
l., 56, 209 (1971)] etc.
En et al. [Proc. Natl. Acad. Sci. USA., 75, 1927
(1978)], lithium method [J. Bacteriol., 153, 163 (198
3)], etc., the method of Graham [Vi
rology, 52, 456 (1973)], and in the case of insect cells, the method of Summers et al. [Mol. Cell. Biol., 3, 2156].
-2165 (1983)] and the like.

The C-HEP15374 or a fragment thereof of the present invention is obtained by culturing a transformant containing the expression vector prepared as described above in a medium and recovering the protein or peptide from the resulting culture. It can be manufactured.

The medium used preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of host cells (transformants). Examples of carbon sources include glucose, dextran, soluble starch, and sucrose, and examples of inorganic or organic nitrogen sources include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large Examples include soybean meal and potato extract. If desired, it may contain other nutrients [eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)]. .

Culturing is performed by a method known in the art. Specific media and culture conditions used according to the host cells are shown below, but the culture conditions in the present invention are not limited to these.

When the host is bacteria, actinomycetes, yeast, filamentous fungi, etc., for example, a liquid medium containing the above-mentioned nutrient source is suitable. A medium having a pH of 5 to 8 is preferable. When the host is E. coli, preferred medium is LB medium, M9
Medium [Miller. J., Exp. Mol. Genet, p. 431, Cold Spr
ing Harbor Laboratory, New York (1972)] and the like. Culturing can be carried out usually at 14 to 43 ° C. for about 3 to 24 hours, with aeration and stirring if necessary. When the host is Bacillus subtilis, it can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours with aeration and stirring if necessary.
When the host is yeast, examples of the medium include Burkholder's minimal medium [Bostian. KL et al, Proc. Natl. Acad. Sci.
USA, 77, 4505 (1980)], and the pH is preferably 5-8. Culturing is usually performed at about 20 to 35 ° C for about 14
It is carried out for about 144 hours, and if necessary, aeration and stirring can be carried out.

When the host is an animal cell, the medium is, for example, a minimum essential medium containing about 5 to 20% fetal bovine serum (M
EM) [Science, 122, 501 (1952)], Dulbecco's modified minimal essential medium (DMEM) [Virology, 8, 396 (195
9)], RPMI 1640 medium [J. Am. Med. Assoc., 19
9, 519 (1967)], 199 medium [Proc. Soc. Exp. Biol.
Med., 73, 1 (1950)] and the like can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually about 30.
It is carried out at -40 ° C for about 15 to 72 hours, and if necessary, aeration and stirring can be carried out.

When the host is an insect cell, the medium is, for example, Grace's medium containing fetal bovine serum [Proc. Natl. Acad.
Sci. USA, 82, 8404 (1985)], and the like.
Is preferably about 5-8. Culture is usually about 20-4
It is carried out at 0 ° C. for 15 to 100 hours, and if necessary, aeration and stirring can be carried out.

Purification of C-HEP15374 or its fragment can be carried out by appropriately combining various commonly used separation techniques depending on the fraction in which the protein or peptide is present.

In a preferred embodiment of the invention,
C-HEP15374 is present on the cell membrane of recombinant mammalian cells. In this case, the culture is centrifuged or filtered to collect the cells, which are suspended in a suitable buffer solution, eg sonicated,
The cells are disrupted (lysed) by freeze-thawing, osmotic shock, etc., and then centrifuged (eg, about 10,000 × g) to collect the supernatant, and further centrifuged (eg, about 1 ×).
The membrane fraction is obtained by recovering the sediment by performing the treatment at about 0,000 × g. Then, after the membrane was destroyed by treatment with a surfactant, etc., anti-C-HEP1 immobilized on an appropriate insoluble carrier
C-HEP15374 can be purified by affinity chromatography using the 5374 antibody.

As another means for rapidly and conveniently obtaining recombinant C-HEP15374 or a fragment thereof, His-ta
Modification of DNA encoding C-HEP15374 or its fragment with g, GST-tag, MBP-tag, etc. and purification by affinity chromatography using metal ion chelate immobilization column, glutathione immobilization column, maltose immobilization column, etc. The method of doing is preferably illustrated.

The present invention also provides an antibody having a specific affinity for C-HEP15374. The antibody may be a polyclonal antibody or a monoclonal antibody, and can be produced by a well-known immunological technique.
Anti-C-HEP15374 antibody is not only a complete antibody molecule, but also C-HE
Any fragment may be used as long as it has an antigen-binding site (CDR) for P15374. For example, Fab, F (a
b ') ₂ , ScFv, minibody and the like.

For example, a polyclonal antibody is C-HEP153
74 protein or fragments thereof (optionally, bovine serum albumin, KLH (K eyhole L impet H emocyani
n) or other carrier protein cross-linked complex) can be used as an antigen, and a commercially available adjuvant (for example,
Complete or incomplete Freund's adjuvant)
From the final immunization, subcutaneously or intraperitoneally administered to an animal about 2 to 4 times every 2 to 3 weeks (the antibody titer of the partially collected serum is measured by a known antigen-antibody reaction, and its increase is confirmed). It can be obtained by collecting whole blood after about 3 to 10 days and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rat, mouse, rabbit, goat, guinea pig and hamster.

Monoclonal antibodies can be synthesized by the cell fusion method (for example, Takeshi Watanabe, principle of cell fusion method and preparation of monoclonal antibody, Akira Taniuchi, Toshitaka Takahashi, "Monoclonal Antibody and Cancer-Basic and Clinical-", No. 1). 2-14, Sanence Forum Publishing, 1985). For example, in mice the factor is
It is administered 4 times subcutaneously or intraperitoneally, and 3 days after the final administration, the spleen or lymph node is collected and leukocytes are collected. This leukocyte and myeloma cell (for example, NS-1, P3X63Ag8 etc.)
Are fused to obtain a hybridoma producing a monoclonal antibody against the factor. Cell fusion is PEG method [ J.
Immunol. Methods , 81 (2): 223-228 (1985)] or the voltage pulse method [ Hybridoma , 7 (6): 627-633 (1988)]. A hybridoma that produces the desired monoclonal antibody can be selected by detecting an antibody that specifically binds to the antigen in the culture supernatant using the well-known EIA or RIA method or the like. Cultivation of the hybridoma producing the monoclonal antibody can be carried out in vitro or in vivo such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and ascites of the animal, respectively.

The antibody of the present invention can be used to detect C-HEP1 in animal tissues.
It can be used to detect the expression of 5374,
It can also be used as a therapeutic agent for diseases associated with abnormal expression of C-HEP15374. In this case, considering the therapeutic effect and safety in humans, the anti-C-HEP15374 antibody of the present invention is preferably a chimeric antibody of human and other animals (for example, mouse), and is a humanized antibody. Is more preferable.
Here, "chimeric antibody" refers to an antibody having a variable region (V region) derived from an immunized animal and a constant region (C region) derived from human, and "humanized antibody" refers to all other regions except CDR. The antibody replaced with a human antibody. The chimeric antibody and the humanized antibody are prepared by, for example, the V region or C
A sequence encoding DR is excised, and a chimeric gene fused with a DNA encoding the C region of an antibody derived from human myeloma is cloned into an appropriate expression vector, and this is introduced into an appropriate host cell to obtain the chimeric gene. It can be obtained by expressing.

The present invention is also an antisense nucleic acid of C-HEP15374 mRNA (having a sequence in which thymine is replaced with uracil in the nucleotide sequence of SEQ ID NO: 1) or an early transcription product. The “antisense nucleic acid” is composed of a nucleotide sequence that can hybridize with the target mRNA (initial transcription product) under physiological conditions of cells expressing the target mRNA (or initial transcription product), and in the hybridized state. It refers to a nucleic acid capable of inhibiting the translation of the polypeptide encoded by the target mRNA (early transcript). Preferably, the antisense nucleic acid of the present invention is a nucleic acid consisting of a base sequence complementary to the base sequence of SEQ ID NO: 1, or a nucleic acid consisting of the base sequence of SEQ ID NO: 1 or the base sequence by post-transcriptional processing (however, Thymine in the sequence is replaced by uracil), resulting in an initial transcript
It is a nucleic acid which comprises a nucleotide sequence that can hybridize under physiological conditions in mammalian cells and can inhibit the translation of the protein encoded by the nucleotide sequence of SEQ ID NO: 1 in the hybridized state. The type of antisense nucleic acid is DN
A or RNA, or DNA
/ RNA chimera.

Such an antisense nucleic acid is C-HEP153.
C-HEP153, as well as the anti-C-HEP15374 antibody described above, as well as a probe and primer for investigating the expression of 74.
It can also be used as a therapeutic agent for diseases associated with abnormal expression of 74. In this case, since the phosphodiester bond of the natural antisense nucleic acid is easily decomposed by the nucleolytic enzyme existing in the cell, the antisense nucleic acid of the present invention has a thiophosphate type ( Phosphate-bonded P
═O is replaced with P═S) or modified nucleotides such as 2′-O-methyl type can be used for synthesis. Other important factors in the design of antisense nucleic acids include enhancing water solubility and cell membrane permeability, which can be overcome by devising the dosage form such as using liposomes and microspheres. .

The length of the antisense nucleic acid of the present invention is C-HE.
There is no particular limitation as long as it can specifically hybridize with the mRNA of P15374 or the early transcription product.
It may be a sequence having a length of about 5 bases and containing a sequence complementary to the entire sequence of mRNA (initial transcription product).
From the viewpoint of easiness of synthesis and antigenicity, preferably about 15
An oligonucleotide consisting of about 30 bases is exemplified. When the antisense nucleic acid is an oligo DNA of about 25 mer, the base sequence that can hybridize with C-HEP15374 mRNA under physiological conditions usually has about 90% or more identity, although it varies depending on the base composition of the target sequence. Anything will do.

The target sequence of the antisense nucleic acid of the present invention is
By hybridizing the antisense nucleic acid, C-
There is no particular limitation as long as it is a sequence that inhibits the translation of the HEP15374 protein, and it may be the entire sequence or a partial sequence of C-HEP15374 mRNA, or the intron part of the initial transcription product. However, when using an oligonucleotide as the antisense nucleic acid, the target sequence is C-HE
It is preferably located from the 5'end of the mRNA of P15374 to the C terminus of the coding region. It is preferably a region from the 5'end to the N-terminal side of the coding region, and most preferably a base sequence near the start codon. Further, the target sequence is preferably selected such that the antisense nucleic acid complementary thereto does not form a secondary structure such as a hairpin structure.

Furthermore, the antisense nucleic acid of the present invention comprises C-
Not only inhibits translation by hybridizing with HEP15374 mRNA or early transcription product, but also binds with double-stranded DNA C-HEP15374 gene to form triplex (triplex) and inhibits transcription of mRNA. It may be obtained.

Another preferred embodiment of the substance that suppresses the expression of the C-HEP15374 gene is that the C-HEP15374 mRNA or the initial transcript is specific within the coding region (including the intron portion in the case of the initial transcript). It is a ribozyme that can be cleaved into. The term "ribozyme" refers to RNA having an enzymatic activity for cleaving nucleic acid. Recently, it has been revealed that an oligo DNA having a base sequence of the enzyme active site also has a nucleic acid cleaving activity. Will be used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme is the self-splicing RN found in infectious RNAs such as viroids and virusoids.
A is known, and a hammerhead type, a hairpin type, etc. are known. The hammerhead type exhibits enzymatic activity with about 40 bases, and several bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are converted into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage that it does not attack genomic DNA because it uses only RNA as a substrate. When C-HEP15374 mRNA has a double-stranded structure by itself, the target sequence is made single-stranded by using a hybrid ribozyme in which an RNA motif derived from a viral nucleic acid capable of specifically binding to RNA helicase is linked. You can [ Proc. Natl. Acad. Sci. USA ,
98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, it may be a hybrid ribozyme in which tRNA-modified sequences are further linked in order to promote translocation to the cytoplasm. Nucleic Acids Re
s. , 29 (13): 2780-2788 (2001)].

Of a substance that suppresses the expression of the C-HEP15374 gene
In yet another embodiment, C-HEP15374 mRNA or
Partial sequence within the coding region of the early transcript (of the early transcript
Double-stranded oligo complementary to the intron part)
It is RNA. Introduce short double-stranded RNA into cells
And RNA whose mRNA complementary to the RNA is degraded
The phenomenon called interference (RNAi) has long been known as nematode and kelp.
It was known for insects and plants, but recently this phenomenon has
Since it was confirmed that it also occurs in cellsNature,411
(6836): 494-498 (2001)], as an alternative technology of ribozyme
Is attracting attention.

The antisense oligonucleotides and ribozymes of the present invention determine the target sequence of mRNA or early transcription product based on the cDNA sequence of C-HEP15374 or the genomic DNA sequence, and a commercially available automatic DNA / RNA synthesizer (Applied. (Biosystems, Beckman, etc.) and synthesize a sequence complementary thereto. A double-stranded oligo RNA having RNAi activity has a sense strand and an antisense strand
Synthesized by an automatic NA / RNA synthesizer, denatured in a suitable annealing buffer at about 90 to about 95 ° C for about 1 minute, and then annealed at about 30 to about 70 ° C for about 1 to about 8 hours. Can be prepared by Alternatively, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide chains so that they overlap with each other, annealing them, and ligating them with ligase.

The antisense nucleic acid and ribozyme can also be provided as an expression vector containing DNA encoding them. The expression vector is the above antisense RN.
A or ribozyme may be functionally linked to a promoter capable of exhibiting promoter activity in a mammalian target cell, or may be changed to a form that is functionally linked in a target cell under certain conditions. Must be placed in the correct position. The promoter to be used is not particularly limited as long as it can function in the target cells of the mammal to be administered, and examples thereof include SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR. , Viral promoters such as adenovirus-derived early promoter, and mammalian constitutive protein gene promoters such as β-actin gene promoter, PGK gene promoter and transferrin gene promoter. The phrase "arranged at a position capable of changing to a form functionally linked under certain conditions" means, for example, that a promoter and an antisense RNA or a DNA encoding a ribozyme is the antisense from the promoter. R
It has a structure divided by two recombinase recognition sequences arranged in the same direction, which are separated by a spacer sequence having a length sufficient to prevent the expression of NA or the ribozyme, and specifically recognizes the recognition sequence. It means that the spacer sequence is excised in the presence of a recognizing recombinase, and the antisense RNA or the DNA encoding the ribozyme is arranged so as to be operably linked to a promoter.

The vector used as an expression vector encoding the antisense nucleic acid or ribozyme of the present invention is not particularly limited, but as a vector suitable for administration to mammals such as humans, retrovirus, adenovirus, adeno-associated Viral vectors such as virus, herpes virus, vaccinia virus, pox virus, polio virus, Sindbis virus, Sendai virus and the like can be mentioned. Adenovirus has advantages such as extremely high gene transfer efficiency and can be introduced into non-dividing cells. However, since the integration of the transgene into the host chromosome is extremely rare, gene expression is transient and usually lasts only about 4 weeks. Considering the persistence of the therapeutic effect,
Also preferred is the use of adeno-associated virus, which has a relatively high gene transfer efficiency, can be introduced into non-dividing cells, and can be integrated into the chromosome via the inverted terminal repeat sequence (ITR).

The present invention also provides DNA encoding the above-mentioned C-HEP15374 or a fragment thereof, or anti-C-HEP153.
74. A method for diagnosing various diseases such as cancer using the antibody is provided. Diseases that can be diagnosed by the method of the present invention include cancers of various organs, liver cirrhosis and Alzheimer's disease, and particularly, brain, thyroid, lung, stomach, pancreas, kidney, colon,
Effective for, but not limited to, the diagnosis of cancers in the prostate, testis and uterus, including C-HEP15374 protein or C-HE
The expression of the P15374 transcript was measured in the tissue at the lesion site or the site of onset of a given disease and the corresponding tissue in normal animals, and when a significant difference in the expression level was observed between the two, C- HEP15374 is considered to be a marker for the disease. Therefore, the present invention also provides a method for selecting a disease in which C-HEP15374 can be a disease marker.

The expression of C-HEP15374 protein is detected by contacting an anti-C-HEP15374 antibody obtained as described above with a biological sample such as a predetermined tissue homogenate or body fluid, and detecting an antigen-antibody reaction with an appropriate label or the like. You can check by doing. On the other hand, the expression of the C-HEP15374 transcript was obtained by using Northern or Southern high as a template with mRNA or cDNA extracted from a predetermined tissue homogenate or body fluid, or a sample thereof, and a DNA encoding C-HEP15374 or a fragment thereof. Hybridization or PCR using the DNA as a primer
It is possible to detect and quantify by carrying out.

If it is determined that C-HEP15374 can serve as a disease marker as a result of the above selection method, the tissue derived from the predicted lesion site or onset causative site of an animal that may be affected by the disease is selected. C-HEP1 in biological samples
The presence or absence of morbidity can be diagnosed by measuring the expression level of the 5374 protein or C-HEP15374 transcript and comparing it with the normal value. Also, depending on the degree of disease progression, C-HEP153
If it turns out that the expression distribution of 74 is different,
By examining the expression profile of C-HEP15374 in a sample from a patient's tissue of interest, the degree of disease progression can be determined. Also, for diseases in which there are different types depending on the medical condition, etc., when a correlation is found between the medical condition and the expression profile of C-HEP15374, the expression profile of C-HEP15374 in a sample derived from a predetermined tissue of the patient is examined. This also makes it possible to type the disease. Furthermore, C-HEP15374 in tumors of each organ
Since the expression of γ is significantly different from that in normal tissue, the method of the present invention is considered to be useful for diagnosing cancer metastasis.

The present invention also provides a reagent for use in the above-mentioned method for diagnosing a disease. Such a reagent is C-HEP1
DNA encoding 5374 or a fragment thereof, C-
It contains, as a main component, a primer pair capable of specifically amplifying all or part of the DNA encoding HEP15374, or an antibody having a specific affinity for C-HEP15374.

C-HEP15374 of the present invention is
It is particularly strongly expressed in the colon, olfactory brain, testis, etc. The distribution of expression in the brain shows that the expression is relatively high in the olfactory brain and cerebral cortex. Therefore, homogenates and secretions of these tissues are considered to be effective sources for screening physiological ligands of C-HEP15374.

The present invention also provides a method for screening a ligand for C-HEP15374, which comprises contacting a test sample with a peptide that is a C-HEP15374 protein or a fragment thereof and selecting a substance that binds to the protein or the peptide. provide. The test sample may be any known compound or novel compound in addition to the above-mentioned tissue homogenate or secretory product, for example, a compound library prepared by using combinatorial chemistry technology, solid phase synthesis or phage. Examples include random peptide libraries prepared by the display method, natural components derived from microorganisms, plants and animals, marine organisms, and the like.

In a preferred embodiment, C-HEP15374
Or a peptide containing its ligand binding domain,
A ligand for C-HEP15374 can be screened by immobilizing a conventionally used insoluble carrier on an affinity column or the like by a well-known method and contacting it with a test sample. If desired, the test sample can be labeled and the binding of the ligand can be measured by detecting the label that binds to the solid phase.

The ligand obtained as described above can be used to screen a substance that inhibits or promotes the activity of the C-HEP15374 receptor. This method is C-HEP1
5374 protein or a peptide containing a ligand-binding domain thereof is contacted with a ligand for C-HEP15374 in the presence or absence of a test substance, and the binding activity between the protein or the peptide and the ligand under both conditions is shown. Including comparing. It is determined that if the binding activity increases in the presence of the test substance, the test substance promotes the activity of C-HEP15374, and if the binding activity decreases, it inhibits the activity of C-HEP15374.

Screening for a substance that inhibits or promotes the activity of C-HEP15374 is carried out by contacting a cell expressing the protein with a ligand for the protein in the presence and absence of a test sample, and the protein and the ligand. It can also be carried out by comparing the change in the cell due to the binding with and under both conditions. The α subunit of G protein coupled with GPCR can be roughly classified into three families depending on the type of effector on which it acts. Of these, Gs
Promotes the activity of adenylate cyclase (AC) when activated by GPCR, resulting in an increase in intracellular cAMP level. On the other hand, Gi suppresses AC activity, and intracellular c
Decrease the amount of AMP. Therefore, C-HEP15374 is Gs
Or, in the case of coupling with Gi, whether the activity of C-HEP15374 is inhibited or promoted by examining the change in adenylate cyclase activity, intracellular cAMP amount or expression of a reporter gene under the control of cAMP response element. Can be determined.

When Gq is activated by GPCR, it promotes the activity of phospholipase C (PLC), and as a result, the intracellular calcium ion concentration increases. Thus, when C-HEP15374 is coupled to Gq, a 12-O-tetradecanoylphorbol-13-acetate (TPA) response element that responds to phospholipase C activity, intracellular calcium ion concentration, or increased calcium ion concentration. Whether or not the activity of C-HEP15374 is inhibited or promoted can be determined by measuring the change in the expression of the reporter gene under the control of.

A substance that inhibits or promotes the activity of C-HEP15374 can be screened without the need for a ligand for C-HEP15374. Therefore, the present invention also provides a lipid bilayer membrane containing the C-HEP15374 protein, as well as a G protein α subunit (Gα) belonging to a certain family.
The present invention provides a screening system in which a system containing a polypeptide containing at least the receptor binding region of Gα and a guanine nucleotide binding region of Gα as a constituent is used as one constituent unit, and the constituent unit is present for the receptor binding region of each family of Gα Then, using the screening system, C-HEP15374
The present invention provides a method for screening for a ligand to, or a substance that inhibits or promotes the activity of the protein.

Hereinafter, substances that promote the activity of C-HEP15374 will be described.
C-HEP15374 agonists, substances that inhibit the same activity C-HE
Sometimes it is an antagonist of P15374. Here, the “agonist activity” refers to the property of specifically binding to the C-HEP15374 receptor and shifting the equilibrium state between the active and inactive forms of C-HEP15374 to the more active side. Therefore, substances having agonistic activity include so-called full agonists and partial agonists as well as physiological ligands of C-HEP15374. On the other hand, "antagonist activity" means that the compound binds competitively to the ligand binding site of C-HEP15374, but has little or no effect on the equilibrium state between active and inactive forms, or any of C-HEP15374. Bound to the site, C-HE
The property of shifting the equilibrium state between the active and inactive forms of P15374 to the more inactive side. Therefore, the “antagonist” in the present invention is defined as a concept including both so-called “neutral antagonist” and “inverse agonist”.

The screening system of the present invention is C-HEP15374.
A lipid bilayer membrane containing a protein, and a polypeptide containing at least a Gα receptor binding region belonging to a family and an arbitrary Gα guanine nucleotide binding region,
A system including essential components is defined as one structural unit, and the structural unit is used as each family of Gα (that is, Gα _s , Gα _i , Gα).
_It is a series of receptor-G protein co-expression systems obtained by constructing the receptor binding region of _q ). The C-HEP15374 protein is as described above. The origin of the lipid bilayer membrane holding the C-HEP15374 protein is not particularly limited as long as the receptor protein can take the original three-dimensional structure on the membrane, but is preferably human, bovine, pig, monkey, mouse. , A fraction containing a cell membrane of mammalian cells such as rat, for example, an intact cell, a cell homogenate, or a cell membrane fraction fractionated from the homogenate by centrifugation or the like. However, for example, an artificial lipid bilayer membrane prepared by a conventional method from a solution in which various lipids such as phosphatidylcholine, phosphatidylserine, and cholesterol are mixed at an appropriate ratio, preferably a ratio close to that in the cell membrane of mammalian cells, is also used. It can be preferably used in one embodiment of the invention.

G_sGα belonging to the family (Gα_s) Is
Uses adenylate cyclase as an effector and promotes its activity
For example, Gα_s-1~ Gα_s-4, Or G
_olfEtc. G_iGα belonging to the family (Gα
_i) Uses adenylate cyclase as an effector and its activity
For example, Gα_i-1~ Gα_i-3,Ah
R is G_zEtc. G_qGα belonging to the family
(Gα_q) Uses phospholipase C as an effector and
Which promotes sex, for example, Gα_qOr G₁₆
Etc. Gα of this screening system_sPolypep
Chid, Gα_iPolypeptide and Gα_qPolypeptide is it
Each is a region (RB region) involved in binding with its own GPCR.
Region) and any Gα guanine nucleotide binding
As long as you have the area to give (GB area),
You may lack it. Is it the result of X-ray crystal structure analysis of Gα?
Et al., The GPCR binding region is near the C-terminus, while
The nin nucleotide binding region is the nucleoprotein of the ras protein.
A region homologous to the tide binding site (from the N-terminal side,
Called G, box, G'box, G "box
Amino acid motif and highly helical domain
The top of the αE helix in the main and the αF helix
It is clear that it is. Against C-HEP15374
A physiological ligand or agonist that binds to the receptor
Then, the Gα activation domain of the receptor is coupled to the receptor.
That interacts with the RB region of Gα
Change and the GDP dissociates from the GB region to accelerate.
GTP is bound gently. Gα-GTP acts on the effector
Then, its activity is promoted or suppressed. Meanwhile, inverse
When a gonist binds, the conformational change of the receptor occurs.
Activation deactivates the Gα activation domain,
The sexualized Gα-GTP level is decreased, and the effector is affected.
Is hindered. Here, instead of GTP, for example, ³⁵
Depending on the GTPase activity of Gα such as S-labeled GTPγS,
Add a GTP analog that does not undergo hydrolysis to the system
If bound, bound to the membrane in the presence and absence of the test substance
By measuring and comparing radioactivity, G in Gα
Evaluate the effect of test substance on DP / GTP exchange reaction
Ligand for C-HEP15374 (or
A substance having nyst or antagonist activity.
Can be cleaned. That is, the presence of the test substance
If the radioactivity increases under the
If it has an agonistic activity against it and the radioactivity decreases,
The test substance has an antagonistic activity against C-HEP15374.
Have.

Once the ligand (or agonist or antagonist) for C-HEP15374 is screened, the family of Gα that couples to the receptor becomes clear. Therefore, the subsequent screening is carried out by selecting Gα polypeptide belonging to the family. It can be carried out using only the system containing the components.

The activity of the ligand for C-HEP15374 is G
It is also possible to measure the action of the α polypeptide on the effector as an index. In this case, the screening system of the present invention needs to include a lipid bilayer membrane including an effector as a constituent in addition to the C-HEP15374 protein. Also, G
The α polypeptide should further include a region for interacting with the effector. Since Gαs belonging to each family have different effector types or directions of action, it is more preferable that all Gα polypeptides have a common effector interaction region than each has its own effector interaction region. preferable. Therefore, in the present screening system, at least two kinds of Gα polypeptides are chimeric polypeptides containing the effector interaction region of Gα belonging to another family. For example, when PLC is used as the effector, the Gα _q polypeptide may be native, but the Gα _s and Gα _i polypeptides are chimeric polypeptides in which the effector interaction region is replaced with that of Gα _q. It must be a peptide. The simplest example of a chimeric polypeptide containing a Gα effector interaction region belonging to another family is about 5 amino acids at the C-terminus of Gα belonging to another family (ie, RB region).
In which the C-terminal sequence is replaced with.

In this screening system, three types of Gα
When the polypeptide contains a Gα _s or Gα _i effector interaction region, a lipid bilayer membrane containing adenylate cyclase is used as an effector. On the other hand, Gα polypeptide
When the effector interaction region of Gα _q is included, it is necessary to use a lipid bilayer membrane containing phospholipase C as an effector.

Adenylate cyclase (A
In the screening system including C), the action of the Gα polypeptide on the effector can be evaluated by directly measuring the AC activity. Any known method may be used to measure the AC activity. For example, GTP is added to the membrane fraction containing AC, and the amount of cAMP produced is determined by anti-cA.
RI (eg, ³² P), enzyme (eg, alkaline phosphatase, peroxidase, etc.) using MP antibody,
Examples include, but not limited to, a method of measurement by a competitive immunoassay with cAMP labeled with a fluorescent substance (eg, FITC, rhodamine, etc.). When the Gα polypeptide contains the effector interaction region of Gα _s , the AC activity is measured and compared in the presence and absence of the test substance, and if the AC activity increases in the presence of the test substance, the test substance is C-
It has agonistic activity against HEP15374. On the contrary, AC
If the activity is decreased, the test substance has an antagonist activity to C-HEP15374. On the other hand, when the Gα polypeptide contains the effector interaction region of Gα _i , if the AC activity is increased in the presence of the test substance, the test substance is C-HEP15374.
It has an antagonistic activity against. On the contrary, if the AC activity is decreased, the test substance has an agonist activity to C-HEP15374.

When intact animal cells are used as the screening system, the action of Gα polypeptide on AC can also be evaluated by measuring the amount of intracellular cAMP. The intracellular cAMP amount can be measured by incubating the cells in the presence and absence of the test substance for an appropriate period of time, and then performing the above competitive immunoassay on the extract obtained by disrupting the cells. However, any other known method can be used.

As another embodiment, there is also a method of evaluating the cAMP level by measuring the expression level of a reporter gene under the control of the cAMP response element (CRE). The expression vector used here will be described in detail later, but in brief, an animal cell into which a vector containing an expression cassette in which a DNA encoding a reporter protein is ligated downstream of a promoter containing CRE is introduced is used as a test substance. It is said that the amount of intracellular cAMP is evaluated by measuring and comparing the expression of the reporter gene in the extract obtained by culturing the cells in the presence or absence for an appropriate time and crushing the cells using a known method. It is a thing.

Therefore, when the Gα polypeptide contains the effector interaction region of Gα _s , if the intracellular cAMP amount (or the expression amount of the reporter gene under CRE control) increases in the presence of the test substance, Test substance is C-HEP15374
Have agonistic activity against. On the contrary, if the amount of cAMP (or the amount of reporter gene expression) decreases,
The test substance has an antagonistic activity against C-HEP15374. On the other hand, when the Gα polypeptide contains the effector interaction region of Gα _i , intracellular cAM in the presence of the test substance
When the P amount (or the expression amount of the reporter gene under CRE control) increases, the test substance has an antagonist activity against C-HEP15374. On the contrary, if the amount of cAMP (or the amount of reporter gene expression) decreases, the test substance has an agonistic activity against C-HEP15374.

On the other hand, phospholipase C (P
LC) in a screening system (that is, when the Gα polypeptide contains the effector interaction region of Gα _q ), the effect of the Gα polypeptide on the effector should be evaluated by directly measuring PLC activity. You can PL
C activity is evaluated by, for example, adding ³ H-labeled phosphatidylinositol-4,5-diphosphate to the PLC-containing membrane fraction and measuring the amount of diacylglycerol produced by a known method. You can PLC activity is measured and compared in the presence and absence of the test substance, and if the PLC activity increases in the presence of the test substance, the test substance has an agonist activity to C-HEP15374. On the contrary, if the PLC activity is decreased, the test substance is C-HEP15374.
It has an antagonistic activity against.

When intact animal cells are used as the screening system, the action of Gα polypeptide on PLC can also be evaluated by measuring the intracellular Ca ²⁺ amount. The amount of intracellular Ca ^{2+ was} determined by incubating the cells in the presence and absence of the test substance for an appropriate time, and then measuring the amount of fluorescent probe (fura-2, indo-1, fluor-3, Calcium-GreenI).
Etc.) or by using aequorin, which is a calcium-sensitive photoprotein, etc., but any other known method may be used.
An apparatus suitable for spectroscopic measurement using a fluorescent probe is a FLIPR (Molecular Devices) system.

In another embodiment, the expression level of a reporter gene under the control of TPA (12-O-tetradecanoylphorbol-13-acetate) response element (TRE) that is up-regulated by Ca ²⁺ is measured. There is also a method of evaluating the amount of Ca ²⁺ . Although the expression vector used here will be described in detail later, in summary, an animal cell into which a vector containing an expression cassette in which a DNA encoding a reporter protein is linked downstream of a promoter containing TRE is introduced is used as a test substance. Evaluate the intracellular Ca ²⁺ amount by measuring and comparing the expression of the reporter gene in the extract obtained by culturing the cells in the presence and absence for an appropriate period of time using a known method Is to do.

Therefore, intracellular Ca ²⁺ in the presence of the test substance
If the amount (or the expression amount of the reporter gene under the control of TRE) increases, the test substance has an agonist activity to C-HEP15374. On the contrary, when the intracellular Ca ²⁺ amount (or the reporter gene expression amount) is decreased, the test substance has an antagonist activity against C-HEP15374.

The test substance to be used in the screening method of the present invention may be any known compound or novel compound. The random peptide library prepared by the above, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.

A preferred embodiment of a system containing a C-HEP15374 protein-containing lipid bilayer membrane and a Gα polypeptide, which are one structural unit of the screening system of the present invention, encodes a C-HEP15374 protein. Expression vector containing the DNA to be expressed and R of Gα belonging to a family
A host animal cell, a homogenate of the cell, or a membrane fraction derived from the cell, which is transfected with an expression vector containing a DNA encoding a polypeptide containing at least the B region and the GB region of arbitrary Gα.

The DNA encoding the C-HEP15374 protein is as described above. On the other hand, DNAs encoding three types of Gα polypeptides are at least G of each family.
As long as it has a sequence encoding the RB region of α and a sequence encoding the GB region of any Gα, a part of the entire coding sequence of Gα may be deleted. The sequences of various Gα genes are known, and as described above, the RB region and GB region are well known from the results of X-ray crystal structure analysis of Gα. Therefore, a person skilled in the art can easily construct a fragment in which a part of the coding sequence of Gα is deleted, if desired.

In the screening system using the action of the Gα polypeptide on the effector as an index, the DNA encoding the Gα polypeptide needs to further include a nucleic acid sequence encoding the effector interaction region. As mentioned above, 3
Since the Gα polypeptides of one species have a common effector interaction region, at least two Gα polypeptides are chimeras having another family of effector interaction regions.
The simplest example of the DNA encoding the chimeric polypeptide is Gα having an effector interaction region of interest.
A sequence in which about 5 amino acids at the C-terminal of the cDNA of cDNA is replaced with a DNA sequence encoding the C-terminal sequence of Gα of another family by using a known method such as PCR.

The DNA encoding the C-HEP15374 protein and the DNA encoding the Gα polypeptide must be operably linked to a promoter capable of exhibiting promoter activity in host mammalian cells. The promoter used is not particularly limited as long as it can function in mammalian cells, and examples thereof include SV40-derived early promoter, cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived LTR, and adenovirus-derived early promoter. And promoters of mammalian constitutive protein genes such as β-actin gene promoter, PGK gene promoter and transferrin gene promoter. The expression vector used preferably contains, in addition to the above promoter, a transcription termination signal, that is, a terminator region, downstream thereof, so that a coding DNA can be inserted between the promoter region and the terminator region. It is desirable to have an enzyme recognition site, preferably a unique restriction enzyme recognition site that cuts the vector at only one site. Furthermore, the expression vector may further contain a selection marker gene (drug resistance gene such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, auxotrophic mutation complementary gene, etc.).

Vectors used in the screening system of the present invention include retroviruses, adenoviruses, adeno-associated viruses suitable for use in mammals such as humans,
Examples include viral vectors such as herpes virus, vaccinia virus, pox virus, polio virus, Sindbis virus, Sendai virus.

DNA encoding the C-HEP15374 protein and G
The DNA encoding the α polypeptide may be carried on two separate expression vectors and co-transfected into host cells, or it may be dicistronically or monocistronically inserted on one vector. , May be introduced into the host cell.

The host cell is not particularly limited as long as it is a mammalian cell such as human, monkey, mouse, rat and hamster. Specifically, COP, L, C127, Sp2 / 0, NS-1, NIH3T
3, mouse-derived cells such as ST2, rat-derived cells, BHK, CHO
Hamster-derived cells such as COS1, COS3, COS7, CV1, Ver
Examples include monkey-derived cells such as o and human-derived cells such as HeLa and 293.

Gene transfer into host cells may be carried out by any known method that can be used for gene transfer into mammalian cells, for example, calcium phosphate coprecipitation method, electroporation method, liposome method, microinjection method. Etc.

The host cell into which the gene has been introduced is, for example,
Minimum essential medium (ME containing about 5-20% fetal bovine serum)
M) [Science, 122, 501 (1952)], Dulbecco's modified minimal essential medium (DMEM) [Virology, 8, 396 (1959)], RPM
I1640 medium [J. Am. Med. Assoc., 199, 519 (1967)],
199 medium [Proc. Soc. Exp. Biol. Med., 73, 1 (195
0)] and the like. The pH of the medium is preferably about 6 to about 8, and the culture temperature is usually about 30.
Is about 40 ° C.

The animal cells introduced with the DNA encoding the C-HEP15374 protein and the DNA encoding the Gα polypeptide obtained as described above may be used as intact cells depending on the screening method used. Or, a cell homogenate obtained by disrupting the cells in an appropriate buffer or by centrifuging the homogenate under appropriate conditions (for example, about 10,000 xg
After centrifuging at about 100 ℃ and collecting the supernatant, about 100,000 ×
It may be in the form of a membrane fraction to be isolated.

When the ligand property of the test substance is evaluated by, for example, the GTPγS binding assay or the activity of the effector is directly measured, the screening system used is preferably prepared from cells as described above. It is the membrane fraction to be processed. On the other hand, intracellular cAMP amount (or cA
When the ligand property of the test substance is evaluated by measuring the expression level of MP-responsive reporter) or the intracellular Ca ²⁺ amount (or the expression level of Ca ²⁺ -responsive reporter), the screening system to be used is It is an intact animal cell.

The ligand activity was evaluated by the cAMP-responsive reporter (when the effector is adenylate cyclase).
Alternatively, when the expression level of a Ca ²⁺ responsive reporter (when the effector is phospholipase C) is used as an index, the host animal cell has a promoter region containing a cAMP response element (CRE) or TPA response element (TRE). It is necessary that a vector containing an expression cassette in which a DNA encoding a reporter protein is functionally linked is introduced downstream. CRE is a cis element that activates gene transcription in the presence of cAMP, and a consensus sequence includes a sequence containing TGACGTCA. As long as it retains cAMP responsiveness, CRE is partially deleted, substituted, or inserted. Alternatively, the sequence may include an addition.
On the other hand, TRE is a cis element that activates gene transcription in the presence of Ca ²⁺ , and TG is a consensus sequence.
Examples thereof include a sequence containing ACTCA, but may be a sequence containing a deletion, substitution, insertion or addition in a part of the sequence as long as it retains Ca ²⁺ responsiveness. As the promoter sequence containing CRE or TRE, the above-mentioned viral promoter or mammalian constitutive protein gene promoter can be similarly used, and a restriction enzyme and DNA ligase can be used, or PCR can be used to A CRE or TRE sequence can be inserted downstream of the promoter sequence. As the reporter gene under the control of CRE or TRE, any known gene whose gene expression can be detected and quantified rapidly and easily may be used, and examples thereof include luciferase, β-galactosidase, β-glucuronidase, and alkaline phosphatase. , And DNA encoding a reporter protein such as peroxidase, but not limited thereto. It is more preferable that a terminator sequence is arranged downstream of the reporter gene. As a vector carrying such a CRE (or TRE) -reporter expression cassette, a known plasmid vector or viral vector can be used.

Another preferred embodiment of a system containing a C-HEP15374 protein-containing lipid bilayer membrane and a Gα polypeptide, which is one structural unit of the screening system of the present invention, is a C-HEP15374 protein. On the C-terminal side, the RB region of Gα belonging to a family and the G of arbitrary Gα
A host animal cell, a homogenate of the cell, or a membrane fraction derived from the cell, which is transfected with an expression vector containing a DNA encoding a fusion protein in which a polypeptide containing at least the B region is linked.

The DNA encoding the C-HEP15374 protein and the DNA encoding the polypeptide containing the Gα RB binding region and the arbitrary Gα GB region of each family can be obtained as described above. Those skilled in the art can easily construct a DNA encoding a fusion protein of C-HEP15374 and Gα polypeptide by appropriately combining known genetic engineering techniques based on these DNA sequences. For example, by using PCR etc., C-HEP1
Examples of the method include ligation using a DNA ligase so that the DNA coding for Gα polypeptide is aligned with the DNA coding for 5374 by removing the termination codon.

DNA encoding the obtained fusion protein
Is inserted into an expression vector as described above and introduced into a host mammalian cell using the gene transfer technique described above.
When the fusion protein was expressed on the membrane of the obtained animal cell, the Gα activation domain on the intracellular third loop of the receptor and the RB region of Gα were in the absence of a physiological ligand for C-HEP15374. Interacting with GDP in Gα
It can accelerate the GTP exchange reaction. Therefore, Gα is in a constantly activated state.

Also for such fusion protein-expressing cells, any form of intact cells, cell homogenates, and membrane fractions can be appropriately selected and used according to the screening method used.

In yet another aspect of the present invention, C-HE
As a lipid bilayer membrane containing P15374 protein, and a screening system containing Gα polypeptide as a constituent,
A purified C-HEP15374 protein and Gα polypeptide, or a purified fusion protein of the receptor and Gα reconstituted in an artificial lipid bilayer membrane can be used. The C-HEP15374 protein can be purified from a membrane fraction obtained from an appropriate tissue of human or other mammal by affinity chromatography using an anti-C-HEP15374 antibody. Alternatively, the receptor is an anti-C-HEP15374 antibody, His-tag, or GST from a recombinant cell into which an expression vector containing a DNA encoding a C-HEP15374 protein has been introduced.
It can also be purified by affinity chromatography or the like using -tag or the like. Similarly, a fusion protein of the receptor and Gα is also obtained from recombinant cells into which an expression vector containing a DNA encoding the fusion protein has been introduced.
It can be purified by affinity chromatography using 5374 antibody, His-tag, GST-tag and the like.

Examples of lipids constituting the artificial lipid bilayer membrane include phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol (Ch), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and the like. A mixture of one kind or two kinds or more in an appropriate ratio is preferably used.

For example, receptor and Gα, or receptor-Gα
The artificial lipid bilayer membrane (proteoliposome) incorporating the fusion protein can be prepared as follows. First, an appropriate amount of a mixed lipid / chloroform solution of PC: PS: Ch = 3: 1: 0.8 is dispensed into a glass tube,
After chloroform is evaporated to dry the lipid into a film, an appropriate buffer solution is added and suspended, and then ultrasonically treated to uniformly disperse, and a buffer solution containing a surfactant such as CHAPS and SDS is further added. Completely dissolve the lipids.
The purified receptor and Gα or the receptor-Gα fusion protein were added to the mixture so that the concentration would be about 1/200 of the lipid, and the mixture was incubated for 20 to 30 minutes in ice with occasional stirring, and then appropriate. Dialyzed against different buffers. About 1
A desired proteoliposome can be prepared by collecting the precipitate by centrifuging at 0,000 xg for 30 to 60 minutes.

The substance which inhibits or promotes the activity of C-HEP15374, which is selected by the screening system and the screening method as described above, can be mixed with an arbitrary pharmaceutically acceptable carrier so that the functional abnormality of C-HEP15374 can be obtained. It can be used as a therapeutic drug for diseases associated with. Therefore, the present invention provides a therapeutic agent for a disease containing an agonist or antagonist for C-HEP15374 selected by the screening method of the present invention as an active ingredient.

The pharmaceutically acceptable carrier is, for example,
Excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methyl cellulose, hydroxypropyl cellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose , Binders such as starch, starch, carboxymethyl cellulose, hydroxypropyl starch,
Disintegrating agents such as sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, magnesium stearate, aerosil, talc,
Lubricants such as sodium lauryl sulfate, citric acid, menthol, glycyrrhizin ammonium salt, glycine, aromatic agents such as orange powder, sodium benzoate, sodium bisulfite, preservatives such as methylparaben, propylparaben, citric acid, sodium citrate, etc. Stabilizers such as acetic acid,
Suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants,
Examples thereof include, but are not limited to, water, physiological saline, a diluent such as orange juice, cocoa butter, polyethylene glycol, and a base wax such as white kerosene.

Formulations suitable for oral administration include solutions prepared by dissolving an effective amount of a ligand (or agonist or antagonist) in a diluent such as water, physiological saline or orange juice, or an effective amount of the ligand (or agonist or antagonist). ) As a solid or granule, a sachet or a tablet, a suspension in which an effective amount of a ligand is suspended in an appropriate dispersion medium, and an effective amount of the ligand (or an agonist or antagonist) is dissolved. An emulsion in which a solution is dispersed in an appropriate dispersion medium and emulsified is used.

Formulations suitable for parenteral administration (eg subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions,
It may contain an antioxidant, a buffer solution, a bacteriostatic agent, an isotonicity agent and the like. Also included are aqueous and non-aqueous sterile suspensions, which include suspending agents, solubilizing agents, thickening agents,
Stabilizers, preservatives and the like may be included. In the case of local injection, an injection solution in which a ligand (or an agonist or an antagonist) as an active ingredient is dissolved or suspended is preferable. Alternatively, a sustained-release preparation can be prepared by using a biocompatible material such as collagen. Since pluronic gel gels at body temperature and is liquid at lower temperatures, it should be long-lasting by locally injecting the ligand (or agonist or antagonist) together with the pluronic gel to gel around the target tissue. You can The ligand (or agonist or antagonist) preparation can be enclosed in a container such as an ampoule or a vial in a single dose or multiple doses. Alternatively, the ligand (or agonist or antagonist) and a pharmaceutically acceptable carrier may be lyophilized and stored in a state in which they may be dissolved or suspended in an appropriate sterile vehicle immediately before use.

The dose of the ligand (or agonist or antagonist) preparation of the present invention depends on the activity of the ligand (whether it is a full agonist or a partial agonist, or a neutral antagonist or an inverse agonist), the severity of the disease, and the species of animal to be administered. Generally, the amount of the ligand is about 0.001 mg to about 100 m per day for an adult, although it varies depending on the drug acceptability of the administration target, body weight, age, etc.
It can be appropriately selected within the range of g / kg.

[0108]

The present invention will be described in more detail with reference to the following examples, but these are merely examples and do not limit the scope of the present invention.

Example 1 Preparation of cDNA Library from Liver Cancer Cell Line by Oligocap Method From Human Liver Cancer Cell Line, Molecular Cloning, A Laborator
y Manual, Second Edition, Cold Spring Harbor Labor
mRNA was extracted by the method described in atory Press (1989). Furthermore, according to the method described in the literature, oligo (d
T) Poly (A) + RNA was purified using a cellulose column (Collaborative labs). The poly
From (A) + RNA, oligo-cap method [M. Maruyama
and S. Sugano, Gene, 138: 171-174 (1994)] was used to prepare a cDNA library. That is, using an oligo cap linker (synthetic RNA) and an oligo (dT) adaptor, the literature [Suzuki Sugano, Protein Nucleic Acid Enzyme,
41: 197-201 (1996); Y. Suzuki et al., Gene, 200: 1
49-156 (1997)], BAP (Bacteri
al Alkaline Phosphatase) treatment, TAP (Tobacco Aci
d Pyrophosphatase) treatment, RNA ligation, first strand cDNA synthesis and RNA removal. Then, it was converted to a double-stranded cDNA by PCR, and the obtained D
The NA fragment was cut with Sfi I. Then, the cDNA was directionally cloned into the expression vector pME18S-FL3 (GenBank accession number: AB009864, full length 3.4 kb) cut with Dra III, and cloned into the downstream of the SRα promoter.
A cDNA expression library in which a DNA clone was functionally inserted was prepared. Escherichia coli DH5α was transformed with this to obtain a recombinant. One c in this library
The DNA clone was named C-HEP15374 and used for the following experiments.

Example 2 Determination of nucleotide sequence of C-HEP15374 cDNA (1) Extraction of plasmid After culturing the recombinant Escherichia coli containing the C-HEP15374 expression plasmid obtained in Example 1 on Luria broth (LB) agar medium, Single colony is Terrific Broth (TB) medium 2
The cells were inoculated in ml and cultured at 37 ° C. with shaking overnight. 2 cultures
The cells were transferred to a ml microtube and centrifuged to collect the cells.
P1 buffer (RNase A) supplied with QIAprep® Spin Miniprep Kit (QIAGENN, Hilden, Germany)
250 μl) was added to the pellet and resuspended.
After 250 μl of P2 buffer (supplied with the kit) was added to lyse the cells, N3 buffer (supplied with the kit) was added to neutralize. After centrifuging the tube at 12,000 rpm for 10 minutes, the supernatant was layered on a spin column (attached to the same kit) and centrifuged at 12,000 rpm for 1 minute. After washing the column with 750 μl of PE buffer (containing ethanol; attached to the same kit), plasmid DNA was added to TE buffer (1
0 mM Tris-HCl, 1 mM EDTA, pH 8.0) 1
Elution was performed with 00 μl. 30 μl of the obtained plasmid DNA solution was diluted 20-fold with TE buffer (570 μl), and the absorbance at 260 nm was measured to calculate the DNA concentration.

(2) Analysis of nucleotide sequence As a reagent kit for nucleotide sequence analysis, ABI PRISM (registered trademark) BigDye TerminatorCycle Sequencing Kit (PE Bios
ystems, Emeryville, Calif., USA). In addition, the following oligonucleotides were designed as primers for nucleotide sequence determination (the synthesis of each primer was outsourced to Amersham Pharmacia Biotech (Tokyo)). CHEP464F 5'-ATGAAGGACGAGGACAAGCC-3 '(SEQ ID NO: 3) CHEP567F 5'-AGAATGGTGACGGCCAAGCG-3' (SEQ ID NO: 4) CHEP935F 5'-GTCTCCATGATCTTCCTGGC-3 '(SEQ ID NO: 5) CHEP1272F 5'-AGGGACAACCATGTACGCCC-3' (SEQ ID NO :) 6) CHEP1372R 5'-AGACCTTCCAGACCACCAGGG-3 '(SEQ ID NO: 7) About 500 ng of the plasmid DNA prepared in (1) above
Premix in PCR tube (included in kit)
PCR was carried out by mixing 8 μl, 3.2 pmol of the above primer and purified water to a total volume of 20 μl. The reaction is
Denaturation: 96 after initial incubation at 96 ° C. for 3 minutes
℃, 10 seconds, annealing: 50 ℃, 5 seconds, extension: 60
A cycle of 4 minutes at 4 ° C was performed 25 times, and then the mixture was left at 4 ° C. The PCR product was layered on a 96-well column set on a 0.2 ml PCR tube, centrifuged at 2,000 rpm for 5 minutes to remove unreacted dNTPs, and then dried under reduced pressure. The 96-well column is a 96-well filter plate (multiscreen-HV plate; Millipore, Tokyo).
A certain amount of column particles (Sephadex G-50 Medium; Amersham Pharmacia Biotech) were fractionated and packed with a column loader (multiscreen 45 μl column loader; Millipore), and 300 μl of purified water was added for about 2 hours. After swelling, it was centrifuged at 2,000 rpm for 5 minutes and used. Template suppres for each PCR tube
After adding 20 μl of sion reagent (PE Biosystems) to dissolve the dried PCR product, it was heated at 96 ° C. for about 2 hours and rapidly cooled in ice. Nucleotide sequence analyzer for each tube
After setting in PRISM (ABI310 Genetic Analyzer; PEBiosystems), the operation for nucleotide sequence analysis was performed according to the instruction manual prepared by the manufacturer of the instrument.
As a result, it was revealed that the C-HEP15374 cDNA clone contained a 1650 bp ORF encoding a novel protein consisting of 549 amino acids (SEQ ID NO: 1). As a result of a homology search, it was found that the sequence of the coding region of this clone had about 50% identity at the base level with a known GPCR gene (Table 1).

[0112]

[Table 1]

Example 3 C-HEP15374 Homology Search The amino acid sequence of C-HEP15374 was determined using NCBI (http: //www.n
Database N published from cbi.nlm.nih.gov/)
For the known sequences disclosed in RDB (updated August 15, 2001), analysis programs (BLAST2.1.2; Nucleic Acid
s Res., 25, 3389-3402, 1997), and found that 6 known GPCR sequences shown in Table 2 and 33-35%
It became clear that they have the same identity. That is, C-HEP1
5374 was found to be a novel protein having homology with GPCR.

[0114]

[Table 2]

Example 4 Hydropathy plot of C-HEP15374 Using the amino acid sequence of C-HEP15374, Kyte and Doolittl
Hydropathy plot was prepared by the method of e (J. Mol. Biol., 157, 105-132, 1982) to predict the transmembrane site. As a result, C-HEP15374 showed 7 transmembrane sites (T
It was found to have M1 to TM7) (FIG. 1).

Example 5 C-HEP15374 motif search The PFAM search (HMMPFAM; Nucleic Aci) using the hidden Markov model with the amino acid sequence of C-HEP15374 as the query
ds Res., 26 (1), 320-322, 1998). Hidden Markov model is HMMER version 2.1 (http: //hmmer.wustl.
edu /), PFAM database is Pfam 6.5 (http: // ww
w.sanger.ac.uk/Software/Pfam/index.shtml) was used. As a result, it was revealed that C-HEP15374 has a 7-transmembrane domain belonging to the selectin family (Table 3).

[0117]

[Table 3]

Example 6 Alignment of C-HEP15374 with similar GPCR C-HEP15374 was aligned with known sequences listed in Table 1 using the multiple sequence alignment program Clustalw 1.81 (FIG. 2). As a result, C-HEP15374 has 7 transmembrane sites (### TMn ###; n = 1-7), and
It was found to have a cysteine residue (marked with @) that is thought to make a disulfide bond specific to PCR.

Example 7 Expression distribution of C-HEP15374 gene in human normal and diseased tissues C-HE using cDNA derived from human normal and diseased tissues as a template
Amplification of P15374 cDNA was performed, and the PCR product was used for TaqMa
The tissue specificity of C-HEP15374 gene expression and the difference in C-HEP15374 gene expression in normal and diseased tissues were examined by quantitative and real-time detection using an n probe.

(1) Reagents and template cDNA PCR primers and TaqMan probe were used for gene analysis software Primer Express version 1.0 (PE Biosystem
s). PCR primer is Amersham Pharmacia Biotech, TaqMan probe is PE B
Production was outsourced to iosystems Japan. In addition, TaqMa
The n probe has a reporter dye FAM at the 5'end,
Quencher tamra was attached to each of the 3'ends.
The base sequences of the primer and TaqMan probe are shown below. PCR primer: CHEP1272F 5'-AGGGACAACCATGTACGCCC-3 '(SEQ ID NO: 6) CHEP1372R 5'-AGACCTTCCAGACCACCAGGG-3' (SEQ ID NO: 7) TaqMan probe: CHEP1315T 5'-TTCCTCATCACCTTCCTCTTTGGCATGGT-3 '(SEQ ID NO: 8) lung, stomach, colon CDNA derived from normal and tumor tissues of ovary, prostate, uterus and kidney were prepared from the same patient.
Ontech's Matched cDNA Pairs were used. CDNA derived from the brain, thyroid gland, pancreas and testis of tumor patients and normal adults, liver of cirrhosis patients and normal adults, kidney of lupus disease patients, hippocampus and frontal lobes of Alzheimer's disease patients and normal adults
Was purchased from BioChain Institute and used. Normal adult placenta, skeletal muscle, heart, spleen, thymus, cerebral cortex, parietal lobe, temporal lobe, occipital lobe, cerebral peduncle, cerebellum, diencephalon, thalamus, corpus callosum, pons, olfactory brain, tonsil, medulla oblongata and CDNA derived from spinal cord
Was purchased from BioChain Institute and used. The PCR reaction reagent is TaqMan Universal PCR Master Mix (PE Bi
osystems) was used. Also, for internal standard measurement, Ta
qMan β-actin Control Reagents (PEBiosystems) was used.

(2) Quantitative PCR reaction A reaction solution having the following composition was prepared. In addition, template cDNA
Were used after being diluted 5 times with water for those obtained from Clontech and 50 times with water for those obtained from BioChain Institute. Reaction volume 2 × Master mix 12.5 μl 50 μM Sense primer 0.5 μl 50 μM Antisense primer 0.5 μl 5 μM TaqMan probe 1 μl Template cDNA 2.5 μl Purified water 8 μl Total amount 25 μl After adding 6 μl of template to 54 μl of master mix solution 25 μl was dispensed into the sample wells (2 wells for each sample) on a PCR plate for a quantitative PCR device. 25 μl of the above master mix solution was dispensed into 2 wells for no template control. PCEP4 vector (Invitrogen; full length 10.4 for standard curve construction)
C-HEP15374 cDNA subcloned into kb, CMV promoter) was prepared by serially diluting 1/10 by 8 steps starting from 100 pg / μl to prepare a standard solution series. After adding 5 μl of each standard solution to 54 μl of master mix, 25 μl Aliquots were dispensed into standard solution wells (2 wells for each standard solution). Therefore, the amount of template vector contained in the reaction solution is 250 pg to 25 ag (= 2.5 × 1).
It was 0 ^-10 g to 2.5 × 10 ^-18 g). After attaching eight caps, light centrifugation was performed to remove air bubbles. The plate was used for quantitative PCR (GeneAmp 5700 Sequence Detection
System; PE Biosystems), reacted according to the following operation program, quantitatively analyzed according to the operation manual of this equipment, and output measurement data. 50 ° C, 2 minutes 95 ° C, 10 minutes 95 ° C, 15 seconds → 60 ° C, 1 minute: 50 cycles

The results are shown in Tables 4 and 5. Regarding the expression profile of the C-HEP15374 gene, a relative ratio was obtained using the expression level of the actin gene as an internal standard, and the average value of two experiments was recorded in the table. In Table 4, it was considered significant when a 3-fold or more expression change was reproduced between normal tissue and diseased tissue. The C-HEP15374 gene showed particularly high expression in the thyroid gland, colon and testis in normal adults. C-HEP15374
A summary of changes in gene expression due to disease revealed that the gene was strongly expressed in normal colon, but the expression was not detected due to carcinogenesis. It was also expressed in the prostate, uterus, and brain, but was rarely expressed in each cancer. Thyroid,
In the lung, testis, and stomach, high normal expression was markedly reduced by carcinogenesis. On the other hand, the expression was remarkably enhanced in the carcinogenesis of the pancreas and kidney, liver cirrhosis, and in the frontal lobe and hippocampus of Alzheimer's disease.

[0123]

[Table 4]

The distribution of expression in the brain showed the highest expression in the olfactory brain, followed by strong expression in the cerebral cortex. Furthermore, expression was also clearly observed in the cerebral peduncle and medulla oblongata (Table 5).

[0125]

[Table 5]

[0126]

EFFECT OF THE INVENTION C-HEP15374 of the present invention is a novel orphan GPCR and can be a new drug target. Therefore, a ligand for C-HEP15374 of the present invention, or
The screening system and screening method for C-HEP15374 agonists or antagonists are useful as tools for developing therapeutic agents for diseases associated with abnormal expression of the receptor. Further, since the receptor shows remarkably different expression in normal and diseased tissues, the DNA encoding the receptor and the antibody against the receptor are useful for diagnosing various diseases.

[0127]

[Sequence listing free text] SEQ ID NO: 3: C-HEP15374 c
An oligonucleotide designed to function as a primer for sequencing DNA. SEQ ID NO: 4: Oligonucleotide designed to function as a primer for sequencing C-HEP15374 cDNA. SEQ ID NO: 5: An oligonucleotide designed to function as a primer for sequencing C-HEP15374 cDNA. SEQ ID NO: 6: Oligonucleotide designed to function as a primer for sequencing C-HEP15374 cDNA. SEQ ID NO: 7: Oligonucleotide designed to function as a primer for sequencing C-HEP15374 cDNA. SEQ ID NO: 8: for quantifying the expression of C-HEP15374 gene
An oligonucleotide designed to function as a TaqMan probe.

[0128]

[Sequence list]                           SEQUENCE LISTING <110> Mitsubishi Pharma Corporation <120> Novel GPCR, Gene Encoding Same and Use Thereof <130> A-4901 <140> JP 2001- <141> 2001-10-22 <160> 14 <170> PatentIn version 3.0 <210> 1 <211> 1650 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (1647) <400> 1 atg gcg acg ccc agg ggc ctg ggg gcc ctg ctc ctg ctc ctc ctg ctc 48 Met Ala Thr Pro Arg Gly Leu Gly Ala Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 ccg acc tca ggt cag gaa aag tcc acc gaa ggg cca aga aac acc tgc 96 Pro Thr Ser Gly Gln Glu Lys Ser Thr Glu Gly Pro Arg Asn Thr Cys             20 25 30 ctg ggg agc aac aac atg tac gac atc ttc aac ttg aat gac aag gct 144 Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu Asn Asp Lys Ala         35 40 45 ttg tgc ttc acc aag tgc agg cag tcg ggc agc gac tcc tgc aat gtg 192 Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp Ser Cys Asn Val     50 55 60 gaa aac ttg cag aga tac tgg cta aac tac gag gcc cat ctg atg aag 240 Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala His Leu Met Lys 65 70 75 80 gaa ggt ttg acg cag aag gtg aac acg cct ttc ctg aag gct ttg gtc 288 Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe Leu Lys Ala Leu Val                 85 90 95 cag aac ctc agc acc aac act gca gaa gac ttc tat ttc tct ctg gag 336 Gln Asn Leu Ser Thr Asn Thr Ala Glu Asp Phe Tyr Phe Ser Leu Glu             100 105 110 ccc tct cag gtt ccg agg cag gtg atg aag gac gag gac aag ccc cct 384 Pro Ser Gln Val Pro Arg Gln Val Met Lys Asp Glu Asp Lys Pro Pro         115 120 125 gac aga gtg cga ctt ccc aag agc ctt ttt cga tcc ctg cca ggc aac 432 Asp Arg Val Arg Leu Pro Lys Ser Leu Phe Arg Ser Leu Pro Gly Asn     130 135 140 agg tct gtg gtc cgc ttg gcc gtc acc att ctg gac att ggt cca ggg 480 Arg Ser Val Val Arg Leu Ala Val Thr Ile Leu Asp Ile Gly Pro Gly 145 150 155 160 act ctc ttc aag ggc ccc cgg ctc ggc ctg gga gat ggc agc ggc gtg 528 Thr Leu Phe Lys Gly Pro Arg Leu Gly Leu Gly Asp Gly Ser Gly Val                 165 170 175 ttg aac aat cgc ctg gtg ggt ttg agt gtg gga caa atg cat gtc acc 576 Leu Asn Asn Arg Leu Val Gly Leu Ser Val Gly Gln Met His Val Thr             180 185 190 agg ctg gct gag cct ctg gag atc gtc ttc tct cac cag cga ccg ccc 624 Arg Leu Ala Glu Pro Leu Glu Ile Val Phe Ser His Gln Arg Pro Pro         195 200 205 cct aac atg acc ctc acc tgt gta ttc tgg gat gtg act aaa ggg acc 672 Pro Asn Met Thr Leu Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr     210 215 220 act gga gac tgg tct tct gag ggc tgc tcc acg gag gtc aga cct gag 720 Thr Gly Asp Trp Ser Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu 225 230 235 240 ggg acc gtg tgc tgc tgt gac cac ctg acc ttt ttc gcc ctg ctc ctg 768 Gly Thr Val Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu                 245 250 255 aga ccc acc ttg gac cag tcc acg gtg cat atc ctc aca cgc atc tcc 816 Arg Pro Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser             260 265 270 cag gcg ggc tgt ggg gtc tcc atg atc ttc ctg gcc ttc acc att att 864 Gln Ala Gly Cys Gly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile         275 280 285 ctt tat gcc ttt ctg agg ctt tcc cgg gag agg ttc aag tca gaa gat 912 Leu Tyr Ala Phe Leu Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp     290 295 300 gcc cca aag atc cac gtg gcc ctg ggt ggc agc ctg ttc ctc ctg aat 960 Ala Pro Lys Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn 305 310 315 320 ctg gcc ttc ttg gtc aat gtg ggg agt ggc tca aag ggg tct gat gct 1008 Leu Ala Phe Leu Val Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala                 325 330 335 gcc tgc tgg gcc cgg ggg gct gtc ttc cac tac ttc ctg ctc tgt gcc 1056 Ala Cys Trp Ala Arg Gly Ala Val Phe His Tyr Phe Leu Leu Cys Ala             340 345 350 ttc acc tgg atg ggc ctt gaa gcc ttc cac ctc tac ctg ctc gct gtc 1104 Phe Thr Trp Met Gly Leu Glu Ala Phe His Leu Tyr Leu Leu Ala Val         355 360 365 agg gtc ttc aac acc tac ttc ggg cac tac ttc ctg aag ctg agc ctg 1152 Arg Val Phe Asn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu     370 375 380 gtg ggc tgg ggc ctg ccc gcc ctg atg gtc atc ggc act ggg agt gcc 1200 Val Gly Trp Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala 385 390 395 400 aac agc tac ggc ctc tac acc atc cgt gat agg gag aac cgc acc tct 1248 Asn Ser Tyr Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser                 405 410 415 ctg gag cta tgc tgg ttc cgt gaa ggg aca acc atg tac gcc ctc tat 1296 Leu Glu Leu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr             420 425 430 atc acc gtc cac ggc tac ttc ctc atc acc ttc ctc ttt ggc atg gtg 1344 Ile Thr Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val         435 440 445 gtc ctg gcc ctg gtg gtc tgg aag gtc ttc acc ctg tcc cgt gct aca 1392 Val Leu Ala Leu Val Val Trp Lys Val Phe Thr Leu Ser Arg Ala Thr     450 455 460 gcg gtc aag gag cgg ggg aag aac cgg aag aag gtg ctc acc ctg ctg 1440 Ala Val Lys Glu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr Leu Leu 465 470 475 480 ggc ctc tcg agc ctg gtg ggt gtg aca tgg ggg ttg gcc atc ttc acc 1488 Gly Leu Ser Ser Leu Val Gly Val Thr Trp Gly Leu Ala Ile Phe Thr                 485 490 495 ccg ttg ggc ctc tcc acc gtc tac atc ttt gca ctt ttc aac tcc ttg 1536 Pro Leu Gly Leu Ser Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu             500 505 510 caa ggt gtc ttc atc tgt tgc tgg ttc acc atc ctt tac ctc cca agt 1584 Gln Gly Val Phe Ile Cys Cys Trp Phe Thr Ile Leu Tyr Leu Pro Ser         515 520 525 cag agc acc aca gtc tcc tct tct act gca aga ttg gac cag gcc cac 1632 Gln Ser Thr Thr Val Ser Ser Ser Thr Ala Arg Leu Asp Gln Ala His     530 535 540 tcc gca tct caa gaa tag 1650 Ser Ala Ser Gln Glu 545 <210> 2 <211> 549 <212> PRT <213> Homo sapiens <400> 2 Met Ala Thr Pro Arg Gly Leu Gly Ala Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Pro Thr Ser Gly Gln Glu Lys Ser Thr Glu Gly Pro Arg Asn Thr Cys             20 25 30 Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu Asn Asp Lys Ala         35 40 45 Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp Ser Cys Asn Val     50 55 60 Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala His Leu Met Lys 65 70 75 80 Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe Leu Lys Ala Leu Val                 85 90 95 Gln Asn Leu Ser Thr Asn Thr Ala Glu Asp Phe Tyr Phe Ser Leu Glu             100 105 110 Pro Ser Gln Val Pro Arg Gln Val Met Lys Asp Glu Asp Lys Pro Pro         115 120 125 Asp Arg Val Arg Leu Pro Lys Ser Leu Phe Arg Ser Leu Pro Gly Asn     130 135 140 Arg Ser Val Val Arg Leu Ala Val Thr Ile Leu Asp Ile Gly Pro Gly 145 150 155 160 Thr Leu Phe Lys Gly Pro Arg Leu Gly Leu Gly Asp Gly Ser Gly Val                 165 170 175 Leu Asn Asn Arg Leu Val Gly Leu Ser Val Gly Gln Met His Val Thr             180 185 190 Arg Leu Ala Glu Pro Leu Glu Ile Val Phe Ser His Gln Arg Pro Pro         195 200 205 Pro Asn Met Thr Leu Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr     210 215 220 Thr Gly Asp Trp Ser Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu 225 230 235 240 Gly Thr Val Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu                 245 250 255 Arg Pro Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser             260 265 270 Gln Ala Gly Cys Gly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile         275 280 285 Leu Tyr Ala Phe Leu Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp     290 295 300 Ala Pro Lys Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn 305 310 315 320 Leu Ala Phe Leu Val Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala                 325 330 335 Ala Cys Trp Ala Arg Gly Ala Val Phe His Tyr Phe Leu Leu Cys Ala             340 345 350 Phe Thr Trp Met Gly Leu Glu Ala Phe His Leu Tyr Leu Leu Ala Val         355 360 365 Arg Val Phe Asn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu     370 375 380 Val Gly Trp Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala 385 390 395 400 Asn Ser Tyr Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser                 405 410 415 Leu Glu Leu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr             420 425 430 Ile Thr Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val         435 440 445 Val Leu Ala Leu Val Val Trp Lys Val Phe Thr Leu Ser Arg Ala Thr     450 455 460 Ala Val Lys Glu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr Leu Leu 465 470 475 480 Gly Leu Ser Ser Leu Val Gly Val Thr Trp Gly Leu Ala Ile Phe Thr                 485 490 495 Pro Leu Gly Leu Ser Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu             500 505 510 Gln Gly Val Phe Ile Cys Cys Trp Phe Thr Ile Leu Tyr Leu Pro Ser         515 520 525 Gln Ser Thr Thr Val Ser Ser Ser Thr Ala Arg Leu Asp Gln Ala His     530 535 540 Ser Ala Ser Gln Glu 545 <210> 3 <211> 20 <212> DNA <213> Artificial / Unknown <220> <221> misc_feature <222> () .. () <223> Oligonucleotide designed to act as primer for sequencing        C-HEP15374 cDNA. <400> 3 atgaaggacg aggacaagcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial / Unknown <220> <221> misc_feature <222> () .. () <223> Oligonucleotide designed to act as primer for sequencing        C-HEP15374 cDNA. <400> 4 agaatggtga cggccaagcg 20 <210> 5 <211> 20 <212> DNA <213> Artificial / Unknown <220> <221> misc_feature <222> () .. () <223> Oligonucleotide designed to act as primer for sequencing        C-HEP15374 cDNA. <400> 5 gtctccatga tcttcctggc 20 <210> 6 <211> 20 <212> DNA <213> Artificial / Unknown <220> <221> misc_feature <222> () .. () <223> Oligonucleotide desighed to act as primer for sequencing        C-HEP15374 cDNA. <400> 6 agggacaacc atgtacgccc 20 <210> 7 <211> 21 <212> DNA <213> Artificial / Unknown <220> <221> misc_feature <222> () .. () <223> Oligonucleotide designed to act as primer for sequencing        C-HEP15374 cDNA. <400> 7 agaccttcca gaccaccagg g 21 <210> 8 <211> 29 <212> DNA <213> Artificial / Unknown <220> <221> misc_feature <222> () .. () <223> Oligonucleotide designed to act as TaqMan probe for quantifying        expression of C-HEP15374 gene. <400> 8 ttcctcatca ccttcctctt tggcatggt 29 <210> 9 <211> 687 <212> PRT <213> Homo sapiens <400> 9 Met Thr Pro Gln Ser Leu Leu Gln Thr Thr Leu Phe Leu Leu Ser Leu 1 5 10 15 Leu Phe Leu Val Gln Gly Ala His Gly Arg Gly His Arg Glu Asp Phe             20 25 30 Arg Phe Cys Ser Gln Arg Asn Gln Thr His Arg Ser Ser Leu His Tyr         35 40 45 Lys Pro Thr Pro Asp Leu Arg Ile Ser Ile Glu Asn Ser Glu Glu Ala     50 55 60 Leu Thr Val His Ala Pro Phe Pro Ala Ala His Pro Ala Ser Arg Ser 65 70 75 80 Phe Pro Asp Pro Arg Gly Leu Tyr His Phe Cys Leu Tyr Trp Asn Arg                 85 90 95 His Ala Gly Arg Leu His Leu Leu Tyr Gly Lys Arg Asp Phe Leu Leu             100 105 110 Ser Asp Lys Ala Ser Ser Leu Leu Cys Phe Gln His Gln Glu Glu Ser         115 120 125 Leu Ala Gln Gly Pro Pro Leu Leu Ala Thr Ser Val Thr Ser Trp Trp     130 135 140 Ser Pro Gln Asn Ile Ser Leu Pro Ser Ala Ala Ser Phe Thr Phe Ser 145 150 155 160 Phe His Ser Pro Pro His Thr Ala Ala His Asn Ala Ser Val Asp Met                 165 170 175 Cys Glu Leu Lys Arg Asp Leu Gln Leu Leu Ser Gln Phe Leu Lys His             180 185 190 Pro Gln Lys Ala Ser Arg Arg Pro Ser Ala Ala Pro Ala Ser Gln Gln         195 200 205 Leu Gln Ser Leu Glu Ser Lys Leu Thr Ser Val Arg Phe Met Gly Asp     210 215 220 Met Val Ser Phe Glu Glu Asp Arg Ile Asn Ala Thr Val Trp Lys Leu 225 230 235 240 Gln Pro Thr Ala Gly Leu Gln Asp Leu His Ile His Ser Arg Gln Glu                 245 250 255 Glu Glu Gln Ser Glu Ile Met Glu Tyr Ser Val Leu Leu Pro Arg Thr             260 265 270 Leu Phe Gln Arg Thr Lys Gly Arg Arg Gly Glu Ala Glu Lys Arg Leu         275 280 285 Leu Leu Val Asp Phe Ser Ser Gln Ala Leu Phe Gln Asp Lys Asn Ser     290 295 300 Ser His Val Leu Gly Glu Lys Val Leu Gly Ile Val Val Gln Asn Thr 305 310 315 320 Lys Val Ala Asn Leu Thr Glu Pro Val Val Leu Thr Phe Gln His Gln                 325 330 335 Leu Gln Pro Lys Asn Val Thr Leu Gln Cys Val Phe Trp Val Glu Asp             340 345 350 Pro Thr Leu Ser Ser Pro Gly His Trp Ser Ser Ala Gly Cys Glu Thr         355 360 365 Val Arg Arg Glu Thr Gln Thr Ser Cys Phe Cys Asn His Leu Thr Tyr     370 375 380 Phe Ala Val Leu Met Val Ser Ser Val Glu Val Asp Ala Val His Lys 385 390 395 400 His Tyr Leu Ser Leu Leu Ser Tyr Val Gly Cys Val Val Ser Ala Leu                 405 410 415 Ala Cys Leu Val Thr Ile Ala Ala Tyr Leu Cys Ser Arg Arg Lys Pro             420 425 430 Arg Asp Tyr Thr Ile Lys Val His Met Asn Leu Leu Leu Ala Val Phe         435 440 445 Leu Leu Asp Thr Ser Phe Leu Leu Ser Glu Pro Val Ala Leu Thr Gly     450 455 460 Ser Glu Ala Gly Cys Arg Ala Ser Ala Ile Phe Leu His Phe Ser Leu 465 470 475 480 Leu Thr Cys Leu Ser Trp Met Gly Leu Glu Gly Tyr Asn Leu Tyr Arg                 485 490 495 Leu Val Val Glu Val Phe Gly Thr Tyr Val Pro Gly Tyr Leu Leu Lys             500 505 510 Leu Ser Ala Met Gly Trp Gly Phe Pro Ile Phe Leu Val Thr Leu Val         515 520 525 Ala Leu Val Asp Val Asp Asn Tyr Gly Pro Ile Ile Leu Ala Val His     530 535 540 Arg Thr Pro Glu Gly Val Ile Tyr Pro Ser Met Cys Trp Ile Arg Asp 545 550 555 560 Ser Leu Val Ser Tyr Ile Thr Asn Leu Gly Leu Phe Ser Leu Val Phe                 565 570 575 Leu Phe Asn Met Ala Met Leu Ala Thr Met Val Val Gln Ile Leu Arg             580 585 590 Leu Arg Pro His Thr Gln Lys Trp Ser His Val Leu Thr Leu Leu Gly         595 600 605 Leu Ser Leu Val Leu Gly Leu Pro Trp Ala Leu Ile Phe Phe Ser Phe     610 615 620 Ala Ser Gly Thr Phe Gln Leu Val Val Leu Tyr Leu Phe Ser Ile Ile 625 630 635 640 Thr Ser Phe Gln Gly Phe Leu Ile Phe Ile Trp Tyr Trp Ser Met Arg                 645 650 655 Leu Gln Ala Arg Gly Gly Pro Ser Pro Leu Lys Ser Asn Ser Asp Ser             660 665 670 Ala Arg Leu Pro Ile Ser Ser Gly Ser Thr Ser Ser Ser Arg Ile         675 680 685 <210> 10 <211> 693 <212> PRT <213> Homo sapiens <400> 10 Met Thr Pro Gln Ser Leu Leu Gln Thr Thr Leu Phe Leu Leu Ser Leu 1 5 10 15 Leu Phe Leu Val Gln Gly Ala His Gly Arg Gly His Arg Glu Asp Phe             20 25 30 Arg Phe Cys Ser Gln Arg Asn Gln Thr His Arg Ser Ser Leu His Tyr         35 40 45 Lys Pro Thr Pro Asp Leu Arg Ile Ser Ile Glu Asn Ser Glu Glu Ala     50 55 60 Leu Thr Val His Ala Pro Phe Pro Ala Ala His Pro Ala Ser Arg Ser 65 70 75 80 Phe Pro Asp Pro Arg Gly Leu Tyr His Phe Cys Leu Tyr Trp Asn Arg                 85 90 95 His Ala Gly Arg Leu His Leu Leu Tyr Gly Lys Arg Asp Phe Leu Leu             100 105 110 Ser Asp Lys Ala Ser Ser Leu Leu Cys Phe Gln His Gln Glu Glu Ser         115 120 125 Leu Ala Gln Gly Pro Pro Leu Leu Ala Thr Ser Val Thr Ser Trp Trp     130 135 140 Ser Pro Gln Asn Ile Ser Leu Pro Ser Ala Ala Ser Phe Thr Phe Ser 145 150 155 160 Phe His Ser Pro Pro His Thr Ala Ala His Asn Ala Ser Val Asp Met                 165 170 175 Cys Glu Leu Lys Arg Asp Leu Gln Leu Leu Ser Gln Phe Leu Lys His             180 185 190 Pro Gln Lys Ala Ser Arg Arg Pro Ser Ala Ala Pro Ala Ser Gln Gln         195 200 205 Leu Gln Ser Leu Glu Ser Lys Leu Thr Ser Val Arg Phe Met Gly Asp     210 215 220 Met Val Ser Phe Glu Glu Asp Arg Ile Asn Ala Thr Val Trp Lys Leu 225 230 235 240 Gln Pro Thr Ala Gly Leu Gln Asp Leu His Ile His Ser Arg Gln Glu                 245 250 255 Glu Glu Gln Ser Glu Ile Met Glu Tyr Ser Val Leu Leu Pro Arg Thr             260 265 270 Leu Phe Gln Arg Thr Lys Gly Arg Ser Gly Glu Ala Glu Lys Arg Leu         275 280 285 Leu Leu Val Asp Phe Ser Ser Gln Ala Leu Phe Gln Asp Lys Asn Ser     290 295 300 Ser Gln Val Leu Gly Glu Lys Val Leu Gly Ile Val Val Gln Asn Thr 305 310 315 320 Lys Val Ala Asn Leu Thr Glu Pro Val Val Leu Thr Phe Gln His Gln                 325 330 335 Leu Gln Pro Lys Asn Val Thr Leu Gln Cys Val Phe Trp Val Glu Asp             340 345 350 Pro Thr Leu Ser Ser Pro Gly His Trp Ser Ser Ala Gly Cys Glu Thr         355 360 365 Val Arg Arg Glu Thr Gln Thr Ser Cys Phe Cys Asn His Leu Thr Tyr     370 375 380 Phe Ala Val Leu Met Val Ser Ser Val Glu Val Asp Ala Val His Lys 385 390 395 400 His Tyr Leu Ser Leu Leu Ser Tyr Val Gly Cys Val Val Ser Ala Leu                 405 410 415 Ala Cys Leu Val Thr Ile Ala Ala Tyr Leu Cys Ser Arg Val Pro Leu             420 425 430 Pro Cys Arg Arg Lys Pro Arg Asp Tyr Thr Ile Lys Val His Met Asn         435 440 445 Leu Leu Leu Ala Val Phe Leu Leu Asp Thr Ser Phe Leu Leu Ser Glu     450 455 460 Pro Val Ala Leu Thr Gly Ser Glu Ala Gly Cys Arg Ala Ser Ala Ile 465 470 475 480 Phe Leu His Phe Ser Leu Leu Thr Cys Leu Ser Trp Met Gly Leu Glu                 485 490 495 Gly Tyr Asn Leu Tyr Arg Leu Val Val Glu Val Phe Gly Thr Tyr Val             500 505 510 Pro Gly Tyr Leu Leu Lys Leu Ser Ala Met Gly Trp Gly Phe Pro Ile         515 520 525 Phe Leu Val Thr Leu Val Ala Leu Val Asp Val Asp Asn Tyr Gly Pro     530 535 540 Ile Ile Leu Ala Val His Arg Thr Pro Glu Gly Val Ile Tyr Pro Ser 545 550 555 560 Met Cys Trp Ile Arg Asp Ser Leu Val Ser Tyr Ile Thr Asn Leu Gly                 565 570 575 Leu Phe Ser Leu Val Phe Leu Phe Asn Met Ala Met Leu Ala Thr Met             580 585 590 Val Val Gln Ile Leu Arg Leu Arg Pro His Thr Gln Lys Trp Ser His         595 600 605 Val Leu Thr Leu Leu Gly Leu Ser Leu Val Leu Gly Leu Pro Trp Ala     610 615 620 Leu Ile Phe Phe Ser Phe Ala Ser Gly Thr Phe Gln Leu Val Val Leu 625 630 635 640 Tyr Leu Phe Ser Ile Ile Thr Ser Phe Gln Gly Phe Leu Ile Phe Ile                 645 650 655 Trp Tyr Trp Ser Met Arg Leu Gln Ala Arg Gly Gly Pro Ser Pro Leu             660 665 670 Lys Ser Asn Ser Asp Cys Ala Arg Leu Pro Ile Ser Ser Gly Ser Thr         675 680 685 Ser Ser Ser Arg Ile     690 <210> 11 <211> 686 <212> PRT <213> Mus musculus <400> 11 Met Ala Val Gln Val Leu Arg Gln Met Val Tyr Phe Leu Leu Ser Leu 1 5 10 15 Phe Ser Leu Val Gln Gly Ala His Ser Gly Ser Pro Arg Glu Asp Phe             20 25 30 Arg Phe Cys Gly Gln Arg Asn Gln Gln Gln Ser Thr Leu His Tyr Asp         35 40 45 Gln Ser Ser Glu Pro His Ile Phe Val Trp Asn Thr Glu Glu Thr Leu     50 55 60 Thr Ile Arg Ala Pro Phe Leu Ala Ala Pro Asp Ile Pro Arg Phe Phe 65 70 75 80 Pro Glu Pro Arg Gly Leu Tyr His Phe Cys Leu Tyr Trp Ser Arg His                 85 90 95 Thr Gly Arg Leu His Leu Arg Tyr Gly Lys His Asp Tyr Leu Leu Ser             100 105 110 Ser Gln Ala Ser Arg Leu Leu Cys Phe Gln Lys Gln Glu Gln Ser Leu         115 120 125 Lys Gln Gly Ala Pro Leu Ile Ala Thr Ser Val Ser Ser Trp Gln Ile     130 135 140 Pro Gln Asn Thr Ser Leu Pro Gly Ala Pro Ser Phe Ile Phe Ser Phe 145 150 155 160 His Asn Ala Pro His Lys Val Ser His Asn Ala Ser Val Asp Met Cys                 165 170 175 Asp Leu Lys Lys Glu Leu Gln Gln Leu Ser Arg Tyr Leu Gln His Pro             180 185 190 Gln Lys Ala Ala Lys Arg Pro Thr Ala Ala Phe Ile Ser Gln Gln Leu         195 200 205 Gln Ser Leu Glu Ser Lys Leu Thr Ser Val Ser Phe Leu Gly Asp Thr     210 215 220 Leu Ser Phe Glu Glu Asp Arg Val Asn Ala Thr Val Trp Lys Leu Pro 225 230 235 240 Pro Thr Ala Gly Leu Glu Asp Leu His Ile His Ser Gln Lys Glu Glu                 245 250 255 Glu Gln Ser Glu Val Gln Ala Tyr Ser Leu Leu Leu Pro Arg Ala Val             260 265 270 Phe Gln Gln Thr Arg Gly Arg Arg Arg Asp Asp Ala Lys Arg Leu Leu         275 280 285 Val Val Asp Phe Ser Ser Gln Ala Leu Phe Gln Asp Lys Asn Ser Ser     290 295 300 Gln Val Leu Gly Glu Lys Val Leu Gly Ile Val Val Gln Asn Thr Lys 305 310 315 320 Val Thr Asn Leu Ser Asp Pro Val Val Leu Thr Phe Gln His Gln Pro                 325 330 335 Gln Pro Lys Asn Val Thr Leu Gln Cys Val Phe Trp Val Glu Asp Pro             340 345 350 Ala Ser Ser Ser Thr Gly Ser Trp Ser Ser Ala Gly Cys Glu Thr Val         355 360 365 Ser Arg Asp Thr Gln Thr Ser Cys Leu Cys Asn His Leu Thr Tyr Phe     370 375 380 Ala Val Leu Met Val Ser Ser Thr Glu Val Glu Ala Thr His Lys His 385 390 395 400 Tyr Leu Thr Leu Leu Ser Tyr Val Gly Cys Val Ile Ser Ala Leu Ala                 405 410 415 Cys Val Phe Thr Ile Ala Ala Tyr Leu Cys Ser Arg Arg Lys Ser Arg             420 425 430 Asp Tyr Thr Ile Lys Val His Met Asn Leu Leu Ser Ala Val Phe Leu         435 440 445 Leu Asp Val Ser Phe Leu Leu Ser Glu Pro Val Ala Leu Thr Gly Ser     450 455 460 Glu Ala Ala Cys Arg Thr Ser Ala Met Phe Leu His Phe Ser Leu Leu 465 470 475 480 Ala Cys Leu Ser Trp Met Gly Leu Glu Gly Tyr Asn Leu Tyr Arg Leu                 485 490 495 Val Val Glu Val Phe Gly Thr Tyr Val Pro Gly Tyr Leu Leu Lys Leu             500 505 510 Ser Ile Val Gly Trp Gly Phe Pro Val Phe Leu Val Thr Leu Val Ala         515 520 525 Leu Val Asp Val Asn Asn Tyr Gly Pro Ile Ile Leu Ala Val Arg Arg     530 535 540 Thr Pro Glu Arg Val Thr Tyr Pro Ser Met Cys Trp Ile Arg Asp Ser 545 550 555 560 Leu Val Ser Tyr Val Thr Asn Leu Gly Leu Phe Ser Leu Val Phe Leu                 565 570 575 Phe Asn Leu Ala Met Leu Ala Thr Met Val Val Gln Ile Leu Arg Leu             580 585 590 Arg Pro His Ser Gln Asn Trp Pro His Val Leu Thr Leu Leu Gly Leu         595 600 605 Ser Leu Val Leu Gly Leu Pro Trp Ala Leu Val Phe Phe Ser Phe Ala     610 615 620 Ser Gly Thr Phe Gln Leu Val Ile Leu Tyr Leu Phe Ser Ile Ile Thr 625 630 635 640 Ser Tyr Gln Gly Phe Leu Ile Phe Leu Trp Tyr Trp Ser Met Arg Phe                 645 650 655 Gln Ala Gln Gly Gly Pro Ser Pro Leu Lys Asn Asn Ser Asp Ser Ala             660 665 670 Lys Leu Pro Ile Ser Ser Gly Ser Thr Ser Ser Ser Arg Ile         675 680 685 <210> 12 <211> 541 <212> PRT <213> Homo sapiens <400> 12 Met Asp Phe Glu Ser Gly Gln Val Asp Pro Leu Ala Ser Val Ile Leu 1 5 10 15 Pro Pro Asn Leu Leu Glu Asn Leu Ser Pro Glu Asp Ser Val Leu Val             20 25 30 Arg Arg Ala Gln Phe Thr Phe Phe Asn Lys Thr Gly Leu Phe Gln Asp         35 40 45 Val Gly Pro Gln Arg Lys Thr Leu Val Ser Tyr Val Met Ala Cys Ser     50 55 60 Ile Gly Asn Ile Thr Ile Gln Asn Leu Lys Asp Pro Val Gln Ile Lys 65 70 75 80 Ile Lys His Thr Arg Thr Gln Glu Val His His Pro Ile Cys Ala Phe                 85 90 95 Trp Asp Leu Asn Lys Asn Lys Ser Phe Gly Gly Trp Asn Thr Ser Gly             100 105 110 Cys Val Ala His Arg Asp Ser Asp Ala Ser Glu Thr Val Cys Leu Cys         115 120 125 Asn His Phe Thr His Phe Gly Val Leu Met Asp Leu Pro Arg Ser Ala     130 135 140 Ser Gln Leu Asp Ala Arg Asn Thr Lys Val Leu Thr Phe Ile Ser Tyr 145 150 155 160 Ile Gly Cys Gly Ile Ser Ala Ile Phe Ser Ala Ala Thr Leu Leu Thr                 165 170 175 Tyr Val Ala Phe Glu Lys Leu Arg Arg Asp Tyr Pro Ser Lys Ile Leu             180 185 190 Met Asn Leu Ser Thr Ala Leu Leu Phe Leu Asn Leu Leu Phe Leu Leu         195 200 205 Asp Gly Trp Ile Thr Ser Phe Asn Val Asp Gly Leu Cys Ile Ala Val     210 215 220 Ala Val Leu Leu His Phe Phe Leu Leu Ala Thr Phe Thr Trp Met Gly 225 230 235 240 Leu Glu Ala Ile His Met Tyr Ile Ala Leu Val Lys Val Phe Asn Thr                 245 250 255 Tyr Ile Arg Arg Tyr Ile Leu Lys Phe Cys Ile Ile Gly Trp Gly Leu             260 265 270 Pro Ala Leu Val Val Ser Val Val Leu Ala Ser Arg Asn Asn Asn Glu         275 280 285 Val Tyr Gly Lys Glu Ser Tyr Gly Lys Glu Lys Gly Asp Glu Phe Cys     290 295 300 Trp Ile Gln Asp Pro Val Ile Phe Tyr Val Thr Cys Ala Gly Tyr Phe 305 310 315 320 Gly Val Met Phe Phe Leu Asn Ile Ala Met Phe Ile Val Val Met Val                 325 330 335 Gln Ile Cys Gly Arg Asn Gly Lys Arg Ser Asn Arg Thr Leu Arg Glu             340 345 350 Glu Val Leu Arg Asn Leu Arg Ser Val Val Ser Leu Thr Phe Leu Leu         355 360 365 Gly Met Thr Trp Gly Phe Ala Phe Phe Ala Trp Gly Pro Leu Asn Ile     370 375 380 Pro Phe Met Tyr Leu Phe Ser Ile Phe Asn Ser Leu Gln Gly Leu Phe 385 390 395 400 Ile Phe Ile Phe His Cys Ala Met Lys Glu Asn Val Gln Lys Gln Trp                 405 410 415 Arg Gln His Leu Cys Cys Gly Arg Phe Arg Leu Ala Asp Asn Ser Asp             420 425 430 Trp Ser Lys Thr Ala Thr Asn Ile Ile Lys Lys Ser Ser Asp Asn Leu         435 440 445 Gly Lys Ser Leu Ser Ser Ser Ser Ile Gly Ser Asn Ser Thr Tyr Leu     450 455 460 Thr Ser Lys Ser Lys Ser Ser Ser Thr Thr Tyr Phe Lys Arg Asn Ser 465 470 475 480 His Thr Asp Ser Ala Ser Met Asp Lys Ser Leu Ser Lys Leu Ala His                 485 490 495 Ala Asp Gly Asp Gln Thr Ser Ile Ile Pro Val His Gln Val Ile Asp             500 505 510 Lys Val Lys Gly Tyr Cys Asn Ala His Ser Asp Asn Phe Tyr Lys Asn         515 520 525 Ile Ile Met Ser Asp Thr Phe Ser His Ser Thr Lys Phe     530 535 540 <210> 13 <211> 512 <212> PRT <213> Homo sapiens <400> 13 Met Asp Phe Glu Ser Gly Gln Val Asp Pro Leu Ala Ser Val Ile Leu 1 5 10 15 Pro Pro Asn Leu Leu Glu Asn Leu Ser Pro Glu Asp Ser Val Leu Val             20 25 30 Arg Arg Ala Gln Phe Thr Phe Phe Asn Lys Thr Gly Leu Phe Gln Asp         35 40 45 Val Gly Pro Gln Arg Lys Thr Leu Val Ser Tyr Val Met Ala Cys Ser     50 55 60 Ile Gly Asn Ile Thr Ile Gln Asn Leu Lys Asp Pro Val Gln Ile Lys 65 70 75 80 Ile Lys His Thr Arg Thr Gln Glu Val His His Pro Ile Cys Ala Phe                 85 90 95 Trp Asp Leu Asn Lys Asn Lys Ser Phe Gly Gly Trp Asn Thr Ser Gly             100 105 110 Cys Val Ala His Arg Asp Ser Asp Ala Ser Glu Thr Val Cys Leu Cys         115 120 125 Asn His Phe Thr His Phe Gly Val Leu Met Asp Leu Pro Arg Ser Ala     130 135 140 Ser Gln Leu Asp Ala Arg Asn Thr Lys Val Leu Thr Phe Ile Ser Tyr 145 150 155 160 Ile Gly Cys Gly Ile Ser Ala Ile Phe Ser Ala Ala Thr Leu Leu Thr                 165 170 175 Tyr Val Ala Phe Glu Lys Leu Arg Arg Asp Tyr Pro Ser Lys Ile Leu             180 185 190 Met Asn Leu Ser Thr Ala Leu Leu Phe Leu Asn Leu Leu Phe Leu Leu         195 200 205 Asp Gly Trp Ile Thr Ser Phe Asn Val Asp Gly Leu Cys Ile Ala Val     210 215 220 Ala Val Leu Leu His Phe Phe Leu Leu Ala Thr Phe Thr Trp Met Gly 225 230 235 240 Leu Glu Ala Ile His Met Tyr Ile Ala Leu Val Lys Val Phe Asn Thr                 245 250 255 Tyr Ile Arg Arg Tyr Ile Leu Lys Phe Cys Ile Ile Gly Trp Gly Leu             260 265 270 Pro Ala Leu Val Val Ser Val Val Leu Ala Ser Arg Asn Asn Asn Glu         275 280 285 Val Tyr Gly Lys Glu Ser Tyr Gly Lys Glu Lys Gly Asp Glu Phe Cys     290 295 300 Trp Ile Gln Asp Pro Val Ile Phe Tyr Val Thr Cys Ala Gly Tyr Phe 305 310 315 320 Gly Val Met Phe Phe Leu Asn Ile Ala Met Phe Ile Val Val Met Val                 325 330 335 Gln Ile Cys Gly Arg Asn Gly Lys Arg Ser Asn Arg Thr Leu Arg Glu             340 345 350 Glu Val Leu Arg Asn Leu Arg Ser Val Val Ser Leu Thr Phe Leu Leu         355 360 365 Gly Met Thr Trp Gly Phe Ala Phe Phe Ala Trp Gly Pro Leu Asn Ile     370 375 380 Pro Phe Met Tyr Leu Phe Ser Ile Phe Asn Ser Leu Gln Gly Leu Phe 385 390 395 400 Ile Phe Ile Phe His Cys Ala Met Lys Glu Asn Val Gln Lys Gln Trp                 405 410 415 Arg Arg His Leu Cys Cys Gly Arg Phe Arg Leu Ala Asp Asn Ser Asp             420 425 430 Trp Ser Lys Thr Ala Thr Asn Ile Ile Lys Lys Ser Ser Asp Asn Leu         435 440 445 Gly Lys Ser Leu Ser Ser Ser Ser Ile Gly Ser Asn Ser Thr Tyr Leu     450 455 460 Thr Ser Lys Ser Lys Ser Ser Ser Thr Thr Tyr Phe Lys Arg Asn Ser 465 470 475 480 His Thr Asp Asn Val Ser Tyr Glu His Ser Phe Asn Lys Ser Gly Ser                 485 490 495 Leu Arg Gln Cys Phe His Gly Gln Val Leu Val Lys Thr Gly Pro Cys             500 505 510 <210> 14 <211> 1014 <212> PRT <213> Homo sapiens <400> 14 Met Val Phe Ser Val Arg Gln Cys Gly His Val Gly Arg Thr Glu Glu 1 5 10 15 Val Leu Leu Thr Phe Lys Ile Phe Leu Val Ile Ile Cys Leu His Val             20 25 30 Val Leu Val Thr Ser Leu Glu Glu Asp Thr Asp Asn Ser Ser Leu Ser         35 40 45 Pro Pro Pro Ala Lys Leu Ser Val Val Ser Phe Ala Pro Ser Ser Asn     50 55 60 Glu Val Glu Thr Thr Ser Leu Asn Asp Val Thr Leu Ser Leu Leu Pro 65 70 75 80 Ser Asn Glu Thr Glu Lys Thr Lys Ile Thr Ile Val Lys Thr Phe Asn                 85 90 95 Ala Ser Gly Val Lys Pro Gln Arg Asn Ile Cys Asn Leu Ser Ser Ile             100 105 110 Cys Asn Asp Ser Ala Phe Phe Arg Gly Glu Ile Met Phe Gln Tyr Asp         115 120 125 Lys Glu Ser Thr Val Pro Gln Asn Gln His Ile Thr Asn Gly Thr Leu     130 135 140 Thr Gly Val Leu Ser Leu Ser Glu Leu Lys Arg Ser Glu Leu Asn Lys 145 150 155 160 Thr Leu Gln Thr Leu Ser Glu Thr Tyr Phe Ile Met Cys Ala Thr Ala                 165 170 175 Glu Ala Gln Ser Thr Leu Asn Cys Thr Phe Thr Ile Lys Leu Asn Asn             180 185 190 Thr Met Asn Ala Cys Ala Ala Ile Ala Ala Leu Glu Arg Val Lys Ile         195 200 205 Arg Pro Met Glu His Cys Cys Cys Ser Val Arg Ile Pro Cys Pro Ser     210 215 220 Ser Pro Glu Glu Leu Gly Lys Leu Gln Cys Asp Leu Gln Asp Pro Ile 225 230 235 240 Val Cys Leu Ala Asp His Pro Arg Gly Pro Pro Phe Ser Ser Ser Gln                 245 250 255 Ser Ile Pro Val Val Pro Arg Ala Thr Val Leu Ser Gln Val Pro Lys             260 265 270 Ala Thr Ser Phe Ala Glu Pro Pro Asp Tyr Ser Pro Val Thr His Asn         275 280 285 Val Pro Ser Pro Ile Gly Glu Ile Gln Pro Leu Ser Pro Gln Pro Ser     290 295 300 Ala Pro Ile Ala Ser Ser Pro Ala Ile Asp Met Pro Pro Gln Ser Glu 305 310 315 320 Thr Ile Ser Ser Pro Met Pro Gln Thr His Val Ser Gly Thr Pro Pro                 325 330 335 Pro Val Lys Ala Ser Phe Ser Ser Pro Thr Val Ser Ala Pro Ala Asn             340 345 350 Val Asn Thr Thr Ser Ala Pro Pro Val Gln Thr Asp Ile Val Asn Thr         355 360 365 Ser Ser Ile Ser Asp Leu Glu Asn Gln Val Leu Gln Met Glu Lys Ala     370 375 380 Leu Ser Leu Gly Ser Leu Glu Pro Asn Leu Ala Gly Glu Met Ile Asn 385 390 395 400 Gln Val Ser Arg Leu Leu His Ser Pro Pro Asp Met Leu Ala Pro Leu                 405 410 415 Ala Gln Arg Leu Leu Lys Val Val Asp Asp Ile Gly Leu Gln Leu Asn             420 425 430 Phe Ser Asn Thr Thr Ile Ser Leu Thr Ser Pro Ser Leu Ala Leu Ala         435 440 445 Val Ile Arg Val Asn Ala Ser Ser Phe Asn Thr Thr Thr Phe Val Ala     450 455 460 Gln Asp Pro Ala Asn Leu Gln Val Ser Leu Glu Thr Gln Ala Pro Glu 465 470 475 480 Asn Ser Ile Gly Thr Ile Thr Leu Pro Ser Ser Leu Met Asn Asn Leu                 485 490 495 Pro Ala His Asp Met Glu Leu Ala Ser Arg Val Gln Phe Asn Phe Phe             500 505 510 Glu Thr Pro Ala Leu Phe Gln Asp Pro Ser Leu Glu Asn Leu Ser Leu         515 520 525 Ile Ser Tyr Val Ile Ser Ser Ser Val Ala Asn Leu Thr Val Arg Asn     530 535 540 Leu Thr Arg Asn Val Thr Val Thr Leu Lys His Ile Asn Pro Ser Gln 545 550 555 560 Asp Glu Leu Thr Val Arg Cys Val Phe Trp Asp Leu Gly Arg Asn Gly                 565 570 575 Gly Arg Gly Gly Trp Ser Asp Asn Gly Cys Ser Val Lys Asp Arg Arg             580 585 590 Leu Asn Glu Thr Ile Cys Thr Cys Ser His Leu Thr Ser Phe Gly Val         595 600 605 Leu Leu Asp Leu Ser Arg Thr Ser Val Leu Pro Ala Gln Met Met Ala     610 615 620 Leu Thr Phe Ile Thr Tyr Ile Gly Cys Gly Leu Ser Ser Ile Phe Leu 625 630 635 640 Ser Val Thr Leu Val Thr Tyr Ile Ala Phe Glu Lys Ile Arg Arg Asp                 645 650 655 Tyr Pro Ser Lys Ile Leu Ile Gln Leu Cys Ala Ala Leu Leu Leu Leu             660 665 670 Asn Leu Val Phe Leu Leu Asp Ser Trp Ile Ala Leu Tyr Lys Met Gln         675 680 685 Gly Leu Cys Ile Ser Val Ala Val Phe Leu His Tyr Phe Leu Leu Val     690 695 700 Ser Phe Thr Trp Met Gly Leu Glu Ala Phe His Met Tyr Leu Ala Leu 705 710 715 720 Val Lys Val Phe Asn Thr Tyr Ile Arg Lys Tyr Ile Leu Lys Phe Cys                 725 730 735 Ile Val Gly Trp Gly Val Pro Ala Val Val Val Thr Ile Ile Leu Thr             740 745 750 Ile Ser Pro Asp Asn Tyr Gly Leu Gly Ser Tyr Gly Lys Phe Pro Asn         755 760 765 Gly Ser Pro Asp Asp Phe Cys Trp Ile Asn Asn Asn Ala Val Phe Tyr     770 775 780 Ile Thr Val Val Gly Tyr Phe Cys Val Ile Phe Leu Leu Asn Val Ser 785 790 795 800 Met Phe Ile Val Val Leu Val Gln Leu Cys Arg Ile Lys Lys Lys Lys                 805 810 815 Gln Leu Gly Ala Gln Arg Lys Thr Ser Ile Gln Asp Leu Arg Ser Ile             820 825 830 Ala Gly Leu Thr Phe Leu Leu Gly Ile Thr Trp Gly Phe Ala Phe Phe         835 840 845 Ala Trp Gly Pro Val Asn Val Thr Phe Met Tyr Leu Phe Ala Ile Phe     850 855 860 Asn Thr Leu Gln Gly Phe Phe Ile Phe Ile Phe Tyr Cys Val Ala Lys 865 870 875 880 Glu Asn Val Arg Lys Gln Trp Arg Arg Tyr Leu Cys Cys Gly Lys Leu                 885 890 895 Arg Leu Ala Glu Asn Ser Asp Trp Ser Lys Thr Ala Thr Asn Gly Leu             900 905 910 Lys Lys Gln Thr Val Asn Gln Gly Val Ser Ser Ser Ser Asn Ser Leu         915 920 925 Gln Ser Ser Ser Asn Ser Thr Asn Ser Thr Thr Leu Leu Val Asn Asn     930 935 940 Asp Cys Ser Val His Ala Ser Gly Asn Gly Asn Ala Ser Thr Glu Arg 945 950 955 960 Asn Gly Val Ser Phe Ser Val Gln Asn Gly Asp Val Cys Leu His Asp                 965 970 975 Phe Thr Gly Lys Gln His Met Phe Asn Glu Lys Glu Asp Ser Cys Asn             980 985 990 Gly Lys Gly Arg Met Ala Leu Arg Arg Thr Ser Lys Arg Gly Ser Leu         995 1000 1005 His Phe Ile Glu Gln Met     1010

[Brief description of drawings]

FIG. 1 is a diagram showing a hydropathy plot of C-HEP15374. In the figure, TM1 to TM7 indicate predicted transmembrane regions.

FIG. 2 shows an alignment of GPCRs similar to C-HEP15374. In the figure, “*” indicates the position where amino acids are completely conserved in all sequences, and “:” indicates the following groups ([STA] [NEQK] [NDBQ] [QHRK] [MILV] [MILF].
The position where any of the amino acids in [HY] [FYW]) is conserved is indicated by “•” in the following groups ([CSA] [ATV]
[SAG] [STNK] [STPA] [SGND] [SNDEQK] [NDEQH
K] [NEQHRK]) indicates the position where any amino acid is conserved. Also, ### TMn ### (n
= 1-7) indicates a predicted transmembrane region, and @ indicates a cysteine residue predicted to contribute to a disulfide bond specific to GPCR.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12P 21/02 C 4C084 5/10 C12Q 1/02 4H045 C12P 21/02 1/68 A C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/566 33/50 33/574 Z 33/566 A61K 45/00 33/574 A61P 1/16 // A61K 45/00 25/28 A61P 1/16 35/00 25/28 C12N 15/00 ZNAA 35/00 5/00 A (72) Inventor God ▲ Saki ▼ Koji Ⅱ 2-6 Nihonbashihonmachi, Chuo-ku, Tokyo Mitsubishi Welpharma Co., Ltd. Tokyo head office (72) Inventor Shinichiro Yasuda 2-6 Nihonbashihonmachi, Chuo-ku, Tokyo Mitsubishi Welpharma Co., Ltd. Tokyo headquarters (72) Inventor Toshimitsu Kishimoto 2-6 Nihonbashihonmachi, Chuo-ku, Tokyo Mitsubishi Welpharma Co., Ltd In-house F-term (reference) 2G045 AA40 DA36 FB03 4B024 AA01 AA11 BA63 CA01 CA04 CA09 CA11 DA02 DA05 DA11 EA 02 EA03 EA04 FA02 GA01 GA11 HA01 HA03 HA12 4B063 QA01 QA18 QA19 QQ01 QQ05 QQ42 QQ52 QR08 QR33 QR42 QR55 QR62 QR80 QS05 QS25 QS34 QS36 QX02 4B064 AG20 AG27 CA01 A19 A24 A01 A90 4A0A02 4C084 AA17 NA14 ZA16 ZA75 ZB26 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA40 DA50 DA76 EA20 EA50 FA72 FA74

Claims (33)

[Claims] 1. The following protein (a) or (b): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2 (b) an amino acid sequence of SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted, inserted, added or modified, A protein that exhibits G protein-coupled receptor activity by coupling with the same G protein as the protein of (a) couples 2. A peptide which is a fragment of the protein of claim 1. 3. A DNA containing a nucleotide sequence encoding the protein of claim 1. 4. The DNA according to claim 3, which is the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence of SEQ ID NO: 1 (b) Hybridizing with the DNA of (a) above under stringent conditions and coupling of the protein encoded by the DNA of (a) above G coupled with the same G protein
DN encoding a protein exhibiting protein-coupled receptor activity
A 5. A DNA containing a nucleotide sequence encoding the peptide according to claim 2. 6. The DNA according to claim 5, wherein the base sequence is a partial base sequence of the base sequence of SEQ ID NO: 1. 7. The DNA according to any one of claims 3 to 6.
A recombinant vector containing. 8. The recombinant vector according to claim 7, wherein the DNA is under the control of a promoter functional in a given host cell. 9. A transformant obtained by introducing the recombinant vector according to claim 7 or 8 into a host cell. 10. An expression vector containing the DNA according to claim 3 or 4 placed under the control of a promoter functional in a predetermined host cell is introduced into the host cell, and the resulting transformant is cultured. A method for producing the protein, comprising recovering the protein according to claim 1 from the culture. 11. An expression vector containing the DNA according to claim 5 or 6 placed under the control of a promoter functional in a predetermined host cell is introduced into the host cell, and the resulting transformant is cultured. A method for producing the peptide, which comprises recovering the peptide according to claim 2 from the culture. 12. An antibody having a specific affinity for the protein according to claim 1 or the peptide according to claim 2. 13. The expression of the protein according to claim 1 or the DNA according to claim 3 or 4 is measured in a tissue at a lesion site or an onset causative site of a predetermined disease and a tissue corresponding to a normal animal, respectively, and both of them are measured. The protein or the D, characterized in that the protein or the DNA is judged to be a marker for the disease when a significant difference in expression level is observed between the proteins.
A method for selecting a disease in which NA can serve as a marker. 14. The protein according to claim 1, in a biological sample derived from a tissue at a predicted lesion site or onset causative site of an animal that may be affected by a disease selected by the method according to claim 13. Or claim 3 or 4
A method for diagnosing the disease, which comprises measuring the expression level of the described DNA. 15. The method of claim 14, wherein the disease is selected from the group consisting of cancer, cirrhosis and Alzheimer's disease. 16. The method of claim 15, wherein the cancer is cancer in an organ selected from the group consisting of brain, thyroid, lung, stomach, pancreas, kidney, colon, prostate, testis and uterus. 17. A diagnostic agent for a disease selected by the method according to claim 13, which comprises any of the following (a) to (c) as a main component. (A) DNA according to any one of claims 3 to 6 (b) Primer pair capable of specifically amplifying all or part of the DNA according to claim 3 (c) Antibody according to claim 12 18. The diagnostic agent according to claim 17, wherein the disease is selected from the group consisting of cancer, cirrhosis and Alzheimer's disease. 19. The diagnostic agent according to claim 18, wherein the cancer is cancer in an organ selected from the group consisting of brain, thyroid, lung, stomach, pancreas, kidney, colon, prostate, testis and uterus. 20. A method comprising contacting a test sample with the protein according to claim 1 or the peptide according to claim 2 and selecting a substance that binds to the protein or the peptide.
A method for screening a ligand for the described protein. 21. A reagent for screening a ligand for the protein according to claim 1, which contains the protein according to claim 1 or the peptide according to claim 2. 22. A method for screening a substance that inhibits or promotes the activity of the protein according to claim 1, wherein the protein or the peptide according to claim 2 is provided with a ligand for the protein in the presence or absence of a test substance. Contact in the presence,
A method for comparing the binding activities of the protein or the peptide and the ligand under both conditions. 23. A method for screening a substance that inhibits or promotes the activity of the protein according to claim 1, wherein cells expressing the protein are treated with a ligand for the protein in the presence and absence of a test substance. A method comprising contacting and comparing changes in cells due to binding of the protein and the ligand under both conditions. 24. The method according to claim 23, wherein the change in the cell is a change in adenylate cyclase activity, intracellular cAMP amount or expression of a reporter gene under the control of a cAMP response element. 25. The change in the cell is a change in phospholipase C activity, intracellular calcium ion content or expression of a reporter gene under the control of a 12-O-tetradecanoylphorbol-13-acetate response element.
The method of claim 23. 26. A lipid bilayer membrane containing the protein according to claim 1, and a poly containing at least a receptor binding region of a G protein α subunit belonging to a family and a guanine nucleotide binding region of an arbitrary G protein α subunit. A system containing a peptide as a constituent element is defined as one constituent unit, and the constituent unit is provided for the receptor binding region of each family of G protein α subunits,
A screening system for a ligand for the protein according to claim 1, or a substance that inhibits or promotes the activity of the protein. 27. An expression vector, wherein the constituent unit comprises the DNA according to claim 3 or 4, a receptor binding region of a G protein α subunit belonging to a family, and an arbitrary G
27. A host animal cell, a homogenate of the cell, or a membrane fraction derived from the cell, which has been transfected with an expression vector containing a DNA encoding a polypeptide containing at least a guanine nucleotide-binding region of a protein α subunit. Screening system. 28. The structural unit comprises, on the C-terminal side of the protein according to claim 1, a receptor-binding region of a G protein α subunit belonging to a family and a guanine nucleotide-binding region of an arbitrary G protein α subunit. D encoding a fused polypeptide of at least the containing polypeptide
27. The screening system according to claim 26, which is a host animal cell transfected with an expression vector containing NA, a homogenate of the cell, or a membrane fraction derived from the cell. 29. The polypeptide comprising the receptor binding region of the G protein α subunit and the guanine nucleotide binding region of any G protein α subunit of each constituent unit further comprises the same effector interaction region, The screening system according to any one of claims 26 to 28, wherein the lipid bilayer membrane further comprises an effector that interacts with the region. 30. In each constituent unit of the screening system according to any one of claims 26 to 28, a labeled GTP analog is added in the presence or absence of a test substance to bind to the guanine nucleotide binding region. The method for screening a ligand for a protein according to claim 1, or a substance that inhibits or promotes the activity of the protein, which comprises comparing the labeled amounts under both conditions. 31. A ligand for the protein according to claim 1, wherein the activity of the effector in each constituent unit of the screening system according to claim 29 is compared in the presence and absence of a test substance. Alternatively, a method for screening a substance that inhibits or promotes the activity of the protein. 32. A nucleic acid according to any of the following (a) or (b): (A) Nucleic acid having a base sequence complementary to the base sequence shown in SEQ ID NO: 1 (b) Nucleic acid having the base sequence shown in SEQ ID NO: 1 or an initial transcription product that produces the base sequence by post-transcriptional processing, and mammalian cells Nucleic acid comprising a base sequence capable of hybridizing under the physiological conditions of SEQ ID NO: 1 and capable of inhibiting the translation of the protein encoded by the base sequence of SEQ ID NO: 1 in the hybridized state 33. An expression vector encoding the nucleic acid according to claim 32.