JPH0984581A - Production of protein of recombinant human melatonin receptor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject protein useful for the development of a prophylactic or a treating agent for prostatic carcinoma, uterine cancer, etc., a pregnancy-controlling agent, etc., by culturing a CHO cell having an expression vector containing a DNA coding for the protein of a human melatonin receptor under conditions in which the gene can be expressed. SOLUTION: A human genome DNA is amplified by a PCR method using a primer prepared by the synthesis based on the base sequence of the cDNA of a known human melatonin receptor and cloned. An expression vector pAKK0- hMe1R7 having a DNA coding for a human melatonin receptor protein is obtained by integrating the DNA coding for a human melatonin receptor protein having the base sequence expressed by the formula into a vector. Subsequently, the expression vector is introduced into a CHO cell deficient in a DHFR gene to transform the cell. The transformed cell is cultured under conditions in which the genes can be expressed to obtain the objective protein useful as a prophylactic or a treating agent for prostatic carcinoma, uterine cancer, benign prostatic hypertrophy, myoma of the uterus, etc., a pregnancy-controlling agent, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a CHO cell or a cell membrane fraction thereof capable of continuously producing a human melatonin receptor protein, a recombinant human melatonin receptor protein or a partial peptide thereof, the CHO cell or A method for screening a compound having affinity for a melatonin receptor or a salt thereof using the cell membrane fraction or the recombinant human melatonin receptor protein or a partial peptide thereof, the screening kit, the screening method or the screening kit The present invention relates to a melatonin receptor affinity compound or a salt thereof, and a pharmaceutical composition containing the compound.

[0002]

BACKGROUND OF THE INVENTION Melatonin (N-acetyl-5-methoxytryptamine) is a substance secreted by the pineal gland and retina, and its secretory reaction is known to be deeply involved in circadian rhythm [Krause, DN Annual.・ Review of Pharmacology and Toxicology (Annu. Rev. Pharmacol. Toxicol.) 31, 549-568 (1
991)]. The action of melatonin has been shown to play an important role in the regulation of the reproductive system such as antigonadotrophic action, and in the mechanism that synchronizes the physiological rhythm / endocrine rhythm with the external environment (light, temperature, etc.). [Repper
t, SM et al. Neuron 13, 1177-1185
(1994), and Ebisawa, T. et al. Proceedings of the National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA).
91, 6133-6137 (1994)]. Attempts have also been made to apply the action of melatonin to the treatment of diseases associated with abnormal reproductive system or in vivo rhythm. It has been shown so far that a decrease in the amount of melatonin secretion in a living body is deeply involved in a certain sleep disorder, and it is considered that the improvement can be achieved by changing the blood concentration of melatonin. Conventionally, oral and transdermal administration of melatonin have been attempted from such a viewpoint, but since the half-life of melatonin in vivo is short and it is easily decomposed and metabolized, stable and effective agonists and antagonists are developed. Was desired.

In general, when developing an agonist or an antagonist of a physiologically active substance, a compound having a high affinity with a receptor to which the substance specifically binds is searched for. In order to efficiently screen for compounds that act on the human melatonin receptor and to develop them into medicines, it is essential to obtain cells that stably express the human melatonin receptor. The cDNA encoding the human melatonin receptor has already been cloned, and COS-
Expression in 7 cells has also been reported, but it is considered to be inappropriate for use in screening because the expression is transient. Therefore, it has been desired to establish a more practical method for producing a human melatonin receptor protein. It was reported that the melatonin receptor protein coding region was divided into two regions by one intron on the human genome [Reppert, SM et al. Neuron (Neuron) 13, 1177-1185 (1994)]. . Therefore, in order to obtain the complete coding region efficiently, cDNA is
It was necessary to use it as a template for CR. But,
It is known that the expression site of melatonin receptor is localized in the supraoptic nucleus of the hypothalamus in the human brain [Casson
e, VM Trends in Neuroscience (Trend
s Neurosci.) 13, 457-464 (1990)], a messenger R encoding a melatonin receptor using this portion as a material.
It was difficult to prepare NA or cDNA using it as a template. Further, a human-derived cell line expressing a messenger RNA encoding a melatonin receptor has not been reported so far.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a CHO cell highly expressing a human melatonin receptor protein cDNA or a cell membrane fraction thereof, a recombinant human melatonin receptor protein or a partial peptide thereof, the CHO cell or Using the cell membrane fraction or the recombinant human melatonin receptor protein or its partial peptide, a method for screening a human melatonin receptor affinity compound or a salt thereof, the screening kit, the screening method or the screening kit The thus-obtained melatonin receptor affinity compound or a salt thereof, and a pharmaceutical composition containing the compound are provided.

[0005]

In view of the above-mentioned circumstances, the present inventors have earnestly studied a method for screening a compound effective for a human melatonin receptor, and as a result, the present inventors have found that a novel synthetic DNA is synthesized. PCR using primers
By carrying out the above, we succeeded in obtaining a portion encoding the melatonin receptor protein from human genomic DNA. In the synthetic DNA primer used in the present invention, a restriction enzyme cleavage site is artificially introduced by modifying a part of the nucleotide sequence, and the fragmented exon can be efficiently linked and inserted into an expression vector. Designed to be. In the present invention, by using these, DNA encoding the same sequence as the amino acid sequence encoded by cDNA was obtained. Furthermore, the present inventors have succeeded in producing a CHO cell line that highly expresses human melatonin receptor protein using the obtained DNA. Furthermore, the present inventors have found that by using the CHO cell line, it is possible to efficiently and surely screen a compound effective for melatonin receptor or a salt thereof effective for humans. Then, the present inventors have completed the present invention as a result of further research based on these findings.

That is, the present invention provides: (1) a CHO cell containing an expression vector carrying a DNA encoding a human melatonin receptor protein, (2) the CHO cell according to item (1) Is produced by culturing under a condition capable of expressing a human melatonin receptor protein, or a CHO cell containing a recombinant human melatonin receptor protein or a cell membrane fraction thereof, (3) DN encoding human melatonin receptor protein
PAKKO-hMelR characterized by retaining A
Expression vector (Escherichia coli DH5
/ PAKKO-hMelR7 (FERM BP-523
Expression vector pAKKO-hMelR contained in 6)
7), (4) A recombinant human melatonin receptor having substantially the same activity as a natural human melatonin receptor protein, characterized in that it is isolated from the CHO cells according to (2). Protein, partial peptide thereof or salt thereof, (5) Recombinant human melatonin characterized by culturing CHO cells according to (1) under conditions capable of expressing human melatonin receptor protein (6) A method for producing a receptor protein, (6) a method for screening a compound having affinity for a melatonin receptor or a salt thereof, which comprises contacting a test compound with a CHO cell or a cell membrane fraction thereof according to (2), (7) ) The test compound is contacted with the recombinant human melatonin receptor protein according to item (4), a partial peptide thereof or a salt thereof. A method for screening a melatonin receptor affinity compound or a salt thereof,

(8) A CHO cell or a cell membrane fraction thereof according to item (2), or the recombinant human melatonin receptor protein according to claim 4, a partial peptide thereof or a salt thereof. A kit for screening a compound having affinity for a melatonin receptor or a salt thereof, which is obtained by using the screening method according to (9) (6) or (7) or the screening kit according to (8). Melatonin receptor affinity compound or salt thereof (10) Melatonin receptor affinity compound or salt thereof according to item (9), wherein the melatonin receptor affinity compound is a melatonin receptor agonist, (11) Melatonin receptor affinity The compound having affinity for melatonin receptor according to item (9), wherein the compound is a melatonin receptor antagonist. Is a salt thereof, (12) a pharmaceutical composition comprising the melatonin receptor agonist according to (10), (13) sleep-wake rhythm disorder, jet lag, insomnia, seasonal depression, reproduction and Neuroendocrine disease, Alzheimer's disease, cerebrovascular dementia, various disorders associated with aging, cerebral circulation disorder,
The pharmaceutical composition according to item (12), which is an agent for preventing or treating stress, anxiety, convulsions, Parkinson's disease, hypertension, cancer or glaucoma, or an ovulation regulator, (14) Melatonin receptor antagonist according to item (11). (15) Prevention of depression, anxiety, neurosis or disturbance of consciousness
The pharmaceutical composition according to item (14), which is a therapeutic agent or therapeutic agent.

More specifically, the present invention provides: (16) a CHO cell deficient in the DHFR gene according to item (1), and (17) the CHO cell is designated by CHO-hMelR7. The CHO cell according to item (1) or (2), which is a CHO cell (FERMBP-5242), (18) the DNA encoding the human melatonin receptor protein has the nucleotide sequence represented by SEQ ID NO: 1. The expression vector according to item (3), which is a DNA-containing DNA, (19) the recombinant human melatonin receptor protein, wherein the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequence represented by SEQ ID NO: 2 Amino acid sequence in which 1 or 2 or more amino acids are deleted, or 1 or 2 or more amino acids are added to the amino acid sequence represented by SEQ ID NO: 2. Or a protein having an amino acid sequence in which one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 are substituted with other amino acids, or the N-terminal signal peptide of these proteins is cleaved Proteins, proteins in which the side chains of amino acids in the molecules of these proteins are protected by appropriate protecting groups (eg, C _1-6 acyl groups such as formyl group, acetyl group, etc.), or sugars in those proteins. The recombinant human melatonin receptor protein according to item (4), which is a protein in which chains or the like are bound, a partial peptide thereof or a salt thereof,

(20) (i) When the CHO cell or the cell membrane fraction thereof described in (2) is contacted with a ligand for a melatonin receptor, (ii) the CHO cell described in (2) or A method for screening a melatonin receptor affinity compound or a salt thereof according to item (6), wherein the cell membrane fraction is compared with a case where a ligand for a melatonin receptor and a test compound are contacted. 21) (i) the case where a labeled ligand for a melatonin receptor is brought into contact with the CHO cell or the cell membrane fraction thereof described in (2), and (ii) the CH described in (2)
When the labeled ligand for the melatonin receptor and a test compound are contacted with O cells or their cell membrane fractions, the amount of binding of the labeled ligand for the melatonin receptor to the CHO cells or its cell membrane fraction is measured and compared. (22) (i) A test compound for the CHO cell or cell membrane fraction thereof according to item (2), wherein the melatonin receptor affinity compound or salt thereof is screened. Recombinant human melatonin receptor-mediated cell stimulating activity when contacted (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ concentration fluctuation, intracellular cAMP production, intracellular c
Promotes GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, cell migration activity, hormone secretion, G protein activation, cell growth, etc. Activity or inhibitory activity, etc.), and a method for screening a melatonin receptor agonist, characterized by comparing

(23) (i) When the melatonin receptor agonist is contacted with the CHO cells or the cell membrane fraction thereof described in (2), and (ii) the CHO described in (2).
Recombinant human melatonin receptor-mediated cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ² ) when a cell or its cell membrane fraction is contacted with a melatonin receptor agonist and a test compound. ⁺ Fluctuation of concentration, intracellular cAMP production, intracellular cGM
P production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH
Decrease, cell migration activity, hormone secretion, G protein activation, activity promoting or suppressing cell growth, etc.) are measured and compared, a method for screening melatonin receptor antagonists, 24) (i) a case where a recombinant human melatonin receptor protein, a partial peptide thereof or a salt thereof according to the above (4) is contacted with a ligand for a melatonin receptor, and (ii) (4) The recombinant human melatonin receptor protein, the partial peptide thereof, or a salt thereof according to the above item is compared with a case where a ligand for the melatonin receptor and a test compound are contacted with each other, (7). A method for screening a compound having affinity for a melatonin receptor or a salt thereof, and the recombinant according to (25) (i) (4). When the labeled human melatonin receptor protein, a partial peptide thereof or a salt thereof is contacted with a labeled ligand for the melatonin receptor, and (ii) the recombinant human melatonin receptor according to (4). When a labeled ligand for the melatonin receptor and a test compound are contacted with a protein, a partial peptide thereof or a salt thereof,
The amount of binding of a labeled ligand for a melatonin receptor to the recombinant human melatonin receptor protein, its partial peptide or salts thereof is measured and compared, and the melatonin receptor according to (7) is characterized. A method for screening an affinity compound or a salt thereof is provided.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, when an expression vector carrying a DNA encoding a human melatonin receptor protein is prepared, the DNA encoding the human melatonin receptor protein may be, for example, a human melatonin receptor protein. Although a cDNA or a genomic DNA that encodes a somatic protein has been used, it has a nucleotide sequence that encodes a human melatonin receptor protein or a partial peptide that has a ligand binding activity substantially equivalent to that of the human melatonin receptor protein. If it is a thing, it is not necessarily restricted to this. For example, a cDNA encoding a known human melatonin receptor protein
And genomic DNA can be used, but synthetic DN
It may be A or the like. Specific examples thereof include a DNA encoding a human melatonin receptor protein having the amino acid sequence represented by SEQ ID NO: 2, and, for example, a DNA having the nucleotide sequence represented by SEQ ID NO: 1 (FIG. 1) and the like. Used. These DNAs can be cloned using a genetic engineering technique known per se, or can be produced using a nucleotide synthesizer. For example, cloning can be performed according to the method of Reference Example 1 described below or a method analogous thereto.

Examples of expression vectors include pAKK
O-111, pAKKO-111H, pXT1, pRc
/ CMV, pRc / RSV, pcDNAINEo and the like are used, and among them, pAKKO-111H and the like are preferable. As the promoter, any promoter can be used so long as it can efficiently function in a host cell, such as SV40 promoter, CMV promoter, HSV-TK promoter,
Examples include SRα promoter and RSV promoter, and among them, CMV promoter, SRα promoter, RSV promoter and the like are preferable, and SRα promoter is particularly preferable. It is preferable to use an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker and the like in addition to the above. Examples of the selection marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as DHFR or dhfr) gene, a neomycin resistance gene [G418 resistance], and the like, and among them, the DHFR gene is preferable. In particular, when the DHFR gene is used as a selection marker using CHO cells deficient in the DHFR gene, selection can also be performed using a thymidine-free medium.

The expression vector carrying the DNA encoding the human melatonin receptor protein of the present invention includes, specifically, the above promoter (particularly, in particular, upstream of the DNA encoding the human melatonin receptor protein).
CMV promoter, SRα promoter, RSV promoter, etc.), a poly A addition signal is inserted downstream of the DNA encoding human melatonin receptor protein, and a selection marker such as DHFR gene or neomycin resistance gene is inserted downstream thereof. However, it is preferable that the ampicillin resistance gene is inserted further downstream. More specifically, for example, in the expression vector that can be produced by the method shown in FIG. 2, the SRα promoter is inserted upstream of the DNA encoding the human melatonin receptor protein to obtain the human melatonin receptor protein. PAKKO-h in which a poly-A addition signal was inserted downstream of the DNA encoding DNA, the DHFR gene was inserted downstream thereof, and the ampicillin resistance gene was inserted further downstream thereof.
An expression vector indicated by MelR7 [Fig. 3] and the like are preferable. A cell capable of highly expressing the DNA encoding the human melatonin receptor protein can be produced by introducing the expression vector having the DNA encoding the human melatonin receptor protein thus produced into a host cell. it can.

The host CHO cells are CHO cells [Journal of Experiment of Medison (J. EXP. Med.), 108, 945 (19).
58)] and the like are preferable. Furthermore, among CHO cells, particularly CHO cells deficient in the DHFR gene (hereinafter sometimes abbreviated as CHO (dhfr ⁻ ) cells) [Procedures of the National Academy of Science]・ USA (Proc.
Natl. Acad. Sci. USA), 77, 421
6-4220 (1980)], CHO K-1 cells [Procedures of the National Academy.
Of Science USA (Proc. Nat
l. Acad. Sci. USA), 60, 1275 (1
968)] and the like are preferable. Especially in the expression vector D
When inserting the HFR gene as a selectable marker, C
HO (dhfr ⁻ ) cells are preferred. When an expression plasmid having the DHFR gene as a selection marker is introduced, the transformants can be selected very easily by simply culturing in a medium containing no nucleic acid. As the combination of the expression vector and the host cell, a preferable combination can be appropriately selected, and for example, pAKKO-h
CHO (dhfr ⁻ ) cells and the like are suitable as host cells for the expression vector [FIG. 3] designated by MelR7. As a method for introducing an expression vector into a host cell, a known method, for example, the calcium phosphate method [Graham, FL
and van der Eb, AJ Virology 52, 4
56-467 (1973)], electroporation [Neumann, E. et al. EMBO J., 1, 841-845 (1982)] and the like.

A cell capable of highly expressing the human melatonin receptor protein can be obtained by clonally selecting a cell in which the above-described expression vector is integrated in the chromosome. Specifically, first, a transformant is selected using the above selection marker as an index. Furthermore, by repeatedly performing clonal selection on the transformant thus obtained using the selectable marker, it is possible to obtain a cell line stably having a high expression ability of the human melatonin receptor protein. . When the DHFR gene is used as a selection marker, methotrexate (M
It is also possible to obtain a cell line with higher expression by amplifying the transgene in cells by gradually increasing the concentration of TX) and culturing to select resistant cells. In addition,
Even when CHO (dhfr ⁻ ) cells are used as a host, for example, since the DHFR gene has been introduced into the expression vector indicated by pAKKO-hMelR7 [FIG. 3], the expression indicated by pAKKO-hMelR7 is shown. The CHO (dhfr ⁻ ) cells containing the vector will consequently have the DHFR gene. In the present specification, an expression vector containing the DHFR gene (for example, pAKKO-hMelR7 etc.) is used as CH.
The CHO cells obtained by containing them in O (dhfr ⁻ ) cells may be referred to as “CHO (dhfr ⁺ ) cells”.

In the present invention, the CHO cells capable of highly expressing the DNA encoding the human melatonin receptor protein include, for example, pA obtained in Example 1 described later.
CH the expression vector designated by KKO-hMelR7
CHO obtained by containing O (dhfr ⁻ ) cells
(Dhfr ⁺ ) cells and the like. Specifically, C
CHO (dhfr ⁺ ) labeled with HO-hMelR7
Cells and the like are preferred. These CHO (dhfr ⁺ )
The cells are about 10 to 3 as compared with the tissue containing the natural melatonin receptor protein (for example, chicken forebrain).
It has a receptor activity (eg, ligand binding activity) of 00-fold, preferably about 200-300 fold. The cells containing the expression vector carrying the DNA encoding the human melatonin receptor protein of the present invention were transformed into D cells encoding the human melatonin receptor protein.
Recombinant human melatonin receptor protein can be produced by culturing under conditions capable of expressing NA. When culturing cells containing an expression vector carrying a DNA encoding a human melatonin receptor protein, the medium is EMEM medium containing about 0.5 to 20% fetal bovine serum, DMEM medium, RPMI1640.
Examples of the medium include α-MEM medium. Especially CHO
When (dhfr ⁻ ) cells and the DHFR selectable marker gene are used, it is preferable to use DMEM medium or α-MEM medium containing thymidine-free dialyzed fetal bovine serum. Preferably, the pH is between about 6-8. Culturing is usually performed at about 30 to 40 ° C. for about 15 to 200 hours,
If necessary, change the medium, add aeration, stirring, etc. Thus, CH containing the expression vector carrying the DNA encoding the human melatonin receptor protein
CHO cells containing recombinant human melatonin receptor protein can be produced from O cells.

The cell membrane fraction of CHO cells containing the recombinant human melatonin receptor protein is obtained by disrupting the cells containing the recombinant human melatonin receptor protein described above and then using a method known per se. Refers to a fraction containing a large amount of cell membrane. Examples of the cell crushing method include crushing the cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or Polytron (manufactured by Kinematica), crushing with ultrasonic waves, from a fine nozzle while crushing cells with a French press, etc. Examples include crushing by jetting. For fractionation of cell membranes, fractionation by centrifugal force, such as fractionation centrifugation and density gradient centrifugation, is mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (generally about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15,000 to 30000 rpm) for 30 minutes.
After centrifugation for 2 minutes to 2 hours, the obtained precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed human melatonin receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of human melatonin receptor protein in the cells containing the recombinant human melatonin receptor protein of the present invention or the cell membrane fraction thereof is 10 ³ per cell.
It is preferably 10 ⁸ molecules, and more preferably 10 ⁵ 10 ⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which not only enables the construction of a highly sensitive screening system,
A large number of samples can be measured in the same lot.

Isolation of the recombinant human melatonin receptor protein from the CHO cells containing the recombinant human melatonin receptor protein obtained above can be carried out, for example, by the following method. When extracting the recombinant human melatonin receptor protein from CHO cells, after culturing, the cells are collected by a known method, suspended in an appropriate buffer solution, and the cells are subjected to ultrasonic waves, a homogenizer or freeze-thawing. The method of obtaining a crude extract of recombinant human melatonin receptor protein by centrifuging or filtering after destroying the protein is appropriately used. PMSF (phenylmethanesulfonyl fluoride) in the buffer,
Proteolytic enzyme inhibitors such as pepstatin and leupeptin, and CHAPS (3-[(3-cholamidopropyl) dimethyl ammo
nio] propanesulfonic acid), digitonin, Triton X
A surfactant such as -100 (registered trademark; hereinafter sometimes abbreviated as TM) may be included. The recombinant human melatonin receptor protein contained in the obtained extract can be purified by an appropriate combination of separation / purification methods known per se. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and the like. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific novelty such as affinity chromatography, use of difference in hydrophobicity such as reversed-phase high-performance liquid chromatography And a method utilizing the difference in isoelectric points such as isoelectric focusing.

When the recombinant human melatonin receptor protein thus obtained is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely obtained as a salt. In some cases, the free form or other salt can be converted by a method known per se or a method analogous thereto. In addition, the recombinant human melatonin receptor protein produced by the recombinant is treated with an appropriate protein-modifying enzyme before or after purification to arbitrarily modify or partially remove the polypeptide. You can also As the protein modifying enzyme, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glucosidase and the like are used. As described above, a recombinant type that can be produced by culturing CHO cells containing an expression vector carrying a DNA encoding a human melatonin receptor protein under conditions capable of expressing the human melatonin receptor protein The human melatonin receptor protein has substantially the same activity as the naturally occurring human melatonin receptor protein. Examples of substantially the same activity include ligand binding activity and signal transduction. As the ligand binding activity, melatonin,
2,3-Dihydromelatonin (Aust.J.Biol.Sci.39,427-4
33 (1986)), S-20098 (JP-A-7-4833).
1), GR-131663 (JP-A-3-169840)
Such as agonists and Luzindol (J.Pharmcol.Exp.The
r.246,902-910 (1988)), ML-23 (Eur.J.Pharmcol.
136,259-260 (1987)), GR-13347 (JP-A-3-
169840) and the like, and binding activity with an antagonist and the like. "Substantially the same quality" means that the ligand binding activity and the like are qualitatively the same. Therefore, quantitative factors such as the molecular weight of the receptor protein may be different.

The recombinant human melatonin receptor protein of the present invention includes, for example, a recombinant human melatonin receptor protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2. Can be mentioned. Specifically, in addition to the recombinant human melatonin receptor protein having the amino acid sequence represented by SEQ ID NO: 2, 1 or 2 or more (preferably 2) in the amino acid sequence represented by SEQ ID NO: 2 30 or less,
More preferably from 2 to 10 amino acids), 1 in the amino acid sequence represented by SEQ ID NO: 2.
Alternatively, two or more (preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less) amino acids are added, or 1 in the amino acid sequence represented by SEQ ID NO: 2.
Alternatively, those in which 2 or more (preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less) amino acids are substituted with other amino acids, etc. Furthermore, these recombinant human melatonin receptor proteins are
The terminal signal peptide may be cleaved, or the side chain of the amino acid in the molecule may be protected with an appropriate protecting group (for example, C _1-6 acyl group such as formyl group and acetyl group). Alternatively, a sugar chain or the like may be bound. The salt of the recombinant human melatonin receptor protein of the present invention is preferably a physiologically acceptable acid addition salt. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid),
Alternatively, organic acids (eg, acetic acid, formic acid, propionic acid,
Fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid,
Malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like salts are used.

The partial peptide of the recombinant human melatonin receptor protein of the present invention is, for example, a portion of the molecule of the recombinant human melatonin receptor protein of the present invention which is exposed outside the cell membrane. Is used.
Specifically, it is a partial peptide analyzed to be an extracellular region (hydrophilic region) in a hydrophobicity plot analysis. Further, a peptide partially containing a hydrophobic (Hydrophobic) site can also be used. Furthermore, a peptide containing each domain individually may be used, but a partial peptide containing multiple domains simultaneously may be used. As the salt of the partial peptide of the recombinant human melatonin receptor protein of the present invention, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid).

The partial peptide of the recombinant human melatonin receptor protein of the present invention or a salt thereof can be prepared according to a method known per se for peptide synthesis, or the recombinant human melatonin receptor protein of the present invention can be prepared with an appropriate peptidase. It can be manufactured by cutting. The peptide synthesis method may be either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion and, when the product has a protecting group, removing the protecting group. Examples of known condensation methods and removal of protecting groups include:
The methods described in the following are mentioned. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervision of Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten The partial peptide of the present invention can be purified and isolated by a combination of purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained by a salt, it can be converted to a free form by a known method. Can be.

CHO cells containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof, or the recombinant human melatonin receptor protein of the present invention, a partial peptide thereof or a salt thereof is melatonin. It is useful as a reagent for screening a receptor affinity compound (preferably a human melatonin receptor affinity compound) or a salt thereof. The human melatonin receptor affinity compound includes a melatonin receptor agonist and a melatonin receptor antagonist. That is, the present invention provides (1) a compound having affinity for a melatonin receptor or a compound thereof, which comprises contacting a test compound with a CHO cell containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof. Method for screening salt, and (2) contacting a test compound with a recombinant human melatonin receptor protein of the present invention, a partial peptide thereof or a salt thereof, and a compound having affinity for a melatonin receptor or a salt thereof A screening method is provided. Specifically, the present invention relates to (1) (i) the case where a ligand for a melatonin receptor is brought into contact with CHO cells containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof. And (ii) a comparison with a case where a CHO cell containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof is contacted with a ligand for a melatonin receptor and a test compound. And (2) (i) Recombinant human melatonin receptor protein of the present invention, a partial peptide thereof or a salt thereof is contacted with a ligand for a melatonin receptor. And when
(Ii) Melatonin receptor characterized in that the recombinant human melatonin receptor protein of the present invention, a partial peptide thereof or a salt thereof is contacted with a ligand for the melatonin receptor and a test compound A method for screening a body-affinity compound or a salt thereof is provided.

More specifically, in the screening method of the present invention, in the cases of (i) and (ii), for example, the recombinant human melatonin receptor protein, its partial peptide or a salt thereof, or a combination thereof is used. It is characterized in that the amount of ligand bound to the melatonin receptor with respect to CHO cells containing the altered human melatonin receptor protein or its cell membrane fraction, the cell stimulating activity by the ligand, etc. are measured and compared. .
That is, the present invention provides (1a) (i) a case where a labeled ligand for a melatonin receptor is contacted with a CHO cell containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof, (Ii) CHO cells containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof are labeled with a melatonin receptor when the labeled ligand for melatonin receptor and a test compound are contacted A method for screening a compound having affinity for a melatonin receptor or a salt thereof, which comprises measuring and comparing the amount of a ligand bound to the CHO cell or a cell membrane fraction thereof, (2a) (i) the recombinant human of the present invention・ For melatonin receptor protein, its partial peptides or their salts
When the labeled ligand for the melatonin receptor is brought into contact, (ii) the recombinant human melatonin receptor protein of the present invention, a partial peptide thereof or a salt thereof is provided with the labeled ligand for the melatonin receptor and a test compound. A melatonin receptor-affinity compound or its salt characterized by measuring and comparing the amount of binding of a labeled ligand to the melatonin receptor to the receptor protein, its partial peptide or their salts when contacted Screening method,

(1b) (i) CHO cells containing the recombinant human melatonin receptor protein of the present invention or human melatonin receptor-mediated cells when a test compound is contacted with a cell membrane fraction thereof Stimulatory activity (eg, arachidonic acid release, acetylcholine release, fluctuation of intracellular Ca ²⁺ concentration, intracellular cAMP production, intracellular cGM
P production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH
Decrease, cell migration activity, hormone secretion, G protein activation, activity that promotes or suppresses cell growth, etc.) is measured and compared, screening of human melatonin receptor agonist And (1c) (i) contacting a melatonin receptor agonist with a CHO cell containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof, and (ii) Human melatonin receptor-mediated cell stimulating activity (eg, arachidone) when a CHO cell containing a recombinant human melatonin receptor protein or a cell membrane fraction thereof is contacted with a melatonin receptor agonist and a test compound. Acid release, acetylcholine release, fluctuation of intracellular Ca ²⁺ concentration, intracellular cAMP
Production, intracellular cGMP production, inositol phosphate production,
Cell membrane potential fluctuation, intracellular protein phosphorylation, cf
os activation, pH decrease, cell migration activity, hormone secretion, G protein activation, activity that promotes or suppresses cell growth, etc.) is measured and compared, and the melatonin receptor is characterized. A method for screening a body antagonist is provided.

In the screening method of (1a) or (2a) above, the recombinant human melatonin receptor protein of the present invention or a partial peptide thereof or the CHO cell of the present invention or a cell membrane fraction thereof is bound,
A compound that inhibits the binding between a ligand for the melatonin receptor and a recombinant human melatonin receptor protein or a partial peptide thereof or the CHO cell of the present invention or a cell membrane fraction thereof is a candidate compound for a melatonin receptor affinity compound or a salt thereof. Can be selected as
Moreover, in the screening method of (1b) above, a cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ concentration fluctuation, intracellular cAMP generation,
Intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, cell migration activity, hormone secretion, G protein activation, cell growth, etc. A compound having a stimulating activity or a suppressing activity) can be selected as a melatonin receptor agonist. further,
In the screening method of (1c) above, an agonist for the melatonin receptor and the recombinant human
A compound that inhibits binding to CHO cells containing a melatonin receptor protein or a cell membrane fraction thereof but does not have the above cell stimulating activity can be selected as a melatonin receptor antagonist.

Before the CHO cells containing the recombinant human melatonin receptor protein of the present invention were obtained, there was no animal cell capable of continuously high-expressing the human melatonin receptor protein. It was not possible to efficiently screen a compound (particularly, a compound having affinity for human melatonin receptor) or a salt thereof. However, CHO cells containing the recombinant human melatonin receptor protein of the present invention are
Since a large amount of melatonin receptor protein can be expressed,
It is useful for screening a compound having affinity for melatonin receptor or a salt thereof. In particular, the suspension culturable C of the present invention
HO cells are suitable for large-scale culture of recombinant human melatonin receptor protein. The specific description of the screening method of the present invention is as follows. When CHO cells containing the human melatonin receptor protein are used in the screening method of the present invention, the CHO cells can be immobilized with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a method known per se.

Examples of the ligand for the melatonin receptor used in the screening method of the present invention include known melatonin receptor agonists (eg, melatonin, 2,3-dihydromelatonin, S-2009).
8, GR-131663, etc.), melatonin receptor antagonists (eg, Luzindol, ML-23, GR
-13347) and the like. Examples of the labeled ligand for the melatonin receptor include, for example,
The aforementioned ligands labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like can be used. Specifically, melatonin labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like can be used. These labels can be produced according to a method known per se, but for example, [ ¹²⁵ I]-or [ ³ H] -labeled melatonin is commercially available (Amersham and DuPont).
So you can use them. Examples of test compounds used in the screening method of the present invention include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. The test compound of 1 may be a novel compound or a known compound.

Specifically, the above (1a) or (2
To carry out the screening method of a), first, a CHO cell containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof, or a recombinant human melatonin receptor protein or a partial peptide thereof. Is suspended in a buffer suitable for screening to prepare a receptor preparation. Any buffer may be used as long as it does not inhibit the binding between melatonin and the receptor, and for example, a phosphate buffer having a pH of 6 to 8 or a Tris-hydrochloric acid buffer is used. For the purpose of reducing non-specific binding, Chaps or Tween 80 (TM),
Surfactants such as digitonin can also be added. Further, a proteinase inhibitor such as PMSF, pepstatin, leupeptin or the like can be added to suppress the degradation of the melatonin receptor protein by proteinase. On the other hand, when the receptor-expressing CHO cells are adherent cells,
Melatonin and the melatonin receptor can be bound to each other while being adhered to the incubator, that is, while the cells are grown. In this case, Hank's solution or the like is used as the buffer solution. About 0.01 to 10 ml of the receptor-expressing cells or the receptor preparation is added to a fixed amount (about 5,000 to 1,000,0).
(00 cpm) of the labeled substance was added, and at the same time, about 10 ^-4 to 10
^{-Coexist with 10} M sample compounds and fermentation products. In order to know the non-specific binding, a substance in which a large excess of unlabeled substance coexists is also prepared. The reaction is carried out at about 0 to 50 ° C (desirably,
About 4 to 37 ° C., about 0.5 to 24 hours (desirably,
About 0.5 to 3 hours). After the reaction, the mixture is filtered with glass fiber filter paper or the like, washed with an appropriate amount of the same buffer solution, and the amount of radiation remaining on the glass fiber is measured with a gamma counter or a liquid scintillation counter. When the value (specific binding amount) obtained by subtracting the nonspecific binding amount from the binding amount (total binding amount) in the absence of coexisting substances is taken as 100%, for example,
A test compound that suppresses this to 50% or less can be selected as a candidate compound for a compound having affinity for a melatonin receptor or a salt thereof.

Further, in order to carry out the screening methods of (1b) and (1c) above, cell stimulating activities mediated by human melatonin receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ concentration Fluctuation, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH decrease, cell migration activity, hormone secretion, G protein (Activation, cell growth, etc. promoting or suppressing activity) can be measured using a known method or a commercially available measurement kit. Specifically, first, CHO cells containing the recombinant human melatonin receptor protein are cultured in a multiwell plate or the like. Before screening, replace with fresh medium or an appropriate buffer that does not show toxicity to cells, add test compounds, etc., and incubate for a certain period of time, then extract cells or collect supernatant and generate The amount of the obtained product is quantified according to each method. Substance as an indicator of cell stimulating activity (for example,
When the production of arachidonic acid or the like is difficult to be assayed by a degrading enzyme contained in cells, an assay may be performed by adding an inhibitor against the degrading enzyme. Also, cAMP
The activity such as production inhibition can be detected as a production inhibition effect on cells whose basic production has been increased by forskolin or the like.

The screening kit of the present invention comprises a CHO cell containing the recombinant human melatonin receptor protein of the present invention or a cell membrane fraction thereof, or a recombinant human melatonin receptor protein of the present invention, or a portion thereof. It contains a peptide or a salt thereof. Examples of the screening kit of the present invention include the following. [Screening reagent] Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. Recombinant human melatonin receptor protein preparation CHO cells containing recombinant human melatonin receptor protein were subcultured to a 12-well plate at 2.25 × 10 ⁵ cells / well, 37 ° C, 5% What was cultured for 2 days in CO ₂ and 95% air. Labeled ligand for melatonin receptor Commercially available [ ³ H], [ ¹²⁵ I] for melatonin receptor,
A ligand labeled with [ ¹⁴ C], [ ³⁵ S] or the like (eg,
Melatonin etc.) Store the solution state at 4 ℃ or -20 ℃,
When using, dilute to 10 nM with the measurement buffer. Standard solution of a ligand for the melatonin receptor A ligand for the melatonin receptor (for example, melatonin) is dissolved in 50% DMSO (manufactured by Sigma) to 1 mM and stored at -20 ° C.

[Measurement Method] CHO cells containing the recombinant human melatonin receptor protein cultured in a 12-well tissue culture plate were washed twice with 0.75 ml of the measurement buffer, and then 49
Add 0 μl assay buffer to each well. After adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M, 10 nM labeled ligand (eg, melatonin)
5 μl is added and the mixture is reacted at room temperature for 1 hour. To determine the amount of non-specific binding, 5 μl of 10 ⁻³ M ligand (eg, melatonin, iodomelatonin, chloromelatonin, hydroxymelatonin, NAS (N-acetyl-5-hydroxytryptamine), etc.) was used instead of the test compound. I will add it. Remove the reaction solution and wash 3 times with 0.75 ml of wash buffer. 0.5 ml of labeled melatonin bound to cells
Dissolved in 0.2 N NaOH-1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Measure the radioactivity using the
ng (PMB) is obtained by the following equation (Equation 1). In addition, [ ¹²⁵
When labeled with I], it can be measured directly by a gamma counter without mixing with a liquid scintillator.

[0033]

[Equation 1] PMB: Percent of Maximum Binding B: Value when a sample is added NSB: Non-specific Binding B ₀ : Maximum binding amount

A melatonin receptor affinity compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a test compound (for example, peptide, protein, etc.) prepared by using the screening method or the screening kit of the present invention. Non-peptidic compound,
Examples thereof include synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like, and these test compounds may be new compounds or known compounds. And a salt thereof, which inhibits the binding between melatonin and the recombinant human melatonin receptor protein of the present invention. The compound includes a compound having a cell stimulating activity via the human melatonin receptor (so-called melatonin receptor agonist), a compound having no such cell stimulating activity (so-called melatonin receptor antagonist) or a salt thereof. Has been. Further, the melatonin receptor affinity compound obtained by using the screening method or the screening kit of the present invention is chemically modified or substituted, or a compound designed based on the structural formula of the compound. Also included in the melatonin receptor affinity compound obtained by using the screening method or the screening kit of the present invention. As the salt of the melatonin receptor affinity compound, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid,
For example, a salt with benzenesulfonic acid) is used.

Among the melatonin receptor affinity compounds or salts thereof obtained by using the screening method or the screening kit of the present invention, the melatonin receptor agonist is a safe and non-toxic compound, and sleep / wake rhythm disorder, time difference Blurring, insomnia, seasonal depression, reproductive and neuroendocrine disorders, Alzheimer's disease, cerebrovascular dementia,
It is useful as a preventive / therapeutic agent for various disorders associated with aging, cerebral circulation disorder, stress, anxiety, convulsions, Parkinson's disease, hypertension, cancer or glaucoma, or an ovulation regulator. The melatonin receptor antagonist is a safe and nontoxic compound, and is useful as a prophylactic / therapeutic agent for depression, anxiety, neurosis or consciousness disorder. Further, the melatonin receptor affinity compound or its salt obtained by the screening method of the present invention, since it can bind to the melatonin receptor, for detecting or quantifying the melatonin receptor protein in the cells expressing the receptor or in vivo. It is also useful as a reagent.

The melatonin receptor affinity compound or its salt obtained by using the screening method or the screening kit of the present invention is treated with an animal (bird, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, dog, cat). Etc.) or a pharmaceutical composition for human and the like, it can be carried out according to a conventional method. For example, orally as tablets, capsules, elixirs, microcapsules and the like, which are optionally sugar-coated,
Alternatively, it can be used parenterally in the form of an injectable solution such as a sterile solution or suspension with water or other pharmaceutically acceptable liquid. For example, a physiologically acceptable carrier, flavoring agent, excipient, vehicle, or
It can be manufactured by admixture with preservatives, stabilizers, binders and the like in a unit dosage form generally required for accepted pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained. Examples of additives that can be incorporated into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry are used. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices such as dissolving or suspending the active substance in a vehicle such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil, etc. .

Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. You may use together with adjuvants, such as alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol), a nonionic surfactant (for example, polysorbate 80 (TM), HCO-50) and the like. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservation Agents (eg, benzyl alcohol,
Phenol, etc.), antioxidants and the like.
The adjusted injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, for example, warm-blooded mammals (for example, rat, mouse, chicken, rabbit, sheep, pig, cow, cat, dog, monkey, baboon, chimpanzee, human) Etc., especially for humans). The dose of the melatonin receptor-affinitive compound or a salt thereof varies depending on the symptoms and the like, but in the case of oral administration, it is generally about 0.1 to 1 per day for an adult (as about 60 kg).
00 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc., but in the form of an injection, it is usually about 1 day in an adult (as 60 kg). About 0.01 to 30 mg, preferably about 0.1 to 20
It is convenient to administer about mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose converted per 60 kg can be administered.

In the present specification and drawings, when abbreviations for bases and amino acids are used, IUPAC-IUB
Abbreviations based on Commision on Biochemical Nomenclature or common abbreviations in the field,
An example is given below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid

DATP: Deoxyadenosine triphosphate dTTP: Deoxythymidine triphosphate dGTP: Deoxyguanosine triphosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate EIA: Enzyme Immunoassay Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine

Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylphenylalanine Tyr: Tyrosine Trp: Glytin: Proptophanin Pron: Asphalin: Para: Tryptophan A para: Protamine: Asparaginic acid. Serum albumin

The plasmid pA obtained in Example 1 below.
Transformant Escherichia coli DH5 / pAKKO-h Carrying KKO-hMelR7
MelR7 has been deposited with the deposit number FERM BP-5236 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (NIBH) since September 20, 1995. In addition, CHO-hMelR7 obtained in Example 2 described later
Has been depositing with the deposit number FERM from the Ministry of International Trade and Industry, Institute of Industrial Science and Technology (NIBH) since September 26, 1995.
Deposited as BP-5242.

Each sequence number in the sequence listing in the present specification will be described below. [SEQ ID NO: 1] This shows the base sequence of DNA encoding human melatonin receptor protein. [SEQ ID NO: 2] This shows the amino acid sequence of human melatonin receptor protein. [SEQ ID NO: 3] D for PCR used for cloning DNA encoding human melatonin receptor protein
This shows the base sequence of NA oligomer. [SEQ ID NO: 4] D for PCR used for cloning DNA encoding human melatonin receptor protein
This shows the base sequence of NA oligomer. [SEQ ID NO: 5] PCR D used for cloning of DNA encoding human melatonin receptor protein
This shows the base sequence of NA oligomer. [SEQ ID NO: 6] D for PCR used for cloning DNA encoding human melatonin receptor protein
This shows the base sequence of NA oligomer. [SEQ ID NO: 7] This shows the cloning site introduced into pAKKO-111H. [SEQ ID NO: 8] This shows the cloning site introduced into pAKKO-111H.

[0043]

The present invention will be described in more detail below with reference to Reference Examples and Examples, but the present invention is not limited thereto. In addition, the gene manipulation method using Escherichia coli is referred to as molecular cloning [Molecular clonin
g] was followed.

[0044]

[Reference Example 1] Amplification of melatonin receptor protein coding region from human genomic DNA by PCR method Known nucleotide sequence of human melatonin receptor cDNA (Ge
Based on nBank, Accession Number U14108), the following four kinds of synthetic DNA primers were prepared. (1) 5'-GG (GTCGAC) AACAGGGGACCATGCAGGGCAA-3 '(SalI recognition sequence is shown in parentheses) (SEQ ID NO: 3) (2) 5'-GG (TGCGCA) TTCCTAAGCTTTTGTTCC-3' (FspI recognition in parentheses) (SEQ ID NO: 4) (3) 5'-TT (CCCGGG) AAACATCTTTGTGGGTGAGCTT A-3 '(SmaI recognition sequence in parentheses) (SEQ ID NO: 5) (4) 5'-TT (TCTAGA) ) TTAAACGGAGTCCACCTTTAC TA-3 ′ (XbaI recognition sequence is shown in parentheses) (SEQ ID NO: 6)

The above-mentioned primers (1) and (2),
PCR was performed using human genomic DNA (Clontech) as a template using (3) and (4) as a set. For the reaction using primers (1) and (2),
10 μl of human genomic DNA (46 ng / μl), Taq D
NA polymerase (Takara Shuzo) 1 μl, Taq DNA polymerase
10x reaction buffer and dNTP attached to each
1 μl each of the primers prepared to a concentration of 20 μM
It was used and the liquid volume was adjusted to 100 μl with distilled water. For the reaction using the primers (3) and (4), 10 μl of human genomic DNA (46 ng / μl) and Taq DNA p
Olymerase (Takara Shuzo) 0.5 μl, Taq DNA polymerase
10x reaction buffer and dNTP attached to each
5 μl each of the primers prepared to a concentration of 20 μM
It was used and the liquid volume was adjusted to 100 μl with distilled water. PC
For R, three steps of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 1 minute were repeated 35 times for one cycle. After completion of the reaction, agarose gel electrophoresis was performed using a part of the reaction solution. [Figure 4] shows human genomic DNA
The result of PCR amplification of the melatonin receptor gene using is shown. Lane 1 is a DNA marker, λDN
A is digested with the restriction enzyme StyI. Lane 2 contains primer (1) (SEQ ID NO: 3) and primer (2)
PC of human genomic DNA using (SEQ ID NO: 4)
The result of performing R amplification is shown. Lane 3 shows the result of PCR amplification of human genomic DNA using primer (3) (SEQ ID NO: 5) and primer (4) (SEQ ID NO: 6). Lane 4 is a DNA marker in which φλ174 DNA was digested with the restriction enzyme HincII. By ethidium bromide staining, a band of about 0.2 kb was confirmed with the combination of primers (1) and (2), and a band of about 0.8 kb was confirmed with the combination of primers (3) and (4) [FIG. 4]. Below, 0.2
The kb PCR product is called fragment 1 and the 0.8 kb PCR product is called fragment 2.

[0046]

[Reference Example 2] Insertion of PCR product into plasmid vector and analysis of base sequence Agarose gels of the band portions of about 0.2 kb (fragment 1) and about 0.8 kb (fragment 2) detected in Reference Example 1 were analyzed. Separated using a razor. Put each into a centrifuge tube with a filter (Ultra Free C3HV, Millipore), freeze in a freezer, then thaw at room temperature, then centrifuge the tube for 2 minutes at 9000 rpm,
The solution containing the PCR product was eluted at the bottom of the filter. This solution was subjected to phenol / chloroform (1: 1) extraction and diethyl ether extraction according to a conventional method to remove impurities, and then DNA was precipitated by ethanol precipitation. The obtained DNA was used as a plasmid vector pCR ^™ II using TA Cloning Kit (Invitrogen).
(Invitrogen) and used for E. coli J
M109 was transformed. The obtained transformant was cultured overnight in 2 ml of LB medium containing ampicillin (50 μg / ml), and plasmid DNA was prepared by an automatic plasmid extractor (Kurabo). The obtained plasmid DN
A part of A was digested with EcoRI (Takara Shuzo) and subjected to agarose gel electrophoresis to select only the plasmid in which the above-mentioned fragment 1 and fragment 2 were inserted. Less than,
The plasmid in which the fragment 1 was inserted is called pCRII-M1 and the plasmid in which the fragment 2 was inserted is called pCRII-M2. The remaining plasmid DNA was used as a template for the analysis of the nucleotide sequence after removing the mixed RNA by RNase (Nippon Gene) digestion. The base sequence is
Fluorescent DNA Sequencer and Taq DyeDeoxy ^™ Term
Decoding was performed using an inator Cycle Sequencing Kit (PerkinElmer Applied Biosystems). As a result of the nucleotide sequence analysis, it was confirmed that fragment 1 and fragment 2 obtained by PCR code for human melatonin receptor [Fig. 1]. [FIG. 1] shows the expression vector pAK
The nucleotide sequence of the complete coding region of the human melatonin receptor constructed on KO-hMelR7 and the amino acid sequence encoded thereby are shown. In the figure, underlined primers (1), (2), (3) and (4) indicate parts corresponding to parts of the respective primer sequences. The black dots (●) are
Since a restriction enzyme recognition site was introduced on the primer sequence, a portion different from the known base sequence is shown, and (*)
Indicates a portion different from the known base sequence. Here, the publicly known nucleotide sequence is in the data bank Gen Bank Accession No. U1410.
It is a quotation of 8.

[0047]

Example 1 Construction of Human Melatonin Receptor Expression Vector pCRII-M1 is a restriction enzyme FspI (Takara Shuzo) and S
alI (Takara Shuzo) and pCRII-M2 with SmaI
It was cut with (Takara Shuzo) and XbaI (Takara Shuzo). The cut sample was subjected to agarose gel electrophoresis, stained with ethidium bromide, and the band portion thereof was cut out with a razor. Put each in a centrifuge tube with a filter (Millipore), freeze in a freezer, then thaw at room temperature, centrifuge the tube at 9000 rpm for 2 minutes,
The solution containing the DNA fragment was eluted at the bottom of the filter. This solution was subjected to phenol / chloroform (1: 1) extraction and diethyl ether extraction according to a conventional method to remove impurities, and then DNA was precipitated by ethanol precipitation. On the other hand, pAKKO- which is an expression vector for animal cells
111H is pA which is an expression vector in CHO cells.
It was constructed as follows using KKO-111. First, p
AKKO-111 was constructed as follows. From pTB1417 described in JP-A-5-076385 to Hi
A 1.4 kb DNA fragment containing the SRα ′ promoter and poly (A) signal was obtained by treatment with ndIII and ClaI. In addition, pTB348 [Naruo, K. et
al. Biochemical and Biophysical Research Communications (Biochem. Biophys. Re
s. Commun.), 128, 257-264 (1985)] to ClaI.
And SalI treatment yielded a 4.5 kb DNA fragment containing the dihydrofolate reductase (dhfr) gene. These DNA fragments were blunt ended by T4 polymerase treatment, and then ligated by T4 ligase,
The pAKKO-111 plasmid was constructed. Then pA
Sal is the only cloning site of KKO-111.
At the I site, the following synthetic DNA and cloning sites were introduced. As a result, SalI as a multi-cloning site downstream of the SRα ′ promoter,
The plasmid pAKKO-111H to which ClaI, SpeI and NheI sites were added was constructed.

5′-GTCGACGAATTCATCGATACACTAGTGCTAGC-3 ′ (SEQ ID NO: 7) 5′-TCGAGCTAGCACTAGTATCGATGAATTCGTC GAC-3 ′ (SEQ ID NO: 8) and pAKKO-111H with restriction enzymes NheI (Takara Shuzo) and SalI. Then, the vector portion was separated and extracted from the agarose gel by the same method as described above. Fragment 1, fragment 2, pA thus obtained
Various restriction enzyme digests of KKO-111H were reacted overnight at 16 ° C using a ligation system (Takara Shuzo). Escherichia coli DH5 was transformed with a part of the ligation reaction product to obtain a transformant Escherichia coli.
DH5 / pAKKO-hMe1R7 was obtained. Obtained transformant Escherichia coli DH5 / pAKKO-hM
The e1R7 was cultured overnight in 2 ml of LB medium containing ampicillin (50 μg / ml), and plasmid DNA (pAKKO-hMe was used by an automatic plasmid extractor (Kurabo).
1R7) was prepared. Fluorescent Sequencer and Taq D
Fragment 1, Fragment 2, pAKKO using yeDeoxy ^™ Terminator Cycle Sequencing Kit (Perkin Elmer)
The nucleotide sequence of the ligation site of -111H was analyzed, and the expression vector pAKKO-h as shown in FIGS. 2 and 3 was obtained.
It was confirmed that the construction of Me1R7 was completed.

[0049]

Example 2 Introduction of Human Melatonin Receptor Expression Vector into CHOdhfr ^- Cells and Confirmation of Expression by Binding Experiment 1 × 10 ⁶ CHs in a tissue culture dish having a diameter of 10 cm.
Odhfr ⁻ cells were seeded and cultured for 24 hours. Human melatonin receptor expression vector pA obtained in Example 1
Using 5 μg of KKO-hMe1R7, a DNA / calcium phosphate precipitate was formed using a gene transfer kit (CellPhect, Pharmacia) by the calcium phosphate method. The medium was replaced with a fresh one, and the DNA / calcium phosphate precipitate was added thereto and incubated for 7 hours, followed by glycerol shock according to the protocol attached to the kit (composition: 50 mM HEPES (N-2-h
ydroxyethylpiperazine-N'-2-ethane sulfonic acid) p
H7.5 15% glycerol). The medium was replaced with a fresh one and the cells were further cultured for 1 day. Then, the medium for transformant selection was replaced and the cells were cultured for 2 days. In addition, trypsin-EDT
The cells in the petri dish were collected by the treatment A, and recultured in a state where the cell density was low, whereby the proportion of transformants was increased. Thereby, the cell line CHO-hMelR7 stably stably expressing the human melatonin receptor
A clone of 2.2 cells grown in selective culture
A 12-well tissue culture plate was seeded at a density of 5 × 10 ⁵ cells / well and cultured for 2 days. Remove the culture solution to 0.1
Hanks' solution containing 0.5% bovine serum albumin 0.75 m
After washing twice with l, 100 pM [ ¹²⁵ I] melatonin (specific activity 2200 Ci / mmol, DuPont) was added to 0.5 ml of the same solution, and the mixture was allowed to stand at 37 ° C. for 1 hour. To some wells, 10 μM unlabeled melatonin was added at the same time in order to know the degree of nonspecific binding. As a control
CHO cells transformed with a vector not incorporating a foreign gene were prepared and treated in the same manner. In order to remove unbound [ ¹²⁵ I] melatonin, each well was washed three times with 0.75 ml of Hanks's solution containing 0.1% bovine serum albumin, and then the cells in the well were washed with 0.
It was dissolved in 2N NaCl and 1% SDS, and the residual radioactivity was measured by a gamma counter [Fig. 5]. [FIG. 5] shows the results of binding experiments performed using the human melatonin receptor protein-expressing CHO cells obtained in Example 2 and [ ¹²⁵ I] melatonin. Lanes 1 and 2 on the horizontal axis represent CHO-hMelR7, and lanes 3 and 4 represent mock CHO (negative control). Lanes 1 and 3 are added with [ ¹²⁵ I] melatonin, and lanes 2 and 4 are added with [ ¹²⁵ I] melatonin and cold melatonin. The vertical axis represents the amount of [ ¹²⁵ I] melatonin bound to each CHO cell in each of lanes 1 to 4 on the horizontal axis. FIG. 5 shows the average of duplicate experiments. C obtained from FIG.
Specific binding of [ ¹²⁵ I] melatonin was confirmed in HO-hMelR7 cells.

[0050]

The CHO cells having the ability to continue producing the human melatonin receptor protein of the present invention are CHO cells capable of highly expressing the human melatonin receptor protein. Further, by using the CHO cell or the cell membrane fraction thereof having the ability to continuously produce the human melatonin receptor protein of the present invention, or the recombinant human melatonin receptor protein or its partial peptide, a melatonin receptor affinity compound or a compound thereof The compound can be advantageously selected by screening a salt.
Therefore, for example, sleep-wake rhythm disorder, jet lag,
Prevention and treatment of insomnia, seasonal depression, reproductive and neuroendocrine diseases, Alzheimer's disease, cerebrovascular dementia, aging-related disorders, cerebral circulation disorders, stress, anxiety, convulsions, Parkinson's disease, high blood pressure, cancer or glaucoma Agents, ovulation regulators, prophylactic / therapeutic agents for depression, anxiety, neurosis or consciousness disorder can be provided at an early stage.

[0051]

[Sequence list]

[SEQ ID NO: 1] Sequence length: 1050 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Characterization method: S sequence ATGCAGGGCA ACGGCAGCGC GCTGCCCAAC GCCTCCCAGC CCGTGCTCCG CGGGGACGGC 60 GCGCGGCCCT CGTGGCTGGC GTCCGCCCTG GCCTGCGTCC TCATCTTCAC CATCGTGGTG 120 GACATCCTGG GCAACCTCCT GGTCATCCTG TCGGTGTATC GGAACAAGAA GCTTAGGAAT 180 GCGGGAAACA TCTTTGTGGT GAGCTTAGCG GTGGCAGACC TGGTGGTGGC CATTTATCCG 240 TACCCGTTGG TGCTGATGTC GATATTTAAC AACGGGTGGA ACCTGGGCTA TCTGCACTGC 300 CAAGTCAGTG GGTTCCTGAT GGGCCTGAGC GTCATCGGCT CCATATTCAA CATCACCGGC 360 ATCGCCATCA ACCGCTACTG CTACATCTGC CACAGTCTCA AGTACGACAA ACTGTACAGC 420 AGCAAGAACT CCCTCTGCTA CGTGCTCCTC ATATGGCTCC TGACGCTGGC GGCCGTCCTG 480 CCCAACCTCC GTGCAGGGAC TCTCCAGTAC GACCCGAGGA TCTACTCGTG CACCTTCGCC 540 CAGTCCGTCA GCTCCGCCTA CACCATCGCC GTGGTGGTTT TCCACTTCCT CGTCCCCATC 600 ATCATAGTCA TCTTCTGTGT CCTGAGAATA TGGATCCTGG TTCTCCAGGT CAGACAGAGG 660 GTGAAACCTGACTACCGACAGA A CCACAGGACT TCAGGAATTT TGTCACCATG 720 TTTGTGGTTT TTGTCCTTTT TGCCATTTGC TGGGCTCCTC TGAACTTCAT TGGCCTGGCC 780 GTGGCCTCTG ACCCCGCCAG CATGGTGCCT AGGATCCCAG AGTGGCTGTT TGTGGCCAGT 840 TACTACATGG CGTATTTCAA CAGCTGCCTC AATGCCATTA TATACGGGCT ACTGAACCAA 900 AATTTCAGGA AGGAATACAG GAGAATTATA GTCTCGCTCT GTACAGCCAG GGTGTTCTTT 960 GTGGACAGCT CTAACGACGT GGCCGATAGG GTTAAATGGA AACCGTCTCC ACTGATGACC 1020 AACAATAATG TAGTAAAGGT GGACTCCGTT 1050

[0052]

[SEQ ID NO: 2] Sequence length: 350 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Gln Gly Asn Gly Ser Ala Leu Pro Asn Ala Ser Gln Pro Val Leu 1 5 10 15 Arg Gly Asp Gly Ala Arg Pro Ser Trp Leu Ala Ser Ala Leu Ala Cys 20 25 30 Val Leu Ile Phe Thr Ile Val Val Asp Ile Leu Gly Asn Leu Leu Val 35 40 45 Ile Leu Ser Val Tyr Arg Asn Lys Lys Leu Arg Asn Ala Gly Asn Ile 50 55 60 Phe Val Val Ser Leu Ala Val Ala Asp Leu Val Val Ala Ile Tyr Pro 65 70 75 80 Tyr Pro Leu Val Leu Met Ser Ile Phe Asn Asn Gly Trp Asn Leu Gly 85 90 95 Tyr Leu His Cys Gln Val Ser Gly Phe Leu Met Gly Leu Ser Val Ile 100 105 110 Gly Ser Ile Phe Asn Ile Thr Gly Ile Ala Ile Asn Arg Tyr Cys Tyr 115 120 125 Ile Cys His Ser Leu Lys Tyr Asp Lys Leu Tyr Ser Ser Lys Asn Ser 130 135 140 Leu Cys Tyr Val Leu Leu Ile Trp Leu Leu Thr Leu Ala Ala Val Leu 145 150 155 160 Pro Asn Leu Arg Ala Gly Thr Leu Gln Tyr Asp Pro Arg Ile Tyr Ser 165 170 175 Cys Thr Phe Ala Gln Ser Val Ser Ser Ala Tyr Thr Ile Ala Val Val 180 185 190 Val Phe His Phe Leu Val Pro Met Ile Ile Val Ile Phe Cys Tyr Leu 195 200 205 Arg Ile Trp Ile Leu Val Leu Gln Val Arg Gln Arg Val Lys Pro Asp 210 215 220 Arg Lys Pro Lys Leu Lys Pro Gln Asp Phe Arg Asn Phe Val Thr Met 225 230 235 240 Phe Val Val Phe Val Leu Phe Ala Ile Cys Trp Ala Pro Leu Asn Phe 245 250 255 Ile Gly Leu Ala Val Ala Ser Asp Pro Ala Ser Met Val Pro Arg Ile 260 265 270 Pro Glu Trp Leu Phe Val Ala Ser Tyr Tyr Met Ala Tyr Phe Asn Ser 275 280 285 Cys Leu Asn Ala Ile Ile Tyr Gly Leu Leu Asn Gln Asn Phe Arg Lys 290 295 300 Glu Tyr Arg Arg Ile Ile Val Ser Leu Cys Thr Ala Arg Val Phe Phe 305 310 315 320 Val Asp Ser Ser Asn Asp Val Ala Asp Arg Val Lys Trp Lys Pro Ser 325 330 335 Pro Leu Met Thr Asn Asn Asn Val Val Lys Val Asp Ser Val 340 345 350

[0053]

[SEQ ID NO: 3] Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GGGTCGACAA CAGGGACCAT GCAGGGCAA 29

[0054]

[SEQ ID NO: 4] Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GGTGCGCATT CCTAAGCTTC TTGTTCC 27

[0055]

[SEQ ID NO: 5] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence TTCCCGGGAA ACATCTTTGT GGTGAGCTTA 30

[0056]

[SEQ ID NO: 6] Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence TTTCTAGATT AAACGGAGTC CACCTTTACT A 31

[0057]

[SEQ ID NO: 7] Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence GTCGGACGAAT TCATCGATAC TAG
TGCTAGC 30

[0058]

[SEQ ID NO: 8] Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence TCGAGCTAGC ACTAGTATCCG ATG
AATTCGT CGAC 34

[0059]

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of the complete coding region of the human melatonin receptor constructed on the expression vector pAKKO-hMelR7 and the amino acid sequence encoded thereby.
In the figure, underlined primer (1), (2), (3), (4)
Indicates a part corresponding to a part of each primer sequence. Further, a black dot (●) indicates a portion different from the known base sequence because the restriction enzyme recognition site was introduced on the primer sequence, and (*) indicates a portion different from the known base sequence. Here, the known base sequence is the data bank Ge
n Quoted from Bank Accession No. U14108.

FIG. 2 Human Melatonin Receptor Expression Vector pAKK
The construction method of O-hMelR7 is shown.

FIG. 3 Human Melatonin Receptor Expression Vector pAKK
A construction drawing of O-hMelR7 is shown. Amp ^r represents the ampicillin resistance gene. DHFR indicates the dihydrofolate reductase gene.

FIG. 4 shows the results of PCR amplification of the melatonin receptor gene using human genomic DNA. Lane 1 is a DNA marker, which is obtained by digesting λDNA with the restriction enzyme StyI. Lane 2 is primer (1) (SEQ ID NO: 3)
And primer (2) (SEQ ID NO: 4)
The results of PCR amplification of genomic DNA are shown. Lane 3 shows the result of PCR amplification of human genomic DNA using primer (3) (SEQ ID NO: 5) and primer (4) (SEQ ID NO: 6). Lane 4 is a DNA marker in which φλ174 DNA was digested with the restriction enzyme HincII.

FIG. 5 shows the results of a binding experiment performed using the human melatonin receptor protein-expressing CHO cells obtained in Example 2 and [ ¹²⁵ I] melatonin. Lanes 1 and 2 on the horizontal axis are CHO-hMelR7, and lanes 3 and 4 are m.
ock CHO (negative control) is shown.
Lanes 1 and 3 are added with [ ¹²⁵ I] melatonin, and lanes 2 and 4 are added with [ ¹²⁵ I] melatonin and cold melatonin. The vertical axis represents the amount of [ ¹²⁵ I] melatonin bound to each CHO cell in each of the lanes 1 to 4 on the horizontal axis. This figure shows the average of duplicate experiments.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 38/04 AAY A61K 37/43 AAB ABL AAE ABN AAL ABU AAM ACV AAY ADU ABL C07H 21/04 ABN C07K 14/72 ABU C12N 15/09 ZNA ACV C12P 21/02 ADU C12Q 1/02 9162-4B C12N 15/00 ZNAA // (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12Q 1/02 C12R 1:91)

Claims (18)

[Claims] 1. A CHO cell containing an expression vector carrying a DNA encoding a human melatonin receptor protein. 2. The CHO according to claim 1, wherein the DNA encoding the human melatonin receptor protein is a DNA containing a DNA having the nucleotide sequence represented by SEQ ID NO: 1.
cell. 3. The CHO cell according to claim 1, which is a CHO cell lacking the DHFR gene. 4. A recombinant human CHO cell according to claim 1, which is produced by culturing the CHO cell under conditions capable of expressing a human melatonin receptor protein.
A CHO cell containing a melatonin receptor protein or a cell membrane fraction thereof. 5. The CHO cell or the cell membrane fraction thereof according to claim 4, which is a CHO cell labeled with CHO-hMelR7. 6. A pAKKO-retaining DNA encoding a human melatonin receptor protein.
An expression vector labeled with hMelR7. 7. A recombinant human melatonin receptor protein having substantially the same activity as the natural human melatonin receptor protein, which is isolated from the CHO cell according to claim 4. Partial peptides or their salts. 8. A method for producing a recombinant human melatonin receptor protein, which comprises culturing the CHO cell according to claim 1 under conditions capable of expressing the human melatonin receptor protein. 9. A method for screening a compound having affinity for a melatonin receptor or a salt thereof, which comprises contacting a test compound with the CHO cell or cell membrane fraction thereof according to claim 4. 10. A method for screening a compound having affinity for a melatonin receptor or a salt thereof, which comprises contacting a test compound with the recombinant human melatonin receptor protein according to claim 7, a partial peptide thereof or a salt thereof. . 11. A melatonin containing the CHO cell according to claim 4 or a cell membrane fraction thereof, or the recombinant human melatonin receptor protein according to claim 7, a partial peptide thereof or a salt thereof. A kit for screening a compound having affinity for a receptor or a salt thereof. 12. A melatonin receptor affinity compound or a salt thereof obtained by using the screening method according to claim 9 or 10, or the screening kit according to claim 11. 13. The melatonin receptor affinity compound or a salt thereof according to claim 12, wherein the melatonin receptor affinity compound is a melatonin receptor agonist. 14. The melatonin receptor affinity compound or a salt thereof according to claim 12, wherein the melatonin receptor affinity compound is a melatonin receptor antagonist. 15. A pharmaceutical composition comprising the melatonin receptor agonist according to claim 13. 16. Sleep / wake rhythm disorder, jet lag, insomnia,
Seasonal depression, reproductive and neuroendocrine diseases, Alzheimer's disease, cerebrovascular dementia, various disorders associated with aging, cerebral circulation disorder, stress, anxiety, convulsions, Parkinson's disease, hypertension,
The pharmaceutical composition according to claim 15, which is a preventive / therapeutic agent for cancer or glaucoma or an ovulation regulator. 17. A pharmaceutical composition comprising the melatonin receptor antagonist according to claim 14. 18. The pharmaceutical composition according to claim 17, which is a prophylactic / therapeutic / therapeutic agent for depression, anxiety, neurosis or consciousness disorder.