WO2003072780A1

WO2003072780A1 - Novel proteins, dnas thereof and use of the same

Info

Publication number: WO2003072780A1
Application number: PCT/JP2003/002133
Authority: WO
Inventors: Tsukasa Sugo; Masaaki Mori
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2002-02-27
Filing date: 2003-02-26
Publication date: 2003-09-04
Also published as: AU2003211722A1

Abstract

Novel proteins of ape-, feline- and canine-origins or salts thereof, polynucleotides encoding these proteins, antibodies against these proteins, etc. are useful as, for example, diagnostic markers for inappetence, uterine inertia and so on. Compounds obtained by screening with the use of these proteins and MCH are usable as appetite enhancers and preventives/remedies for inappetence, etc.

Description

Description New protein, its DNA and application Technical field

The present invention relates to a novel G protein-coupled receptor protein, a DNA encoding the same, and uses thereof. For more information, anorexia, anemia associated with anorexia, prevention and treatment of hypoproteinemia, weak labor, laxative bleeding, uterine remodeling failure or milk stasis, or obesity, hyperphagia, affective disorder, sex Dysfunction, labour, tonic contractions, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease, fatty liver, infertility or The present invention relates to screening for compounds having a prophylactic and therapeutic effect on osteoarthritis. Background art

Obesity is known to be a risk factor for the development of many lifestyle-related diseases such as diabetes and heart disease. With changes in eating habits and lifestyles, fertility is increasing, especially in developed countries, and it has become a very socially significant problem from the health economic perspective. Obesity is known to be caused by various factors.Recently, several peptides located in the hypothalamus, the feeding center, are involved in promoting or suppressing feeding behavior in mammals. Thus, the contribution of these peptides to obesity has received particular attention. This is because drugs that inhibit or activate the action of these peptides are considered to be potential central antiobesity drugs. One such hypothalamic peptide, melanin-concentrating hormone (Melanin

Concentrating Hormone) (hereinafter sometimes referred to as MCH) is a peptide originally found as a color changing hormone that aggregates pigment granules of fish melanophores. It is clear that it exists in mammals such as. Recently, several facts have been reported indicating that MCH plays an important role in feeding behavior and energy metabolism in mammals.

In mammals, MCH is localized in the feeding center, the hypothalamic lateral area and the irregular zone. And were more likely to be involved in eating and drinking behavior, but Qu et al. First showed that MCH was involved in eating behavior as follows (Qu, D. et al., Nature, vol. 380). , 243-247, 1996). Expression of altered mRNA levels in the hypothalamus of ob / ob mice and wild-type mice, which are genetically obese models, was searched for by the di ferent al al di sp lay method. Was found to be significantly increased. Increased MCH gene expression was observed in both wild-type and ob / ob mice after a 24-hour fast. These results suggested that MCH functioned as a food-stimulating peptide, but in fact, administration of MCH directly to the lateral ventricle of rats significantly increased food intake. In addition, it was reported that MCH knockout mice lost significant weight due to reduced food intake and increased metabolism.

(Nature, 396, 670-674, 1998). Remarkably, no change in behavior or other biochemical parameters other than weight loss was observed in the mice compared to the wild type. It has also recently been reported that genetically modified mice that overproduce MCH exhibit an obese phenotype and exhibit insulin resistance (J. Clin. Invest., 107, 3793-86, 2001). Year) . These facts imply that MCH plays a central role in feeding behavior and energy metabolism. In the hypothalamus, in addition to MCH, various feeding-promoting peptides represented by neuropeptide Y, galanin or orexin are present. However, suppression of eating behavior and weight loss observed in mice deficient in the MCH gene have not been reported in animals knocked down by these peptides, and MCH functions as a particularly important regulator in eating behavior. it seems to do. In addition, since the MCH gene-deficient mice did not show any prominent phenotype other than weight loss, it was suggested that suppressing the effect of MCH could lead to the development of anti-obesity drugs with few side effects.

SLC-1 (MCHK MCHR1) was previously known as a functional receptor for MCH (Nature, 400, 26-1265, 1999, Nature, 400, 265-269, 1999, Bi ochei. Bi ophys. Res. Commun., 261, 622-626, 1999, Nature Cell Biol., 1st, 267-271, 1999, FEBS Lett. 524, 1999, WO 00/40725, etc.), and recently, SLT (MCH2, CHR2) has been cloned as the second receptor subtype of MCH (J. Biol. Chem. , Volume 276, 20125-20129 Co., 2001, Biochem. Biophys. Res.Co, Vol. 283, 1013- 1018, 2001, Proc. Natl. Acad. Sci. USA, 98, 7576-7581, 2001, J. Biol. Chem., 276, 34664-34670, 2001, Mol. Pharmacol., 60, 632-639 2001, W002 / 03070, W00 / 49046, etc.). It is not clear at this time which of these two MCH receptors is involved in the physiological effects of MCH, particularly on feeding. SLC-1 receptor is particularly strongly expressed in the hypothalamus region involved in feeding (arcus nucleus, ventral nucleus, dorsolateral nucleus, paraventricular nucleus) and in the nucleus accumbens which is thought to be involved in the reward system On the other hand, SLT is expressed in the hippocampus and tonsils and is low in the hypothalamus, so SLC-1 is mainly involved in the inducing action of MCH, and SLT is It has been suggested that they may be involved in memory and emotion's effects. On the other hand, SLT is distributed in the hypothalamus in areas different from SLC-1 (pronucleus, lateral nucleus, etc.), and is involved in feeding because it is located near the causative gene of childhood obesity. Some reports have reported that this may be done (Proc. Natl. Acad. Sci. USA, 98, 75664-7569, 2001). However, while SLC-1 has its gene in rats as well as humans and its tissue distribution analysis has been reported, SLT has been reported in appropriate non-human experimental animals to date. The mechanism of MCH's involvement in physiological effects such as increased food intake has not always been fully elucidated.

By elucidating the involvement of SLT, which is an MCH receptor, in the physiological effects of MCH, such as increased food intake, and using a drug screening system, it is hoped that new drugs, such as anti-obesity drugs, having a novel mechanism of action will be developed. Was rare. Disclosure of the invention

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that genes encoding monkey, canine, and feline SLTs showing homology to human SLT can be obtained from monkey, canine, and feline brain cDNA. More successfully found and cloned. In addition, these receptors bind to MCH. As a result of further study based on these findings, the present invention has been completed.

That is, the present invention (1) a protein or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 14 or SEQ ID NO: 24;

(2) a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 or a salt thereof;

(3) a protein comprising the amino acid sequence represented by SEQ ID NO: 14 or a salt thereof;

(4) a protein consisting of the amino acid sequence represented by SEQ ID NO: 24 or a salt thereof;

((55)) a partial peptide of the protein according to (1) or a salt thereof,

(6) a polynucleotide containing a polynucleotide encoding the protein of (1) or the partial peptide of (5),

(7) the polynucleotide according to (6), which is a DNA,

(8) The polynucleotide according to the above (7), comprising the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23,

(9) a recombinant vector containing the polynucleotide according to (8),

(10) a transformant transformed with the recombinant vector according to the above (9),

(11) culturing the transformant described in (10) above, producing and accumulating the protein described in (1) or the partial peptide described in (5) above, and collecting this. A method for producing the protein according to the above (1) or the partial peptide or the salt thereof according to the above (5),

(12) a medicine comprising the protein of (1) or the partial peptide of (5) or a salt thereof,

(13) a medicament comprising the polynucleotide according to the above (6),

(14) a diagnostic agent comprising the polynucleotide according to (6),

(15) an antibody against the protein according to (1) or the partial peptide according to (5) or a salt thereof,

(16) the antibody according to (15), which is a neutralizing antibody that inactivates the signal transduction of the protein according to (1); (17) The antibody according to (15), which is an antibody that activates signal transduction of the protein according to (1).

(18) a diagnostic agent comprising the antibody according to the above (15),

(19) a pharmaceutical comprising the antibody according to the above (16),

(20) a medicine comprising the antibody according to the above (17),

(21) The protein described in the above (1) or the partial peptide or a salt thereof described in the above (5) and a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25, or A peptide comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25, characterized by using a salt thereof, and a peptide according to the above (1) or a salt thereof. Screening method for a compound or a salt thereof that alters the binding of

(22) The protein described in the above (1) or the partial peptide or a salt thereof described in the above (5) and a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25, or A peptide comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, characterized by containing a salt thereof, and a protein according to the above (1) or a salt thereof. A kit for screening a compound that changes binding property or a salt thereof,

(23) An amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, which can be obtained using the screening method described in (21) or the screening kit described in (22). A compound or a salt thereof that alters the binding property between the peptide or a salt thereof and the protein or the salt thereof according to (1) above,

(24) The compound described in (23) above, which is an agonist, or a salt thereof,

(25) The compound according to the above (23) or a salt thereof, which is an antagonist; (26) a medicine comprising the compound or the salt thereof according to the above (24); (27) the compound according to the above (25) or a salt thereof (28) an antisense polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of (6) or a part thereof,

(29) A medical comprising the antisense polynucleotide according to (28). (30) a method for quantifying the mRNA of the protein according to (1), which comprises using the polynucleotide or a part thereof according to (6);

(31) The method for quantifying a protein according to (1), wherein the antibody according to (15) is used.

(32) A method for diagnosing a disease associated with the function of the protein according to (1), which comprises using the quantification method according to (30) or (31).

(33) A method for screening a compound or a salt thereof that alters the expression level of the protein described in (1) above, which comprises using the quantification method described in (30) above. A method for screening a compound or a salt thereof, which changes the amount of a protein in a cell membrane according to the above (1), characterized by using a quantification method;

(35) A compound or a salt thereof that alters the expression level of the protein according to (1), which can be obtained by using the screening method according to (33).

(36) a compound or a salt thereof, which alters the amount of the protein according to (1) above in a cell membrane obtainable by using the screening method according to (34);

(37) A medicament comprising the compound according to (35) or (36) or a salt thereof,

(38) a polynucleotide that hybridizes to the polynucleotide according to (6) under conditions of high stringency,

(39) a non-human transgenic animal having a DNA encoding the protein of (1) or a mutant DNA thereof,

(40) The above (12), (13), (20) which is an agent for the prevention and treatment of anorexia, anemia associated with anorexia, hypoproteinemia, weak labor, bleeding blood, uterine remodeling failure or milk stasis ) Or the medicament according to (26),

(41) Tomomiasis, eating disorders, affective disorders, sexual dysfunction, overwork labor, ankylosing contractions, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary The medicament according to (19), (27) or (29), which is a prophylactic or therapeutic agent for arteriosclerosis gout, respiratory disease, fatty liver, infertility or osteoarthritis; (42) Anorexia, anemia associated with anorexia, hypoproteinemia, weak labor, and relaxation characterized by administering to a mammal an effective amount of the compound or a salt thereof according to (24). Prevention and treatment of bleeding, uterine repair failure or milk stasis

(43) An obesity, hyperphagia, affective disorder, sexual dysfunction, or hyperactivity, comprising administering to a mammal an effective amount of the compound according to (25) or a salt thereof. Labor, ankylosing uterine contractions, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease, fatty liver, infertility or osteoarthritis Prevention and treatment of

(44) The compound according to (24) for the manufacture of a prophylactic or therapeutic agent for anorexia, anemia associated with anorexia, hypoproteinemia, weak labor, lax blood, uterine remodeling failure or milk stasis. Or the use of its salts, '

(45) Obesity, hyperphagia, affective disorders, sexual dysfunction, overwork labor, ankylosing uterine contractions, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary Provide the use of the compound described in (25) or a salt thereof for the manufacture of a prophylactic or therapeutic agent for arteriosclerosis, gout, respiratory disease, fatty liver, infertility or osteoarthritis. I do. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the intracellular Ca ion elevating activity of various concentrations of MCH on cat SLT-expressing CH0 cells measured using FLIPR.

FIG. 2 is a graph of the expression level of the cat SLT gene in various parts of the cat brain. The expression level was expressed as 1000 times the value obtained by dividing the expression level of the cat SLT gene for the same type II RNA level by the expression level of the cat GAPDH gene. BEST MODE FOR CARRYING OUT THE INVENTION _

The protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 14 or SEQ ID NO: 24 (hereinafter, may be referred to as the protein of the present invention) is It is a G protein-coupled receptor protein and is used in human warm-blooded animals (eg, guinea pig, rat, mouse, chicken, Cells of egrets, bushes, sheep, sheep, monkeys, monkeys, dogs, cats, etc. Epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, Neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, or these cells Progenitor cells, stem cells or cancer cells, etc.) or any tissue in which those cells are present, such as the brain, each part of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus) , Cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, ligament, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), It may be a protein derived from blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., or may be a synthetic protein. .

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 includes amino acids having 98% or more, preferably about 99% or more homology with the amino acid sequence represented by SEQ ID NO: 4. And the like.

Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 4 include, for example, a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by the aforementioned SEQ ID NO: 4 However, a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 4 is preferred.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14 includes 92% or more, preferably about 95% or more, more preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 14. Amino acid sequences having a homology of 97% or more, more preferably about 99% or more, may be mentioned.

Examples of proteins containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 14 include, for example, amino acids substantially the same as the amino acid sequence represented by the aforementioned SEQ ID NO: 14 A protein containing a sequence and having substantially the same activity as a protein having the amino acid sequence represented by SEQ ID NO: 14 is preferred. 03

9

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 includes 92% or more, preferably about 95% or more, more preferably about 95% or more of the amino acid sequence represented by SEQ ID NO: 24. Amino acid sequences having a homology of 97% or more, more preferably about 99% or more, and the like can be mentioned.

Examples of proteins having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 24 include, for example, amino acids substantially the same as the amino acid sequence represented by SEQ ID NO: 24 above. A protein containing a sequence and having substantially the same activity as a protein having the amino acid sequence represented by SEQ ID NO: 24 is preferred.

Substantially the same activity includes, for example, a binding activity to MCH, a signal information transduction effect, and the like. Substantially the same means that their activities are the same in nature. Therefore, it is preferable that the activities such as the binding activity to MCH and the activity of signal transduction are equivalent (eg, about 0.5 to 2 times), but the quantitative factors such as the degree of these activities and the molecular weight of the protein are It may be different.

The activity such as the binding activity and the signal transduction action can be measured according to a method known per se or a method analogous thereto.

Examples of the protein of the present invention include (1) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 4 (eg, about 1 to 8, preferably about 1 to 5, (Ii) preferably one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 4 (for example, about 1 to 200, preferably 1 to 1); About 50, preferably about 1 to 100, preferably about;! To about 50, preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 (5) an amino acid sequence to which the amino acid of (iii) is added; (iii) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 4 (eg, about 1 to 200, preferably 1 to 15) About 0, preferably about 1 to 100, preferably about 1 to 50, preferably about 1 to 30, preferably 1 to 10 An amino acid sequence into which about, more preferably a number (1 to 5) amino acids have been inserted; (iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 4 (eg, 1 to 200) About 1, preferably about 1 to 150, preferably about 1 to 100, preferably 1 An amino acid sequence in which up to about 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) of amino acids have been substituted with another amino acid; or (V) a so-called mutin such as a protein containing an amino acid sequence obtained by combining them; (2) (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 14 (for example, about 1 to 25 An amino acid sequence in which about 1 to about 10 amino acids have been deleted, more preferably about 1 to 5 amino acids, and (ii) one or two amino acids in the amino acid sequence represented by SEQ ID NO: 14. (For example, about 1 to 200 pieces, preferably about 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably Is about 1 to 10 amino acids, more preferably about 1 to 5 amino acids Sequence, (iii) SEQ ID NO: 1 or 2 or more (for example, about 1 to 200, preferably about 1 to 150, preferably 1 to 100) About 1 amino acid, preferably about 1 to 5.0 amino acids, preferably about 1 to 30 amino acids, more preferably about 1 to 10 amino acids, and even more preferably (1 to 5) amino acids. Sequence, (iv) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 14 (for example, about 1 to 200, preferably about 1 to 150, and preferably 1 to 1 About 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably the number (1 to 5) of amino acids is other amino acids. (3) (i) a so-called mutin such as a protein containing a substituted amino acid sequence or (V) a protein containing an amino acid sequence combining them; 1 or 2 or more (for example, about 1 to 25, preferably about 1 to 10, and more preferably about 1 to 5) amino acids in the amino acid sequence represented by SEQ ID NO: 24; (Ii) one or two or more amino acid sequences represented by SEQ ID NO: 24 (for example, about 1 to 200, preferably about 1 to 150, and preferably About 100, preferably about 1 to 50, preferably about 1 to 30, preferably about 1 to 10, and more preferably about (1 to 5) amino acids (Iii) one or two or more amino acid sequences represented by SEQ ID NO: 24 (for example, about 1 to 200, preferably about 1 to 150, and preferably 1 to 1 About 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, preferably about 1 to 10 pieces, and more preferably Or (iv) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 24 (for example, about 1 to 200 amino acids). Degree, preferably about 1 to 150 pieces, preferably about 1 to 100 pieces, preferably about 1 to 50 pieces, preferably about 1 to 30 pieces, and preferably about 1 to 10 pieces. Preferably, a so-called mutin such as a protein containing an amino acid sequence in which a number (1 to 5) of amino acids are substituted with another amino acid, or (V) an amino acid sequence obtained by combining them is also included.

When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

Specific examples of the protein of the present invention include, for example, a protein containing the amino acid sequence represented by SEQ ID NO: 4, a protein containing the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 24 And proteins containing the amino acid sequence represented by

The partial peptide of the protein of the present invention (hereinafter sometimes abbreviated as the partial peptide of the present invention) is a partial peptide of the protein of the present invention described above, and is preferably the same as the above-described protein of the present invention. Any material having properties may be used.

For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more of the constituent amino acid sequences of the protein of the present invention. For example, a peptide having the amino acid sequence of

In addition, the partial peptide of the present invention lacks one or more (preferably about 1 to 10, more preferably, about 1 to 5) amino acids in its amino acid sequence, Alternatively, one or more (preferably, about 1 to 20, more preferably, about 1 to 10, and more preferably, about 1 to 5) amino acids are added to the amino acid sequence. Or 1 or 2 or more amino acids in the amino acid sequence thereof (preferably, about 1 to 20; more preferably, about 1 to 10;

5) amino acids, or one or more (preferably about 1 to 10, more preferably several, and more preferably 1 to (About 5 amino acids) may be substituted with another amino acid.

As the partial peptide of the present invention, for example, in the amino acid sequence represented by SEQ ID NO: 4, for example, the 1st to 41st amino acids, the 31st to 34th amino acids, SEQ ID NO: 1 For example, in the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 24, the 1st to 41st amino acid sequence, the 31st amino acid sequence to the 34th amino acid sequence, SEQ ID NO: 24 The amino acid sequences of the 1st to 41st and the 31st to 330th amino acids are preferred.

A peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25 (hereinafter sometimes referred to as the peptide used in the present invention or the peptide of the present invention) is a human Cells of warm-blooded animals (eg, guinea pigs, rats, mice, chicks, egrets, pigs, higgies, lions, monkeys, dogs, cats, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells) 3 cells, bone marrow cells, mesangial cells, Langer's cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (Eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone Cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells) or any tissue in which these cells are present, for example, brain, brain Anatomy (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, kidney, kidney, liver, gonad, thyroid, gall bladder, Bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, It may be a protein derived from skeletal muscle or the like, or may be a synthetic protein.

Examples of the peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 25 Peptides having substantially the same activity as the peptide having the amino acid sequence represented by SEQ ID NO: 25 are preferred.

'Substantially equivalent activities include, for example, SLT binding activity, etc. It is. Substantially the same indicates that the activity is the same in nature. Therefore, it is preferable that the binding activity to SLT is equivalent (eg, about 0.5 to 2 times), but quantitative factors such as the degree of this activity and the molecular weight of the protein may be different. No.

The binding activity can be measured according to a method known per se.

As the peptide of the present invention, a peptide containing the amino acid sequence represented by SEQ ID NO: 25 is preferably used.

Examples of the peptide of the present invention include: (i) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 (for example, about 1 to 30, preferably about 1 to 10; More preferably, an amino acid sequence in which a number (1 to 5) of amino acids are deleted, and (ii) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 25 (for example, about 1 to 30 amino acids, Preferably about 1 to 10 amino acids, more preferably about 1 to 5 amino acids, and (iii) one or more amino acid sequences in the amino acid sequence represented by SEQ ID NO: 25. (For example, about 1 to 30 amino acids, preferably about 1 to 10 amino acids, and more preferably about 1 to 5 amino acids). (Iv) SEQ ID NO: 25 1 or 2 or more in the amino acid sequence (for example, about 1 to 30, preferably about 1 to 10, more preferably Preferably, a peptide containing an amino acid sequence in which a number (1 to 5) of amino acids are substituted with another amino acid, or (V) an amino acid sequence obtained by combining them is also included.

Specific examples include a peptide containing the 5th to 19th partial sequences from the N-terminal of the amino acid sequence represented by SEQ ID NO: 25. More specifically, it has the amino acid sequence represented by SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31. Peptides are examples. Preferably, a peptide having the amino acid sequence represented by SEQ ID NO: 28 is used.

Substantially identical substitutions of amino acids in the amino acid sequence may be selected, for example, from other amino acids of the class to which the amino acid belongs. Non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valin, proline, phenylalanine, tributofan, methionine, and the like. Polar (neutral) amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. , Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. '

The position at which the above-mentioned other amino acid is deleted or substituted is preferably a position other than Cys in the amino acids constituting the peptide of the present invention.

Further, the peptide of the present invention may be labeled. Specific examples include peptides labeled with an isotope by a method known per se, fluorescently labeled peptides (for example, fluorescently labeled with fluorescein), biotinylated peptides, enzyme-labeled peptides, and the like. .

Specifically, for example, by known methods, [¾], ⁽¹²⁵ 1], it can be utilized, such as a peptide of ^[l4 C], the present invention labeled with etc. ^[35 S]. Moreover, Porton A labeled product of the peptide of the present invention prepared by a known method using one Hunter reagent may be used.

Specific examples of the labeled peptide of the present invention include, for example,

(1) [ ¹²⁵ 1 • [N- (3- (4-hydroxy-3_dodophenyl) -pulpionyl) -Asp ¹ ] .MCH,

^{(2) [I25 I [N} - (3- (4- hydroxy-3 - ® one Dofueniru) propionic ^{sulfonyl) - Phe 2] 'MCH (} 2- 19),

(3) [ ¹²⁵ I- [N- (3- (4-hydroxy-3-iodophenyl) propionyl) -Asp ³ ] ′ MCH (3-19),

(4) [ ¹²⁵ I— [N- (3- (4-hydroxy-3-iodophenyl) propionyl) -Met ⁴ ] ′ MCH (4-19),

(5) [ ¹²⁵ I — [N- (3- (4-hydroxy-3-iodophenyl) propionyl) -Leu ⁵ ]. MCH (5-19),

(6) [ ¹²⁵ I-[N- (3- (4-hydroxy-3-iodophenyl) -pulpionyl) -Arg ⁶ ] ■ MCH (6-19),

(7) ^[I25 I] one [N_ (3- (4- hydroxy - 3 - Yodofueniru) propionyl) -Cys ^7] - such as MCH (7-19) can be mentioned. '

Of these, [ ^| 251]-[N- (3- (4-hydroxy-3-iodophenyl) propionyl) -Met ⁴ ] -MCH (4-19) is preferably used.

In the present specification, the left end of the protein and the peptide is the N-terminus (amino end) and the right end is the C-terminus (potassium terminal) according to the convention of peptide labeling. This onset Ming proteins and peptides, C-terminal, carboxyl group (-C00H), carbo Kishireto (-C00-), amide (- C0M ₂₎ or ester (- C00R) any der connexion may be of.

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, isopropyl, n - C, such as butyl, _ ₆ alkyl groups, such as cyclopentyl, such as hexyl cyclo 〇 ₃ - ₈ cycloalkyl group, for example, phenyl, alpha-naphthyl of which C ₆ _ ₁₂ 7 aryl group, e.g., benzyl, phenyl, such as phenethyl - C, C ₇ _ such as alpha-Nafuchiru alkyl groups such as _ ₂ Al kill group or carry one naphthylmethyl _{A 14} aralkyl group, a bivaloyloxymethyl group and the like are used.

When the protein and peptide of the present invention have a lipoxyl group (or lipoxylate) other than at the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the proteins and peptides of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Furthermore, in the proteins and peptides of the present invention, the amino acid residues at the N-terminus (eg, Mechionin residues) Amino group protecting groups (e.g., formyl group, etc. C _M Ashiru group such Arukanoiru such Asechiru group) Protected, N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, Substituent on the side chain of amino acid in the molecule (for example, -OH, -SH, amino group, imidazo Ichiru group, indole group, etc. Guanijino group) is also protected with a suitable protecting group (e.g., formyl group, etc. c, _ ₆ Ashiru groups such Arukanoiru groups such as § Se butyl group), or Also included are complex proteins such as so-called glycoproteins to which sugar chains are bound. In the partial peptide of the present invention, C-terminal, the force Rupokishiru group (-C00H), the force Lupo - Kishireto (-C00-), amide (- C0Nh ₂₎ or may be filed in any of ester (-C00R).

Furthermore, the partial peptide of the present invention includes, as in the case of the protein of the present invention, those having a carbonyl group (or carboxylate) other than the C-terminus, N-terminal amino acid residues (eg, methionine). Residue is protected with a protecting group, the glutamine residue formed by cleavage of the N-terminal in vivo is a pyroglutamate, and the substituent on the side chain of the amino acid in the molecule is appropriate. Also included are those protected with a protecting group or complex peptides such as so-called glycopeptides to which sugar chains are bonded.

Salts of the protein or partial peptide of the present invention and salts of the peptide of the present invention include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts). Preference is given to the use of, especially the physiologically acceptable acid addition salts. Such salts include, for example, 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) Salts with formic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) are used.

The protein of the present invention or a partial peptide thereof, the peptide of the present invention or a salt thereof can be produced from the above-mentioned human or warm-blooded animal cell or tissue by a known protein purification method. Can also be produced by culturing a transformant containing DNA encoding It can also be produced according to the peptide synthesis method described below.

When producing from human or mammalian tissues or cells, the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the resulting extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be achieved by combining the above chromatography.

For the synthesis of the peptide of the present invention, the protein of the present invention, its partial peptide, their amides or their salts, commercially available resins for protein / peptide synthesis can be used. As such a resin, for example, Methyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamide Methyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy resin, 41- (2', 4'-dimethoxyphenyl Fmocaminoethyl) phenoxy resin, etc. Can be. Using such a resin, an amino acid appropriately protected with an α-amino group and a side chain functional group is condensed on the resin in accordance with the sequence of the desired protein / peptide according to various known condensation methods. Let it. At the end of the reaction, proteins and peptides are cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the desired protein, peptide or amide thereof.

Regarding the condensation of the protected amino acids described above, various activating reagents that can be used for protein / peptide synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimides, DCC, N, N'-diisopropyl carbopimide. N-ethyl-N '-(3-dimethylaminoprolyl) carbopimide is used. For these activations, the protected amino acid may be added directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt) or pre-protected as a symmetrical acid anhydride or HOBt ester or HOOBt ester. It can be added to the resin after activation of the amino acid.

The solvent used for activating the protected amino acid or for condensing with the resin may be appropriately selected from solvents known to be usable for the protein-peptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol , Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. Can be The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about 120 ° (: to 5 Ot :). The sensitized amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When a sufficient condensation cannot be obtained by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protecting group for the amino group of the starting material include Z, Boc, Yuichi Sharipentyloxycarbonyl, Isoporonyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C11Z, Br-Z , Adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

Lepoxyl groups can be, for example, alkyl esterified (e.g., methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.). Or cyclic alkyl esterification), aralkyl esterification (e.g., benzyl ester, 412 trobenzyl ester, 4-methoxybenzyl ester, 4-methyl benzyl ester, benzhydryl esterification), phenacyl esterification , Benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for this esterification, for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarponyl group, and the like are used. Examples of a group suitable for etherification include a benzyl group, a tetrahydroviranyl group, and a t-butyl group.

The protecting group of the phenolic hydroxyl group of tyrosine, for example, B zl, C 1 ₂ -

Bzl, 2-nitrobenzyl, Br-Z, Yuichi Sharybutyl and the like are used. As the protecting group for imidazole of histidine, for example, Tos, 4-methoxy-12,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used. Examples of activated carboxylic acid groups of the raw material include, for example, corresponding acid anhydrides, azides, and activated esters [alcohols (for example, pentachlorophenol, 24,5-trichloromouth phenol, 2,4-dinitrophenol) , Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt). As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.

Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid. Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof; base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc .; Reduction by a system is also used. The elimination reaction by the above acid treatment is generally carried out at a temperature of about 120 ° C. (: up to 40 ° C.) In the acid treatment, for example, anisol, phenol, thioanisole, methacresol, paracresol It is effective to add a cation scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc. The 2,4-dinitrophenyl group used as an imidazole protecting group for histidine is effective. The formyl group which is removed by thiophenol treatment and used as an indole protecting group for tributofan is not only protected by acid treatment in the presence of 1,2-ethanedithiol and 1,4-butanedithiol as described above, but also dilute hydroxylated. Also removed by alkali treatment with sodium solution, dilute ammonia, etc.

The protection of the functional group which should not be involved in the reaction of the raw materials, the protective group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.

As another method for obtaining an amide of a protein or peptide, for example, first, after amidating and protecting the α-hydroxyl group of the amino acid at the terminal end of the amino acid, a peptide chain is extended to a desired chain length on the amino side. After that, the protein / peptide obtained by removing only the protecting group for the α-amino group at the Ν-terminal of the peptide chain and the C-terminal lipoxyl group are removed. A protein and a peptide from which only the protecting group has been removed are produced, and the peptide and the protein are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected peptide / protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude peptide / crude protein can be obtained. The crude peptide / crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein / peptide.

In order to obtain an ester of protein / peptide, for example, after condensing the α-hydroxyloxyl group of a carboxy-terminal amino acid with a desired alcohol to form an amino acid ester, the same procedure as in the amide of protein / peptide is carried out. It is possible to obtain an ester of a protein and a peptide.

The peptide of the present invention and the protein of the present invention can be produced according to a peptide synthesis method known per se. Further, the partial peptide of the protein of the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein of the present invention with an appropriate peptidase.

As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the peptide of the present invention is condensed with the peptide of the present invention or the partial peptide or amino acid capable of constituting the protein of the present invention and the remaining portion, and when the product has a protecting group, the protecting group is eliminated to remove the target peptide. Can be manufactured. Examples of the known condensation method and elimination of the protecting group include the methods described in the following (i) to (V).

(i) M. Bodanszky and M.A. Ondet t i, Peptide Synthes is, Interscience Publ i shers, New York (1966) ''

(ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

(iii) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd. (1975)

(iv) Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)

(V) Supervised by Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten After the reaction, the peptide of the present invention or the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, laffification, liquid chromatography, and recrystallization. . When the peptide or partial peptide obtained by the above method is in a free form, it can be converted to an appropriate salt by a known method. Conversely, when the peptide or partial peptide is obtained in a salt form, it can be converted to a free form by a known method Can be converted.

The polynucleotide encoding the protein of the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein of the present invention. Preferably it is DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the cells and tissues described above, cDNA library derived from the cells and tissues described above, and synthetic DNA.

The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, a reverse RNA was directly prepared using the total RNA or mRNA fraction prepared from the cells and tissues described above.

It can also be amplified by Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method).

The DI ^ A encoding the protein of the present invention includes, for example, (i) a DNA containing the nucleotide sequence represented by SEQ ID NO: 3 or a nucleotide sequence represented by SEQ ID NO: 3 under highly stringent conditions. A DNA encoding a protein having substantially the same properties as a protein having an amino acid sequence represented by SEQ ID NO: 4, having a base sequence hybridizing with: (ii) SEQ ID NO: 13 And a protein having a nucleotide sequence that hybridizes under high stringent conditions with the nucleotide sequence represented by SEQ ID NO: 13 or a protein comprising the amino acid sequence represented by SEQ ID NO: 14 (Iii) a DNA containing the nucleotide sequence represented by SEQ ID NO: 23, or a DNA encoding a protein having substantially the same properties. Any DNA encoding a protein having a nucleotide sequence that hybridizes under stringent conditions and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 24 It may be. Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 3 under high stringency conditions include, for example, 98% or more, preferably about 99% or more of the nucleotide sequence represented by SEQ ID NO: 3 DNA containing a nucleotide sequence having homology of

Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 13 under high stringency conditions include, for example, a nucleotide sequence represented by SEQ ID NO: 13 and 92% or more, preferably about 9% or less. A DNA containing a nucleotide sequence having a homology of 5% or more, more preferably about 97% or more, more preferably about 99% or more is used.

Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 23 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 23 and 92% or more, preferably about 9% or less. DNA containing a base sequence having a homology of 5% or more, more preferably about 97% or more, more preferably about 99% or more is used.

Hybridization is performed by a known method or a method equivalent thereto, such as the method described in Molecular Cloning (Molecular Cloning) 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). It can be done according to. If a commercially available library is still used, it can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringency conditions.

High stringency conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70, preferably about 60 to 65. The conditions at ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.

More specifically, DNAs encoding the protein containing the amino acid sequence represented by SEQ ID NO: 4 include DNA containing the base sequence represented by SEQ ID NO: 3, and SEQ ID NO: 38. The DNA encoding the protein having the amino acid sequence represented by SEQ ID NO: 14 has the nucleotide sequence represented by SEQ ID NO: 13 DNA, represented by SEQ ID NO: 39 Examples of the DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 24, such as a DNA having a nucleotide sequence represented by SEQ ID NO: 24, include a DNA having a base sequence represented by SEQ ID NO: 23, DNA containing the base sequence represented by 40 or the like is used.

The polynucleotide encoding the partial peptide of the present invention may be any polynucleotide as long as it contains a base sequence encoding the above-described partial peptide of the present invention. DNA is preferred. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA. '

Examples of the DNA encoding the partial peptide of the present invention include, for example, DNA having a part of the DNA having the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23, or SEQ ID NO: 3 A DN having a nucleotide sequence that hybridizes under high stringent conditions to the nucleotide sequence represented by SEQ ID NO: 1.3 or SEQ ID NO: 23, and encoding a protein having substantially the same activity as the protein of the present invention. DNA containing a part of A is used.

The DNA that can be hybridized with the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23 has the same significance as described above.

The same hybridization method and high stringency conditions as described above are used.

The DNA or DNA encoding the protein or partial peptide of the present invention (hereinafter, these may be simply referred to as the protein of the present invention in the description of the cloning and expression of the DNA encoding the same). As a means of cloning, the DNA of the present invention is amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA of the present invention is incorporated into an appropriate vector. Selection can be performed by hybridization with a DNA fragment encoding a part or all of the region or labeled with a synthetic DNA. The hybridization can be performed according to, for example, the method described in Molecular Cloning 2nd (J. Sabrook et al., Cold Spring Harbor Lab. Press, 1989). Ma When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted by PCR or a known kit such as Mutan ™ -Super Express Km (Takara Shuzo) or Mutan ™ -K (Takara Shuzo) using the 0DA-LA PCR method or the like. It can be performed according to a known method such as the gapped duplex method or the Kunkel method, or a method analogous thereto.

The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5, terminal side, and TAA, TGA or TAG as a translation termination codon on the 3, terminal side. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.

The expression vector of the protein of the present invention can be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) converting the DNA fragment into a promoter in an appropriate expression vector. It can be manufactured by connecting downstream of

Examples of the vector include a plasmid derived from E. coli (eg, pBR322, PBR32 5, pUC 12, pUC 13), a plasmid derived from Bacillus subtilis (eg, pUB 110, pTP 5, pC 194), a yeast-derived plasmid (eg, pSH19, pSH15) In addition to pacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus, pAl-11, pXTl, pR cZCMV, pRc / RSV, pc DNA I ZNe o and the like are used.

The promoter used in the present invention may be any suitable promoter corresponding to the host used for gene expression. For example, when animal cells are used as hosts, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc. may be mentioned. Of these, CMV (cytomegalovirus) promoter It is preferable to use SRo! When the host is Escherichia, the trp promoter, lac promoter, recA promoter, λPL promoter If the host is a Bacillus genus, SP01 promoter, SP02 promoter, penP promoter, etc.If the host is yeast, the host is yeast. , PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. The expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV400 ri), if desired. Anything can be used. The selection marker one, for example, dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [Mesotorekise Ichito (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp ^r ), Neomai, Shin-resistant gene (hereinafter sometimes abbreviated as Ne o ^r, include G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using Chinese hamster cells deficient in the dh 遺伝子 r gene, the target gene can be selected using a thymidine-free medium.

If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. If the host is Escherichia, the Pho A signal sequence, Omp A signal sequence, etc., if the host is Bacillus, the amylase signal sequence, subtilisin signal sequence, etc. In the case of yeast, MFa signal sequence and SUC2 signal sequence can be used. When the host is an animal cell, insulin signal sequence, high interferon signal sequence, antibody molecule and signal sequence can be used, respectively. .

Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant can be produced.

Examples of the host include Escherichia, Bacillus, yeast, insect cells, insects, animal cells, and the like.

Specific examples of the genus Escherichia include, for example, Escherichia coli.

(Escherichia coli) K12-DH1 (Proc. Natl. Acad. Sci. USA, 60, 160 (1968)), JM103 (Nucleic Acids Research, 9, 309 (1981)), JA2 21 (Journal of Molecular Biology, 120, 517 (1978)), HB101 [Journal of 'Molecular' Biology, 41, 459 (1969)], C600 (Genetics, 39, 440 (1954)) )) Is used.

Examples of Bacillus bacteria include, for example, Bacillus subtilis (Bacillus

subtilis) MI114 (Gene, 24, 255 (1983)), 207-21 (Journal of Biochemistry, 95, 87 (1984)) and the like are used. '' Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R—, ΝΑ87-11A, DKD-5D, 20B-12, Schizosaccharomyces porabe, NC YC 1913, NCYC 2036 Pastoris (Pichia pastoris) K # 71 or the like is used.

As insect cells, for example, when the virus is Ac NPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, and egg derived from Trichoplusia ni egg High Five ™ cells, cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, a cell line derived from a silkworm (Bombyx mori N cell; BmN cell) is used. Examples of the Sf cells include Sf9 cells (ATCC

CRL1711), Sf21 cells (Vaughn, J.L. et al., In.

Vivo), 13, 213-217, (1977)).

As insects, for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient chinini—zhamster cell CH〇 (hereinafter CHO (dh fr)). Cells), mouse L cells, mouse AtT-20, mouse myeoma cells, rat GH3, human FL cells, and the like.

Transformation of the genus Escherichia can be performed, for example, according to the method described in Proc. Natl; Acad. Sci. USA, 69, 2110 (1972) or Gene, 17, 107 (1982). it can. Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Volume 168, 111 (1979).

'' The yeast is transformed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). be able to.

Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

To transform animal cells, for example, see Cell Engineering Annex 8 New Cell Engineering Experimental Protocol. 263—267 (1995) (published by Shujunsha), Virology, 52

Volume, 456 (1973).

Thus, a transformant transformed with the expression vector containing DNA encoding the protein can be obtained.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn chip lica, peptone, zein, meat extract, soybean meal, and potato extract. Examples of the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

Examples of a medium for culturing Escherichia bacteria include, for example, an M9 medium containing glucose and casamino acids [Miller, Journal “Obexperiment”, “Molecular”, Journal of Experiments in

Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. Here, if necessary, a drug such as 3; 6-indolylacrylic acid can be added to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually carried out at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.

When the host is a bacterium belonging to the genus Bacillus, cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours.

When culturing a transformant in which the host is yeast, for example, Burkholder's minimal medium [Bostian, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980) And a SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The cultivation is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added as necessary.

When culturing an insect cell or a transformant whose host is an insect, the medium used is Grace's Insect Medium (Grace, TC, Nature, 195, 788 (1962)). Those to which additives such as serum are appropriately added are used. Preferably, the ρΗ of the culture medium is adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum (Science, 122, 501 (1952)), a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73] , 1 (1950)]. Preferably, the pH is about 6-8. Cultivation is usually performed at about 30 ° C (: up to 40 ° C for about 15 to 60 hours, and aeration and agitation are added as necessary.

As described above, the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.

The protein of the present invention can be separated and purified from the culture by, for example, the following method.

When extracting the protein of the present invention from cultured cells or cells, after the culture, the cells or cells are collected by a known method, suspended in an appropriate buffer, After lysing cells or cells by lysozyme and Z or freeze-thawing, a method of obtaining a crude protein extract by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-1000 ^™ . When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.

Purification of the protein contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods. These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Methods that utilize differences in charge, methods that use differences in charge, such as ion-exchange chromatography, methods that use specific affinities, such as affinity chromatography, and hydrophobicity, such as reverse-phase high-performance liquid chromatography. A method using the difference between the isoelectric points, such as an isoelectric focusing method, and the like, may be used.

When the protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in the form of a salt, a known method or a method analogous thereto Can be converted into a free form or another salt.

The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The presence of the protein of the present invention thus produced can be measured by, for example, enzymatic immunoassay western blotting using a specific antibody. The antibody against the protein or partial peptide of the present invention or a salt thereof may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide of the present invention or a salt thereof. An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, these may be simply abbreviated to the protein of the present invention in the description of the antibody) is obtained by using the protein of the present invention as an antigen. The antibody or antiserum can be produced according to the following method.

[Preparation of monoclonal antibody]

(a) Preparation of monoclonal antibody-producing cells

The protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site capable of producing an antibody upon administration. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of the warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them. By fusing the antibody-producing cells obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein Nature, 256, 495 (1975)]. 'Fusion promoters include, for example, polyethylene glycol (PEG), Sendai virus and the like, and preferably PEG is used.

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is used at a concentration of about 10 to 80%. Cell fusion can be carried out efficiently by adding the mixture and incubating at 20 to 40 ° C, preferably 30 to 37 ° C, for 1 to 10 minutes. Various methods can be used to screen monoclonal antibody-producing hybridomas.For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A labeled with a radioactive substance or an enzyme, and a monoclonal antibody bound to the solid phase Antibody detection method, Add the hybridoma culture supernatant to a solid phase to which anti-immunoglobulin antibody or protein A is adsorbed, add proteins labeled with radioactive substances, enzymes, etc. Examples include a method for detecting a single antibody.

The selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, and GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd. )) Alternatively, serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20 to 40, preferably about 37 ° C. The culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(b) Purification of monoclonal antibody

Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Adsorption-desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which the antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody). Can be done.

(Preparation of polyclonal antibody)

The polyclonal antibody of the present invention is produced according to a known method or a method analogous thereto. can do. For example, a immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting the contents and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier-protein used for immunizing a warm-blooded animal, the type of the carrier protein and the mixing ratio between the carrier and the hapten are different from those of the hapten immunized by cross-linking the carrier. Any antibody may be cross-linked at any ratio as long as it can be efficiently produced.For example, serum albumin, thyroglobulin, hemocyanin, etc., in a weight ratio of about 0.1 to hapten per hapten, may be used. -20, preferably about 1-5.

Various condensing agents can be used for force coupling between the hapten and the carrier, but glutaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. .

The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.

The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.

The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.

Complementary to the nucleotide sequence of DNA encoding the protein or partial peptide of the present invention (hereinafter, these DNAs may be abbreviated as the DNA of the present invention in the description of antisense polynucleotide), or An antisense polynucleotide having a substantially complementary base sequence or a part thereof includes a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof. Any antisense having an action capable of suppressing the expression of the DNA. Although it may be a polynucleotide, antisense DNA is preferred.

The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). And about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. In particular, of the total nucleotide sequence of the complementary strand of the DNA of the present invention, the complementary strand of the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention (for example, the nucleotide sequence near the start codon) is about 70%. %, Preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95%.

Specifically, a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23 Or a part thereof, preferably, for example, a nucleotide sequence complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23, or Antisense polynucleotides having a part thereof are exemplified.

An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.

To prevent degradation by hydrolases such as nucleases, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. May be substituted. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, an antisense polynucleotide (nucleic acid) corresponding to the protein gene of the present invention, which can inhibit the replication or expression of the gene, is cloned or the determined protein is encoded. It can be designed and synthesized based on DNA base sequence information. Such an antisense polynucleotide can hybridize to RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or bind to the protein-related RNA of the present invention. Can regulate and control the expression of the protein gene of the present invention through the interaction of Polynucleotides complementary to the selected sequence of the protein-related RNA of the present invention, and polynucleotides capable of specifically hybridizing with the protein-related RNA of the present invention, include the protein of the present invention in vivo and in vitro. It is useful for regulating and controlling gene expression, and is also useful for treating or diagnosing diseases. The term "corresponding" means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. The "correspondence" between a nucleotide, nucleotide sequence or nucleic acid and a protein usually refers to the amino acids of the (directed) protein derived from the nucleotide (nucleic acid) sequence or its complement. The 5 'end hairpin loop of the protein gene,

5 'end 6—base pair' repeat, 5 'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 'end palindrome region or 3' end Although a hairpin loop or the like can be selected as a preferred target region, any region in a protein gene can be selected as a target. Regarding the relationship between a target nucleic acid and a polynucleotide complementary to at least a part of the target region, If the nucleic acid of interest can hybridize to the target region, the nucleic acid of interest can be said to be "antisense" to the polynucleotide of the target region. Antisense polynucleotides can be polydexoxy liponucleotides containing 2-deoxy D-reports, polyliponucleotides containing D-reports, N-glycosides of purine or pyrimidine bases and other types of polynucleotides. Other polymers having nucleotide or non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that such polymers are found in DNA or RNA) Base pairing and nucleotides having a configuration that allows base attachment). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, as well as unmodified polynucleotides (or unmodified oligonucleotides), Related modifications with known modifications, e.g., those with labels, caps, methylated, one or more natural nucleotides known in the art Substituted, Intramolecular nucleotide modification, eg uncharged binding

(Eg, having a methylphosphonate, phosphotriester, phosphoramidate, potassium salt, etc.), having a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), eg, protein

(Eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc. Eg, containing acridine, psoralen, etc., containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, modified bonds (Eg, a anomeric nucleic acid). Here, "nucleoside", "nucleotide", and "nucleic acid" may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with a halogen, an aliphatic group, or the like, or a functional group, such as an ether or an amine. Conversion.

The antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include a sulfur derivative of a nucleic acid, a thiophosphate derivative, a polynucleoside amide / a nucleic acid that is resistant to degradation of oligonucleoside amide, and the like. The antisense polynucleotide of the present invention can be designed, for example, as follows. In other words, the antisense polynucleotide is more stable in the cell, the cell permeability of the antisense polynucleotide is increased, the affinity for the target sense strand is increased, and If so, reduce the toxicity of the antisense polynucleotide. Many such modifications are reported, for example, in Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Appli cations, CRC Press, 1993. The antisense polynucleotides of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes, microspheres, or may be applied by genetic therapy. Can be given in an added form. Thus, the addition forms include polycations such as polylysine, which acts to neutralize the charge on the phosphate backbone, and lipids, which enhance the interaction with cell membranes and increase the uptake of nucleic acids ( For example, phosphoric acid, hydrophobic, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside linkage. Other groups are cap groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonuclease and RNase. No. Examples of such a capping group include, but are not limited to, hydroxyl-protecting groups known in the art, such as diols such as polyethylene glycol and tetraethylene glycol.

The inhibitory activity of the antisense polynucleotide can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. Can be. Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as the D NA), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter may be abbreviated as the antibody of the present invention), and an antisense polynucleotide of the DNA of the present invention (hereinafter may be referred to as an antibody). , Which may be abbreviated as the antisense polynucleotide of the present invention).

[1] Agent for preventing and treating various diseases related to the protein of the present invention

The protein of the present invention is a G protein-coupled receptor protein, MC By binding to H, it plays an important role in promoting appetite (feeding) and promoting oxytocin secretion.

Therefore, when the DNA encoding the protein of the present invention is abnormal or defective, or when the expression level of the protein of the present invention is reduced, for example, anorexia (eg, nervous appetite) Anemia or hypoproteinia due to anorexia, weak labor, laxative bleeding, uterine remodeling failure, milk stasis, etc. develop various diseases.

Therefore, the protein of the present invention and the DNA of the present invention include, for example, an appetite (feeding) enhancer, anorexia (eg, anorexia nervosa), anemia or hypoproteinemia associated with anorexia, weak labor pain It can be used as a medicament such as a preventive and remedy for flaccid hemorrhage, uterine remodeling failure, and milk stasis.

For example, if the protein of the present invention is reduced or deficient in the living body, and there is a patient in which the appetite (feeding) enhancement or oxytocin secretion promoting activity of MCH is not sufficiently or normally exerted, B) by administering the DNA of the present invention to the patient and expressing the protein of the present invention in vivo, by inserting the DNA of the present invention into cells and expressing the protein of the present invention. By transplanting the cells into a patient, or (8) administering the protein of the present invention to the patient, etc., to fully or normally exert the role of the protein of the present invention in the patient. it can.

When the DNA of the present invention is used as the above-described prophylactic or therapeutic agent, the DNA is used alone or in an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. Thereafter, it can be administered to humans or warm-blooded animals according to conventional means. The DNA of the present invention can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered using a gene gun or a catheter such as a hide mouth gel catheter.

When the protein of the present invention is used as the prophylactic / therapeutic agent, the protein purified to at least 90%, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more. It is preferred to use. The protein of the present invention can be used, for example, in the form of tablets, capsules, elixirs, microcapsules, and the like, which are sugar-coated as necessary, orally, or aseptically with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as aqueous solutions or suspensions. For example, the protein of the present invention may be used together with physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form required for generally accepted pharmaceutical practice. It can be manufactured by mixing. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained. '' Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, Swelling agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cellulose. 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 standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.

Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). Agents, for example, alcohols (eg, alcohol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), non-ionic surfactants (eg, Polysorbate 80 ^™ , HC Ο—50 etc.). Examples of the oily liquid include sesame oil, soybean, and oil, and may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate, buffer, etc.), soothing agents (eg, benzalkonium chloride, proforce hydrochloride, etc.)

), Stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. 'May be combined. The prepared injection is usually filled in an appropriate ampoule. The vector into which the DNA of the present invention has been inserted is also formulated in the same manner as described above, and is usually used parenterally.

The preparations obtained in this way are safe and have low toxicity, for example, in warm-blooded animals (e.g., humans, rats, mice, guinea pigs, egrets, birds, higgies, bush, pests, pests, cats, Dogs, monkeys, chimpanzees, etc.).

The dosage of the protein of the present invention varies depending on the target disease, the administration subject, the administration route, and the like. For example, when the protein of the present invention is orally administered for the purpose of treating renal failure, generally, the adult (as 60 kg) is used. Is administered in an amount of about 0.1 to 100 mg, preferably about 1. (! To 50 mg, more preferably about 1.0 to 20 mg. Per day. The single dose varies depending on the administration subject, the target disease, and the like. For example, when the protein of the present invention is administered to an adult (with a body weight of 60 kg) in the form of an injection for the purpose of treating anorexia nervosa, It is convenient to administer the protein by injecting about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the protein per day into the affected area. 60 kg for other animals Amount converted to have enough can be administered.

[2] Screening of drug candidate compounds for disease

'The protein of the present invention is useful as a reagent for screening a compound or a salt thereof that changes the binding between the peptide of the present invention (eg, MCH) and the protein of the present invention.

A compound or a compound that alters the binding between the peptide of the present invention and the protein of the present invention, which comprises using the peptide of the present invention and the protein of the present invention (including a partial peptide of the protein of the present invention). A method for screening a salt thereof, or a kit for screening a compound or a salt thereof, which changes the binding property between the peptide of the present invention and the protein of the present invention, which comprises using the peptide of the present invention and the protein of the present invention ( Hereinafter, the screening method of the present invention and the kit for screening of the present invention are abbreviated in detail below. Using the protein of the present invention, or constructing a recombinant expression system of the protein of the present invention, and using a binding assay system (ligand-receptor assay system) with the peptide of the present invention using the expression system. Thus, a compound that alters the binding between the peptide of the present invention and the protein of the present invention, a compound that alters the binding between the peptide of the present invention and the protein of the present invention (for example, peptide, protein, non-peptide) Compounds, synthetic compounds, fermentation products, etc.) or salts thereof.

Such compounds, cell stimulating activity via protein of the present invention (e.g., Arakidon acid release, acetylcholine release, intracellular C a ^{2 +} release, intracellular c AM P producing formation, intracellular c AM P production inhibition , Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos activation, pH decrease, cAMP-dependent protein kinase activation, cGMP Activation of protein-dependent protein kinase, activation of phospholipid-dependent protein kinase, activity of promoting or inhibiting the activation of microtubule-associated protein kinase (MAP kinase), etc. Compounds (agonist) and compounds (antagonists) having no such cell stimulating activity. "Altering the binding between the peptide of the present invention and the protein of the present invention" includes both cases of inhibiting and promoting the binding of the peptide of the present invention to the protein of the present invention. .

That is, the present invention relates to (i) the case where the protein of the present invention is brought into contact with the peptide of the present invention, and (ii) the case where the above-mentioned protein of the present invention is brought into contact with the peptide of the present invention and a test compound. And a method for screening a compound or a salt thereof, which changes the binding property between the peptide of the present invention and the protein of the present invention, characterized in that the comparison is performed.

In the screening method of the present invention, (i) the case where the above-mentioned protein of the present invention is brought into contact with the peptide of the present invention and (ii) the above-mentioned protein of the present invention which comes in contact with the peptide of the present invention and a test compound. In such a case, for example, the amount of the peptide of the present invention bound to the protein of the present invention, the cell stimulating activity and the like are measured and compared.

Specific examples of the screening method of the present invention include, for example, (a) the peptide of the present invention relative to the protein of the present invention when the peptide of the present invention is brought into contact with the protein of the present invention, and when the peptide and the test compound of the present invention are brought into contact with the protein of the present invention; A method for screening a compound or a salt thereof that changes the binding property between the peptide of the present invention and the protein of the present invention, which comprises measuring and comparing the amount of binding;

(b) when the peptide of the present invention is brought into contact with a cell containing the protein of the present invention or a membrane fraction of the cell, and a cell containing the peptide and the test compound of the present invention and the protein of the present invention. Alternatively, the peptide of the present invention and the peptide of the present invention are characterized by measuring and comparing the amount of binding of the peptide of the present invention to the cell or the membrane fraction when brought into contact with the membrane fraction of the cell. A method for screening a compound or a salt thereof that changes the binding property to a protein, and

(c) the screening method according to the above (b), wherein the protein of the present invention is a protein of the present invention expressed on a cell membrane by culturing a transformant containing a DNA encoding the protein of the present invention;

(d) the peptide of the present invention is a labeled peptide, such as the screening method according to any one of (a) to (c) above,

(e) Cell stimulating activity mediated by the protein of the present invention when the peptide of the present invention is brought into contact with the protein of the present invention and when the peptide of the present invention and a test compound are brought into contact with the protein of the present invention A method of screening a compound or a salt thereof, which changes the binding property between the peptide of the present invention and the protein of the present invention, which comprises measuring and comparing

(f) when the peptide of the present invention is brought into contact with a cell containing the protein of the present invention or a membrane fraction of the cell, and the peptide or test compound of the present invention or the cell containing the protein of the present invention or It changes the binding property between the peptide of the present invention and the protein of the present invention, which is characterized by measuring and comparing the cell stimulating activity mediated by the protein of the present invention when brought into contact with the membrane fraction of the cell. A method for screening a compound or a salt thereof, and

(g) a protein of the present invention expressed on a cell membrane by culturing a transformant containing a DNA encoding the protein of the present invention; A cell stimulating Atsay system such as the screening method of the above (f) and the like can be mentioned.

The specific description of the screening method of the present invention is as follows.

First, the protein of the present invention used in the screening method of the present invention may be any protein containing the above-described protein of the present invention. For example, the protein of the present invention which is expressed in a large amount using a recombinant is suitable. For the production of the protein of the present invention, the aforementioned methods and the like are used.

In the screening method of the present invention, when cells containing the protein of the present invention or the cell membrane fraction are used, the preparation method described later may be followed.

When a cell containing the protein of the present invention is used, the cell may be immobilized with datalaldehyde, formalin, or the like. The immobilization method can be performed according to a known method. '' The cell containing the protein of the present invention refers to a host cell expressing the protein of the present invention. Examples of the host cell include the aforementioned Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like. .

The membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after cell disruption. The cells can be crushed by crushing the cells with a Potter-Elvehj em homogenizer, crushing with a Warlinda blender-Polytron (Kinematica), crushing with ultrasonic waves, or pressing with a French press. Crushing by ejecting cells from thin nozzles. For cell membrane fractionation, centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used. For example, the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30,000 rpm) for 30 minutes to 2 hours to obtain a precipitate. Is the membrane fraction. The membrane fraction is rich in the expressed protein of the present invention and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of the protein of the present invention in the cells or membrane fraction containing the protein of the present invention is preferably 10 ³ to 10 ⁸ molecules per cell, more preferably 10 ⁵ to 10 ⁷ molecules per cell. is there. 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, but also allows the same lot Can measure a large amount of samples.

In order to carry out the above-described screening method for the receptor binding assay system or the cell stimulation assay system, for example, the protein fraction of the present invention and the peptide of the present invention (eg, the labeled peptide of the present invention) and the like are used. Used. The protein fraction of the present invention is preferably a natural protein fraction of the present invention or a recombinant protein fraction of the present invention having an activity equivalent thereto. Here, the equivalent activity means equivalent ligand binding activity and the like. The labeled peptide, such as a radioactive isotope (e.g., ^[125 1], ^[131 1], [¾], [ "C], ^[32 P], ^[33 P], etc. ^[35 s]), Fluorescent substances (eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Bioscience), etc.), fluorescamine, fluorescein sothiosinate, etc.), enzymes (eg, —galactosidase, j6—labeled with darcosidase, alkaline phosphatase, oxidase, malate dehydrogenase, etc., luminescent substances (eg, luminol, luminol derivatives, J-reciferin, lucigenin, etc.), piotin, lanthanide, etc. In particular, the peptide of the present invention prepared by a known method using a Porton-Hunter reagent can be used. It is also possible to use the body.

Specifically, to screen for a compound that alters the binding between the peptide of the present invention and the protein of the present invention, first, cells containing the protein of the present invention or the membrane fraction of the cells are screened. Prepare the receptor standard by suspending it in a buffer suitable for tanning. Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor, such as a phosphate buffer having a pH of 4 to 10 (preferably, a pH of 6 to 8), such as Tris-HCl buffer. Also, for the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ™ (Kao-Atlas), digitonin, and dexcholate can be added to the buffer. In addition, a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptide sutin is added to suppress the degradation of the protein of the present invention and the peptide of the present invention by protease. You can also. To the receptions evening first solution of 0. 01~10ml, added labeled peptide of the present invention a certain amount (5,000 to 500,000 cpm), coexist simultaneously ^10-4 to 10-test compound M. Large to know the amount of non-specific binding (NSB) A reaction tube containing an excess of the unlabeled peptide of the present invention is also prepared. The reaction is carried out at 0 to 50 ° C, preferably 4 to 37 ° C, for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the reaction solution is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured using a liquid scintillation counter or a counter. When the count (B. One NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B.) when there is no antagonistic substance is 100%, the specific binding amount (B-NSB) is For example, a test compound having 50% or less can be selected as a candidate substance having a competitive inhibitory ability.

Further, as a method for measuring the binding between the protein of the present invention and the peptide of the present invention, BIAcore (manufactured by Amersham Pharmacia Biotech) can be used. In this method, the peptide of the present invention is immobilized on a sensor chip by an amino coupling method according to the protocol attached to the device, and contains a cell containing the protein of the present invention or a DNA encoding the protein of the present invention. Phosphate buffer or Tris buffer containing the protein of the present invention purified from the transformant or the membrane fraction containing the protein of the present invention, or the purified protein of the present invention or the membrane fraction containing the protein of the present invention, and the test compound Pass one or more buffers over the sensor chip at a rate of 2-20 ^ I per minute. By observing that the coexisting test compound changes the surface plasmon resonance caused by the binding of the peptide of the present invention and the protein of the present invention on the sensor chip, the protein of the present invention and the present invention are changed. A compound that changes the binding to a peptide can be screened. In this method, the protein of the present invention is immobilized on a sensor chip, and a buffer solution such as a phosphate buffer or a Tris buffer containing the peptide of the present invention or the peptide of the present invention and a test compound is passed over the sensor chip. The same method can be used for measurement. Test compounds include those similar to the above.

To perform the screening methods of the cell stimulating Atsusi system mediated cell stimulating activities proteins of the present invention (e.g., Arakidon acid release, Asechirukori emissions release, intracellular C a ^{2 +} release, intracellular c AM P generated , Inhibition of intracellular cAMP production, Intracellular cGMP production, Wild boar! ^ -Monophosphate production, Cell membrane potential fluctuation, Intracellular protein Phosphorylation, activation of c-fos, decrease of pH, activation of cAMP-dependent protein kinase, activation of cGMP-dependent protein kinase, activation of phospholipid-dependent protein kinase, The activity of promoting or suppressing the activation of microtubule-associated protein kinase (MAP kinase), etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the protein of the present invention are cultured on a multiwell plate or the like. Before performing screening, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant. Then, the generated product is quantified according to each method. When it is difficult to produce a substance (for example, arachidonic acid) as an indicator of cell stimulating activity using a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme may be added to perform the assay. . In addition, activities such as inhibition of cAMP production can be detected as an activity of inhibiting production of cells whose basic production has been increased by forskolin or the like.

For screening by measuring the cell stimulating activity, cells expressing an appropriate protein of the present invention are used. As the cells expressing the protein of the present invention, the above-mentioned recombinant cell lines expressing the protein of the present invention and the like are desirable. The transformant expressing the protein of the present invention may be a stable expression strain or a transient expression strain. In addition, the same kind of animal cells as described above are used.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.

The above-mentioned screening method for the cell-stimulating assay system is described more specifically in the following (1) to (12).

(1) When a receptor-expressing cell is stimulated by a receptor agonist, an intracellular G protein is activated and GTP is bound. This phenomenon is also observed in the membrane fraction of receptor-expressing cells. Normally, GTP is hydrolyzed to GDP, but if GTPaS is added to the reaction solution at this time, GTP7S binds to G protein like GTP, but is hydrolyzed. Without G cells The state of being bonded to the membrane is maintained. If labeled GTPaS is used, the activity of stimulating receptor agonist receptor-expressing cells can be measured by measuring the labeled GTPaS remaining on the cell membrane.

By utilizing this reaction, the stimulatory activity of the peptide of the present invention on the protein-expressing cell of the present invention is measured, whereby a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is obtained. You can screen.

This method is performed using a membrane fraction containing the protein of the present invention. In this assay method, the substance exhibiting the activity of promoting the GTP-A binding to the protein membrane fraction of the present invention is agonist.

Specifically, the case where the peptide of the present invention is brought into contact with the protein cell membrane fraction of the present invention in the presence of labeled GTP TS, and the case where the peptide and test compound of the present invention are brought into contact with the protein cell membrane fraction of the present invention. By measuring and comparing the activity of promoting GTPTS binding to the protein cell membrane fraction of the present invention when it is brought into contact with, a compound that alters the binding property between the peptide of the present invention and the protein of the present invention is determined. Screen.

In this method, the peptide GTP _T S binding promoting activity test compound showing activity to suppress to proteins the cell membrane fraction of the present invention according to the present invention can be selected as Ah Ru candidates competitive inhibition.

On the other hand, screening of agonist is carried out by bringing only the test compound into contact with the protein cell membrane fraction of the present invention and measuring the GTP-AS binding promoting activity to the protein cell membrane fraction of the present invention. You can also.

One example of the screening method is described below more specifically.

The cell membrane fraction containing the protein of the present invention prepared according to known methods, membrane dilution buffer (50 mM T ris, 5 mM M g C 1 2, 150 mM N a C 1, 1 M GDP, 0. Dilute with 1% BSA; pH 7.4). The dilution ratio depends on the expression level of the receptor. This was dispensed by 0. 2 ml min F a 1 c 0 n 2053, a peptide or peptide and a test compound of the present invention of the present invention was added, further to a final concentration of 200 pM added ^[35 S] GTPrS. After incubation for 1 hour at 25 ° C, wash ice-cold buffer (50 mM Tr is, 5mM MgC 1 2, 150 mM NaCl, 0.1% BSA, 0.05% CHAP S; pH 7.4) Add 15 ml, and filter through glass fiber filter paper GFZF. After incubating for 65 and 30 minutes and drying, it was bound to the membrane fraction remaining on the filter paper with a liquid scintillation counter.

The radioactivity of [ ³⁵ S] GTPrS is measured. The radioactivity of the experimental group to which only the peptide of the present invention was added was 100%, and the radioactivity of the experimental group to which the peptide of the present invention was not added was 100%.

Assuming 0%, the effect of the test compound on the GTP _rs binding promoting activity of the peptide of the present invention is calculated. A test compound having a GTPaS binding promoting activity of, for example, 50% or less can be selected as a candidate substance having competitive inhibitory ability.

(2) In the cell expressing the protein of the present invention, the production of intracellular cAMP is suppressed by stimulation of the peptide of the present invention. By utilizing this reaction to measure the stimulating activity of the peptide of the present invention on the protein-expressing cells of the present invention, the binding between the peptide of the present invention and the protein of the present invention is changed. The compound can be screened.

Specifically, the case where the peptide of the present invention is brought into contact with the protein-expressing cell of the present invention in the presence of a substance that increases the amount of intracellular cAMP, the case where the peptide of the present invention and the test compound express the protein of the present invention, A compound that alters the binding between the peptide of the present invention and the protein of the present invention is determined by measuring the activity of inhibiting the production of intracellular cAMP in the cell when the cell is brought into contact with the cell and comparing the results. I do.

As a substance that increases the amount of intracellular cAMP, for example, forskolin, calcitonin and the like are used.

The amount of cAMP produced in the protein-expressing cells of the present invention is determined by comparing the amount of anti-cAMP antibody obtained by immunizing mice, rats, rabbits, goats, rabbits, etc. with [ ¹²⁵ 1] -labeled cAMP (both commercially available products). Can be measured using an RIA system by using EIA or an EIA system combining an anti-cAMP antibody and labeled cAMP. Moreover, the anti-cAMP antibody, using a protein A or beads containing scintillant which is secured using such antibodies to etc. I gG of animals used in the anti-c AMP antibody production ^[125 1]-labeled c AMP Quantification by the SPA (Scintillation Proximity Assay) method is also possible (using a kit manufactured by Amersham Pharmacia Biotech). In the method, c A of the protein-expressing cell of the present invention by the peptide of the present invention A test compound exhibiting an activity of inhibiting MP production inhibitory activity can be selected as a candidate substance having a competitive inhibition ability.

On the other hand, a compound showing an agonist activity can be screened by contacting only the test compound with the protein-expressing cell of the present invention and examining the cAMP production inhibitory activity.

One specific example of the screening method is described below.

Cells expressing the protein of the present invention (eg, animal cells such as CHO cells) are seeded on a 24-well plate at 5 × 10 ⁴ ce 1 LZwe 11 and cultured for 48 hours. Wash the cells with Hank's buffer (pH 7.4) containing 0.2 ηιΜ3-isobutyl-methylxanthine, 0.05% BSA and 2 OmM HEPES (hereinafter abbreviated as reaction buffer 1). Then add 0.5 ml of reaction buffer and incubate in the incubator for 30 minutes. Excluding the reaction buffer, add 0.25 ml of the reaction buffer to the cells, and then add 1 M of the peptide of the present invention or 1 M of the peptide of the present invention and 2 M forskolin to which the test compound is added. Add 0.25 ml of reaction buffer to the cells and incubate at 37 ° C for 24 minutes. Stop the reaction by adding 100 1 of 20% perchloric acid, and then leave it on ice for 1 hour to extract intracellular cAMP. The amount of cAMP in the extract is measured using a cAMP EIA kit (Amersham Pharmacia Biotech). The amount of cAMP produced by the stimulation of forskolin was defined as 100%, and the amount of cAMP suppressed by the addition of 1 / M of the peptide of the present invention was defined as 0%. The effect of the test compound on is calculated. A test compound that inhibits the activity of the peptide of the present invention and has a cAMP production activity of, for example, 50% or more can be selected as a candidate substance capable of competitive inhibition.

In addition, when using the protein-expressing cell of the present invention, which exhibits the property of increasing the amount of intracellular CAMP by stimulation of the peptide of the present invention, the peptide of the present invention was brought into contact with the protein-expressing cell of the present invention. And a test compound obtained by contacting the peptide of the present invention and the test compound with the protein-expressing cell of the present invention, by measuring and comparing the intracellular cAMP production promoting activity of the cell. Compounds that alter the binding to the proteins of the invention can be screened. In this method, a test compound showing an activity of inhibiting the cAMP production promoting activity of the protein-expressing cells of the present invention by the peptide of the present invention can be selected as a candidate substance having an antagonistic inhibitory ability. ''

On the other hand, a compound exhibiting agonist activity can be screened by contacting the test compound alone with the protein-expressing cell of the present invention and examining cAMP production promoting activity.

c The AMP production promoting activity can be measured by adding the peptide of the present invention or the peptide of the present invention and the test compound to the protein-expressing cells of the present invention (eg, animal cells such as CHO cells) without adding forskolin in the above screening method. The amount of cAMP produced by the addition of is determined and measured by the method described above.

(3) By measuring the stimulating activity of the peptide of the present invention on cells expressing the protein of the present invention using the CRE-reporter gene vector, the peptide of the present invention and the protein of the present invention can be measured. Compounds that alter binding can be screened.

DNA containing CRE (cAMP response element) is inserted into the vector upstream of the reporter gene to obtain the CRE—repo overnight gene vector. In the protein-expressing cells of the present invention into which the CRE-reporter gene vector has been introduced, stimulation accompanied by an increase in cAMP is accompanied by CRE-mediated expression of the reporter gene, and subsequent stimulation of the gene product (protein) of the repo overnight gene. Induce production. In other words, by measuring the enzymatic activity of the reporter gene protein, it is possible to detect a change in the amount of CAMP in cells into which the CRE-repo overnight gene vector has been introduced.

Specifically, when the peptide of the present invention is brought into contact with the protein-expressing cell of the present invention into which the peptide of the present invention has been introduced in the presence of a substance that increases the amount of intracellular cAMP, the peptide of the present invention And the test compound was brought into contact with the protein-expressing cell of the present invention when the CRE-repo overnight gene vector was introduced. The enzyme activity of the reporter gene protein was measured and compared. Screening for a compound that alters the binding between the peptide of the present invention and the protein of the present invention. As a substance that increases the amount of intracellular CAMP, for example, forskolin, calcitonin and the like are used. As the vector, for example, Pitka Gene Basic Vector, Pitka Gene Enhancer Vector (Toyo Ink Manufacturing Co., Ltd.) and the like are used. The CRE-containing DNA is inserted into the above-mentioned vector in the repo overnight gene, for example, the multicloning site upstream of the luciferase gene, to obtain the CRE-reporter gene vector.

In this method, a test compound that restores the inhibition of the enzyme activity of the reporter gene protein by the peptide of the present invention can be selected as a candidate substance capable of competitive inhibition.

On the other hand, agonists can be screened by bringing only the test compound into contact with the protein-expressing cells of the present invention and measuring the same inhibition of the amount of luminescence increased by forskolin stimulation as with the peptides of the present invention.

A specific example of this screening method will be described below using an example using luciferase as a reporter gene.

Cells expressing the protein of the present invention into which the CRE-repo overnight gene (luciferase) has been introduced are seeded on a 24-well plate at 5 × 10 ³ ce 1 lZwe 11 and cultured for 48 hours. The cells are washed with Hank's buffer (pH 7.4) containing 0.2 mM 3_isobutyl-methylxanthine, 0.05% BSA and 20 mM HE PES (hereinafter abbreviated as reaction buffer). Then add 0.5 ml of application buffer and incubate for 30 minutes in the incubator. After removing the reaction buffer and adding 0.25 ml of a new reaction buffer to the cells, add 2 μl of forskolin containing 1 μl of the peptide of the present invention or 1 μl of the peptide of the present invention and a test compound. Add 0.25 ml of reaction buffer containing to the cells and incubate for 24 minutes with 37 tubes. Lyse the cells with a cell lysing agent for Pitka Gene (Toyo Ink Mfg. Co., Ltd.) and add a luminescent substrate (Toyo Ink Mfg. Co., Ltd.) to the lysate. Luminescence from luciferase is measured using a luminometer, liquid scintillation counter, or top counter. The amount of luminescence by luciferase when the peptide of the present invention alone is added and when 1 M of the peptide of the present invention and a test compound are added are measured and compared.

The peptides of the present invention provide for luciferase-induced luminescence upon forskolin stimulation. Suppress increase in volume. A compound that restores the inhibition can be selected as a candidate substance having competitive inhibitory ability.

As reporter genes, for example, alkaline phosphatase, chloramphene

Acetyl trans f erase) or a gene such as iS-galactosidase may be used. The enzyme activity of these reporter gene proteins is measured according to a known method or using a commercially available measurement kit. Alkaline phosphatase activity can be measured using, for example, Lumi-Phos 530 manufactured by Wako Pure Chemical, and chloramphenicol acetyltransferase can be measured using, for example, FAST CAT chrol amphenicol manufactured by Wako Pure Chemical.

The i3-galactosidase activity is measured using, for example, Acetyl trans erase erase Assay KiT using, for example, Aurora Ga1-XE manufactured by Wako Pure Chemical Industries.

(4) The protein-expressing cells of the present invention release arachidonic acid metabolites extracellularly by stimulation of the peptides of the present invention. By utilizing this reaction to measure the stimulating activity of the peptide of the present invention on the cells expressing the protein of the present invention, it is possible to screen for a compound that alters the binding property between the peptide of the present invention and the protein of the present invention. .

The arachidonic acid metabolite-releasing activity can be measured by measuring the labeled arachidonic acid metabolite released outside the cell by incorporating the labeled arachidonic acid into the cells expressing the protein of the present invention in advance. can do. Specifically, when the peptide of the present invention is brought into contact with a cell expressing the protein of the present invention containing labeled arachidonic acid, the peptide of the present invention and the test compound are brought into contact with the cell of the present invention containing labeled arachidonic acid. By measuring and comparing the arachidonic acid metabolite release activity when brought into contact with a protein-expressing cell, a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is screened. .

In this method, a test compound that inhibits the arachidonic acid metabolite release activity of the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability. Alternatively, the test compound alone is brought into contact with the protein-expressing cell of the present invention, and the arachidonic acid metabolite releasing activity of the protein-expressing cell of the present invention is examined by a known method. A compound exhibiting agonist activity can also be screened.

One specific example of the screening method is described below.

The protein-expressing cells of the present invention are seeded on a 24-well plate at 5 × 10 ⁴ ce 11 / we 11, and cultured for 24 hours, and [ ³ H] arachidonic acid is added to 0.25 Ci / well. After 16 hours, wash the cells with Hanks buffer (pH 7.4) containing 0.05% BSA and 20 mM HE PES (hereinafter abbreviated as reaction buffer 1). A reaction buffer 500 ^ 1 containing a final concentration of 10 M of the peptide of the present invention or a final concentration of 10 / M of the peptide of the present invention and a test compound is added to each we11. After incubating at 37 ° C for 60 minutes, the reaction solution 4001 is added to the scintillator overnight, and the amount of [ ³ H] arachidonic acid metabolite + released in the reaction solution is measured by scintillation counting.

When only 500 ^ 1 of the reaction buffer was added (without adding the peptide of the present invention and without adding the test compound), the amount of free [ ³ H] arachidonic acid metabolite was reduced to 0%, and 1 OnM of the present invention was added. If the addition of reaction buffer containing peptide free ^[3 H] Arakidon the amount of acid metabolites as 100%, free ^[3 H] Arakidon acid when the test compound added 'to addition of (test compound non-added additive) Calculate the amount of metabolite.

A test compound having an arachidonic acid metabolite releasing activity of, for example, 50% or less can be selected as a candidate substance having an antagonistic ability.

(5) In the cell expressing the protein of the present invention, the intracellular Ca concentration increases by stimulation with the peptide of the present invention. By utilizing this reaction, the stimulating activity of the peptide of the present invention on the protein-expressing cell of the present invention is measured to screen for a compound that changes the binding property between the peptide of the present invention and the protein of the present invention. be able to. '

Specifically, the intracellular calcium concentration in the case where the peptide of the present invention is brought into contact with the protein-expressing cell of the present invention and in the case where the peptide of the present invention and the test compound are brought into contact with the protein-expressing cell of the present invention By measuring and comparing the increasing activity, a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is screened. The measurement is performed according to a known method.

In the method, an increase in intracellular calcium concentration by the peptide of the present invention is suppressed. The test compound to be tested can be selected as a candidate substance capable of competitive inhibition. On the other hand, an agonist can be screened by measuring an increase in fluorescence intensity due to the addition of the test compound alone.

'A specific example of the screening method is described below.

The protein-expressing cells of the present invention are seeded on a sterilized cover glass for a microscope. Two days later, the culture solution is replaced with HBSS suspended in 4 mM Fura-2AM (Dojindo Laboratories), and incubated at room temperature. Leave for 30 minutes. After washing with HBSS, place a coverslip on the cuvette, add the peptide of the present invention or the peptide of the present invention and a test compound, and emit fluorescence at 505 nm at excitation wavelengths of 300 nm and 380 nm. Measure the increase in intensity ratio with a fluorimeter and compare.

Alternatively, FLIPR (manufactured by Molecular Devices) may be used. Add F1uo-3AM (manufactured by Dojindo Laboratories) to the protein-expressing cell suspension of the present invention, allow the cells to take up, and wash the supernatant several times by centrifugation. Seed the cells. 'Set in a FLIPR apparatus, add the peptide of the present invention or the peptide of the present invention and a test compound in the same manner as in the case of Fura_2, and measure the increase in the ratio of the fluorescence intensities using a fluorimeter and compare.

Furthermore, a protein gene (eg, aequorin, etc.) that emits light due to an increase in intracellular Ca ion is co-expressed in the protein-expressing cell of the present invention, and the intracellular Ca ion concentration is increased. By utilizing the fact that the gene protein (eg, aequorin or the like) becomes Ca-bound and emits light, it is also possible to screen for a compound that changes the binding property between the peptide of the present invention and the protein of the present invention. A gene for a protein that emits light when the intracellular Ca ion increases is co-expressed, and the protein-expressing cell of the present invention is seeded on a 96-well plate, and the peptide of the present invention or the Add the peptide and test compound, measure the increase in the ratio of fluorescence intensity with a fluorimeter, and compare.

A test compound that suppresses an increase in fluorescence intensity due to the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability.

One specific example of the screening method is described below.

As a control, clone 24 of ETA (endothelin A receptor) -expressing CH0 cell Abbreviated as ETA24 cells. Journal of Pharmacology and Experimental

Therapeutics, Vol. 279, pp. 675-685, 1996) was used to measure the activity of increasing the intracellular Ca ion concentration of the protein-expressing cells of the present invention and ETA24 cells using FLIPR (manufactured by Molecular Devices). This is performed using The cells expressing the protein of the present invention and ETA24 cells should be subcultured in DMEM supplemented with 10% dialyzed fetal calf serum (hereinafter referred to as dFBS). Protein expressing cells of the present invention, suspended in medium (10% d FBS-DMEM) such that ETA24 cells each 15xl0 ⁴ cells / ml, in 96 well plate for FLIPR (Black plate clear bottom. Coster, Inc.) dispenser by 200 1 each Ueru with implantation ^{(3.0xl0 4 cells / 200 / l} / © E Le), used after overnight incubation at 37C in 5% C0 ₂ incubator in one (hereinafter, cells play ). H / HBSS (Nissy Hanks 2 (Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, adjusted to pH 7.4 with sodium hydroxide solution, filter sterilized) 20 ml, 250 mM Probenecid 200 ゥ Mix 200 fetal serum (FBS). In addition, Fluo 3-AM (Dojindo Research Laboratories) 2 piles (50 xg) was converted to dimethyl sulfoxide 401, 20% Pluronic acid

(Molecular Probes) 40, and added to the above H / HBSS-Probenecid-FBS. After mixing, pipette 100 l of each well into the cell plate from which the culture solution has been removed using an 8-tube pipette. % C0 ₂ incubator base 1 hour at 37 ° C for at one coater in incubate (dye the loading). H / HBSS 1501 containing 2.5 mM Probenecid and 0.1% CHAPS is added to the sample for each assay (each fraction), diluted, and transferred to a 96-well plate for FLIPR (V-Bottom plate, Coster) (hereafter, sample plate) ). After dye loading of the cell plate is completed, wash the cell plate four times using a plate washer (Molecular Devices) with a washing buffer containing 2.5 mM Probenecid in ¾33, and leave 100/1 washing buffer after washing. Set the cell plate and sample plate in the FLIPR and perform the assay (the sample from the sample plate is transferred to the cell plate by the FLIPR).

(6) Addition of receptor agonist to cells expressing the receptor increases intracellular inositol triphosphate concentration. By utilizing the intracellular inositol triphosphate-producing activity of the peptide of the present invention in the protein-expressing cell of the present invention. Thus, a compound that changes the binding property between the peptide of the present invention and the protein of the present invention can be screened.

Specifically, a peptide of the present invention was brought into contact with a protein-expressing cell of the present invention in the presence of labeled inositol, and a peptide and a test compound of the present invention were brought into contact with a protein-expressing cell of the present invention. In this case, the inositol triphosphate-producing activity is measured and compared to screen for a compound that alters the binding property between the peptide of the present invention and the protein of the present invention. The measurement is performed according to a known method. .

In this method, a test compound that suppresses inositol triphosphate-producing activity can be selected as a candidate substance capable of competitive inhibition.

On the other hand, agonists can be screened by bringing only the test compound into contact with the protein-expressing cells of the present invention and measuring the increase in inositol triphosphate production.

One specific example of the screening method is described below.

The cells expressing the protein of the present invention are seeded on a 24-well plate and cultured for one day. Thereafter, the cells are cultured for 1 day in a medium supplemented with myo- [2-¾] inositol (2.5 Ci / well), and the cells are thoroughly washed with a medium without radioactive inositol. After adding the peptide of the present invention or the peptide and the test compound of the present invention, 10% perchloric acid is added to stop the reaction. 1. neutralized with 5M potassium hydroxide and 60 mM HEPES solution and then passed through a column packed with AGlx8 tree fat (Bi o-Rad) in 0. 5 ml, 5 mM sodium tetraborate (Na ₂ B ₄ 0 ₇₎ and 60raM After washing with ammonium formate, the radioactivity eluted with 1 M ammonium formate and 0.1 M formic acid is measured with a liquid scintillation counter. The radioactivity when the peptide of the present invention is not added is 0%, and the radioactivity when the peptide of the present invention is added is 100%. calculate.

A test compound having an inositol triphosphate-producing activity of, for example, 50% or less can be selected as a candidate substance having an antagonistic ability.

(7) Using the TRE-repo overnight gene vector to measure the stimulatory activity of the peptide of the present invention on cells expressing the protein of the present invention, Compounds that alter the binding between the peptide and the protein of the present invention can be screened.

DNA containing a TRE (TPA response element) is inserted upstream of the reporter gene of the vector to obtain a TRE-repo overnight gene vector. In the protein-expressing cells of the present invention into which the TRE-repo overnight gene vector has been introduced, stimulation accompanied by an increase in the intracellular calcium concentration is caused by the TRE-mediated repo overnight gene expression and the subsequent reporter Induces the production of the gene product (protein) of the gene. In other words, by measuring the enzymatic activity of the reporter gene protein, it is possible to detect fluctuations in the amount of calcium in the cells into which the TRE-reporter gene vector has been introduced.

Specifically, when the peptide of the present invention is brought into contact with a TRE-reporter gene vector-introduced protein-expressing cell of the present invention, the peptide of the present invention and the test compound are expressed in the TRE-reporter gene. Vector-introduced Changes in the binding activity between the peptide of the present invention and the protein of the present invention by measuring and comparing the enzymatic activities of the reporter gene protein when brought into contact with the cells expressing the protein of the present invention. Screen compounds.

Examples of the vector include Pitska Gene Basic Vector and Pitska Gene Enhancer Vector (Toyo Ink Mfg. Co., Ltd.). The TRE-containing DNA is inserted into a reporter gene of the above vector, for example, a multi-cloning site upstream of the luciferase gene, to obtain a TRE-reporter gene vector.

In this method, a test compound that suppresses the enzymatic activity of the repo overnight gene protein by the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability.

On the other hand, agonist screening is performed by contacting only the test compound with the TRE-repo allele-introduced gene-introduced protein-expressing cells of the present invention and measuring the increase in luminescence in the same manner as the peptide of the present invention. You can do it.

A specific example of this screening method will be described below using an example using luciferase as a reporter gene. Cells expressing the protein of the present invention into which the TRE-reporter gene (luciferase) has been introduced are seeded on a 24-well plate at 510 ³ 6 1 176 11 and cultured for 48 hours. After washing the cells with Hanks buffer (pH 7.4) containing 0.05% BSA and 20 mM HEPES, 10 nM of the peptide of the present invention or 10 nM of the peptide of the present invention and a test compound are added thereto, and the mixture is added at 37 ° C. And react for 60 minutes. Lyse the cells with a cell lysing agent for Pitka Gene (Toyo Ink Mfg. Co., Ltd.) and add a luminescent substrate (Toyo Ink Mfg. Co., Ltd.) to the lysate. Luminescence from luciferase is measured using a luminometer, liquid scintillation counter or top counter. The amount of luminescence by luciferase when the peptide of the present invention is added and when {と η} the peptide of the present invention and a test compound are added are measured and compared.

The increase in intracellular calcium by the peptide of the present invention increases the amount of luminescence by luciferase. A compound that suppresses this increase can be selected as a candidate substance capable of competitive inhibition.

Reporter genes include, for example, alkaline phosphatase, chloramphenic acid, acetylfurantransferrase ^ chloramphenicol

Genes such as acetyltransferase) and _galactosidase may be used. The enzyme activities of these reporter gene proteins are measured according to a known method or using a commercially available measurement kit. '' Alkali phosphatase activity was measured using, for example, Lumi-Phos 530 manufactured by Wako Pure Chemical, and chloramphenicol acetyltransferase was evaluated using, for example, FAST CAT chrola immediately manufactured by Wako Pure Chemical.

Using Acetyltransferase Assay KiT, | S-galactosidase activity is measured using, for example, Aurora Gat XE manufactured by Wako Pure Chemical Industries.

(8) In the protein-expressing cell of the present invention, MAP kinase is activated and proliferates by stimulation of the peptide of the present invention. By utilizing this reaction to measure the stimulating activity of the peptide of the present invention on the cells expressing the protein of the present invention, it is possible to screen for a compound that alters the binding property between the peptide of the present invention and the protein of the present invention. .

Specifically, the peptide of the present invention was brought into contact with a cell expressing the protein of the present invention. By measuring and comparing the cell proliferation when the peptide of the present invention and the test compound are brought into contact with the protein-expressing cells of the present invention, the binding between the peptide of the present invention and the protein of the present invention can be determined. Screen for compounds that alter the.

Proliferation of the protein-expressing cells of the present invention may be measured, for example, by measuring MAP kinase activity, thymidine uptake activity, cell number, and the like.

As a specific example, for the MAP kinase activity, after adding the peptide of the present invention or the peptide of the present invention and a test compound to the cells expressing the protein of the present invention, an anti-VIAP kinase antibody was used from a cell lysate. After obtaining the MAP kinase fraction by immunoprecipitation, the MAP kinase activity is measured using a known method, for example, MAP Kinase Assay II- [ ³² P] -ATP manufactured by Wako Pure Chemical Industries, and compared.

Regarding the thymidine incorporation activity, the protein-expressing cells of the present invention were seeded on a 24-well plate, cultured, added with the peptide of the present invention or the peptide of the present invention and a test compound, and then labeled with radioactivity. thymidine (e.g., [methy Bok ³ H] - thymidine down, etc.) is added, then the cells were lysed and the radioactivity of the thymidine incorporated into the cells, by counting in a liquid scintillator one Chillon counter, thymidylate ' Measure gin uptake activity and compare.

For the measurement of the cell number, the protein-expressing cells of the present invention were seeded on a 24-well plate, cultured, and after adding the peptide of the present invention or the peptide of the present invention and a test compound, MTT (3- ( Add 4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide). MTT formazan in which MTT has been changed by being taken into cells is dissolved in an aqueous solution of isopropanol acidified with hydrochloric acid, and then measured by absorption at 570 nm for comparison.

In this method, a test compound that suppresses the growth of the protein-expressing cell of the present invention can be selected as a candidate substance having a competitive inhibition ability.

On the other hand, agonists can be screened by bringing only the test compound into contact with the protein-expressing cell of the present invention and measuring the cell proliferation activity similar to that of the peptide of the present invention.

One specific example of the screening method using the thymidine uptake activity is described below. The evening protein-expressing cells of the present invention are seeded at 5,000 cells / well in a 24-well plate and cultured for one day. The cells are then starved in a serum-free medium for 2 days. The peptide or peptide and a test compound of the present invention of the present invention, after incubation was added to the cells 24 hours, [methy Bok ³ H] - thymidine was added Ueru per 0. 015MBq, incubation 6 hours. After washing the cells with PBS, add methanol and let stand for 10 minutes. Next, add 5% trichloroacetic acid and let stand for 15 minutes. Wash the fixed cells four times with distilled water. Lyse the cells with 0.3N sodium hydroxide solution and measure the radioactivity in the lysate with a liquid scintillation counter.

A test compound that suppresses an increase in radioactivity when the peptide of the present invention is added can be selected as a candidate substance capable of competitive inhibition.

(9) In the protein-expressing cell of the present invention, the stimulation of the peptide of the present invention activates the potassium channel, and intracellular K ions flow out of the cell. By utilizing this reaction, the stimulating activity of the peptide of the present invention on cells expressing the protein of the present invention is measured to screen for compounds that alter the binding between the peptide of the present invention and the protein of the present invention. be able to.

Rb ions (rubidium ions), which are homologous to K ions, flow out of the cell through potassium channels without distinction from K ions. Thus, the protein-expressing cells of the present invention, a radioactive isotope R b After allowed incorporation of ^{([8 6 R b])} , the ^{8 6} R b flowing out by stimulation of the peptides of the present invention By measuring the flow (efflux activity), the stimulating activity of the peptide of the present invention on the cells expressing the protein of the present invention is measured.

Specifically, when the peptide of the present invention is brought into contact with the protein-expressing cell of the present invention in the presence of ⁸⁶ Rb, the peptide of the present invention and the test compound are brought into contact with the protein-expressing cell of the present invention. in the case of it is to measure the efflux activity of ^{8 6} R b, by comparing, for screening the compound that changes the binding property between the protein of the peptide and the invention of the present invention.

In this method, the ^{8 6} R b test compound to suppress an increase in efflux activity of by peptide stimulation of the present invention, it can be selected as a candidate substance capable of competitive inhibition. On the other hand, only the test compound is brought into contact with the protein-expressing cell of the present invention, It is also possible to perform the disk re-learning of Agonisu Bok by measuring the increase in peptide similar ^{8 6} R b of efflux activity.

One specific example of the screening method is described below.

The protein-expressing cells of the present invention are seeded on a 24-well plate and cultured for 2 days. Thereafter, the cells are incubated for 2 hours in a medium containing 1 mCiZm1 and ⁸⁶ RbC1. Wash cells thoroughly with medium to completely ^{remove 86} RbC1 in the external solution. The peptide of the present invention or the peptide of the present invention and a test compound are added to the cells, and after 30 minutes, the external solution is collected, and the radioactivity is measured and compared at a local time.

A test compound that suppresses an increase in the ⁸⁶ Rb efflux activity due to peptide stimulation of the present invention can be selected as a candidate substance having a competitive inhibitory ability.

(10) Extracellular pH changes when cells expressing the protein of the present invention react with the peptide of the present invention. By utilizing this reaction to measure the stimulating activity of the peptide of the present invention on cells expressing the protein of the present invention, it is possible to screen for a compound that alters the binding between the peptide of the present invention and the protein of the present invention. it can. Specifically, the extracellular p in the case where the peptide of the present invention is brought into contact with the protein-expressing cell of the present invention and in the case where the peptide of the present invention and the test compound are brought into contact with the protein-expressing cell of the present invention. By measuring and comparing the change in H, a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is screened.

The extracellular pH change is measured using, for example, a Cytosensor device (Molecular Devices). ,

In this method, a test compound that suppresses the extracellular pH change caused by the peptide of the present invention can be selected as a candidate substance capable of competitive inhibition.

On the other hand, agonist screening can be performed by bringing only the test compound into contact with the protein-expressing cell of the present invention and measuring the extracellular pH change similar to that of the peptide of the present invention.

One specific example of the screening method is described below.

The protein-expressing cells of the present invention are cultured overnight in a capsule for a Cytosensor device, set in the chamber of the device, and set to 0.1% for about 2 hours until the extracellular pH is stabilized. Perfuse 1 to 1640 medium (Molecular Devices) containing 83-8. After the pH is stabilized, a medium containing the peptide of the present invention or the peptide of the present invention and the test compound is perfused on the cells. Measure and compare the pH change of the medium caused by perfusion.

A compound that suppresses the extracellular pH change caused by the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability. '

丄 1) The sex pheromone receptor, ST e2, of the haploida-mating type (MAT) of yeast (Sacc aromyces cerevisiae) is conjugated to the G protein, Gpa1, and responds to the sex pheromone mating factor. To activate MAP kinase, followed by activation of Far 1 (cell-cycle arrest) and the transcriptional activator Ste12. Stel 2 induces the expression of various proteins (eg, FUS 1 involved in conjugation). On the other hand, the control factor S st 2 functions in the above process in a suppressive manner. In this system, a yeast into which a receptor gene has been introduced is produced, a signal transduction system in the yeast cell is activated by stimulation of the receptor gene, and the resulting proliferation is used as an index. Attempts have been made to measure the reaction between a protein and a receptor (Trends in Biotechnology, 15, 487-494, 1997). Using the above-described receptor gene-introduced yeast system, a compound capable of changing the binding property between the peptide of the present invention and the protein of the present invention can be screened. '

Specific examples are shown below.

The genes encoding Ste2 and Gpa1 of the MATα yeast are removed, and instead, the protein gene of the present invention and a gene encoding the Gpa1-Gai2 fusion protein are introduced. The gene encoding Far is removed to prevent ceU-cycle arrest from occurring, and the gene encoding Sst is removed to increase the sensitivity of the response to the peptide of the present invention. Furthermore, the FUS1—HIS3 gene in which the histidine biosynthesis gene HIS3 is linked to FUS1 is introduced. This genetic recombination operation can be performed, for example, by replacing the somatostin-tin receptor-type-1 (SSTR2) gene with the protein of the present invention in the method described in Molecular and Cellular Biology, vol. 15, p. 6188-6195, 1995. Can be implemented. The transformed yeast thus constructed reacts with high sensitivity to the peptide of the present invention, resulting in the activation of MAP kinase, the synthesis of histidine biosynthesis enzyme, and the histidine-deficient medium. It is possible to grow in.

Therefore, the above-described protein-expressing yeast of the present invention (the Ste2 gene and the Gpa1 gene were removed, the protein gene of the present invention and the Gpa1-Gai2 fusion protein-encoding gene were introduced, and the Far gene was introduced. Sα yeast with the FUS1-1HIS3 gene removed and the Sst gene removed) is cultured in a histidine-deficient medium, and the peptide of the present invention or the peptide of the present invention and a test compound are contacted with each other; By measuring and comparing the growth of the yeast, it is possible to screen for compounds that alter the binding between the peptide of the present invention and the protein of the present invention. In this method, a test compound that suppresses the growth of the yeast can be selected as a candidate substance having a competitive inhibition ability.

On the other hand, agonist screening can also be performed by bringing only the test compound into contact with the above-described yeast expressing the protein of the present invention and measuring the growth of yeast similar to the peptide of the present invention.

One specific example of the screening method is described below.

The above-described protein-expressing yeast of the present invention is cultured overnight in a liquid medium of a complete synthetic medium, and then added to a dissolved agar medium from which histidine has been removed to a concentration of 2 × 10 ⁴ ce 11 / m 1. Then sow in a 9 x 9 cm square petri dish. After the agar solidifies, a sterile filter paper impregnated with the peptide of the present invention or the peptide of the present invention and the test compound is placed on the agar surface, and cultured at 30 ° C for 3 days. The effect of the test compound is to compare the growth of yeast around the filter paper with the use of sterile filter paper impregnated with only the peptide of the present invention. In addition, the peptide of the present invention is added to the agar medium from which histidine has been removed in advance, and the test compound is soaked in sterile filter paper to culture the yeast. You may observe that they are affected.

A compound that suppresses the growth of yeast can be selected as a candidate substance having a competitive inhibition ability.

(12) Inject the protein gene RNA of the present invention into Xenopus oocytes When stimulated with the peptide of the present invention, the concentration of cellular calcium increases, resulting in calcium-activated chloride current. This can be considered as a change in the membrane potential (even when there is a change in the K ion concentration gradient). By utilizing the above reaction in the Xenopus laevis oocytes transfected with the protein of the present invention caused by the peptide of the present invention, the stimulating activity of the peptide of the present invention on the protein-expressing cell of the present invention is measured. Compounds that alter the binding between the peptide of the present invention and the protein of the present invention can be screened.

Specifically, the case where the peptide of the present invention is brought into contact with the protein gene of the present invention, RNA-introduced African Xenopus oocytes, and the case where the peptide and the test compound of the present invention are brought into contact with the protein gene of the present invention By measuring and comparing changes in cell membrane potential when brought into contact with the introduced Xenopus laevis oocytes, a compound that changes the binding property between the peptide of the present invention and the protein of the present invention is screened. I do.

In this method, a test compound that suppresses a change in cell membrane potential can be selected as a candidate substance having a competitive inhibition ability.

On the other hand, agonists can be screened by bringing only test compounds into contact with the protein gene of the present invention, RNA-introduced African oocytes, and measuring changes in cell membrane potential similar to those of the peptides of the present invention.

One specific example of the screening method is described below.

In ice water was removed from female African Tsu mega El became stuck, the oocytes mass, MBS solution (88mM NaCl, lniM KC1, 0.41mM CaCl 2, 0.33mM Ca (N0 3) 2, 0.82mM MgS0 4 _{, 2.4mM NaHC0 3, lOmM HEPES;} pH7.4) in melted collagenase (0. 5 mg / m 1) 19 ° until the oocytes are loosened by C,. 1 to 6 hours, that processes at 0.99 rpm. The external solution is washed three times by replacing the MBS solution, and the oocyte is microinjected with the protein gene po1yA-added cRNA (50 ng / 50η1) of the present invention using a micromanipulator.

The protein gene mRNΑ of the present invention may be prepared from a tissue or a cell, or may be transcribed from a plasmid in vitro. The protein gene mRNA of the present invention was cultured in MBS solution at 20 ° C for 3 days, and this was cultured in a voltage clamp equipped with Ringer solution. Insert the potential fixing glass microelectrode and the potential measurement glass microelectrode into the cell, and place the electrode outside the cell. When the potential is stabilized, a Ringer solution containing the peptide of the present invention or the peptide of the present invention and a test compound is passed, and the change in potential is recorded. The effect of the test compound can be measured by comparing the change in the cell membrane potential of the oocytes of the present invention into which the protein gene of the present invention has been transfected with RNA, compared to the case where a Ringer solution containing only the peptide of the present invention is applied. .

A compound that suppresses cell membrane electrical changes can be selected as a candidate substance having a competitive inhibition ability.

In the above system, the increase in the amount of change in the potential facilitates the measurement, and thus, the pO1A-added RNA of each G protein gene may be introduced. In addition, by co-injecting polyA-added RNA of a gene of a protein (eg, aequorin, etc.) that produces luminescence in the presence of potassium, it is possible to measure luminescence instead of membrane potential change.

A screening kit for a compound or a salt thereof that alters the binding property between the peptide of the present invention and the protein of the present invention includes a protein of the present invention, a cell containing the protein of the present invention, or a cell containing the protein of the present invention. A membrane fraction, and a peptide of the present invention.

Examples of the screening kit of the present invention include the following. 1. Screening reagent

(i) Measurement buffer and washing buffer

Hanks' Balanced Salt Solution (manufactured by Gibco) plus 0.05% serum albumin (manufactured by Sigma).

Sterilize by filtration with a 0.45 m pore size filter and store at 4 ° C, or prepare freshly before use.

(ii) Sample of the protein of the present invention

CH0 cells expressing the protein of the present invention were subcultured in 12-well plates at ⁵ × 10 ⁵ Z-wells and cultured at 37 ° C., 5% CO ₂ , 95% air for 2 days. .

(i i i) Labeled ligand

[¾], ⁽¹²⁵ 1], ^[14 C], the peptides of the present invention labeled with a ^[35 S]. Dissolve in a suitable solvent or buffer at 4 or at -20 ° C, and dilute to 1 / M with the measuring buffer before use.

(iv) Ligand standard solution

The peptide of the present invention is dissolved to a concentration of lmM in PBS containing 0.1% ゥ serum albumin (Sigma) and stored at -20 ° C.

2. Measurement method-

(i) The cells expressing the protein of the present invention cultured on a 12-well tissue culture plate were washed twice with 1 ml of the measurement buffer, and 490 / l of the measurement buffer was added to each well. Add to ₍

After the test compound solution ^{^{(ii) 10- 3 ~ 1 (}} ) M 5 1 added, the peptide of the present invention labeled 5 1 was added to react at room temperature for one hour. In order to know the amount of non-specific binding, 5/1 of the peptide of the present invention is added instead of the test compound.

(iii) Remove the reaction solution and wash three times with 1 ml of washing buffer. The labeled peptide of the present invention bound to the cells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquid scintillator overnight-A (manufactured by Wako Pure Chemical). ,

(iv) The radioactivity is measured using a liquid scintillation counter (manufactured by Beckman), and the Percent Maximum Binding (PMB) is determined by the following equation (Equation 1).

(Equation 1)

PMB = [(B-NSB) / (B ₀ -NSB)] xl OO

PMB: Percent Ma imum Binding

B: Value when the sample is added

NSB: Non-specific Binding ''

B. : Maximum binding amount

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound that changes the binding (inhibits or promotes the binding) between the peptide of the present invention and the protein of the present invention. Is a compound having a cell stimulating activity via the protein of the present invention or a salt thereof (a so-called agonist of the protein of the present invention), or a compound not having the stimulating activity (a so-called antagonist of the protein of the present invention). . Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like. It may be a novel compound or a known compound.

The specific method for evaluating whether the above-mentioned protein of the present invention is an agonist or an antagonist may be according to the following (Α) or (Β).

(Ii) The binding assay shown in the above screening method is performed to obtain a compound that changes the binding property (particularly, inhibits the binding) between the peptide of the present invention and the protein of the present invention. Is determined whether it has the above-described cell stimulating activity via the protein of the present invention. A compound having a cell stimulating activity or a salt thereof is an agonist of the protein of the present invention, and a compound or a salt thereof having no such activity is an agonist of the protein of the present invention.

(B) (a) A test compound is brought into contact with a cell containing the protein of the present invention, and the cell stimulating activity mediated by the protein of the present invention is measured. The compound having a cell stimulating activity or a salt thereof is an agonist of the protein of the present invention.

(b) when a compound that activates the protein of the present invention (for example, a peptide of the present invention or an agonist of the protein of the present invention) is brought into contact with a cell containing the protein of the present invention; The cell stimulating activity mediated by the protein of the present invention when the compound to be converted and the test compound are brought into contact with the cells containing the protein of the present invention is measured and compared. The compound capable of decreasing the cell stimulating activity of the compound activating the protein of the present invention or a salt thereof is an antagonist of the protein of the present invention.

Since the agonist of the protein of the present invention has the same activity as the physiological activity of the peptide of the present invention on the protein of the present invention, it is useful as a safe and low-toxic drug like the peptide of the present invention. It is.

Conversely, the protein antagonist of the present invention can suppress the physiological activity of the peptide of the present invention on the protein of the present invention, and is therefore useful as a safe and low-toxic drug for suppressing the receptor activity. is there.

The protein of the present invention is involved in an appetite (feeding) promoting action and an oxytocin secretion promoting action. Therefore, among the compounds obtained using the above-mentioned screening method or screening kit, agonists of the protein of the present invention include, for example, an appetite (feeding) enhancer, anorexia (eg, anorexia nervosa), Appetite It can be used as a medicament for the prevention and treatment of anemia or hypoproteinemia due to tremor, weak labor, laxative bleeding, uterine remodeling failure, and milk stasis. The protein antagonists of the present invention include, for example, obesity (eg, malignant mastocytosis), exogenous obesity, hyperinsulin inar obesity, hyperplasma Hyperplasmic obesity ^ hypophyseal adiposity, hypoplasmic obesity> hypothyroid obesity, hypothalamic obesity, hypothalamic obesity, symptoms Obesity (sy immediate tomatic obesity), childhood obesity (infantile obesity), upper body obesity, alimentary obesity, hypogonadal obesity, systemic mastocytosis

(systemic mastocytosis), simple obesity, central obesity

(central obesity)), hyperphagia, affective disorders, sexual dysfunction, overwork, tonic contractions, fetal asphyxia, uterine rupture, tubal lacerations, preterm birth, Prader-Willi syndrome, glucoseuria and Its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease (Pickwick syndrome, sleep apnea syndrome) It can be used as a medicine for prophylactic and therapeutic agents such as fatty liver, infertility, osteoarthritis, etc. (especially antiobesity agents, appetite (feeding) regulators, etc.).

As a salt of the compound obtained by using the above-described screening method or screening kit, for example, a pharmaceutically acceptable salt or the like is used. Examples thereof include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids.

Preferable examples of the salt with an inorganic base include an alkali metal salt such as a sodium salt and a potassium salt, an alkaline earth metal salt such as a calcium salt and a magnesium salt, and an aluminum salt and an ammonium salt.

Preferred examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-alutidine, ethanolamine, genoaluminamine, triethanolamine, cyclohexylamine, dicyclohexane. Examples include salts with xylamine, N, N, and dibenzylethylenediamine. Preferred examples of the salt with an inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like.

Suitable examples of salts with organic acids include, for example, formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid And the like.

Preferable examples of the salt with a basic amino acid include salts with, for example, arginine, lysine, orcinus, and preferable examples of the salt with an acidic amino acid include, for example, salts with aspartic acid, glutamic acid, etc. Is raised.

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned medicament, it can be carried out in the same manner as when the above-mentioned peptide of the present invention is used as a medicament.

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned medicine, it can be carried out according to a conventional method. For example, sterile orally with tablets or capsules, capsules, elixirs, microcapsules, etc., if necessary, coated with sugar or enteric coating, or with water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections, such as aqueous solutions or suspensions. For example, by mixing the compound or a salt thereof with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder and the like in a generally accepted unit dosage form. Can be manufactured. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Such a leavening agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, cocoa oil or cellulose are used. When the preparation unit form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection include active substances in vehicles such as water for injection, natural substances such as sesame oil, coconut oil and the like. It can be formulated according to the usual formulation practice such as dissolving or suspending the produced vegetable oil.

Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.). Agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 ™, HCO-50) and the like may be used in combination. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with a dissolution aid such as benzyl benzoate or benzyl alcohol. Also, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents

(Eg, Shirazidani Benzalkonium, Proforce hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. You may mix. The prepared injection solution is usually filled in a suitable ampoule.

The preparations obtained in this way are safe and low toxic, and can be used, for example, in mammals (eg, humans, mice, rats, guinea pigs, egrets, sheep, sheep, bushus, dogs, cats, dogs, monkeys, chimpanzees, etc. ) Can be administered.

The dose of a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention varies depending on symptoms and the like. About 0.1 to 1000 mg, preferably about 1.0 to 300 mg, more preferably about 3.0 to 50 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. For example, in the case of injection, injection into adult obese patients (with a body weight of 60 kg) is required. In administration, it may be convenient to administer the antagonist by intravenous injection at about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, the dose can be administered in terms of 60 kg.

[3] Quantification of the protein of the present invention, its partial peptide or its salt

Since the antibody of the present invention can specifically recognize the protein of the present invention, It can be used for quantification of the protein of the present invention in a test solution, particularly for quantification by sandwich immunoassay.

That is, the present invention

(i) reacting the antibody of the present invention with a test solution and the labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody. A method for quantifying the protein of the present invention in a test solution, and

(ii) After reacting the test liquid with the antibody of the present invention insolubilized on the carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insolubilized carrier is measured. The present invention provides a method for quantifying the protein of the present invention in a test solution, characterized in that in the quantification method of the above (ii), one of the antibodies is an antibody that recognizes the N-terminal of the protein of the present invention, and the other is It is desirable that the above-mentioned antibody reacts with the C-terminal of the protein of the present invention.

In addition, the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, the anti-body molecule itself may be used, or the F (ab,) ₂ , Fab ′ or Fab fraction of the antibody molecule may be used.

The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. . For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.

Examples of the labeling agent used in the assay method using the labeling substance, for example, radioactive isotopes elemental (eg, ^[125 1], ^[131 1], [¾], [ ^'4 C], ^[32 P], ^[33 P], [ ^35s ], etc.), fluorescent substances (eg, cyanine fluorescent dyes (eg, Cy2, Cy3, Cy5, Cy5.5, Cy7 (manufactured by Amersham Biosciences), etc.), fluorescamine, fluorescenisochi Etc.), enzymes (eg, -galactosidase,) 3-darcosidase, Lucariphosphatase, passoxidase, malate dehydrogenase, etc., luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), piotin, lanthanide elements, etc. are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

In the sandwich method, a test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be in accordance with those described above. Also, in the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.

In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds. Used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method or a nephelometry. (In the competition method, the antigen in the test solution and the labeled antigen are used for the antibody. After reacting competitively, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (BZF separation), and the amount of labeling of either B or F is measured. Quantify the amount of antigen in the test solution You. In this reaction method, a soluble antibody was used as the antibody, B / F separation was performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid phase antibody was used as the first antibody. Alternatively, an immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.

In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. The antigen is allowed to react with an excess amount of the labeled antibody, then the immobilized antigen is added, and the unreacted labeled antibody is bound to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.

In nephrometry, the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser-nephrometry using laser-dispersion is preferably used.

In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, documents, etc.

For example, edited by Hiro Irie, "Radio Nonotsusei" (Kodansha, published in Showa 49), edited by Hiroshi Irie, "Continued Radio Immnoatsusy" (Kodansha, published in 1954), Eiji Ishikawa et al. "Measurement Method" (Medical Shoin, published in 1958), Eiishi Ishikawa et al., "Enzyme Immunoassay" (Second Edition) (Medical Publishing, published in 1977), Eiji Ishikawa, et al., "Enzyme Immunoassay" (3rd edition) (Medical Shoin, published in 1962), "Methods in

ENZYMOLOGYJ Vol. 70 (Immunochemi cal Techniques (Part A)), Ibid.

73 (Immunochemical Techniques (Part B)), ibid.Vol. 7 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part B)

D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)), ibid.

121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies))) (published by Academic Press). As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.

Furthermore, when a decrease in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, anorexia (eg, anorexia nervosa, etc.) Anemia or hypoproteinemia due to anorexia, weak labor, lax hemorrhage, uterine remodeling failure, milk stasis, etc. can be diagnosed as having a high possibility of developing. Conversely, if an increase in the concentration of the protein of the present invention is detected, for example, obesity (eg, vein mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary) Obesity, reduced plasma obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, pediatric obesity, upper body obesity, dietary obesity, hypogonadism, systemic mastocytosis, simple obesity , Central obesity, etc.), hyperphagia, affective disorders, sexual dysfunction, excessive labor, tonic contractions, fetal asphyxia, uterine rupture, facial lacerations, preterm birth, Prader-Willi syndrome, diabetes and its complications Disease (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease (Pickck syndrome, sleep apnea syndrome), Fatty liver, infertility It can be diagnosed that the possibility of osteoarthritis and the like is high.

Further, the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue. In addition, for the preparation of an antibody column used for purifying the protein of the present invention, the detection of the protein of the present invention in each fraction during purification, and the analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.

(4) Gene diagnostics ''

The DNA of the present invention can be used, for example, in humans or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, bushes, horses, cats, cats, dogs, Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the protein of the present invention or a partial peptide thereof in monkeys, chimpanzees, etc.). It is useful as a gene diagnostic agent for mutation or decrease in expression, and increase or excessive expression of the DNA or mRNA.

The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern Hybridization ゃ PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989)) Proceedings of the National Academy of Sciences of The United States of America, Vol. 86, pp. 2766-2770 (1989)). For example, when an increase in expression is detected by Northern hybridization, for example, obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, hypoplasma) Obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity, dietary obesity, hypogonadism obesity, systemic mastocytosis, simple obesity, centrality Obesity, etc.), hyperphagia, affective disorders, sexual dysfunction, excessive labor, tonic contractions, fetal asphyxia, uterine rupture, tubal lacerations, preterm birth, Prader-Willi syndrome, diabetes and its complications ( Eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy), hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease (Pickwick syndrome, sleep apnea syndrome), fat Liver, It can be diagnosed that the possibility of infertility or osteoarthritis is high. Conversely, if down-regulation is detected or if a DNA mutation is detected by PCR-SSCP, for example, anorexia (eg, anorexia nervosa), anorexia associated with anorexia or It can be diagnosed as likely to be low protein, weak labor, lax hemorrhage, uterine remodeling failure, and milk stasis.

[5] a drug containing an antisense polynucleotide

The antisense polynucleotide of the present invention, which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, has low toxicity, and functions of the protein of the present invention or the DNA of the present invention in vivo (eg, Since it can suppress eating (appetite) promoting activity, for example, obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity) , Hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity, dietary obesity, hypogonadism, systemic mastocytosis, simple obesity , Central obesity), Hyperphagia, affective disorders, sexual dysfunction, overwork labor, tonic contractions, fetal asphyxia, ruptured pupils, facial tears, premature birth, Prader-Willi syndrome, diabetes and its complications (eg, diabetic Nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease (Pickwick syndrome; sleep apnea syndrome), fatty liver, infertility, deformity It can be used as a prophylactic / therapeutic agent for osteoarthritis (especially antiobesity agents, appetite (feeding) regulators, etc.).

When the above-mentioned antisense polynucleotide is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a known method.

For example, when the antisense polynucleotide is used, the antisense polynucleotide is inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. It can be administered orally or parenterally to humans or mammals (eg, rats, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.). The antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and can be administered by a gene gun or a catheter such as a hydrogel catheter.

The dosage of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the antisense polynucleotide of the present invention is intravenously administered for the purpose of treating obesity, Generally, in an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense polynucleotide is administered per day.

Further, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.

Further, the present invention provides

(i) a double-stranded RNA containing a part of the RNA encoding the protein of the present invention and an RNA complementary thereto,

(i i) a medicine comprising the double-stranded RNA,

(iii) a lipozyme containing a part of the RNA encoding the protein of the present invention,

(iv) a medicament comprising the lipozyme, (v) An expression vector or the like containing the gene (DNA) encoding the lipozyme is provided.

Like the above-mentioned antisense polynucleotide, double-stranded RNA, lipozyme and the like can disrupt RNA transcribed from the DNA of the present invention or suppress the function thereof, and the protein or the same of the present invention in vivo. Can inhibit the function of DNA encoding, for example, obesity (eg, malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, Plasma obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, pediatric obesity, upper body obesity, dietary obesity, hypogonadism, systemic mastocytosis, simple obesity, centrality Obesity, etc.), hyperphagia, affective disorders, sexual dysfunction, overwork labor, tonic contractions, fetal asphyxia, ruptured uterus, tubal lacerations, preterm birth, Prader-Willi syndrome, diabetes and its complications (Eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary arteriosclerosis, gout, respiratory disease (Pickwick syndrome, sleep apnea syndrome group) ), Fatty liver, infertility, osteoarthritis, etc. (especially anti-obesity agents, appetite (feeding) regulating agents, etc.) can be used as a preventive and therapeutic agent.

The double-stranded RNA can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).

The lipozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by substituting a part of a known lipozyme sequence with a part of RNA encoding the protein of the present invention. As a part of the RNA encoding the protein of the present invention, a consensus sequence NU X (where N represents all bases, X represents a base other than G) which can be cleaved by a known lipozyme, And the like. When the above-mentioned double-stranded RNA or lipozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide. The expression vector (V) is used in the same manner as known gene therapy methods and the like, and is used as the above-mentioned prophylactic / therapeutic agent. [6] A drug containing the antibody of the present invention

Since the antibody of the present invention having the action of neutralizing the activity of the protein of the present invention can suppress the function of the protein of the present invention (eg, inactivate signal transduction), for example, obesity (eg, obesity) Malignant mastocytosis, exogenous obesity, hyperinsulinic obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity , Upper body obesity, dietary obesity, hypogonadism, obesity, systemic mastocytosis, simple obesity, central obesity, etc.), hyperphagia, affective disorder, sexual dysfunction, labour, tonicity Uterine contractions, fetal asphyxia, uterine rupture, ruptured tubules, preterm birth, Prader-Willi syndrome, diabetes and its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, Hyperlipidemia, coronary Pulse sclerosis, gout, respiratory disease (Pickwick syndrome, sleep apnea syndrome), fatty liver, infertility, osteoarthritis, etc. (especially antiobesity agents, appetite (feeding) regulators, etc.) It can be used as a prophylactic and therapeutic agent.

Since the antibody of the present invention having an activity of activating the activity of the protein of the present invention can promote the function of the protein of the present invention (eg, activate signal transduction), for example, increase appetite (feeding) Drugs, anorexia (eg, anorexia nervosa), anemia or hypoproteinemia associated with anorexia, weak labor, lax bleeding, uterine remodeling failure, milk stasis, etc. it can.

The antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.

That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules ( Soft capsules), syrups, emulsions, suspensions and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets. As compositions for parenteral administration, for example, injections, suppositories, etc. are used. Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, etc. Is included. Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. Aqueous liquids for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents, such as alcohol (eg, ethanol) and polyalcohol (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxythylene (50 mol) adduc tof hydrogenated cas tor oil)), etc. Good. As the oily liquid, for example, sesame oil, soybean oil, and the like are used, and benzyl benzoate, benzyl alcohol, and the like may be used as a solubilizing agent. The prepared injection is usually filled in an appropriate ampoule. A suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a conventional suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form adapted to the dose of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like. Usually, each dosage unit dosage form is 5 to 500 mg, especially for injections. Preferably, 5 to 100 mg, and other dosage forms, contain 10 to 25 Omg of the antibody.

Each of the above-mentioned compositions may contain other active ingredients as long as no undesirable interaction occurs due to the combination with the above antibody.

The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form, in humans or mammals (eg, rat, egret, sheep, etc.). Can be administered orally or parenterally (eg, intravenously) to mice, dogs, cats, cats, dogs, monkeys, etc.). The dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like. 0.01 to 20 mg / kg body weight, preferably 0.1 to 10 mg / kg body weight, more preferably 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 to 5 times a day About 3 times as injection It is convenient to give. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.

Further, the antibody of the present invention is also useful, for example, as a diagnostic agent for the above-mentioned diseases and the like.

[7] Creation of an animal having the DNA of the present invention

The present invention has a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). A non-human mammal is provided.

That is, the present invention

(1) a non-human mammal having an exogenous DNA of the present invention or a mutant DNA thereof,

(2) the animal according to (1), wherein the non-human mammal is a rodent;

(3) The animal according to (2), wherein the rodent is a mouse or a rat, and

(4) To provide a recombinant vector or the like containing the exogenous DNA of the present invention or its mutant DNA, which can be expressed in mammals.

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA-transfected animal of the present invention) include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. Preferably, at the stage of embryonic development in non-human mammal development (more preferably, at the stage of a single cell or a fertilized egg and before the general fo 8 cell stage), the calcium phosphate method, the electric pulse method, the Lipofection It can be produced by introducing the target DNA by a method such as a coagulation method, a coagulation method, a microinjection method, a particle gun method, or a DEAE-dextran method. Further, by the DNA introduction method, the exogenous DNA of the present invention intended for somatic cells, organs of living organisms, tissue cells, and the like can be introduced and used for cell culture, tissue culture, and the like. The DNA-introduced animal of the present invention can also be produced by fusing the cells with the above-mentioned germ cells by a known cell fusion method.

As the non-human mammal, for example, porcupine, pig, hidge, goat, magpie, dog, cat, guinea pig, eight-star, mouse, rat and the like are used. Above all, the ontogeny and biological cycle are relatively short in terms of the creation of disease animal model systems. In addition, rodents that are easy to breed, especially mice (for example, pure strains such as C57B LZ6 strain and DBA2 strain, and hybrid strains such as BSC SFi strain, BDFi strain, B6D2 Ft strain, BALBZc strain, ICR strain, etc. ) Or light (eg, Wistar, SD, etc.) is preferred. '

Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention does not mean the DNA of the present invention originally possessed by a non-human mammal but refers to the DNA of the present invention once isolated and extracted from the mammal.

As the mutant DNA of the present invention, DNA having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition, deletion, or substitution of another base to another base DNA that has been used is used, and also includes abnormal DNA.

The abnormal DNA means a DNA that expresses an abnormal protein of the present invention. For example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.

The exogenous DNA of the present invention may be derived from a mammal of the same species or a different species as the target animal. In introducing the DNA of the present invention into a subject animal, it is generally advantageous to use the DNA as a DNA construct linked downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is introduced, it is derived from various mammals having the DNA of the present invention having a high homology with the DNA (eg, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.). By injecting the DNA construct of the present invention (eg, a vector) into a fertilized egg of a target mammal, eg, a mouse fertilized egg, downstream of various promoters capable of expressing the DNA of the present invention. A DNA-introduced mammal that highly expresses the DNA of the present invention can be produced.

Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as λ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus and the like. For example, animal viruses such as baculovirus are used. Among them, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferred. It is used well. ',

Examples of promoters that regulate the DNA expression include, for example, (i) DNA promoters derived from viruses (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.) Yuichi, (ii) Promoters derived from various mammals (humans, egrets, dogs, cats, guinea pigs, mussels, rats, mice, etc.), such as albumin, insulin II, ゥ roplakin II, Erasu evening Protease, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidic protein, dalyuthione S-transferase, platelet-derived growth factor 13, keratin Kl, 10 and 14, collagen I And II, cyclic AMP-dependent protein kinase / 3I subunit, dystrophy Int, tartrate-resistant alkaline phosphatase, atrial sodium diuretic factor, endothelial receptor thymic synthase (generally abbreviated as Tie 2), sodium potassium adenosine 3 kinase (Na, K-ATPase), Neurofilament light chain, meta-oral thionine I and IIA, metallobrotinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine) 3-hydroxylase, thyroid gland peroxida-1 (TPO), '' polypeptide chain elongation factor 1a (EF-1), β-actin, α and) 3 myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Τhy_l, immunoglobulin , H chain variable region (VNP), serum amyloid P component, myoglobin, troponin (:, smooth muscle actin, preproenke Promoters such as farin A and vasopressin are used, among them, cytomegalovirus promoter, which can be highly expressed in the whole body, and human polypeptide chain elongation factor 1ά (EF-1α) promoter. , Human and chicken) 3 actin promoter.

The vector preferably has a sequence that terminates the transcription of the target mRNA in the DNA-transfected mammal (generally referred to as "Yuichi Mineta-1"). For example, it is derived from viruses and various mammals. The sequence of each DNA can be used. Preferably, Simian virus SV40 or the like is used. In addition, the splicing of each DNA for the purpose of further expressing the desired foreign DNA It is also possible to link a licensing signal, an enhancer region, a part of an intron of a eukaryotic DNA, etc., at 5, upstream of the promoter region, between the promoter region and the translation region, or 3 'downstream of the translation region.

The normal translation region of the protein of the present invention is DNA derived from liver, kidney, thyroid cells, fibroblasts derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). And all or part of genomic DNA from various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cells, and fibroblast-derived RNA as raw materials I can do it. In addition, an exogenous abnormal DNA can produce a translation region obtained by mutating a normal polypeptide translation region obtained from the above cells or tissues by a point mutagenesis method.

The translation region is a DNA construct that can be expressed in an introduced animal, which is usually ligated to the downstream of the promoter and optionally the upstream of a transcription termination site.

It can be produced by a DNA engineering technique.

Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after the DNA transfer means that all the progeny of the transgenic animal retain the exogenous DNA of the present invention in all of the germ cells and somatic cells Means that. The offspring of this type of animal that has inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of its germ cells and somatic cells.

A non-human mammal into which the exogenous normal DNA of the present invention has been introduced can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably retained by the crossing. .

Introduction of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after the introduction of the DNA indicates that all the offspring of the produced animal carry the exogenous DNA of the present invention in all of its germ cells and somatic cells. It means every time you have it. The progeny of this type of animal that has inherited the exogenous DNA of the present invention has an excess of the exogenous DNA of the present invention in all of its germinal and somatic cells. By obtaining a homozygous animal having the introduced DNA on both homologous chromosomes and mating the male and female animals, it is possible to breed and passage so that all offspring have the DNA in excess.

The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and finally promotes the function of the endogenous normal DNA, thereby finally obtaining the protein of the present invention. May develop functional hyperplasia, and can be used as a disease model animal. For example, using the normal DNA-introduced animal of the present invention to elucidate the pathological mechanism of hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention, and to examine a method for treating these diseases. Is possible. '' In addition, since a mammal into which the exogenous normal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, a screening test for a therapeutic agent for a disease associated with the protein of the present invention is performed. Is also available.

On the other hand, a non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by mating. I can do it. Furthermore, the desired exogenous DNA can be incorporated into the above-mentioned plasmid and used as a raw material. The DNA construct with the promoter can be prepared by ordinary DNA engineering techniques. Introduction of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the animal produced after the transfer of DNA means that all the offspring of the animal produced have the abnormal DNA of the present invention in all of the germ cells and somatic cells. The progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. 'A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to propagate the offspring so that all offspring have the DNA.

The non-human mammal having the abnormal DNA of the present invention expresses the abnormal DNA of the present invention at a high level, and finally inhibits the function of endogenous normal DNA, thereby ultimately obtaining the protein of the present invention. In some cases, it becomes a functional inactive refractory disease and can be used as a model animal for the disease. For example, using the abnormal DNA-introduced animal of the present invention, It is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.

Further, as a specific possibility, the abnormal DNA-highly expressing animal of the present invention can be used to inhibit the function of a normal protein by the abnormal protein of the present invention in function-inactive refractory disease of the protein of the present invention (dominant negatase). ive effect). In addition, since the mammal into which the foreign abnormal DNA of the present invention has been introduced has an increased symptom of the released protein of the present invention, it can be used in a therapeutic drug screening test for the protein of the present invention or its functionally inactive refractory disease. Is also available.

Further, as other possible uses of the above two kinds of DNA-introduced animals of the present invention, for example,

(Use as a cell source for tissue culture,

(ii) Direct analysis of DNA or RNA in the tissue of the DNA-introduced animal of the present invention or specific expression or activation by the protein of the present invention by analyzing a polypeptide tissue expressed by DNA Analysis of the relationship with proteins,

(iii) cells of tissues having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from generally difficult tissues,

(iv) screening for a drug that enhances cell function by using the cell described in (iii) above, and

(v) It is conceivable to isolate and purify the mutant protein of the present invention and to prepare an antibody thereof. Furthermore, using the DNA-introduced animal of the present invention, The clinical symptoms of the disease related to the protein of the present invention can be examined, and more detailed pathological findings in each organ of the disease model related to the protein of the present invention can be obtained. Furthermore, it can contribute to research and treatment of secondary diseases caused by the disease.

In addition, it is possible to remove each organ from the DNA-introduced animal of the present invention, cut it into small pieces, and then use a protease such as trypsin to obtain the released DNA-introduced cells, culture them, or systematize the cultured cells. It is possible. Further, the specification of the protein-producing cell of the present invention, its relationship with apoptosis, differentiation or proliferation, or They can examine the signal transduction mechanism of them and investigate their abnormalities, etc., and are useful research materials for elucidating the protein of the present invention and its action.

Further, in order to develop a therapeutic agent for a disease associated with the protein of the present invention, including a functionally inactive refractory type of the protein of the present invention, using the DNA-transfected animal of the present invention, Using a quantitative method or the like, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease. In addition, using the animal into which the DNA of the present invention has been introduced or the exogenous DNA expression vector of the present invention, it is possible to study and develop a method for treating DNA associated with the protein of the present invention. In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on the respective abbreviations by the I UPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When there is an optical isomer with respect to the amino acid, the L-form is indicated unless otherwise specified. ,

DNA deoxylipo nucleic acid

c DNA complementary deoxylipo nucleic acid

A adenine

T thymine

G Guanin

C

RNA liponucleic acid

mRNA messenger liponucleic acid

d ATP Deoxyadenosine triphosphate

dTTP Deoxythymidine triphosphate

dGTP Deoxyguanosine triphosphate

d CTP 'triphosphate

ATP 'triphosphate

EDTA ethylenediaminetetraacetic acid

SDS dodecyl sulfate: G 1 y: glycine

A 1 a: Alanine

Va 1: Valine

Le u: Leucine

I 1 e: Isoleucine

S e r: Serine

Th r: Threonine

Cys: Sistine

Me t: Methionin

G 1 u: Glutamic acid

As p: Aspartic acid

L y s: lysine

A r g: Arginine

H i s: Histidine

P he: feniralanin

Ty r: Tyrosine

T r p: Tribute fan

Pro: Proline

A s n: Asparagine

G 1 n: Dal Yu Min

pG1u: pyroglutamic acid

The substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

Me: methyl group

E t: ethyl group

B u: butyl group

P h: phenyl group

TC: Thiazolidine-4 (R) one-pot lipoxamide group

To s: p-toluenesulfonyl CHO Holmill

B z 1

Cl ₂ -Bzl 2,6-dichlorobenzene

Bom benzyloxymethyl

Z benzyloxycarponyl

C 1 Z

B r -Z 2 bromobenzyloxycarponyl

B oc t-butoxycarponyl

DNP dinitrophenyl

Bum t—butoxymethyl

Fmo c N— 9 _fluorenylmethoxycarbonyl

HOB t 1-hydroxybenztriazole

HOOB t 3, 4-dihydro-3-hydroxy-1 4-oxo-1

1, 2, 3-benzotriazine

HONB Tohydroxy-5-norpolene-2,3-dicarboximide

DCC

GAPDH dariceraldehyde phosphate dehydrogenase The sequence numbers in the sequence listing in the present specification show the following sequences.

[SEQ ID NO: 1]

1 shows the nucleotide sequence of primer 11 used in the PCR reaction in Example 1 below. [SEQ ID NO: 2]

1 shows the nucleotide sequence of Primer 12 used in the PCR reaction in Example 1 below. [SEQ ID NO: 3]

1 shows the nucleotide sequence of cDNA encoding the cynomolgus SLT receptor.

[SEQ ID NO: 4]

Fig. 3 shows the amino acid sequence of the cynomolgus monkey SLT receptor.

[SEQ ID NO: 5] 2 shows the nucleotide sequence of primer 11 used in the PCR reaction in Example 2 below. [SEQ ID NO: 6]

The base sequence of primer 2 used in the PCR reaction in Example 112 below is shown. [SEQ ID NO: 7]

FIG. 3 shows the nucleotide sequence of cDNA encoding the C-terminal region of the feline SLT receptor obtained in Example 2 below.

[SEQ ID NO: 8]

The base sequence of primer 1 used in the PCR reaction in Example 3 below is shown. [SEQ ID NO: 9]

3 shows the base sequence of primer 2 used in the PCR reaction in Example 3 below. [SEQ ID NO: 10]

3 shows the nucleotide sequence of cDNA encoding the N-terminal region of the feline SLT receptor obtained in Example 3 below.

[SEQ ID NO: 11]

The base sequence of primer 1 used in the PCR reaction in Example 4 below is shown. [SEQ ID NO: 12]

The base sequence of primer 2 used in the PCR reaction in Example 4 below is shown.

[SEQ ID NO: 13]

1 shows the nucleotide sequence of a cDNA encoding a cat SLT receptor.

[SEQ ID NO: 14]

2 shows the amino acid sequence of the feline SLT receptor.

[SEQ ID NO: 15]

7 shows the base sequence of primer 1 used in the PCR reaction in Example 5 below. [SEQ ID NO: 16]

7 shows the base sequence of primer 2 used in the PCR reaction in Example 5 below. [SEQ ID NO: 17]

5 shows the nucleotide sequence of cDNA encoding the C-terminal region of canine SLT receptor obtained in Example 5 below.

[SEQ ID NO: 18] 7 shows the nucleotide sequence of primer 11 used in the PCR reaction in Example 6 below. [SEQ ID NO: 19]

7 shows the base sequence of primer 2 used in the PCR reaction in Example 6 below. [SEQ ID NO: 20]

7 shows the nucleotide sequence of cDNA encoding the N-terminal region of canine SLT receptor obtained in Example 6 below.

[SEQ ID NO: 21]

7 shows the base sequence of primer 1 used in the PCR reaction in Example 7 below. [SEQ ID NO: 22]

7 shows the nucleotide sequence of primer 2 used in the PCR reaction in Example 7 below. [SEQ ID NO: 23]

1 shows the nucleotide sequence of cDNA encoding canine SLT receptor.

[SEQ ID NO: 24]

2 shows the amino acid sequence of the canine SLT receptor.

[SEQ ID NO: 25]

1 shows the amino acid sequence of MCH. .

[SEQ ID NO: 26]

2 shows the amino acid sequence of Des-Asp′-MCH (MCH (2-19)).

[SEQ ID NO: 27]

² shows the amino acid sequence of Des- [Asp ¹ , Phe ² ] -MCH (MCH (3-19)).

[SEQ ID NO: 28]

² shows the amino acid sequence of Des- [Asp ¹ , Phe ² , Asp ³ ] -MCH (MCH (4-19)).

[SEQ ID NO: 29]

² shows the amino acid sequence of Des- [Asp ¹ , Phe ² , Asp ³ , Met ⁴ ] -MCH (MCH (5-19)).

[SEQ ID NO: 30]

² shows the amino acid sequence of Des- [Asp ¹ , Phe ² , Asp ³ , Met ⁴ , Leu ⁵ ] -MCH (MCH (6-19)). [SEQ ID NO: 31]

^{^{Des- [Asp 1, Phe 2,}} Asp 3, Met 4, Leu 5, Arg 6] - shows the amino acid sequence of the MCH (MCH (7-19)). [SEQ ID NO: 32] 'This shows the base sequence of primer 1 used in EXAMPLE 10 below.

[SEQ ID NO: 33]

The base sequence of primer 2 used in Example 10 below is shown.

[SEQ ID NO: 34]

The base sequence of the probe used in Example 10 below is shown (FAM (6-carboxy-fluorescein) as the reporter dye at the 5 'end, TAMRA (6-carboxy-ietrameihyl-rhodamine as the quencher at the 3' end). )]].

[SEQ ID NO: 35],

The base sequence of primer 13 used in Example 10 below is shown.

[SEQ ID NO: 36]

The base sequence of primer 14 used in Example 10 below is shown.

[SEQ ID NO: 37]

The base sequence of the probe used in Example 10 below is shown [FAM (6-carboxy-fluorescein) as a reporter dye at the 5 'end, TAMRA (6-carboxy-tetramethyl-rhodamine) as a quencher at the 3 end. )]].

[SEQ ID NO: 38]

1 shows the nucleotide sequence of cDNA which encodes the entire length of the cynomolgus monkey SLT receptor obtained in Example 1 below.

[SEQ ID NO: 39]

7 shows the nucleotide sequence of cDNA encoding the full length of cat SLT receptor obtained in Example 4 below.

[SEQ ID NO: 40]

7 shows the nucleotide sequence of cDNA encoding the entire length of the canine SLT receptor obtained in Example 7 below. Escherichia col i T0P10 / pCR4_monSLT obtained in Example 1 described below is an independent administrative corporation of Chuo No. 6 (Zip code 305-8566) 1-1 Toto, Tsukuba-shi, Ibaraki, Japan from June 4, 2002. Accession No. FERM BP-8066 to National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Has been deposited as

Escherichia coli TOPlO / pcDNA-catSLT obtained in Example 4 described below has been administered by Independent Administration of 1-1 Chukoku No. 6 Tsukuba Totoro, Ibaraki, Japan (Postal Code 305-8566) since April 25, 2002. At the National Institute of Advanced Industrial Science and Technology, the Patent Organism Depositary, the deposit number FERM BP-8030, from April 16, 2002, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8686) They have been deposited with the Fermentation Research Institute (IF0) under the accession number IF0 16789.

Escherichia coli TOPlO / pAKKO-dogSLT obtained in Example 11 described below has been established on April 25, 2002, as an independent company of 1-1 Chukoku No. 1-1, Tsukuba Totoro, Ibaraki, Japan (zip code 305-8566). Deposited number at Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology

From April 16, 2002, FERM BP-8031, 2-17-85 Jusanhoncho, Yodogawa-ku, Osaka, Osaka

They have been deposited with the Fermentation Research Institute (IF0) of Japan (zip code 532-8686) under the accession number IF0 16790. Hereinafter, the present invention will be described in more detail with reference to Examples, but these do not limit the scope of the present invention. In addition, the gene manipulation method using Escherichia coli followed the method described in Molecular cloning.

Example 1

Acquisition of RLT cynomolgus SLT gene

After total RNA was prepared from the hypothalamus of the cynomolgus monkey using Isogen (Futtsubon Gene), a poly (A) ⁺ RNA fraction was prepared using Oligotex (dT) ₃₀ (Takara Shuzo).力 oly (A) ^T RNA 2. Power, et al. Superscript reversetranscr iptase

(Gibco BRL) and reverse transcription was performed using random primers according to the manual to prepare cDNA. Using the primer 1 (SEQ ID NO: 1) prepared with reference to the 5 'untranslated region of human SLT and the primer 2 (SEQ ID NO: 2) having the sequence of the human 3' untranslated region, a cynomolgus monkey hypothalamic cDNA A PCR reaction was performed using the 錶 type. The reaction was carried out using a cDNA equivalent to 20 ng of mRNA as a type II in a volume of 201. The composition of the reaction solution was a primer concentration of 0.5 M, a dNTP mixture solution of 0.2 mM, Z-Taq (Takara Shuzo). 1/100 volume, 10-fold concentrated Buffer 1/10 volume. The cycle for amplification is After keeping it at 94 ° C for 120 seconds, a cycle of 98 ° C for 1 second and 72 ° C for 30 seconds was repeated 40 times. The resulting reaction solution 21 was subcloned into a plasmid vector pcr4 using a 1 "0-0 TA cloning kit (Invitrogen), and then introduced into E. coli T0P10. From the resulting transformant, QIA prep8 mini Plasmid DNA was purified using prep (Qiagen) The reaction for nucleotide sequence determination was performed using the BigDye Terminator Cycle Sequence Ready Reaction Kit (PerkinElmer Inc.) Using a fluorescent automatic sequencer As a result, the nucleotide sequence shown in SEQ ID NO: 38 was obtained, and this sequence contained the nucleotide sequence (SEQ ID NO: 3) encoding the entire amino acid sequence of Cynomolgus SLT (SEQ ID NO: 4) Therefore, Escherichia coli TOP10 / pCR4-monSLT was obtained by transforming Escherichia coli TOP10 with this plasmid.

3 '— Acquisition of 3' sequence of cat SLT gene by RACE method

After preparing total RNA from the whole cat brain using Isogen (Futtsubon Gene),

A poly (A) ⁺ RNA fraction was prepared using Oligotex (dT) ₃₀ (Takara Shuzo). Type III for 3'-RACE was prepared based on feline whole brain poly (A) + RNA using SMART RACE cDNA Amplification kit. The primer set used for the RACE PCR reaction was as follows: the first PCR reaction included the Universal Primer Mix and primer 1 (SEQ ID NO: 5) attached to the kit; the second PCR reaction included the Nested Universal Primer and primer 2 (sequence number) attached to the kit. : 6) was used. The reaction was carried out in the form of 铸 with a volume of 201 liquid, ie, 11 * 6 equivalents of the reversely transcribed 00 ^. The composition of the reaction solution was a primer concentration of 0.5 zM, a dNTP mixed solution of 0.2 mM, Advantage? Polymerase Mix (Clontech) 1/50 volume ゝ 10-fold concentrated Buffer 1/10 volume. The cycle for amplification is 94X · 5 seconds for both PCRs,

After repeating the cycle of 60 ° C for 25 seconds and 72 ° C for 120 seconds for 40 times, the _c- reactant incubated at 72 ° C for 10 minutes was electrophoresed on 1.2% Seakein GTG Agarose (Takara Shuzo), and then purified with ethidium bromide. A band around 2000 bp, which was visible when stained with, was extracted with a GeneClean Spin kit (Bio 101), subcloned into a plasmid vector pcr4-T0P0 using a T0P0 TA cloning kit (Invitrogen), and then introduced into E. coli T0P10. Plasmid DNA was isolated from the resulting transformant using QIA prep8 mini prep (Qiagen). Purified. 'The reaction for base sequence determination was performed using the BigDye Terminator Cycle Sequence Ready Reaction Kit (PerkinElmer). As a result of decoding using a fluorescent automatic sequencer, a base sequence represented by SEQ ID NO: 7 including the C-terminal region of cat SLT was obtained. Example 3

Acquisition of 5 'side sequence of cat SLT gene by 5'-RACE method

Type I for 5'-RACE was prepared from feline whole brain poly (A) ⁺ RNA using Marathon ready cDNA Amplification'kit. The set of primers used for the RACE PCR reaction included adapter primer 1 for the Marathon ready cDNA Amplification kit in the first PCR reaction. Primer 2 (SEQ ID NO: 9) and primer 2 (SEQ ID NO: 9) for Marathon ready cDNA Amplification kit were used in the second PCR reaction. The reaction was carried out in the amount of 20 ng of the reverse transcribed cDNA corresponding to 2 ng of mRNA at a volume of 20 / i1. The composition of the reaction solution was primer concentration 0.5 M, dNTP mixture 0.2 mM, Advantage 2 Polymerase Mix (Clontech) 1/50 volume, and 10-fold concentrated Buffer 1/10 volume. The cycle for amplification is 10 cycles of 94 ° C for 5 seconds, 66 ° C for 120 seconds for both PCRs, 94 ° C for 5 seconds, 60 ° C for 20 seconds, 72 ° C for After repeating the cycle of 120 seconds 25 times, it was kept at 72 ° C for 10 minutes. The reaction product was electrophoresed on a 1.2% Seakem GTG Agarose (Takara Shuzo), and a band around 500 bp, which was visible when stained with ethidium amide, was extracted with the GeneClean Spin kit (Bio 101). After subcloning into plasmid vector pcr4-T0P0 using E. coli, Escherichia coli E0 was introduced. Plasmid DNA was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The reaction for base sequence determination was performed using BigDye Terminator Cycle Sequence Ready Reaction Kit (Perkin Elmer). As a result of decoding using a fluorescent automatic sequencer, a base sequence represented by SEQ ID NO: 10 including the N-terminal region of cat SLT was obtained. Example 4

Acquisition of full-length cat SLT sequence In order to obtain a sequence containing the full length of cat SLT predicted from the results of the 5'- and 3'-RACE methods, 5 was used as the forward primer, primer 1 (SEQ ID NO: 11) was added to the untranslated region, and reverse PCR was performed with 3 as a primer and primer 2 (SEQ ID NO: 12) in the untranslated region. The reaction was performed in the form of type II with a volume of 20 ^ 1 corresponding to 2 ng of mRNA of the reverse transcribed cDNA. The composition of the reaction solution was primer concentration 0.5 M, dNTP mixture 0.2 mM, Z-Taq (TaKaRa) 1/50 volume, and 10-fold concentrated Buffer 1/10 volume. The cycle for amplification is as follows: after holding at 94 ° C for 15 seconds, 98t: 1 second, 64 ° C, 15 seconds, 72 ° C Keep warm for a minute. The reaction product was electrophoresed on 1.2% Seakein GTG Agarose (Takara Shuzo), and a band around 1200 bp, which was visible when stained with ethidium bromide, was extracted using the GeneClean Spin kit (Bio 101) and the Eukaryotic T0P0 TA cloning kit (Invitrogen) After subcloning into plasmid vector pcDNA3.1 / V5 / His-T0P0 using Escherichia coli, E. coli TOP10 was introduced. Plasmid DM was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The reaction for base sequence determination was performed using a BigDye Terminator Cycle Sequence Ready Reaction Kit (Pakkin Elma). As a result of decoding using a fluorescent automatic sequencer, the nucleotide sequence represented by SEQ ID NO: 39 was obtained. Since this sequence contained the nucleotide sequence (SEQ ID NO: 13) encoding the entire amino acid sequence of cat SLT (SEQ ID NO: 14), Escherichia coli TOP10 was transformed with this plasmid to transform Escherichia coli TOP10. / pcDNA-catSLT was obtained. Example 5

3 '— RACE method to obtain 3' sequence of canine SLT gene

After preparing total RNA from the canine frontal lobe with Isogen (Futtsubon Gene),

A poly (A) ⁺ RNA fraction was prepared using Oligotex (dT) ₃₀ (Takara Shuzo). A type for 3'_RACE was prepared using SMART RACE cDNA Amplification kit based on canine frontal lobe poly (A) + RNA. The primer set used for the RACE PCR reaction was the Universal Primer Mix and primer 1 (SEQ ID NO: 15) attached to the kit in the fourth PCR reaction, and the Nested Universal Primer and primer 1 in the kit for the second PCR reaction. (SEQ ID NO: 16) was used. The reaction was performed by converting 20 ng of reverse-transcribed cDNA into Performed with 1 liquid volume. The composition of the reaction solution was primer concentration 0.5 / M, dNTP mixture 0.2 mM, Advantage2 Polymerase Mix (Clontech) 1/50 volume ^ 10-fold concentrated Buffer 1/10 volume. The cycles for amplification were as follows: 94 ° C for 5 seconds, 72 ° C for 90 seconds, three cycles for each PCR, 94 ° C for 5 seconds, 70 ° C for 90 seconds, three cycles. After repeating the cycle of ° C for 5 seconds and 68 ° C for 90 seconds 40 times, it was kept at 72 ° C for 10 minutes. 'The reaction product was electrophoresed on 1.2% Seakem GTG Agarose (Takara Shuzo), and a band around 900 bp, which was visible when stained with ethidium bromide, was extracted with the GeneClean Spin kit (Bio 101). ) Was used for subcloning into a plasmid vector pc-TOPO, and then introduced into E. coli T0P10. Plasmid DNA was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The reaction for base sequence determination is BigDye Terminator Cycle Sequence Ready

The reaction was performed using Reaction Kit (Pakkin Elma). As a result of decoding using a fluorescent automatic sequencer, a base sequence represented by SEQ ID NO: 17 including the C-terminal region of canine SLT was obtained. Example 6

Acquisition of 5 'sequence of canine SLT gene by 5'-RACE method

5, -Type II for RACE was prepared from canine frontal lobe poly (A) ⁺ RNA using Marathon ready cDNA Amplification kit. The primer set used for the RACE PCR reaction was the adapter primer 1 and primer 1 (SEQ ID NO: 18) for the Marathon ready cDNA Amplification kit in the first PCR reaction, and the Marathon ready cDNA Amplification kit in the second PCR reaction. The adaptor primer 2 and the primer 1 (SEQ ID NO: 19) were used. The reaction was carried out in the form of type II with a volume of 201, corresponding to 2 ng of mRNA of the reverse-transcribed cDNA. The composition of the reaction solution was primer concentration 0.5 M, dNTP mixture 0.2 mM,

Advantage2 Polymerase Mix (Clontech) 1/50 volume ^ 10 times concentrated Buffer 1/10 volume. The cycle for amplification was 94 ° C for 5 seconds for both PCRs.

A cycle of 66 ° C for 120 seconds was repeated 10 times, a cycle of 94 for 5 seconds, a cycle of 60 ° C for 20 seconds, and a cycle of 72 for 120 seconds was repeated 25 times, followed by incubation at 72 ° C for 10 minutes. Reactants to 1.2% Seakem GTG

Appears when electrophoresed on Agarose (Takara Shuzo) and stained with ethidium bromide A band near 400 bp was extracted with a GeneClean Spin kit (Bio 101), subcloned into a plasmid vector pcr4-T0P0 using a TOPO TA cloning kit (Invitrogen), and then introduced into E. coli TOP10. Plasmid DNA was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The anti-sequence for nucleotide sequencing is the BigDye Terminator Cycle Sequence Ready Reaction Kit

(PerkinElmer). As a result of decoding using a fluorescent automatic sequencer, a nucleotide sequence represented by SEQ ID NO: 20 including the N-terminal region of canine SLT was obtained. '' Example 7

Dog SLT full length sequence acquisition ¹

In order to obtain a sequence containing the full length of canine SLT predicted from the results of the 5,-and 3'-RACE methods, primer 1 (SEQ ID NO: 21) was added to the 5 'untranslated region as a forward primer, and 3 'PCR was performed by setting Primer 2 (SEQ ID NO: 22) to the untranslated region. The reaction was carried out in the form of type II with a volume of 201, corresponding to 2 ng of mRNA of the reverse-transcribed cDNA. The composition of the reaction solution was a primer concentration of 0.5 M, a dNTP mixed solution of 0.2 mL, a Z-Taq (TaKaRa) 1/50 volume, and a 10-fold concentrated Buffer 1/10 volume. The cycle for amplification is as follows: after holding at 94 ° C for 15 seconds, repeat the cycle of 98 ° C for 1 second, 64 ° C for 15 seconds, 72 ° C for 20 seconds 40 times, and then for 72 ° C. For 2 minutes. The reaction product was electrophoresed on a 1.2% Seakem GTG Agarose (Takara Shuzo), and a band around 1200 bp, which was visible when stained with ethidium umide, was extracted with the GeneClean Spin kit (Bio 101). Eukaryotic T0P0 TA cloning kit (Invitrogen) After subcloning into the plasmid vector PCDNA3.1 / V5 / His-TOP0 using Escherichia coli, Escherichia coli T0P10 was introduced. Plasmid DNA was purified from the resulting transformant using QIA prep8 mini prep (Qiagen). The reaction for base sequence determination was carried out using BigDye Terminator Cycle Sequence Ready Reaction Kit (Pakkin Elma). As a result of decoding using a fluorescent automatic sequencer, the base sequence represented by SEQ ID NO: 40 was obtained. This sequence contained the nucleotide sequence (SEQ ID NO: 23) encoding the entire amino acid sequence of canine SLT (SEQ ID NO: 24). Bacterium TOP10 was transformed to obtain Escherichia coli TOPlO / pcDNA-dogSLT. Example 8

Preparation of feline SLT expressing CHO cells

Transformation with a plasmid encoding the full-length amino acid sequence of the feline SLT prepared in Example 4 with a SalI recognition sequence added to the 5 'side and a SpeI recognition sequence added to the 3' side. Plasmids were prepared from the cloned Escherichia coli using the Plasmid Midi Kit (Qiagen) and cut with restriction enzymes Sal I and Spe I to cut out the insert. After the electrophoresis, the insert DNA was cut out from an agarose gel with a force razor, and then recovered by performing fragmentation, phenol extraction, phenol-chloroform extraction, and ethanol precipitation. This insert DNA was cut with Sal I and Spe I for animal cell expression vector Plasmid FpAKKO-llH (Hinuma, S. et al., Biochim. Biophys. Acta, Vol. 1219, pp. 251-259, 1994, pAKKOl. In addition to ligation using T4 ligase (Takara Shuzo) in addition to the same vector—plasmid as 11H, a plasmid pAKKO-catSLT for protein expression was constructed.

After culturing Escherichia coli TOP10 co-immediate etent cells (Invitrogen) transformed with pAKKO-catSLT, plasmid DNA of pAKKO-catSLT was prepared using Plasmid Midi Kit (Qiagen). This was added to the CHO dhfr cell 500,000 cells / 150 1 O tiMEM (Gibco) cell suspension at 50 g / ml, and 240 V 960 F pulse was applied to the cells by Genepulsa (Bio-Rad). after culturing for 1 day ΜΕΜα medium containing the introduced _t 10% © shea calf serum and passaged and cultured in a nucleic acid-free MEM a medium containing 10% dialyzed © shea calf serum selective medium. Transformed cells that were feline SLT-expressing CH0 cells growing in the selection medium were selected. Example 9

Measurement of intracellular Ca ion concentration increasing activity of cat SLT expressing CHO cells by MCH using FLIPR

Measurement of the intracellular Ca ion concentration increasing activity of the feline SLT-expressing CH0 cells obtained in Example 8 by MCH (SEQ ID NO: 25, Peptide Research Laboratories) was performed using FLIPR (Molecular Device Company).

CHO / catSLT cells are suspended in DMEM containing 10% dialyzed fetal serum to a concentration of 15 x 10 ⁴ cells / ml and dispensed into a 96-well plate for FLIPR (Black plate clear bottom, Costar). 200 1 by implantation into the Ueru using (3.0X10 ⁴ cells / 200 l / © El), 5% C0 ₂ was 37 ° C De晚cultured at incubator base one coater in was used to Atsusi (hereinafter This plate is called a cell plate). HANKS '/ HBSS (Nissy Hanks 2

(Nissui Pharmaceutical Co., Ltd.) 9.8 g, sodium hydrogencarbonate 0.35 g, HEPES 4.77 g, adjusted to PH7.4 with 6 M sodium hydroxide solution, filter " ¹ sterilized" 20 ml, 250 mM Probenecid 200 / zKゥ Two vials (50) of Fluo 3-A (Dojindo Laboratories) are mixed with fetal serum (FBS) 200 1 and 40 l of dimethyl sulfoxide and 20% Pluronic acid (Molecular Probes) 40 after mixing added dissolved in 1, after dispensed at each Ueru 100 the cell plate except the culture solution using an 8 channel pipette, incubated for 1 hour at 37 ° C for at 5% C0 ₂ incubator in one Each well of a 96-well plate for FLIPR (V-Bottom plate, Coaster) was loaded with 150 l of 2.5 mM Probenecid, HAMS / HBSS containing 0.05% BSA, and various other components. A sample plate was prepared by adding MCH at a concentration of 1. The color of the cell plate After finishing the washing, wash the cell plate 4 times using a plate washer (Molecular Devices) with HANKS '/ HBSS and 2.5 mM Probenecid using a washing buffer. The cell plate and the sample plate were set on the FLIPR and assembled (the sample from the sample plate was transferred to the cell plate by the FLIPR).

The results showed that MCH increased the intracellular Ca ion concentration of feline SLT-expressing CH0 cells in a concentration-dependent manner (Fig. 1). The increase in intracellular Ca ion concentration is indicated by the increase in fluorescence of the dye loaded on the cell caused by Ca.

, Example 10

Distribution of Feline SLT Gene Expression in Feline Brain and Quantification of Its Expression Using TaqMan PCR

From cat whole brain, frontal lobe, temporal lobe, occipital lobe, parietal lobe, pons, cerebellum, medulla oblongata, hippocampus, flat The amygdala, thalamus, corpus callosum, cerebral peduncle, spinal cord, substantia nigra, caudate nucleus, and hypothalamus were removed, and a total RNA fraction was prepared using ISOGEN (Futtsubon Gene). The obtained RNA was digested with RNase by digestion with proteinase K (Invitrogen), DNase I digested genomic DNA using the Message Clean Kit (GenHunter), and total RNA lxg at each site was extracted. Reverse transcription was carried out using Superscript II reversetranscriptase (Invitrogen) as a template and random primers according to the attached manual to produce cDNA. A reverse transcript equivalent to 25 ng of the obtained total RNA or a standard cDNA prepared as described later, 1 × Universsal PCR Master Mix (PE Biosystems), the primer 1 represented by SEQ ID NO: 32 and the sequence Primer 1 represented by No. 33, 100 nM each, and SEQ ID NO: 34 (Fam-accaggtact tggctctcgtccaaccat t'tc-Tamra; in the column, Fam is 6-carboxy-fluorescein, Tamra is 6- PCR was carried out using ABI PRISM 7700 Sequence Detector (PE Biosystems) for a reaction mixture 25 ^ 1 containing 100 nM of a TaqMan probe represented by carboxy-tetramethy and rhodamine. PCR was performed by treating the cells at 50 ° C for 2 minutes and 95 ° C for 10 minutes, and then repeating the cycle of 95 for 15 seconds and 60 ° C for 60 seconds 40 times.

The standard cDNA was prepared by PCR using the primers 1 and 2 (SEQ ID NO: 32 and SEQ ID NO: 33) in the form of 200 xl using primers 1 and 2 (SEQ ID NO: 32 and SEQ ID NO: 33) with 50 pg of the plasmid pcDNA-catSLT obtained in Example 4. Done and created. The composition was performed using primers 1 and 2, 0.5 M, 2.5 mM MgCl ₂ , dNTP 0.2 niM, AmliTaq Gold (Pakinkin Elmer) 1/100 volume, and 10-fold concentrated Ampl iTaq Gold Buffer 1/10 volume. The reaction was incubated at 95 ° C for 10 minutes, and then repeated at 95 ° C for 15 seconds, 60 at 15 seconds, and 72 ° C for 10 seconds 40 times. The amplification product is purified from the reaction mixture using the PCR purification kit (Qiagen), the absorbance at 260 nm is measured, the concentration is calculated, the exact copy number is calculated, and the copy is diluted with distilled water. A standard cDNA solution of ⁷ copies of lxlO was prepared. Also,

TaqMan PCR probes and primers were designed using Primer Express (Version I.O) (PE Biosystems).

The expression level was calculated using ABI PRISM 7700 SDS software. The number of cycles at the moment when the fluorescence intensity of the reporter reaches the set value is plotted on the vertical axis. A standard curve was created using the logarithmic value of the initial concentration as the horizontal axis. The initial concentration of each reverse transcription product was calculated from the standard curve, and the expression amount of the cat SLT gene per 25 ng of total RNA at each site was determined. Further, the expression level of the feline GAP DH gene in each sample was determined using the primers # 3 (SEQ ID NO: 35), primer 4 (SEQ ID NO: 36) and the probe (Fam-ccaggagcgagatcccgcca-Tamura) shown in SEQ ID NO: 37. The expression of the feline SLT gene was corrected by the TaqMan: PCR method in the same manner as in the analysis of the SLT gene expression level.

As a result, the expression level of the feline SLT gene relative to the feline GAPDH gene was 0.084% in the frontal lobe, 0.32% in the temporal lobe, 0.046% in the occipital lobe, 0.056% in the parietal lobe, 0.086% in the pons, and 0.012% in the cerebellum. 0.0075% in medulla oblongata, 0.092% in hippocampus, 0.15% in amygdala, 0.020% in thalamus, 0.035% in corpus callosum, 0.016% in cerebral peduncle, 0.014% in spinal cord, 0.018% in substantia nigra, 0.039% in caudate nucleus In the hypothalamus, it was 0.012%. From the above, it was found that the feline SLT gene was particularly highly expressed in the frontal lobe, temporal lobe, pons, hippocampus, and amygdala in the cat brain.

A graph of the expression level of cat SLT in each part of the cat brain is shown in FIG. Example 1 1

Preparation of canine SLT animal cell expression vector

A plasmid into which a gene encoding the full-length amino acid sequence of canine SLT to which the sequence was confirmed in Example 7 and a Sal I recognition sequence was added on the 5 'side and a Spe I recognition sequence was added on the 3' side was added. E. coli was transformed with pcDNA-dogSLT. Plasmids were prepared from the E. coli clones using Plasmid Midi Kit (Qiagen) and cut with restriction enzymes Sal I and Spe I to cut out the insert. Insert DNA after electrophoresis, excised with a force Misori from Agarosugeru, then morselized, Fueno - Le extraction, phenol 'black hole Holm extraction, it is recovered by performing an ethanol precipitation _c Sal This insert DNA I and Spe Vector plasmid for animal cell expression pAKKO-111H (Biochim. Biophys. Acta, Vol. 1219, pp. 251-259, same vector plasmid as described in PAKK01.11H described in 1994, 1994) cut with T4 Using Takara Shuzo to construct the protein expression plasmid pAKKO-dogSLT. I built it. This 1¾0- (1 (½3 Escherichia coli 1 "01 ⁾ 10 competent cell (Invitrogen) was transformed to obtain Escherichia coli TOPlO / pAKKO-dogSLT.

The proteins, polynucleotides, antisense polynucleotides and antibodies of the present invention include, for example, anorexia (eg, anorexia nervosa), anemia or hypoproteinemia associated with anorexia, weak labor, lax hemorrhage, uterus It is useful as a marker for diagnosis of retro failure, milk stasis, etc. An agonist of the protein obtained by a screening method using the protein and the peptide of the present invention includes an appetite (feeding) enhancer, anorexia (eg, anorexia nervosa), anemia associated with anorexia, As a prophylactic / therapeutic agent for hypoproteinemia, weak labor, laxative bleeding, uterine remodeling failure, milk stasis, etc. Obesity, hyperplasmic obesity, pituitary obesity, hypoplasmic obesity, hypothyroid obesity, hypothalamic obesity, symptomatic obesity, childhood obesity, upper body obesity, dietary obesity, hypogonadism Obesity, systemic mastocytosis, simple obesity, central obesity, etc.), hyperphagia, affective disorders, sexual dysfunction, excessive labor, tonic contractions, fetal distress, uterine rupture , Vascular laceration, premature birth, Prader-Willi syndrome, diabetes and its complications (eg, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, etc.), hypertension, hyperlipidemia, coronary atherosclerosis, Prevention and treatment of gout, respiratory diseases (Pickwick syndrome, sleep apnea syndrome), fatty liver, infertility, osteoarthritis (especially antiobesity agents, appetite (feeding) regulators, etc.) It can be used as an agent.

Claims

The scope of the claims

1. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 4,, SEQ ID NO: 14 or SEQ ID NO: 24, or a salt thereof.

2. A protein comprising an amino acid sequence represented by SEQ ID NO: 4, or a salt thereof.

3. A protein comprising an amino acid sequence represented by SEQ ID NO: 14 or a salt thereof.

4. A protein comprising an amino acid sequence represented by SEQ ID NO: 24 or a protein thereof

■ ΙίΤΤΤ

5. A partial peptide of the protein according to claim 1, or a salt thereof.

6. A polynucleotide comprising the polynucleotide encoding the protein according to claim 1 or the partial peptide according to claim 5.

7. The polynucleotide according to claim 6, which is DNA.

8. The polynucleotide according to claim 7, which comprises the base sequence represented by SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23.

9. A recombinant vector containing the polynucleotide according to claim 8.

10. A transformant transformed with the recombinant vector according to claim 9.

11. The transformant according to claim 10 is cultured to produce and accumulate the protein according to claim 1 or the partial peptide according to claim 5, and to collect the protein. A method for producing the protein of claim 1 or the partial peptide of claim 5 or a salt thereof.

12. A medicament comprising the protein according to claim 1 or the partial peptide according to claim 5 or a salt thereof.

13. A medicine comprising the polynucleotide according to claim 6.

14. A diagnostic agent comprising the polynucleotide according to claim 6.

15. An antibody against the protein according to claim 1, the partial peptide according to claim 5, or a salt thereof.

16. A neutralizing antibody that inactivates the signal transduction of the protein according to claim 1. 16. The antibody according to claim 15, wherein

17. The antibody according to claim 15, which is an antibody that activates signal transduction of the protein according to claim 1.

18. A diagnostic agent comprising the antibody according to claim 15.

19. A pharmaceutical comprising the antibody according to claim 16.

20. A medicament comprising the antibody according to claim 17.

21. The protein according to claim 1 or the partial peptide according to claim 5, or a salt thereof, and a peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 25, or a salt thereof Wherein the peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25 and the protein or the salt thereof according to claim 1 are used. A method for screening a compound or a salt thereof that changes the binding property.

22. The protein according to claim 1 or the partial peptide according to claim 5, or a salt thereof, and a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25, or a peptide thereof. 2. A bond between the protein according to claim 1 and a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 25, characterized by containing a salt. A screening kit for a compound or a salt thereof that changes the sex.

23. The same or substantially the same as the amino acid sequence represented by SEQ ID NO: 25, which can be obtained by using the screening method according to claim 21 or the screening kit according to claim 22. A compound or a salt thereof, which alters the binding between a peptide or a salt thereof containing the amino acid sequence of claim 1 and the protein or a salt thereof according to claim 1.

24. The compound or a salt thereof according to claim 23, which is an agonist.

25. The compound or a salt thereof according to claim 23, which is an angonist.

26. A medicament comprising the compound according to claim 24 or a salt thereof.

27. A medicament comprising the compound according to claim 25 or a salt thereof.

28. An antisense polynucleotide comprising a nucleotide sequence complementary to the polynucleotide according to claim 6, or a part thereof.

29. A medicament comprising the antisense polynucleotide according to claim 28.

30. The method for quantifying mRNA of evening protein according to claim 1, wherein the polynucleotide according to claim 6 or a part thereof is used.

31. The method for quantifying a protein according to claim 1, wherein the antibody according to claim 15 is used.

32. A method for diagnosing a disease associated with the function of the protein according to claim 1, wherein the quantification method according to claim 30 or 31 is used.

33. The method for screening a compound or a salt thereof that changes the expression level of a protein according to claim 1, wherein the method for quantification according to claim 30 is used.

3 4. The method for screening a compound or a salt thereof that changes the mass of protein according to claim 1, wherein the compound or the salt thereof is used in a cell membrane, characterized by using the quantification method according to claim 30.

35. A compound or a salt thereof that changes the expression level of the protein according to claim 1, which can be obtained by using the screening method according to claim 33.

36. A compound or a salt thereof that alters the amount of the protein according to claim 1 in a cell membrane obtainable by using the screening method according to claim 34.

37. It comprises the compound according to claim 35 or claim 36 or a salt thereof.

38. A polynucleotide that hybridizes with the polynucleotide of claim 6 under high stringency conditions.

39. A non-human transgenic animal having exogenous DNA encoding the protein of claim 1 or a mutant DNA thereof.

40. Claim 12, Claim 13, Claim 20 which is a preventive / therapeutic agent for anorexia, anemia associated with anorexia, hypoproteinemia, faint labor, lax hemorrhage, uterine remodeling failure or milk stasis. Or the medicament according to claim 26.

4 1. Obesity, hyperphagia, affective disorders, sexual dysfunction, overwork labor, ankylosing uterine atrophy, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary The medicament according to claim 19, claim 27, or claim 29, which is a prophylactic and / or therapeutic agent for arteriosclerosis, gout, respiratory disease, fatty liver, infertility or osteoarthritis.

42. Anorexia, anemia associated with anorexia, hypoproteinemia, weak labor, and relaxation characterized by administering an effective amount of the compound according to claim 24 or a salt thereof to a mammal. Prevention and treatment of bleeding, uterine reconstruction failure or milk stasis.

43. An obesity, anorexia nervosa, affective disorder, sexual dysfunction, overwork, characterized by administering an effective amount of the compound according to claim 25 or a salt thereof to a mammal. Pain, ankylosing uterine contractions, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary atherosclerosis, gout, respiratory disease, fatty liver, infertility or deformed bone How to prevent and treat arthropathy.

44. The compound according to claim 24 or a compound thereof for producing a prophylactic or therapeutic agent for anorexia, anemia associated with anorexia, hypoproteinemia, weak labor, laxative bleeding, uterine remodeling failure or milk stasis. Use of salt.

4 5. Obesity, hyperphagia, affective disorders, sexual dysfunction, overwork labor, ankylosing uterine atrophy, preterm birth, Prader-Willi syndrome, diabetes and its complications, hypertension, hyperlipidemia, coronary Use of the compound according to claim 25 or a salt thereof for the manufacture of a prophylactic or therapeutic agent for arteriosclerosis, gout, respiratory disease, fatty liver, infertility or osteoarthritis.