JP2002335976A - New physiologically active peptide and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new peptide and to provide use thereof. SOLUTION: This invention provides a new peptide and a DNA encoding the peptide; medicines containing the peptide or the DNA; a screening method/a screening kit for a compound or its salt promoting or inhibiting the activity of the peptide; compounds or salts thereof obtained by the screening; medicines containing the compounds or salts thereof, and the like. The peptide and the DNA encoding the peptide are usable in, for example, diagnosing, treating and preventing digestive diseases, or the like. Moreover, the peptide is useful as a reagent for screening a compound or its salt promoting or inhibiting the activity of the peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to SEQ ID NO: 20.
Alternatively, a peptide or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 21, and SEQ ID NO: 1 or SEQ ID NO :, which is an orphan receptor protein, It contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21, and a protein or a salt thereof having the same or substantially the same amino acid sequence as the represented amino acid sequence. And a method for screening a compound or a salt thereof useful as a prophylactic / therapeutic agent for gastrointestinal diseases, which comprises using a peptide or a salt thereof characterized by the above.

[0002]

2. Description of the Related Art Main functions of maintenance of homeostasis of a living body, reproduction, development of an individual, metabolism, growth, nervous system, circulatory system, immune system, digestive system, metabolic system, sensory reception, and other important functions are regulated by: Cells receive various endogenous factors such as hormones and neurotransmitters, and sensory stimuli such as light and odor through specific receptors on the cell membranes of living organisms. It is done by reacting. Many of the hormone and neurotransmitter receptors involved in such function regulation are guanine nucleotide-bindin
It is characterized by being conjugated to g protein (hereinafter sometimes abbreviated as G protein) and transmitting a signal into cells by activation of the G protein to express various functions. In addition, these receptor proteins have seven transmembrane regions in common. From these facts, these receptors are G protein-coupled receptors or 7
Collectively referred to as transmembrane receptors. Thus, various hormones and neurotransmitters and their receptor proteins are known to exist and interact with each other to play an important role in the regulation of biological functions. (Such as neurotransmitters) and whether receptors for them are present are often still unknown. In recent years, the accumulation of sequence information by random sequencing of human genomic DNA or cDNA derived from various human tissues, and rapid advances in gene analysis techniques have led to the rapid elucidation of human genes. Along with that, the existence of many genes predicted to encode proteins of unknown function has been revealed. G protein-coupled receptors not only have seven transmembrane domains but also have many common sequences in their nucleic acids or amino acids, and are clearly classified as G protein-coupled receptors among such proteins. be able to. On the other hand, polymerase chain
Reaction (Polymerase Chain Reaction)
Such a G protein-coupled receptor gene has also been obtained by the (CR) method (Nature Cell Biolog).
y, Vol. 2, 703-708 (2000)). Among the G protein-coupled receptors thus obtained, some of them are subtypes having a high structural homology with known receptors, and their ligands can be easily predicted. Although, in most cases, the endogenous ligand is unpredictable, these receptors have no corresponding ligand found. For this reason, these receptors are called orphan receptors. Such unidentified endogenous ligands for orphan receptors may be involved in biological phenomena that have not been fully analyzed due to unknown ligands. When such ligands are associated with important physiological actions or disease states, the development of receptor agonists or antagonists is expected to lead to the creation of innovative drugs (Stadel, J . et al., T
iPS, 18, 430-437, 1997, Marchese, A. et a
l., TiPS, 20, 370-375, 1999, Civelli, O. et.
al., Brain Res., 848, 63-65, 1999). However, there are not so many examples of actually identifying ligands for orphan G protein-coupled receptors. Recently,
Several groups have attempted to search for ligands for such orphan receptors, and reports on the isolation and structure determination of ligands, which are new bioactive peptides. Re
Insheid et al. and Meunier et al. independently report on orphan G protein-coupled receptor LC132 or OR
The cDNA encoding L1 was introduced to express the receptor, and the response was used as an index to identify orphanin FQ or nocicep.
A novel peptide named tin was isolated from porcine or rat brain extracts and sequenced (Reinsheid,
RK et al., Science, 270, 792-794, 1995,
Meunier, J.-C. et al., Nature, 377, 532-535, 1
995). Although this peptide was reported to be involved in nociception, further studies in receptor knockout mice revealed that it was involved in memory (Ma
nabe, T. et al., Nature, 394, 577-581, 1998.
Year). After that, PrR was obtained by the same method as described above.
P (prolactin releasing peptide), orexin,
apelin, ghrelin and GALP (galani
Novel peptides such as n-like peptide have been isolated as ligands for orphan G protein-coupled receptors (Hi
numa, S. et al., Nature, 393, 272-276, 1998.
Year, Sakurai, T. et al., Cell, 92, 573-585, 199
8 years, Tatemoto, K. et al., Bichem. Biophys. Res. Co.
mmun., Volume 251, pp. 471-476, 1998, Kojima, M. et a
l., Nature, 402, 656-660, 1999, Ohtaki, T. e.
t al., J. Biol. Chem., 274, 37041-37045, 1999.
Year). On the other hand, a receptor for a physiologically active peptide, which has not been clarified, may be elucidated by a similar method. The motilin receptor involved in intestinal contraction is GP
R38 (Feighner, SD
et al., Science, 284, 2184-2188, 1999), and SLC-1 was identified as a receptor for melanin-concentrating hormone (MCH) (Chambers, J. et al., Nature, 4).
00, 261-265, 1999, Saito, Y. et al., Natur
e, 400, 265-269, 1999, Shimomura, Y. et al.,
Biochem. Biophys. Res. Commun., 261, 622-626,
1999, Lembo, PMC et al., Nature Cell Bio
l. 1, 267-271, 1999, Bachner, D. et al., FE
BS Lett., 457, 522-524, 1999), and GPR1
4 (SENR) was reported to be a receptor for urotensin II (Ames, RS et al., Nature, 401, 282-286).
, 1999, Mori, M. et al., Biochem. Biophys. Re.
s. Commun., 265, 123-129, 1999, Nothacker,
H.-P. et al., Nature Cell Biol., 1, 383-385, 1
999, Liu, Q. et al., Biochem. Biophys. Res. Comm
un., 266, 174-178, 1999). MCH has been shown to be involved in obesity because its knockout mice exhibit an emulsified phenotype (Shimada, M. et a.
L., Nature, 396, 670-674, 1998), and the identification of its receptor has made it possible to search for receptor antagonists that have potential as antiobesity agents. Also urot
It has also been reported that ensin II exerts a potent effect on the cardiovascular system by inducing cardiac ischemia by intravenous administration to monkeys (Ames, RS et al., Nature, 401, 2).
82-286, 1999). As described above, the orphan receptor and its ligand are often involved in a new physiological action, and the elucidation thereof is expected to lead to the development of a new drug. However, the search for ligands for orphan receptors involves many difficulties, and only a small number of orphan receptors have had their ligands revealed so far. . The present inventors have proposed a novel receptor ZAQ which is an orphan G protein-coupled receptor (a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 in the present specification: Hereinafter, in this specification, simply Z
(Sometimes referred to as AQ), but it has not been known what the ligand is.

[0003]

DISCLOSURE OF THE INVENTION Search for a ligand for ZAQ, an orphan receptor protein, and ZAQ
It has been an issue to establish a screening method for compounds and the like characterized by using such ligands.

[0004]

Means for Solving the Problems The present inventors have found that a substance having a ligand activity specific to ZAQ is present in a milk extract, separated the substance and determined its structure. Furthermore, a gene encoding a human peptide of the present active ingredient was found and expressed in animal cells. As a result, it was confirmed that a peptide substance that activates ZAQ-expressing cells was secreted into the culture supernatant. . The present inventors, based on such findings, using a screening system using ZAQ and ZAQ ligand peptide, ZAQ
It has been found that it is possible to screen for a therapeutic agent (such as a ZAQ antagonist or agonist, specifically a prophylactic / therapeutic agent for a gastrointestinal disease) for a disease mediated by the disease.

That is, the present invention relates to (1) SEQ ID NO: 2
0 or a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21 or a salt thereof, (2) the same or substantially the same amino acid as the amino acid sequence represented by SEQ ID NO: 21 The peptide according to the above (1), which is a peptide containing a sequence or a salt thereof, (3) the peptide according to the above (1) or a salt thereof containing an amino acid sequence represented by SEQ ID NO: 20, (4) SEQ ID NO: The peptide or the salt thereof according to the above (1), comprising the amino acid sequence represented by 21;
(5) The peptide according to (1) or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 22 or SEQ ID NO: 23; (1) a polynucleotide containing a polynucleotide encoding the peptide of (1), (7) a polynucleotide of (6) which is DNA, (8) a base sequence represented by SEQ ID NO: 26 or SEQ ID NO: 27 The DNA according to the above (7), comprising:
(9) The DNA according to the above (7), comprising the base sequence represented by SEQ ID NO: 28 or SEQ ID NO: 29, (10)
(6) a recombinant vector containing the polynucleotide according to (6), (11) a transformant transformed with the recombinant vector according to (10), and (12) culturing the transformant according to (11). And producing and accumulating the peptide according to (1), a method for producing the peptide according to (1) or a salt thereof, (13) an antibody against the peptide or salt thereof according to (1), 14) The peptide or a salt thereof according to the above (1) and SEQ ID NO: 1
A protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by the following formula (1): A method for screening for a compound or a salt thereof that alters the binding property to a protein or a salt thereof containing the same or substantially the same amino acid sequence as the represented amino acid sequence, (15) the peptide or the salt thereof according to the above (1), A peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, a partial peptide thereof, or a salt thereof; An amino acid identical or substantially identical to the amino acid sequence represented by No. 36 A method for screening a compound or a salt thereof which alters the binding property to a protein containing the sequence or a salt thereof, (16) a peptide or a salt thereof according to the above (1) and the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1 Identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 with the peptide or the salt thereof according to the above (1), characterized by containing a protein or a partial peptide thereof or a salt thereof, which contains the same amino acid sequence. A kit for screening a compound or a salt thereof that changes the binding property to a protein or a salt thereof, which contains a substantially identical amino acid sequence, (17) the peptide or a salt thereof according to (1) above and SEQ ID NO: 3
6. A peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by No. 6 or a partial peptide thereof or a salt thereof, and the peptide according to the above (1) or a salt thereof and SEQ ID NO: 36
(18) a screening method for a compound or a salt thereof which changes the binding property to a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 and the peptide or the salt thereof according to (1), which can be obtained by using the screening kit according to (16). Or a compound or a salt thereof that alters the binding to a salt thereof, (19) the peptide according to (1), which can be obtained using the screening method according to (15) or the screening kit according to (17). Or the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 36, or a salt thereof. A compound having an amino acid sequence or a salt thereof which alters the binding to a protein or a salt thereof, (20) a medicament comprising the compound of the above (18) or (19) or a salt thereof, The medicament according to the above (20), which is a prophylactic / therapeutic agent for a disease, (2)
2) a medicament comprising the peptide of (1) or a salt thereof, (23) a medicament of (22), which is a prophylactic / therapeutic agent for gastrointestinal diseases, and (24) a mammal, (14) The screening method according to (1) or (1) above.
6) A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a protein thereof, which can be obtained by using the screening kit according to the above (1) or a salt thereof. A method for preventing or treating gastrointestinal diseases, which comprises administering an effective amount of a compound that changes the binding property to a salt or a salt thereof, (25) a method for screening a mammal according to the above (15), A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, which can be obtained by using the screening kit according to (17) or the peptide or salt thereof according to (1). Or a compound capable of changing the binding to a salt thereof or an effective amount of a salt thereof, for preventing or treating gastrointestinal diseases. And (26) the peptide according to (1), which can be obtained by using the screening method according to (14) or the screening kit according to (16) for producing a prophylactic / therapeutic agent for a gastrointestinal disease. Or use of a compound having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a compound or a salt thereof, which alters the binding to a protein or a salt thereof, (27) digestive disorders And the peptide or salt thereof according to (1), which can be obtained by using the screening method according to (15) or the screening kit according to (17) for producing a prophylactic / therapeutic agent according to (1). Changes the binding to a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by Compound or a salt thereof is to provide a like.

Further, the present invention provides (28) SEQ ID NO:
An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21 is at least about 60% (preferably at least about 70%, More preferably about 80%
Or more preferably, about 85% or more, particularly preferably about 90% or more, and most preferably about 95% or more). No: 20 or SEQ ID NO:
An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 21 is one or more (preferably,
1 to 30 amino acids, more preferably about 1 to 20 amino acids), or 1 or 2
Or more (preferably about 1 to 40, more preferably about 1 to 30, especially preferably about 1 to 20)
Amino acid sequence having the amino acid added thereto, SEQ ID NO: 20
Or an amino acid sequence in which one or more (preferably about 1 to 30, more preferably about 1 to 20) amino acids in the amino acid sequence represented by SEQ ID NO: 21 have been substituted with other amino acids; Or the peptide or salt thereof according to the above (1), which is an amino acid sequence obtained by combining them;

(30) (i) A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof with the peptide of the above (1) or a salt thereof And (ii) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a peptide or a salt thereof described in (1) above or a test compound, or The screening method according to (14), wherein the comparison is performed with the case of contacting with a partial peptide or a salt thereof.
1) (i) Contacting the labeled peptide of the above (1) or a salt thereof with a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof And (ii) a protein or a part thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 in which the labeled peptide of (1) or a salt thereof and a test compound are labeled. A protein or a part thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 of the labeled peptide or the salt thereof according to the above (1) when brought into contact with the peptide or the salt thereof The above-mentioned (1), wherein the amount of binding to the peptide or its salt is measured and compared. (32) (A method for screening a compound or a salt thereof that changes the binding property to a protein or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with a peptide or a salt thereof, i) when the labeled peptide according to (1) or a salt thereof is brought into contact with a cell containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1,
(Ii) The labeled peptide of the above (1) or a salt thereof and a test compound were brought into contact with cells containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. In this case, the amount of the labeled peptide or salt thereof described in (1) or its salt bound to the cell is measured and compared, and the peptide or salt thereof described in (1) above is represented by SEQ ID NO: 1. A method for screening a compound or a salt thereof that changes the binding property to a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence, (33) (i) the labeled peptide according to (1) or The salt contains a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. And (ii) the labeled peptide of the above (1) or a salt thereof and the test compound are identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. Measuring the amount of binding of the labeled peptide according to (1) or a salt thereof to the membrane fraction of the cell when the peptide or the salt thereof is brought into contact with the membrane fraction of the cell containing the protein containing the sequence. A compound or a compound thereof which alters the binding property to a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with the peptide or the salt thereof according to the above (1). (34) (i) the labeled peptide of (1) or a salt thereof identical to the amino acid sequence represented by SEQ ID NO: 1 Or a protein expressed on the cell membrane of a transformant obtained by culturing a transformant transformed with a recombinant vector containing a DNA containing a DNA encoding a protein containing a substantially identical amino acid sequence A protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1; (1) when a transformant transformed with a recombinant vector containing a DNA containing a DNA encoding the transformant is brought into contact with a protein expressed on the cell membrane of the transformant, and Measuring the amount of binding of the peptide or a salt thereof to the protein, and comparing them, Screening for a compound or a salt thereof that changes the binding property to a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with the peptide or the salt thereof described in the above (1). (35) (i) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36 or a partial peptide thereof or a salt thereof with the peptide of the above (1) or a salt thereof; And (ii) a protein or a portion thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, or a peptide or salt thereof described in (1) above and a test compound. (15) The method according to the above (15), wherein the comparison is performed with the case where the peptide or the salt thereof is brought into contact. Method of screening,
(36) (i) converting the labeled peptide of the above (1) or a salt thereof to a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, a partial peptide thereof or a salt thereof; And (ii) a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, or (ii) a labeled peptide of the above (1) or a salt thereof and a test compound. A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36 of the labeled peptide of the above (1) or a salt thereof when brought into contact with a partial peptide or a salt thereof, or Measuring the amount of binding to a partial peptide or a salt thereof, and comparing 1) A method for screening a compound or a salt thereof, which changes the binding property to a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, or a salt thereof, (37) (i) The labeled peptide of the above (1) or a salt thereof was contacted with a cell containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36. And (ii) applying the labeled peptide of the above (1) or a salt thereof and a test compound to a cell containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36. The amount of binding of the labeled peptide or salt thereof according to the above (1) to the cell when contacted is Binding to the protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, and the peptide or the salt thereof described in (1) above, (38) (i) labeling the peptide or salt thereof according to (1) with the sequence of SEQ ID NO: 3
And (ii) the above-mentioned (1) in the case of contacting with a membrane fraction of a cell containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by (6).
The labeled peptide or a salt thereof and a test compound were labeled when they were brought into contact with the membrane fraction of a cell containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36. The peptide or salt thereof according to (1) or SEQ ID NO: 36, wherein the amount of binding of the peptide or salt thereof to the membrane fraction of the cells is measured and compared.
(39) (i) a method for screening for a compound or a salt thereof that alters the binding property to a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by the above (1). Or a salt thereof, transformed with a recombinant vector containing a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36. The case where the transformant was cultured and brought into contact with the protein expressed on the cell membrane of the transformant, and (ii) the labeled peptide of the above (1) or a salt thereof and the test compound are represented by SEQ ID NO: 36. DNA encoding a protein containing the same or substantially the same amino acid sequence as The labeled peptide or the salt thereof according to the above (1), when the transformant transformed with the recombinant vector containing the DNA is brought into contact with a protein expressed on the cell membrane of the transformant. Containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36 with the peptide or salt thereof according to (1) above, wherein the amount of binding to the protein is measured and compared. Screening method for a compound or a salt thereof that changes the binding property to a protein or a salt thereof

(40) (i) the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 1 and (i) the peptide or salt thereof described in (1), which can be obtained by the screening method described in (30) to (34) above; A compound or a salt thereof, which alters the binding property to a protein or a salt thereof containing the same amino acid sequence;
(I) The peptide or salt thereof according to (1), which can be obtained by the screening method according to 9), and SEQ ID NO: 36
Or a salt thereof, which changes the binding property to a protein or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence represented by the formula (42); Medicines containing salts,

(43) The screening kit according to (16), which comprises a cell containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. , (4
4) The screening kit according to the above (16), which comprises a membrane fraction of a cell containing a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. (45) a transformant transformed with a recombinant vector containing a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1; The screening kit according to the above (16), which contains a protein expressed in the cell membrane of the transformant by culturing, (46) identical or substantially the same as the amino acid sequence represented by SEQ ID NO: 36 (17) The above (17), which comprises a cell containing a protein containing the same amino acid sequence. Screening kit, (47)
The screening kit according to the above (17), which comprises a membrane fraction of a cell containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, 48) culturing a transformant transformed with a recombinant vector containing a DNA containing a DNA encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36 The screening kit according to the above (17), which further comprises the protein expressed in the cell membrane of the transformant, and (49) the screening kit according to the above (43) to (45). And the amino acid sequence represented by SEQ ID NO: 1 and the peptide according to (1) or a salt thereof. Substantially compound or its salt that changes the binding property between a protein or its salt containing the same amino acid sequence, (50) the (46) - (4
8) A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36 or a protein thereof, which can be obtained by using the screening kit according to the above (1) or a salt thereof. A compound or a salt thereof that changes the binding property to a salt, (5
1) a medicament containing the compound of (49) or (50) or a salt thereof, (52) a medicament of (42) or (51), which is a prophylactic or therapeutic agent for a gastrointestinal disease.

(53) A method for quantifying a peptide or a salt thereof according to (1), wherein the antibody according to (13) is brought into contact with the peptide or salt thereof according to (1). 54) The antibody according to (13), and a test solution and a labeled peptide or salt thereof according to (1) are competitively reacted with each other, and the labeled (1) bound to the antibody is reacted. A method for quantifying the peptide or the salt thereof according to the above (1) in the test solution, which comprises measuring a ratio of the peptide or the salt thereof according to the above (11); ) And the labeled antibody according to (11) are reacted simultaneously or successively, and then the activity of the labeling agent on the insolubilized carrier is measured. The peptide according to the above (1) or a salt thereof Determination, (55) (7), wherein the DNA high stringent polynucleotide hybridizing under conditions, (56) DN of the (7), wherein
A polynucleotide containing a base sequence complementary to the base sequence of A or a part thereof, (57) containing a base sequence complementary to the base sequence represented by SEQ ID NO: 28 or SEQ ID NO: 29 or a part thereof (58) the peptide of (1) or a salt thereof and the amino acid sequence represented by SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49 Or a protein or a partial peptide thereof or a salt thereof containing substantially the same amino acid sequence, characterized by using SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: No .: 48 or a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49, or A method of screening a compound or its salt that alters the binding property between salt, (59) the (1), wherein the peptide or a salt thereof and SEQ ID NO: 4
0, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, characterized by containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence, a partial peptide thereof, or a salt thereof. A protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49 with the peptide according to (1) or a salt thereof Or a screening kit for a compound or a salt thereof that changes the binding property to the salt thereof, (60) the screening method described in the above (58) or the (1) which can be obtained using the screening kit described in the above (59). ) And the salts thereof and SEQ ID NO: 40, SEQ ID NO: 4
7, a compound having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 48 or SEQ ID NO: 49 or a compound capable of changing the binding property to a salt thereof, or a salt thereof, (61) (62) a medicament comprising the compound or salt thereof according to (60), (62) a medicament according to (61), which is a prophylactic / therapeutic agent for digestive diseases, (63) an amino acid sequence represented by SEQ ID NO: 34, Peptides containing the same or substantially the same amino acid sequence or salts thereof and SEQ ID NO: 1, SEQ ID NO: 3
6, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48
Alternatively, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49, a partial peptide thereof, or a salt thereof is used, and the amino acid sequence represented by SEQ ID NO: 34 is used. SEQ ID NO: 1, SEQ ID NO: 3 with a peptide containing the same or substantially the same amino acid sequence or a salt thereof
6, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48
Alternatively, a method for screening for a compound containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49 or a compound capable of changing the binding to a salt thereof or a salt thereof, (64) SEQ ID NO: 34
Or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or the sequence An amino acid sequence identical to or substantially identical to the amino acid sequence represented by SEQ ID NO: 34, comprising a protein having the same or substantially the same amino acid sequence as that represented by SEQ ID NO: 49, a partial peptide thereof, or a salt thereof. Or an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49 with a peptide containing a substantially identical amino acid sequence or a salt thereof Compounds that alter the binding to proteins or salts thereof that contain the same or substantially the same amino acid sequence as Or a salt thereof, or (65) the same or the same as the amino acid sequence represented by SEQ ID NO: 34, which can be obtained by using the screening method described in (63) or the screening kit described in (64). SEQ ID NO: 1, SEQ ID NO: 3 to a peptide containing a substantially identical amino acid sequence or a salt thereof
6, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48
Or a compound or a salt thereof which changes the binding property to a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49, (66) the compound according to the above (65) or (67) The medicament comprising the salt, (67) the medicament according to (66), which is a prophylactic / therapeutic agent for gastrointestinal diseases.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION A peptide or a salt thereof according to the present invention is a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a salt thereof (hereinafter referred to as a protein of the present invention). (Sometimes abbreviated) or a salt thereof, and a peptide or a salt thereof capable of binding and activating the protein of the present invention. Further, the peptide of the present invention or a salt thereof has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49. And a peptide or a salt thereof capable of binding to a protein or a salt thereof, and a peptide or a salt thereof capable of binding and activating the protein. Particularly preferably, the peptide of the present invention or a salt thereof contains the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21, and binds to the protein of the present invention; A peptide or a salt thereof, which has the ability to activate. The ability of the peptide of the present invention or a salt thereof to bind to the protein of the present invention and to activate the protein of the present invention can be measured by the methods described below. Hereinafter, the peptide of the present invention or a salt thereof may be abbreviated as the peptide of the present invention.

The protein (G protein-coupled receptor protein) of the present invention is identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 (the amino acid sequence in FIG. 1 to FIG. 3 or FIG. 4 to FIG. 6). It is a receptor protein containing the same amino acid sequence (hereinafter, the protein of the present invention or a salt thereof may be abbreviated as the protein of the present invention).

The peptide of the present invention and the protein of the present invention may be, for example, humans or mammals (eg, guinea pigs,
Rats, mice, rabbits, pigs, sheep, cattle, monkeys, etc., all cells (eg, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells) 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, osteocytes, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells) and blood cells (For example, MEL, M1, CTLL
-2, HT-2, WEHI-3, HL-60, Josk
-1, K562, ML-1, MOLT-3, MOLT-
4, MOLT-10, CCRF-CEM, TALL-
1, Jurkat, CCRT-HSB-2, KE-3
7, SKW-3, HUT-78, HUT-102, H
9, U937, THP-1, HEL, JK-1, CM
K, KO-812, MEG-01), or any tissue in which those cells are present, for example, the brain, each part of the brain (eg, olfactory bulb, squamous nucleus, basal sphere, hippocampus, thalamus, hypothalamus, Subthalamic nucleus, cerebral cortex, medulla, cerebellum, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary, stomach, pancreas, 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, peripheral blood cells, prostate, testicle, testis, ovary, placenta, uterus, It may be a peptide / protein derived from bone, joint, skeletal muscle, etc. (particularly, brain or various parts of the brain), or may be a synthetic peptide / synthetic protein. When the peptide of the present invention or the protein of the present invention has a signal sequence, the peptide or protein can be efficiently secreted extracellularly.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21 includes, for example, SEQ ID NO: 20 or SEQ ID NO: 21
And about 60% or more (preferably about 70% or more, more preferably about 80% or more, more preferably about 85% or more, particularly preferably about 90% or more, and most preferably about 95% or more. ) And the like. Examples of the peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21 include, for example, a peptide substantially identical to the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21 And a peptide having substantially the same properties as the peptide having the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21. Hereinafter, a peptide containing the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21 may be described as a humanized ZAQ ligand mature peptide.

The peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21 includes, for example, the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21. Contains the same or substantially the same amino acid sequence as
Peptides having substantially the same activity as the peptide having the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21 are preferred, and specifically, SEQ ID NO: 2
0, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO:
And a peptide containing the amino acid sequence represented by No. 23. Hereinafter, SEQ ID NO: 22 or SEQ ID NO: 2
A peptide containing the amino acid sequence represented by 3 may be described as a human ZAQ ligand precursor peptide. Substantially the same activity includes, for example, a binding activity to the protein of the present invention, a signal transduction action via the protein of the present invention, and a gastrointestinal (eg, intestinal) contraction activity. Substantially the same indicates that their activities are the same in nature. Therefore, it is preferable that the activities such as the binding activity to the protein of the present invention, the signal information transduction via the protein of the present invention, and the gastrointestinal contraction activity are equal (eg, about 0.5 to 2 times). Quantitative factors such as the degree of activity and the molecular weight of the peptide may be different. These activities can be measured according to a known method, but can also be measured according to, for example, a screening method described below.

The peptide of the present invention may be one or more (preferably about 1 to 30, more preferably 1 to 20) in the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21. Amino acid sequence, SEQ ID NO: 20 or SEQ ID NO:
An amino acid sequence in which one or more (preferably about 1 to 40, more preferably about 1 to 30, and particularly preferably about 1 to 20) amino acids have been added to the amino acid sequence represented by 21; One or more (preferably about 1 to 30, more preferably 1 to 2) in the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21
A peptide containing an amino acid sequence in which (about 0) amino acids are substituted with another amino acid, or an amino acid sequence obtained by combining them is also used. The peptides of the present invention are preferably human or non-human mammals, more preferably human-derived peptides.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 90% or more, preferably about 95% or more, more preferably about 90% or more of the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequences having about 98% or more homology, and the like. Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1; A protein having substantially the same properties as the protein containing the amino acid sequence represented by 1 is preferred. SEQ ID NO: 1 of the present invention
As a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by, for example,
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 and having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferred. Examples of substantially the same activity include a ligand binding activity and a signal transduction action. Substantially the same indicates that their activities are the same in nature. Therefore, it is preferable that the activities such as the ligand binding activity and the signal transduction activity 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 different. You may. The activity such as the ligand binding activity and signal information transduction can be measured according to a known method. For example, the activity can be measured according to a determination method or a screening method described later. Hereinafter, a protein containing the amino acid sequence represented by SEQ ID NO: 1 may be referred to as ZAQ. Further, as the protein of the present invention, one or two or more (preferably about 1 to 30, more preferably about 1 to 10) in the amino acid sequence represented by SEQ ID NO: 1,
More preferably, an amino acid sequence in which several (1 or 2) amino acids have been deleted, and 1 or 2 or more (preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1.
Amino acid sequence to which about 1, more preferably about 1 to 10, and still more preferably several (one or two) amino acids are added, and one or more (preferably Is an amino acid sequence in which about 1 to 30, more preferably about 1 to 10, and still more preferably several (one or two) amino acids are substituted with another amino acid, or an amino acid sequence obtained by combining them. Contained proteins may also be used.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 36 includes, for example, about 90% or more of the amino acid sequence represented by SEQ ID NO: 36;
Preferably, the amino acid sequence has about 95% or more, more preferably about 98% or more homology. As a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 36, for example,
A protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 36 and having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 36 is preferred. SEQ ID NO: 3
Examples of the protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 6 include the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, :
A protein having substantially the same activity as the protein containing the amino acid sequence represented by No. 36 is preferred, and specific examples include the protein described in WO 98/46620. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 40 includes, for example, about 9 amino acids as the amino acid sequence represented by SEQ ID NO: 40.
Amino acid sequences having a homology of 7% or more, preferably about 98% or more, more preferably about 99% or more, and most preferably about 99.5% or more. SEQ ID NO: 4
As a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 0, for example, a protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 40 and represented by SEQ ID NO: 40 Preferred are proteins having substantially the same properties as the protein containing the amino acid sequence to be used. Examples of the protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 40 include the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 40. And a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 40. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 47 includes, for example, about 95% or more, preferably about 96%, of the amino acid sequence represented by SEQ ID NO: 47
Above, more preferably about 97% or more, most preferably about 98% or more amino acid sequence having homology. As a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 47, for example, a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 47; 4
A protein having substantially the same properties as the protein containing the amino acid sequence represented by 7 is preferred.
Examples of the protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 47 include the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 47. ,
A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 47 is preferred. The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 48 includes, for example, about 95% or more of the amino acid sequence represented by SEQ ID NO: 48;
Preferably, the amino acid sequence has about 96% or more, more preferably about 97% or more, and most preferably about 98% or more homology. Examples of the protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 48 include, for example, a protein containing the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48; A protein having substantially the same properties as the protein containing the amino acid sequence represented by No. 48 is preferred. Examples of the protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48 include, for example, the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48. , A protein having an activity substantially the same as the protein containing the amino acid sequence represented by SEQ ID NO: 48, and the like. Specifically, Biochem.
Acta, Vol. 1491, pages 369-375, 2000, and the like. As the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 49, for example, about 95% or more, preferably about 96% or more, more preferably about 97% of the amino acid sequence represented by SEQ ID NO: 49 As described above, most preferably, an amino acid sequence having about 98% or more homology is exemplified. SEQ ID NO:
As a protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 49, for example, a protein containing the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 49 and represented by SEQ ID NO: 49 Preferred are proteins having substantially the same properties as the protein containing the amino acid sequence to be used. Examples of the protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49 include, for example, the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49. And proteins having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 49, and specific examples thereof include the proteins described in WO 98/46620. Examples of substantially the same activity include a ligand binding activity and a signal transduction action. Substantially the same indicates that their activities are the same in nature. Therefore, activities such as ligand binding activity and signal transduction are equivalent (eg, about 0.5 to 2).
Fold), but the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different. The activities such as the ligand binding activity and the signal transduction action can be measured according to known methods.
The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 34 is about 60% or more, preferably about 70% or more, more preferably about 80% or more as the amino acid sequence represented by SEQ ID NO: 34 , Most preferably about 9
Amino acid sequences having 0% or more homology are exemplified. Examples of the peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 34 include the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 34 , An activity substantially equivalent to that of the peptide containing the amino acid sequence represented by SEQ ID NO: 34 (eg, ileal contraction,
Peptides having a distal colon colon contracting action, a proximal colon relaxing action, etc.) are preferred, and specific examples include MIT1 described below.

In the present specification, peptides and proteins have N-terminal (amino terminal) at the left end and C-terminal (carboxyl terminal) at the right end according to the convention of peptide labeling.
The proteins of the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, have carboxyl groups (-COOH) and carboxylate (-
COO ⁻ ), amide (—CONH ₂ ) or ester (−
COOR). Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl and cyclohexyl, for example, phenyl A C _6-12 aryl group such as α-naphthyl, for example, a phenyl-C _1-2 alkyl group such as benzyl and phenethyl or α
In addition to a C _7-14 aralkyl group such as an α-naphthyl-C _1-2 alkyl group such as -naphthylmethyl, a pivaloyloxymethyl group widely used as an oral ester and the like are used. When the peptide / protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminal, those in which the carboxyl group is amidated or esterified are also included in the peptide / protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. Furthermore, the peptide protein of the invention, C ₁ of the peptide protein as described above, an amino group protecting group of the N-terminal methionine residue (e.g., formyl group, C _2-6 alkanoyl group such as acetyl group _-6 acyl group), a glutamyl group formed by cleavage of the N-terminal side in vivo and pyroglutamine oxidation, a substituent on the side chain of an amino acid in the molecule (eg, -OH,- SH, an amino group, an imidazole group, an indole group, a guanidino group, etc. are suitable protecting groups (for example, C, such as formyl group and acetyl group).
Protected by a C _1-6 acyl group such as a _2-6 alkanoyl group) or a complex peptide or a complex protein such as a so-called glycopeptide or glycoprotein having a sugar chain bonded thereto. Specific examples of the peptide of the present invention include, for example, a human-derived (more preferably human brain-derived) peptide containing the amino acid sequence represented by SEQ ID NO: 20, or an amino acid sequence represented by SEQ ID NO: 21 Peptides derived from humans (more preferably from human brain) and the like can be mentioned. More preferably, a human-derived peptide containing the amino acid sequence represented by SEQ ID NO: 21 can be mentioned. Specific examples of the protein of the present invention include, for example, a human-derived (more preferably human-derived) protein containing the amino acid sequence represented by SEQ ID NO: 1.

The partial peptide of the protein of the present invention (hereinafter sometimes abbreviated as the partial peptide of the present invention) may be any partial peptide of the protein of the present invention described above. For example, among the protein molecules of the present invention, those which are exposed outside the cell membrane and have substantially the same ligand binding activity are used. Specifically, the partial peptide of the protein containing the amino acid sequence represented by SEQ ID NO: 1 was analyzed as an extracellular region (hydrophilic site) in the hydrophobicity plot analysis shown in FIG. Is a peptide comprising Hydrophobic (Hy
A peptide partially containing a drophobic) site can also be used. A peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used. The number of amino acids of the partial peptide of the present invention,
Peptides containing at least 20 or more, preferably 50 or more, more preferably 100 or more amino acid sequences of the constituent amino acid sequences of the protein of the present invention are preferred. A substantially identical amino acid sequence is
More than about 50%, preferably about 70%
%, More preferably about 80% or more, even more preferably about 90% or more, and most preferably about 95% or more homology. Here, “substantially the same ligand binding activity” has the same meaning as described above. The “substantially the same ligand binding activity” can be measured according to a known method.

The partial peptide of the present invention may comprise one or more (preferably 1 to 10)
About, more preferably several (1 or 2) amino acids are deleted, or 1 or 2 or more (preferably about 1 to 20, more preferably 1 to 10) in the amino acid sequence thereof Degree, more preferably several (1 or 2
)) Or 1 or 2 or more (preferably about 1 to 10)
More preferably, about 1 to 5, more preferably several (one or two) amino acids may be substituted with another amino acid. In the partial peptide of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used. further,
Similar to the above-described protein of the present invention, the partial peptide of the present invention includes a peptide in which the amino group of the N-terminal methionine residue is protected by a protecting group, and a Gln formed by cleavage of the N-terminal side in vivo. Glutamine-oxidized products, those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those in which a sugar chain is bonded, such as a so-called glycopeptide, are also included. SEQ ID NO: 36,
The partial peptide of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49 is the aforementioned SEQ ID NO: 36 , SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49 as long as it is a partial peptide of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence. For example, a protein molecule that is exposed outside the cell membrane and has substantially the same ligand binding activity among protein molecules may be used. The number of amino acids in the partial peptide is preferably a peptide containing at least 20 or more, preferably 50 or more, more preferably 100 or more amino acid sequences among the constituent amino acid sequences of the protein. A substantially identical amino acid sequence is defined as about 50% or more, preferably about 70% or more, more preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more, of these amino acid sequences. 1 shows an amino acid sequence having homology.

The salt of the peptide of the present invention or the protein of the present invention or a partial peptide thereof is preferably a physiologically acceptable acid addition salt. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,
For example, salts with methanesulfonic acid and benzenesulfonic acid) are used.

The peptide or protein of the present invention or a salt thereof, and a protein or a salt thereof having the amino acid sequence represented by SEQ ID NO: 36, SEQ ID NO: 40 or SEQ ID NO: 47 can be prepared from the above-mentioned human or mammal. It can be produced from cells or tissues by a known peptide / protein purification method, and is represented by the peptide of the present invention, the protein of the present invention, SEQ ID NO: 36, SEQ ID NO: 40 or SEQ ID NO: 47 described below. It can also be produced by culturing a transformant containing a DNA encoding a protein containing an amino acid sequence. Further, it can also be produced by a peptide / protein synthesis method described below or according to the method. Further, a protein containing the amino acid sequence represented by SEQ ID NO: 48 or a salt thereof is described in Biochem. Biophys. Acta, Vol.
It can be produced according to the method described on pages 9-375, 2000. The protein containing the amino acid sequence represented by SEQ ID NO: 49 or a salt thereof can be produced according to the method described in WO 98/46620. When producing from human or mammalian tissue or cells, the homogenized human or mammalian tissue or cells are extracted with an acid or the like, and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.

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 peptide / protein synthesis can be used. Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin,
Methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin,
-(2 ', 4'-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target peptide / protein according to various known condensation methods. At the end of the reaction, the peptide / protein is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target peptide / protein or an amide thereof. For the condensation of the protected amino acids described above, various activating reagents that can be used for peptide / protein synthesis can be used, and carbodiimides are particularly preferable.
As the carbodiimides, DCC, N, N'-diisopropylcarbodiimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide and the like are used. Activation by these involves adding a protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt), or symmetrical acid anhydride or HOB.
The protected amino acid can be added as a t-ester or HOOBt ester to the resin after activation of the protected amino acid in advance.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the peptide-protein condensation reaction. For example, N, N-dimethylformamide, N, N
Acid amides such as -dimethylacetamide and N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; sulfoxides such as dimethylsulfoxide;
Ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof are used. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually about -20 ° C to 50 ° C.
Is appropriately selected from the range. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protecting group for the amino group of the starting material include Z, Boc, tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, Trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used. The carboxyl group may be, for example, a linear, branched or cyclic alkyl ester such as an alkyl esterified ester (eg, methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). Aralkyl esterification (eg, benzyl ester, 4-
Nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary 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 the esterification, for example, a lower alkanoyl 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 and an ethoxycarbonyl group, and the like are used.
Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group. As a protecting group for the phenolic hydroxyl group of tyrosine,
For example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like are used.
As a protecting group for imidazole of histidine, for example,
Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bu
m, Boc, Trt, Fmoc, etc. are used.

Examples of the activated carboxyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N
-Ester with hydroxyphthalimide, HOBt)]
Are used. As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used. As a method for removing (eliminating) the protecting group, for example, Pd
-Catalytic reduction in a stream of hydrogen in the presence of a catalyst such as black or Pd-carbon, or acid with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof. Treatment, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, and the like, reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the above-mentioned acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol,
It is effective to add a cation scavenger such as paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like. Further, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of the functional group which should not be involved in the reaction of the raw materials, the removal of the protective group, and the activation of the functional group involved in the reaction are performed by known means. The protective group used for protecting the functional group is known. Each is appropriately selected from the groups and applied. As another method for obtaining an amide form of a peptide / protein, for example, first, after amidating and protecting the α-carboxyl group of the carboxy terminal amino acid, after extending the peptide chain to a desired chain length on the amino group side, A peptide / protein in which only the protecting group of the N-terminal α-amino group of the peptide chain is removed and a peptide / protein in which only the protecting group of the C-terminal carboxyl group is removed are produced, The condensation is carried out 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 using various known purification means, and the main fraction is freeze-dried to obtain a desired peptide / protein amide. To obtain an ester of a peptide or protein, for example,
After condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the desired peptide / protein ester can be obtained in the same manner as the peptide / protein amide.

The peptide of the present invention can be produced according to a known peptide synthesis method. The partial peptide of the protein of the present invention or a salt thereof can be produced according to a known peptide synthesis method, 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 or the partial peptide or amino acid that can constitute the protein of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide. it can. Known methods for condensation and elimination of protecting groups include, for example, the methods described in (1) to (4) below. M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptide,
Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd.
(1975) Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemistry 1, Protein Chemistry IV, 205, (1977) Supervision of Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten The peptide of the present invention or the partial peptide of the present invention can be purified and isolated by a combination of purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the 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, and conversely, when it is obtained in a salt form, it can be converted to a free form by a known method. can do.

The DNA encoding the peptide / protein of the present invention may be any DNA containing the above-described nucleotide sequence encoding the peptide / protein of the present invention. In addition, genomic DNA, genomic DNA library, cDN derived from the aforementioned cells and tissues
A, a cDNA library derived from the cells and tissues described above,
Any of synthetic DNAs may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse transcriptase Po is directly used by preparing a total RNA or mRNA fraction from the cells and tissues described above.
It can also be amplified by a lymerase Chain Reaction (hereinafter abbreviated as RT-PCR method).

Specifically, as the DNA encoding the peptide of the present invention, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 26 or SEQ ID NO: 27, or SEQ ID NO: 26 or SEQ ID NO: 27 Contains DNA that hybridizes under high stringent conditions with DNA containing the nucleotide sequence represented by the following formula, and has substantially the same activity as the peptide of the present invention (eg, binding activity to the protein of the present invention, Any DNA may be used as long as it encodes a peptide having a signal transduction action via a protein and a gastrointestinal (eg, intestinal) contraction activity. Examples of the DNA containing the nucleotide sequence represented by SEQ ID NO: 26 include a DNA containing the nucleotide sequence represented by SEQ ID NO: 28. SEQ ID NO: 26
As a DNA that hybridizes with a DNA containing the nucleotide sequence represented by the following under stringent conditions, for example, about 60% or more, preferably about 70% or more, and more preferably the nucleotide sequence represented by SEQ ID NO: 26 Is a DN containing a nucleotide sequence having about 80% or more homology.
A or the like is used. Examples of the DNA containing the nucleotide sequence represented by SEQ ID NO: 27 include a DNA containing the nucleotide sequence represented by SEQ ID NO: 29. DN that hybridizes under high stringent conditions with DNA containing the base sequence represented by SEQ ID NO: 27
As A, for example, a DNA containing a base sequence having homology of about 60% or more, preferably about 70% or more, more preferably about 80% or more with the base sequence represented by SEQ ID NO: 27 is used. .

Further, D encoding the protein of the present invention
As NA, for example, SEQ ID NO: 2 or SEQ ID NO:
A protein of the present invention comprising a DNA containing the nucleotide sequence represented by SEQ ID NO: 3 or a DNA that hybridizes under high stringent conditions to a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 Encoding a protein having substantially the same activity (eg, ligand binding activity, signal transduction action, etc.)
Any one may be used as long as it is A. DN that hybridizes under high stringent conditions with DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3
As A, for example, SEQ ID NO: 2 or SEQ ID NO: 3
About 90% or more, preferably about 9%
DNA containing a base sequence having a homology of 5% or more, more preferably about 98% or more is used.

Hybridization is carried out by a known method or a method analogous thereto, for example, Molecular Cloning 2nd (J. Sambrook et al.).
al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is 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.
In mM, the temperature is about 50-70 ° C, preferably about 60-6
The condition at 5 ° C. is shown. In particular, when the sodium concentration is about 19 mM
Most preferably, the temperature is about 65 ° C.

More specifically, DN encoding a peptide containing the amino acid sequence represented by SEQ ID NO: 20
A includes a DNA containing the nucleotide sequence represented by SEQ ID NO: 26, and a DNA encoding a peptide containing the amino acid sequence represented by SEQ ID NO: 21 includes a nucleotide sequence represented by SEQ ID NO: 27 The DNA encoding the peptide having the amino acid sequence represented by SEQ ID NO: 22 is exemplified by a DNA containing the base sequence represented by SEQ ID NO: 28.
NA, and the DNA encoding a peptide containing the amino acid sequence represented by SEQ ID NO: 23 includes:
DNA containing the base sequence represented by SEQ ID NO: 29
Is raised. Examples of the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 include a DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.

A nucleotide (oligonucleotide) containing a nucleotide sequence complementary to the nucleotide sequence of the DNA encoding the peptide of the present invention or a part thereof is not limited to including the DNA encoding the peptide of the present invention. Not
It is used to include RNA. According to the present invention, an antisense (oligo) nucleotide (nucleic acid) capable of inhibiting the replication or expression of the gene of the peptide of the present invention is cloned or determined. It can be designed and synthesized based on nucleotide sequence information. Such (oligo) nucleotides (nucleic acids) can hybridize with the RNA of the gene of the peptide of the present invention, inhibit the synthesis or function of the RNA, or interact with the peptide-related RNA of the present invention. Through the action, the expression of the gene of the peptide of the present invention can be regulated and controlled. (Oligo) nucleotides complementary to the selected sequence of the peptide-related RNA of the present invention, and (oligo) nucleotides capable of specifically hybridizing with the peptide-related RNA of the present invention, can be used in vivo and in vitro. It is useful for regulating and controlling the expression of the gene of the peptide of the present invention, and is also useful for treating or diagnosing diseases and the like. The term "corresponds"
The term means having homology or being complementary to a specific nucleotide, nucleotide sequence or nucleic acid sequence including a gene. "Corresponding" between a nucleotide, a base sequence or a nucleic acid and a peptide means a nucleotide (nucleic acid)
Or the amino acids of the peptide in the instructions derived from its complement. 5′-end hairpin loop of the gene of the peptide of the present invention, 5′-end 6-base pair
Repeat, 5'-end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon,
The 3′-end untranslated region, the 3′-end palindrome region, and the 3′-end hairpin loop can be selected as preferred target regions, but any region in the gene of the peptide of the present invention can be selected as the target.

The relationship between the target nucleic acid and the (oligo) nucleotide complementary to at least a part of the target region, ie, the relationship between the target nucleic acid and the (oligo) nucleotide capable of hybridizing with the target is referred to as "antisense". be able to. Antisense (oligo) nucleotides
Polydeoxynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or having a non-nucleotide backbone Other polymers (eg,
Commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that the polymers are arranged in a manner that permits base pairing or base attachment as found in DNA or RNA) Containing nucleotides). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can also be unmodified polynucleotides or oligonucleotides, as well as known modifications. Such as labeled, capped, methylated, substituted for one or more naturally occurring nucleotides with analogs, Done, such as uncharged bonds (eg,
Those having methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc., those having a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as proteins (nucleases, nuclease inhibitors) , Toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (eg, monosaccharides), etc., or those having an interactive compound (eg, acridine, psoralen, etc.) ,
Chelating compounds (eg, metals, radioactive metals,
It may be one containing boron, an oxidizing metal, or the like, one containing an alkylating agent, or one having a modified bond (for example, α-anomeric nucleic acid). Here, "nucleoside", "nucleotide" and "nucleic acid" include not only purine and pyrimidine bases, but also
It may include those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
The modified nucleotides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or converted to functional groups such as ethers, amines, etc. May have been.

The antisense nucleic acid of the present invention comprises RNA, D
NA or modified nucleic acid. Specific examples of modified nucleic acids include, but are not limited to, sulfur derivatives and thiophosphate derivatives of nucleic acids, and those that are resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Minimize the toxicity of sense nucleic acids. Thus, many modifications are known in the art, for example, J. Kawakami et al., Pharm
Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395,
1992; ST Crooke et al. Ed., Antisense Research
and Applications, CRC Press, 1993. The antisense nucleic acid of the present invention may contain altered or modified sugars, bases or bonds, may be provided in a special form such as liposomes or microspheres, may be applied by gene therapy, It could be provided in an added form. Thus, in the form of addition, polycations such as polylysine which acts to neutralize the charge of the phosphate skeleton, lipids which enhance the interaction with the cell membrane or increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond.
Other groups include cap groups specifically arranged at the 3 'end or 5' end of nucleic acids, which are for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of an antisense nucleic acid 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 a protein. The nucleic acid can be applied to cells by various known methods.

DNA encoding the partial peptide of the present invention
May be any as long as it contains a base sequence encoding the above-described partial peptide of the present invention. In addition, any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells / tissues, cDNA library derived from the aforementioned cells / tissues, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, using the mRNA fraction prepared from the cells and tissues described above,
Transcriptase Polymerase Chain Reaction (hereinafter, RT
-PCR method). Specifically, D encoding the partial peptide of the present invention
As NA, for example, SEQ ID NO: 2 or SEQ ID NO:
DNA containing a partial base sequence of DNA containing the base sequence represented by SEQ ID NO: 3, or DNA hybridizing under high stringent conditions with DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3
And a DNA containing a partial nucleotide sequence of a DNA encoding a protein having substantially the same activity (eg, ligand binding activity, signal transduction activity, etc.) as the protein peptide of the present invention. SEQ ID NO:
Examples of the DNA that hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 under high stringent conditions include, for example, about 90% of the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3. As described above, DNA containing a base sequence having a homology of about 95% or more, more preferably about 98% or more is used.

DN that completely encodes the peptide of the present invention
As a means for cloning A, the DNA of the present invention may be amplified by PCR using a synthetic DNA primer containing a partial nucleotide sequence of the nucleotide sequence of the DNA encoding the peptide of the present invention, or the DNA of the present invention may be incorporated into an appropriate vector. DNA encoding part or all of the peptide
Selection can be performed by hybridization with fragments or those labeled with synthetic DNA. Hybridization methods are described, for example, in Molecular Cloning 2nd (J. Sambro
ok et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. Cloning of a DNA completely encoding the protein of the present invention or a partial peptide thereof (hereinafter abbreviated as the protein of the present invention) can also be performed in the same manner as cloning of a DNA completely encoding the peptide of the present invention.

The DNA base sequence can be converted by ODA using PCR or a known kit such as Mutan ^™ -superExpress Km (Takara Shuzo) or Mutan ^™ -K (Takara Shuzo).
-It can be carried out according to a known method such as the LAPCR method, the gapped duplex method, the Kunkel method, or a method analogous thereto. The DNA encoding the cloned peptide / protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. The expression vector for the peptide / protein of the present invention may be prepared, for example, by (A) cutting out a DNA fragment of interest from DNA encoding the peptide / protein of the present invention, and (B) converting the DNA fragment into a promoter of an appropriate expression vector. It can be manufactured by connecting downstream.

As a vector, a plasmid derived from E. coli (eg, pBR322, pBR325, pUC12, pUC12
UC13), a plasmid derived from Bacillus subtilis (eg, pUB11)
0, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
A1-11, pXT1, pRc / CMV, pRc / RS
V, pcDNAI / Neo, pcDNA3.1, pRc
/ CMV2, pRc / RSV (Invitrogen) and the like are used. As the promoter used in the present invention,
Any promoter may be used as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, the SRα promoter, SV4
0 promoter, HIV-LTR promoter, CMV
Promoter, HSV-TK promoter and the like. Among them, it is preferable to use the CMV promoter, SRα promoter and the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, etc. When the host is Bacillus, SPO1 promoter, SPO2 promoter and penP promoter, the host is In the case of yeast, PHO5 promoter, PGK promoter, G
An AP promoter, an ADH promoter and the like are preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

[0042] In addition to the above, an expression vector containing an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), and the like, if desired, may be used. Can be used. As a selection marker, for example, dihydrofolate reductase (hereinafter, dhfr)
Gene (sometimes abbreviated as “methotrexate (M
TX) resistance], an ampicillin resistance gene (hereinafter referred to as Amp
^r ), a neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r , G418 resistance), and the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, 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.
When the host is a genus Escherichia, a PhoA signal sequence, an OmpA signal sequence, and the like are used. In some cases, MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule, signal sequence, etc. can be used. Using the vector containing the DNA encoding the peptide and protein of the present invention thus constructed, a transformant can be produced.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used. Specific examples of Escherichia bacteria include
Escherichia coli K12 DH
1 [Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad.. Sci. USA), 60, 160
(1968)], JM103 [Nukuilec Acids
Research, (Nucleic Acids Research), 9, 309
(1981)], JA221 [Journal of Molecular Biolog
y), 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41]
Vol., 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like. Examples of the bacterium belonging to the genus Bacillus include, for example, Bacillus subtilis MI114 [Gene, 2
4, 255 (1983)], 207-21 [Journal of Biochemistr
y), 95, 87 (1984)]. Examples of yeast include Saccharomyces cerevisiae AH22, AH22
^{R -, NA87-11A, DKD-5D} , 20B-1
2. Schizosaccharomy
ces pombe) NCYC1913, NCYC2036, Pichia pastoris and the like.

As insect cells, for example, virus is A
In the case of cNPV, a cell line derived from night larvae of larvae (Spodop
tera frugiperda cell (Sf cell), Trichoplusia ni
MG1 cells from the midgut, H from eggs of Trichoplusia ni
igh Five ^TM cells, cells from Mamestra brassicae or
Estigmena acrea-derived cells are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mor
iN; BmN cells). Examples of the Sf cells include Sf9 cells (ATCC CRL1711), Sf2
One cell (Vaughn, JL et al., In Viv
o), 13, 213-217, (1977)). As insects, for example, silkworm larvae and the like are used [Maeda et al., Nature, 315, 592 (198).
5)]. As animal cells, for example, monkey cells COS-
7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter, CHO (dhf)
r ⁻ ) cells), mouse L cells, mouse AtT-2
0, mouse myeloma cells, rat GH3, human FL cells, and the like.

In order to transform Escherichia, for example, Procesings of the National Academy of Sciences of the USA (Proc. Natl. Acad.. Sci. USA), 69 Vol. 211
0 (1972) and Gene, Vol. 17, 107 (19)
82) and the like.
In order to transform Bacillus, for example, Molecular and General Genetics (Molecula
r & General Genetics), 168, 111 (197
9) and the like. For transforming yeast, for example, Methods in Enzymology, 194,
182-187 (1991), Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. U
SA), Vol. 75, 1929 (1978). For transformation of insect cells or insects, for example, bio / technology (Bio / Te
Chem., 6, 47-55 (1988)). To transform animal cells, for example, see Cell Engineering Separate Volume 8, New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456
(1973). Thus, a transformant transformed with the expression vector containing the DNA encoding the G protein-coupled protein is obtained. The host is Escherichia,
When culturing a transformant of the genus Bacillus, a liquid medium is suitable as a medium to be used for culturing, and includes a carbon source, a nitrogen source, an inorganic substance, and the like necessary for growth of the transformant. It is included. As a carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc.As a nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein,
Meat extract, soybean meal, inorganic or organic substances such as potato extract, as the inorganic substance, for example, calcium chloride,
Examples include 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 to 8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journa)
l of Experiments in Molecular Genetics), 431-
433, Cold Spring Harbor Laboratory, New York 1
972] is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added in order 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 added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, KL
Proc. Natl. Acad. .Sci. USA, 77, 450, "Progressing's of the National Academy of Sciences of the USA."
5 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., "Processings of the National Academy of Sciences of the USA (Proc. Natl. Acad.. Sci. US
A), 81, 5330 (1984)].
Preferably, the pH of the medium is adjusted to about 5-8. The cultivation is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and if necessary, aeration and stirring are added.

When culturing an insect cell or a transformant whose host is an insect, the medium used is Grace's Insect Mediu.
m (Grace, TCC, Nature, 195,788 (196
Those to which an additive such as 10% bovine serum immobilized in 2)) is appropriately added are used. The pH of the medium is about 6.2-6.
Adjustment to 4 is preferred. Culture is usually performed at about 27 ° C for about 3
５5 days, with aeration and / or agitation as needed. When culturing a transformant in which the host is an animal cell, the medium may be, for example, MEM containing about 5 to 20% fetal bovine serum.
Medium [Science, Vol. 122, 501 (19)
52)], DMEM medium [Virology,
8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medica
l Association) 199, 519 (1967)], 19
9 medium [Proceeding of the Society for the Biological Medicine (Proceeding o
fthe Society for the Biological Medicine), 73
Vol., 1 (1950)]. pH is about 6-8
It is preferred that The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and if necessary, aeration or stirring is added. As described above, the peptide / protein of the present invention can be produced in the transformant, in the cell membrane, or outside the cell.

The peptide / protein of the present invention can be separated and purified from the culture by, for example, the following method. When extracting the peptide / protein of the present invention from cultured cells or cells, after culturing,
The cells or cells are collected by a known method, suspended in an appropriate buffer, and disrupted by ultrasonication, lysozyme, and / or freeze-thawing, and then the crude protein is extracted by centrifugation or filtration. A method for obtaining a liquid is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . If peptides and proteins are secreted into the culture,
The supernatant is separated from the cells or cells by a known method, and the supernatant is collected. Purification of the peptide / 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 include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and use of difference in hydrophobicity such as reverse phase high performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.

When the peptide / protein thus obtained is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto. Can be converted into a free form or another salt by the method of or a method analogous thereto. In addition, the peptide / protein produced by the recombinant can be arbitrarily modified or the peptide / polypeptide can be partially removed by applying 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 activity of the peptide of the present invention thus produced can be measured by a binding experiment between the labeled peptide of the present invention and the protein of the present invention, an enzyme immunoassay using a specific antibody, or the like. The activity of the produced protein of the present invention can be measured by a binding experiment with a labeled peptide of the present invention, an enzyme immunoassay using a specific antibody, or the like.

An antibody against the peptide of the present invention, the protein of the present invention or its partial peptide, or a salt thereof may be an antibody capable of recognizing the peptide of the present invention, the protein of the present invention, its partial peptide, or a salt thereof. Any of a polyclonal antibody and a monoclonal antibody may be used. Antibodies against the peptide of the present invention or the protein of the present invention or a partial peptide thereof or a salt thereof (hereinafter, sometimes abbreviated as the peptide / protein of the present invention) may be obtained by using the peptide / protein of the present invention as an antigen, It can be produced according to a known antibody or antiserum production method.

[Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The peptide or protein of the present invention is administered to a mammal at a site where the antibody can be produced by administration to itself or together with a carrier and a diluent. Is done. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times.
As mammals to be used, for example, monkeys, rabbits,
Dogs, guinea pigs, mice, rats, sheep and goats are exemplified, 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 having an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. By fusing the contained antibody-producing cells with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The measurement of the antibody titer in the antiserum can be performed, for example, by reacting the labeled peptide or protein of the present invention described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. it can. The fusion operation is performed in a known manner, for example,
The method of Koehler and Milstein [Natural
e) 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 /
Although 0 is mentioned, 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,
PEG (preferably PEG1000 to PEG600
0) is added at a concentration of about 10 to 80%, and about 20 to 4
By incubating at 0 ° C., preferably at about 30-37 ° C. for about 1-10 minutes, cell fusion can be carried out efficiently.

Various methods can be used to screen the monoclonal antibody-producing hybridoma. For example, the hybridoma culture supernatant can be applied to a solid phase (eg, a microplate) on which the antigen such as the peptide or protein of the present invention is adsorbed directly or together with a carrier. , And then add an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A,
A method for detecting a monoclonal antibody bound to a solid phase, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, and labeling a peptide or protein of the present invention, etc., labeled with a radioactive substance, an enzyme, or the like. In addition, a method of detecting a monoclonal antibody bound to a solid phase can be mentioned. The selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto.
(Thymidine) in a medium for animal cells or the like. As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, an RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, a GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1 to 10% fetal bovine serum, or a serum-free medium for hybridoma culture (SFM-10
1, Nissui Pharmaceutical Co., Ltd.) can be used. The culturing temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. 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 Separation and purification of the monoclonal antibody can be carried out in the same manner as in the separation and purification of the normal polyclonal antibody. Electrophoresis, adsorption / desorption using an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase or active adsorbent such as protein A or protein G to collect only antibody and dissociate the bond Specific purification method for obtaining an antibody).

[Preparation of Polyclonal Antibody] The polyclonal antibody of the present invention can be produced according to known methods or modifications thereof. For example, a complex of an immunizing antigen (antigen such as the peptide or protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. -It can be produced by collecting a substance containing an antibody against a protein or the like and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used to immunize a mammal, the type of the carrier protein and the mixing ratio of the carrier and the hapten should be such that the antibody can be efficiently produced against the hapten immunized by crosslinking the carrier. Any kind may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin, etc. may be used in a weight ratio of about 0.1 to 20 with respect to 1 hapten. Preferably, a method of coupling at a ratio of about 1 to 5 is used. Further, for coupling of the hapten and the carrier, various condensing agents can be used,
Glutaraldehyde, carbodiimide, maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, and the like are 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 or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is
Usually, once every about 2 to 6 weeks, about 3 to 10 times in total. The polyclonal antibody can be collected from blood, ascites, or the like, preferably from blood, of the mammal 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 serum described above. The separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

The peptide of the present invention, a DNA encoding the peptide of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention), and an antibody against the peptide of the present invention (hereinafter, referred to as “DNA of the present invention”).
(May be abbreviated as the antibody of the present invention) is a prophylactic / therapeutic agent for various diseases involving the peptide of the present invention; Quantification of the peptide of the present invention or a salt thereof, a gene diagnostic agent, a drug containing the antisense DNA, a drug containing the antibody of the present invention, production of a non-human animal containing the DNA of the present invention,
It is useful for the implementation of drug design based on comparison with structurally similar ligands / receptors. In particular, compounds (eg, ZAQ agonists) that alter the binding of a ligand to the protein of the present invention specific to humans or mammals by using a receptor binding assay system using the expression system for the recombinant protein of the present invention. ,
ZAQ antagonists) can be screened, and the agonists or antagonists can be used as agents for preventing or treating various diseases. The use of the peptide of the present invention, the DNA of the present invention and the antibody of the present invention will be specifically described below.

(1) Therapeutic / prophylactic agent for various diseases related to the peptide of the present invention As described in Examples below, the peptide of the present invention
It exists as a humoral factor in the living body, activates the protein of the present invention, and increases the intracellular Ca ²⁺ ion concentration of cells expressing the protein of the present invention. Is a ligand of In addition, the peptide of the present invention may be a snake venom Mamba Intestinal Toxin 1 (may be abbreviated as MIT1; SEQ ID NO: 34; Toxicon, vol. 28, 847-856).
FEBS Letters, 461, 183-188, 1999
) And about 56% homology at the amino acid level. It has been reported that MIT1 causes contraction of the ileum and distal colon, or relaxation of the proximal colon, the extent of which is as strong as 40 mM potassium chloride (FEBS Lette
rs, 461, 183-188, 1999), but the point of action and mechanism of action have not been elucidated. We have MI
It was revealed that the action of T1 was also expressed via the protein of the present invention. From the above, the peptide of the present invention has an activity of causing contraction or relaxation of the entire intestinal tract and controlling intestinal motility (digestive activity) (eg, Example 11 described later). Therefore, the DN of the present invention
When A or the like is deficient or the expression level is abnormally decreased, for example, gastrointestinal diseases (eg, enteritis, diarrhea,
Constipation, malabsorption syndrome, etc.). Therefore, the peptide of the present invention and the DN of the present invention
A can be used as a medicament such as an agent for treating or preventing various diseases such as digestive diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.). For example, in the case where there is a patient in whom information transmission in cells expressing the protein of the present invention is not sufficiently or normally exerted due to reduction or deletion of the peptide of the present invention in vivo, (a) By administering the DNA of the present invention to the patient and expressing the peptide of the present invention in vivo, (b) inserting the DNA of the present invention into cells and expressing the peptide of the present invention, The role of the peptide of the present invention in the patient can be sufficiently or normally exerted by transplantation into the patient or (c) administering the peptide of the present invention to the patient. When the DNA of the present invention is used as the above-mentioned therapeutic or prophylactic agent, the DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc. It can be administered to humans or warm-blooded animals. 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, using a gene gun or a catheter such as a hydrogel catheter. When the peptide of the present invention is used as the above therapeutic / prophylactic agent, it is at least 90%, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.
% Or more is preferably used.

The peptide of the present invention can be used, for example, as a sugar-coated tablet, capsule, elixir, microcapsule or the like, orally, or with water or another pharmaceutically acceptable liquid. Can be used parenterally in the form of injections, such as sterile solutions or suspensions. For example, a physiologically acceptable carrier for the peptide of the present invention,
It can be manufactured by mixing with flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like in the unit dosage form generally required for the practice of preparations.
The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained. Examples of additives that can be incorporated into tablets, capsules, and the like include binders such as gelatin, corn starch, tragacanth, and acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. Suitable leavening agents, lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, reddish oil or cherry are used. When the preparation unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice, such as by dissolving or suspending the active substance in vehicles, such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, and the like. Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-sorbitol).
Mannitol, sodium chloride, etc.), and suitable solubilizing agents, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate) 80 ^TM , HCO-50, etc.)
And the like. Examples of the oily liquid include sesame oil and soybean oil, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent. A buffer (eg, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, polyethylene glycol, etc.), You may mix | blend with a preservative (for example, benzyl alcohol, phenol, etc.), an antioxidant, etc. The prepared injection is usually filled in a suitable 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 thus obtained are safe and have low toxicity, and therefore, for example, mammals (eg, human, rat, mouse, guinea pig, rabbit, sheep, pig, cow, horse, cat, dog, monkey, etc.) ) Can be administered. The dose of the peptide of the present invention varies depending on the target disease, the subject of administration, the administration route, and the like. For example, when the peptide of the present invention is orally administered for the purpose of treating gastrointestinal diseases, it is generally adult (60 kg). )) About 1 mg to 1000 mg of the peptide of the present invention per day,
Preferably about 10-500 mg, more preferably about 10 mg
Administer ~ 200 mg. In the case of parenteral administration, the single dose of the peptide of the present invention varies depending on the administration subject, the target disease and the like. For example, the peptide of the present invention may be administered in the form of an injection for the purpose of treating digestive diseases. (As a body weight of 60 kg), the peptide should be used in an amount of about 1 to about
About 1000 mg, preferably about 1 to 200 mg,
More preferably, about 10 to 100 mg is conveniently administered by injecting into the affected area. In the case of other animals, the amount can be administered in terms of 60 kg.

(2) Screening of 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. The present invention is characterized by using the peptide of the present invention, the protein of the present invention, and a partial peptide. A method for screening 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, or the method of using the peptide of the present invention and the protein of the present invention, characterized by using the peptide of the present invention and the protein of the present invention. The screening kit for a compound or a salt thereof that changes the binding property (hereinafter, abbreviated as the screening method of the present invention and the screening kit of the present invention) will be described in detail below.

Using the protein of the present invention or constructing a recombinant expression system of the protein of the present invention, a binding assay system (ligand / receptor assay system) for the peptide of the present invention using the expression system is constructed. A compound that changes the binding between the peptide of the present invention and the protein of the present invention and a compound that changes the binding between the peptide of the present invention and the protein of the present invention (for example, peptide, protein, non-peptidic compound , Synthetic compounds, fermentation products, etc.) or salts thereof. Such compounds have a cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production,
A compound (ZAQ agonist) having intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity for promoting or suppressing pH reduction, etc.) Such compounds that do not have the cell stimulating activity (ZAQ antagonists) and the like are included. “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 peptide of the present invention is brought into contact with the protein of the present invention and (ii) the case where the peptide of the present invention and the test compound are brought into contact with the above-mentioned protein of the present invention. A method for screening a compound or a salt thereof, which alters the binding property between the peptide of the present invention and the protein of the present invention, which is characterized by performing a comparison. 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 case where the above-mentioned protein of the present invention comes into contact with the peptide of the present invention and a test compound. For example, the amount of the peptide of the present invention bound to the protein of the present invention and the cell stimulating activity are measured and compared.

Specifically, the screening method of the present invention includes a case where the labeled peptide of the present invention is brought into contact with the above-mentioned protein of the present invention and a case where the labeled peptide of the present invention and the test compound are added to the protein of the present invention. A compound that changes the binding property between the peptide of the present invention and the protein of the present invention, which is characterized by measuring the amount of binding of the labeled peptide of the present invention to the protein of the present invention, and comparing A method for screening a salt, wherein the labeled 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 labeled peptide of the present invention and the test compound contain the protein of the present invention. The labeled peptide of the present invention when contacted with a cell to be treated or a membrane fraction of the cell. 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 to the membrane fraction, When the transformant containing the DNA encoding the protein of the present invention is cultured and then brought into contact with the protein of the present invention expressed on the cell membrane, the labeled peptide of the present invention and the test compound are combined with the protein of the present invention. Measuring and comparing the amount of the labeled peptide of the present invention to the protein of the present invention when the transformant containing the encoding DNA is brought into contact with the protein of the present invention expressed on the cell membrane by culturing. A compound that changes the binding property between the peptide of the present invention and the protein of the present invention, or a compound thereof The screening method of the present invention, wherein the peptide of the present invention is brought into contact with cells containing the protein of the present invention, and the peptide and test compound of the present invention are brought into contact with cells containing the protein of the present invention. Cell stimulating activity via protein (eg, arachidonic acid release, acetylcholine release,
Intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGM
P production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, pH
And a method for screening 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, characterized by measuring and comparing the activity of promoting or suppressing the reduction of the protein. The peptide of the present invention was brought into contact with the protein of the present invention expressed on the cell membrane by culturing a transformant containing the DNA encoding the protein of the present invention, Cell stimulating activity mediated by the protein of the present invention (for example, arachidonic acid release, etc.) when the transformant containing the DNA encoding the protein of the present invention is brought into contact with the protein of the present invention expressed on the cell membrane by culturing. Acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP
Production, intracellular cGMP production, inositol phosphate production,
Fluctuation of cell membrane potential, phosphorylation of intracellular protein, cf
or a salt thereof, which changes the binding property between the peptide of the present invention and the protein of the present invention, wherein the activity of promoting or suppressing the activation of os, the decrease in pH, etc.) is measured and compared. Screening method.

A specific description of the screening method of the present invention will be given below. 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. However, since it is particularly difficult to obtain human-derived organs, the protein of the present invention and the like, which is expressed in large amounts using recombinants, is suitable for screening. To produce the protein of the present invention, the above-described methods and the like are used. When cells containing the protein of the present invention or the cell membrane fraction are used in the screening method of the present invention, the preparation method described below may be followed. When a cell containing the protein of the present invention is used, the cell may be fixed with glutaraldehyde, 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 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 crushing cells and then by a known method. As a method for crushing cells, a method of crushing cells with a Potter-Elvehjem type homogenizer, crushing with a Waring blender or a polytron (manufactured by Kinematica), crushing with ultrasonic waves, spouting cells from a thin nozzle while applying pressure with a French press, etc. Crushing and the like. For the fractionation of cell membranes, fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (generally about 1 minute to 10 minutes), and the supernatant is further centrifuged at a higher speed (15,000 to 30000 rpm) for 30 minutes.
After centrifugation for 2 minutes to 2 hours, the obtained precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed 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 the membrane fraction containing the protein of the present invention is 10 ³ to 1 per cell.
Is preferably from 0 ⁸ molecule is suitably from ^{105 to 107} molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. . In order to carry out the above-mentioned (1) for screening a compound that alters the binding property between the peptide of the present invention and the protein of the present invention, an appropriate protein fraction of the present invention and a labeled peptide of the present invention are used. The fraction containing the protein of the present invention is preferably a fraction containing the protein of the present invention in a natural form, or a fraction containing a protein of the present invention in a recombinant form having an activity equivalent thereto. Here, the equivalent activity indicates an equivalent ligand binding activity and the like. Examples of the labeled peptide of the present invention include [ ³ H], [
¹²⁵ I], ^[14 C], it can be utilized, such as a peptide of the present invention which is labeled with a ^[35 S]. In particular, a labeled form of the peptide of the present invention prepared by a known method using a Bolton-Hunter reagent can also be used. Specifically, in order to screen for a compound that alters the binding property between the peptide of the present invention and the protein of the present invention, first, cells containing the protein of the present invention or a membrane fraction of the cells are suitable for screening. A receptor preparation is prepared by suspending in a buffer. PH 4 buffer
Phosphate buffer of 10 to 10 (preferably pH 6 to 8),
Any buffer that does not inhibit the binding between the ligand and the receptor, such as a Tris-HCl buffer, may be used. In addition, CHAPS, T
ween-80 ^™ (Kao-Atlas), digitonin,
Surfactants such as deoxycholate can also be added to the buffer. Further, in order to suppress the degradation of the protein of the present invention and the peptide of the present invention by protease, P
Protease inhibitors such as MSF, leupeptin, E-64 (manufactured by Peptide Research Laboratories), and pepstatin can also be added. A certain amount (5,000 cpm to 500,000 cps) is added to 0.01 to 10 ml of the receptor solution.
m) The labeled peptide of the invention is added and simultaneously
^-4 to 10 ^-1 μM of the test compound is allowed to coexist. A reaction tube containing a large excess of the unlabeled peptide of the present invention is also prepared to determine the non-specific binding amount (NSB). Reaction is 0 ° C
To 50 ° C, preferably 4 to 37 ° C for 20 minutes to 2
It is performed for 4 hours, preferably for 30 minutes to 3 hours. After the reaction, the mixture is filtered with 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 with a liquid scintillation counter or a γ-counter. Count (B ₀ −NS) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance
Specific binding amount (B-NSB) assuming that B) is 100%
Is 50% or less, for example, can be selected as a candidate substance having competitive inhibitory ability. BIAcore (Amersham Pharmacia Biotech) can also be used as a method for measuring the binding between the protein of the present invention and the peptide of the present invention. In this method, the peptide of the present invention is immobilized on a sensor chip by the amino coupling method according to the protocol attached to the device, and the cells containing the protein of the present invention or the DNA containing the DNA encoding the protein of the present invention are transformed. A buffer such as a phosphate buffer or a Tris buffer containing the protein of the present invention or a membrane fraction containing the protein of the present invention purified from the body, or a membrane fraction containing the purified protein of the present invention or the protein of the present invention and a test compound. The liquid is passed over the sensor chip at a rate of 2-20 μl per minute. By observing that a test compound coexisting with the change of 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 changes, the protein of the present invention and the peptide of the present invention change. Screening for compounds that alter binding can be performed. This method uses a method in which a protein of the present invention is immobilized on a sensor chip, and a buffer 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. Can be similarly measured. Examples of the test compound include the same compounds as described above. In order to carry out the above-mentioned method (1) for screening a compound that alters the binding property between the peptide of the present invention and the protein of the present invention, cell stimulating activity (for example, arachidonic acid release, acetylcholine release) through the protein of the present invention is required. Activity to promote intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. Activity, 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 in a multiwell plate or the like. Before performing screening, the cells were exchanged with a fresh medium or an appropriate buffer that is not toxic to cells, and a test compound was added and incubated for a certain period of time. The product is quantified according to the respective method. When the production of a substance (for example, arachidonic acid or the like) as an indicator of cell stimulating activity is difficult to be assayed by a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor to the degrading enzyme. In addition, the activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production amount has been increased by forskolin or the like.

For screening by measuring 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 are desirable. The transformant-expressing cells of the protein of the present invention may be either a stable expression strain or a transient expression strain. 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 ligand / receptor assay system is described in more detail as follows (1)
The assay system of (12) is used. (1) When a receptor-expressing cell is stimulated by a receptor agonist, 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 change to GDP. At this time, if GTPγS is added to the reaction solution, GTPγS binds to G protein similarly to GTP, but contains G protein without hydrolysis. The state of being bound to the cell membrane is maintained. When labeled GTPγS is used, the activity of stimulating the receptor-expressing cells of the receptor agonist can be measured by measuring the radioactivity remaining on the cell membrane. Utilizing this reaction, the stimulating activity of the peptide of the present invention on the cells expressing the protein of the present invention can be measured. This method does not use cells containing the protein of the present invention as described above, but is an assay method using a membrane fraction containing the protein of the present invention as described above. In the present assay, a substance exhibiting GTPγS binding promoting activity to a membrane fraction containing the protein of the present invention is an agonist. Here, the peptide of the present invention or the peptide of the present invention and a test compound were added, and the change in the activity of promoting GTPγS binding to the cell membrane fraction containing the protein of the present invention as compared to the case of administering the peptide of the present invention alone was observed. By observing, it is possible to screen for a compound that changes the binding between the peptide of the present invention and the protein of the present invention. At this time, the GT of the peptide of the present invention to the cell membrane fraction containing the protein of the present invention was used.
A compound exhibiting an activity of suppressing the PγS binding promoting activity can be selected as a candidate substance having a competitive inhibitory ability.
On the other hand, an agonist can be screened by administering only the test compound and observing the activity of promoting GTPγS binding to the cell membrane fraction containing the protein of the present invention. One example of the screening method is described below more specifically. The cell membrane fraction containing the protein of the present invention is treated with a membrane dilution buffer (50 mM Tris, 5 mM MgCl ₂ , 150 mM N
Dilute with aCl, 1 μM GDP, 0.1% BSA pH 7.4). The dilution ratio varies depending on the expression level of the protein of the present invention. This is dispensed in 0.2 ml portions into Falcon2053, the peptide of the present invention or the peptide of the present invention and a test compound are added, and [ ³⁵ S] GTPγS is further added to a final concentration of 200 pM. twenty five
After washing at room temperature for 1 hour, wash buffer (50 mM T
ris, 5 mM MgCl ₂ , 150 mM NaCl, 0.1% BSA, 0.05% C
HAPS pH 7.41.5ml) and add glass fiber filter paper GF / F
And filtered. After incubating at 65 ° C. for 30 minutes and drying, the radioactivity of [ ³⁵ S] GTPγS bound to the membrane fraction remaining on the filter paper is measured by a liquid scintillation counter. The radioactivity of the experimental group to which only the peptide of the present invention was added was set to 100%, and the radioactivity of the experimental group to which no peptide of the present invention was added was set to 0%, and the effect of the test compound on the GTPγS binding promoting activity of the peptide of the present invention was determined. calculate. A test compound having a GTPγS binding promoting activity of, for example, 50% or less can be selected as a candidate substance having a competitive inhibition ability.

(2) In cells expressing the protein of the present invention, the amount of intracellular cAMP is reduced by stimulation with the peptide of the present invention. Using this reaction, the stimulating activity of the peptide of the present invention on cells expressing the protein of the present invention can be measured. CAMP production amount of various animal cells expressing the protein of the present invention is a mouse, rat, rabbit, goat, and anti-cAMP antibody obtained by immunizing such as cattle ¹²⁵
By using I-labeled cAMP (both commercially available), it can be measured by RIA or other EIA systems combining anti-cAMP antibody and labeled cAMP. Is a quantitative possible by also SPA method using a beads and ^{12 5} I-labeled cAMP scintillant containing fixed using such antibodies to such animal IgG using an anti-cAMP antibody to protein A or anti-cAMP antibody-producing (Use a kit from Amersham Pharmacia Biotech). In an assay for inhibiting cAMP production, the amount of intracellular cAMP is increased by a ligand such as forskolin or calcitonin, which increases the amount of intracellular cAMP. By observing that the suppression of the amount of intracellular cAMP by the single administration of the peptide of the present invention is changed, a compound that changes the binding between the peptide of the present invention and the protein of the present invention can be screened. At this time, a compound exhibiting an activity of inhibiting the cAMP production inhibitory activity of cells expressing the protein of the present invention by the peptide of the present invention 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 adding only a test compound and examining cAMP production inhibitory activity. The screening method is described more specifically below. CHO cells expressing the protein of the present invention (ZAQC-B
1 cell; Example 3 (3-5) described later) is seeded on a 24-well plate at 5 × 10 ⁴ cells / well, and cultured for 48 hours. Cells were incubated with 0.2 mM 3-isobutyl-methylxanthine and 0.05%
Wash with Hanks buffer (pH 7.4) containing BSA and 20 mM HEPES (hereinafter, Hanks buffer (pH 7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES is referred to as a reaction buffer. ). Then 0.5ml
And the mixture is incubated for 30 minutes in an incubator. After removing the reaction buffer and adding a new 0.25 ml reaction buffer to the cells, contain 2 μM forskolin
0.25 ml of a reaction buffer supplemented with 1 nM of the peptide of the present invention or 1 nM of the peptide of the present invention and a test compound are added to the cells, and the mixture is reacted at 37 ° C. for 24 minutes.
The reaction was stopped by adding 100 μl of 20% perchloric acid,
Next, the cells are placed on ice for one hour to extract intracellular cAMP. The amount of cAMP in the extract is measured using a cAMP EIA kit (Amersham Pharmacia Biotech). CAMP produced by forskolin stimulation
The amount of cAMP inhibited by the addition of 1 nM of the peptide of the present invention is defined as 0%, and the effect of the test compound on the cAMP production inhibitory activity of the peptide of the present invention is calculated. Inhibiting the activity of the peptide of the invention
A test compound having an AMP production activity of, for example, 50% or more can be selected as a candidate substance having competitive inhibitory ability. In order to measure the cAMP production promoting activity, cAMP produced by adding a test compound to the protein-expressing CHO cells of the present invention without adding forskolin is quantified by the above method.

(3) CRE (cAMP response element)
Is inserted into the multiple cloning site upstream of the luciferase gene of the Picker Gene Basic Vector or the Picker Gene Enhancer Vector (Toyo Ink Mfg. Co., Ltd.), and this is used as the CRE-reporter gene vector. In cells transfected with the CRE-reporter gene vector, cAMP
Stimulation with elevation induces CRE-mediated luciferase gene expression and subsequent production of luciferase protein. That is, by measuring the luciferase activity, a change in the amount of cAMP in the cells into which the CRE-reporter gene vector has been introduced can be detected. CR
A compound that alters the binding of the peptide of the present invention to the protein of the present invention can be screened using cells obtained by transfecting the E-reporter gene vector into the cells expressing the protein of the present invention. The specific screening method is described below. Cells expressing the protein of the present invention into which the CRE-reporter gene has been introduced are seeded at 5 × 10 ³ cells / well in a 24-well plate, and cultured for 48 hours. Cells were washed with Hanks buffer (p.p.) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and
(H7.4) (hereinafter, Hanks buffer (pH 7.4) containing 0.2 mM 3-isobutyl-methylxanthine, 0.05% BSA and 20 mM HEPES is referred to as a reaction buffer). Thereafter, 0.5 ml of a reaction buffer is added, and the mixture is kept warm in an incubator for 30 minutes. After removing the reaction buffer and newly adding 0.25 ml of the reaction buffer to the cells, 0.25 ml of the reaction buffer containing 1 nM of the peptide of the present invention or 1 nM of the peptide of the present invention and the test compound and 2 μM forskolin Is added to the cells and reacted at 37 ° C. for 24 minutes. The cells are lysed with a cell lysing agent for Picker Gene (Toyo Ink Mfg. Co., Ltd.), and a luminescent substrate (Toyo Ink Mfg. Co., Ltd.) is added to the solution. Luminescence by luciferase is measured with a luminometer, liquid scintillation counter or top counter. The effect of the compound that alters the binding between the peptide of the present invention and the protein of the present invention can be measured by comparing the amount of luminescence by luciferase as compared with the case where the peptide of the present invention is administered alone. At this time, the administration of the peptide of the present invention suppresses an increase in the amount of luminescence due to forskolin stimulation, and a compound that reverses this suppression can be selected as a candidate substance having competitive inhibitory ability. On the other hand, only the test compound was administered,
An agonist can be screened by observing the same suppression of the amount of luminescence increased by forskolin stimulation as the peptide of the present invention. As a reporter gene, other than luciferase, for example, alkaline phosphatase, chloramphenicol acetyltransferase or β-galactosidase can also be used. The enzyme activities of the gene products of these reporter genes can be easily measured using a commercially available measurement kit as follows. Alkaline phosphatase activity can be measured, for example, by chloramphenicol acetyltransferase (chlo) by Lumi-Phos 530 manufactured by Wako Pure Chemical Industries, Ltd.
ramphenicol acetyltransferase) activity is, for example, FAST CAT chrolamphenicol Acetyltransferase A
With the ssay Kit, β-galactosidase activity can be measured by, for example, Aurora Gal-XE manufactured by Wako Pure Chemical Industries.

(4) The cells expressing the protein of the present invention release arachidonic acid metabolites extracellularly as a result of stimulation with the peptide of the present invention. By previously introducing arachidonic acid having radioactivity into cells, this activity can be measured by measuring radioactivity released outside the cells. At this time, by adding the peptide of the present invention or the peptide of the present invention and a test compound, and examining the effect of the peptide of the present invention on the arachidonic acid metabolite releasing activity, the binding of the peptide of the present invention to the protein of the present invention is examined. Screening for compounds that have an effect can be performed. At this time, a compound that inhibits the arachidonic acid metabolite release activity of the peptide of the present invention can be selected as a candidate substance having competitive inhibitory ability. Alternatively, a compound showing an agonist activity can be screened by adding only a test compound and examining the arachidonic acid metabolite releasing activity of the protein-expressing cells of the present invention. The method for screening a compound that affects the binding between the peptide of the present invention and the protein of the present invention will be described more specifically below. CHO cells expressing the protein of the present invention were placed in a 24-well plate at 5 × 10 ⁴ cells / well.
In seeded, cultured for 24 hours, 0.25 ^[3 H] arachidonic acid
Add to make μCi / well. [ ³ H] Arachidonic acid addition 1
After 6 hours, the cells are washed with Hank's buffer (pH 7.4) containing 0.05% BSA and 20 mM HEPES.
500 μl of a buffer containing 10 nM of the peptide of the present invention or 10 nM of the peptide of the present invention and a test compound dissolved in Hanks buffer (pH 7.4) containing 0 mM HEPES.
Is added. Hereinafter, Hanks buffer (pH 7.4) containing 0.05% BSA and 20 mM HEPES is referred to as a reaction buffer. 3
After incubating at 7 ° C for 60 minutes,
l is added to a scintillator, and the amount of [ ³ H] arachidonic acid metabolite released in the reaction solution is measured by a scintillation counter. The amount of ^[3 H] arachidonic acid metabolites in the medium with non-addition reaction buffer of the peptides of the present invention was 0%, in the medium upon addition of 10 nM of the peptide of the present invention ^[3 H] arachidonic acid metabolite The effect of the test compound on the binding between the peptide of the present invention and the protein of the present invention is calculated based on the amount of the compound as 100%. A test compound having an arachidonic acid metabolite-producing activity of, for example, 50% or less can be selected as a candidate substance capable of competitive inhibition.

(5) By stimulating cells expressing the protein of the present invention with the peptide of the present invention, C
The a ²⁺ ion concentration increases. By utilizing this, the effect of the test compound on the binding between the peptide of the present invention and the protein of the present invention can be examined. Specifically, Example 3 (3-5) and Example 5 (5-3) described later
It can be checked by a method according to -4). The protein-expressing cells of the present invention were inoculated on a sterilized cover glass for a microscope, and after 2 days, the culture solution was replaced with HBSS in which 4 mM Fura-2 AM (Dojindo Laboratories) was suspended, and incubated at room temperature for 2 hours 30 hours.
Leave for a minute. After washing with HBSS, a cover glass was set on the cuvette, and the ratio of the fluorescence intensity of 505 nm at the excitation wavelength of 340 nm and 380 nm when the peptide of the present invention or the peptide of the present invention and the test compound were added was measured with a fluorescence meter. Measure the rise. At this time, by measuring the change in the fluorescence intensity caused by the addition of the test compound as compared to when the peptide of the present invention is administered alone, the compound affecting the binding between the peptide of the present invention and the protein of the present invention is measured. Screening can be performed. Also, FLIPR (manufactured by Molecular Devices) can be used as follows. That is, Fluo-3 AM (manufactured by Dojindo Laboratories) is added to the cell suspension, and the cells are taken up. After washing the supernatant several times by centrifugation, the cells are seeded on a 96-well plate. Set it on the FLIPR device,
As in the case of -2, the peptide of the present invention or the peptide of the present invention and the test compound were added, and the observed fluorescence intensity was changed by the addition of the test compound as compared to when the peptide of the present invention was administered alone. By measuring, a compound that affects the binding between the peptide of the present invention and the protein of the present invention can be screened. In these, a compound that suppresses an increase in fluorescence intensity by the peptide of the present invention can be selected as a candidate substance having an antagonistic ability. On the other hand, an agonist can be screened by observing an increase in fluorescence intensity due to the addition of the test compound alone.
In the protein-expressing cell of the present invention, a gene of a protein that emits light by increasing intracellular Ca ²⁺ ion (eg,
aequorin) is co-expressed and the gene (eg, aequorin, etc.) becomes Ca ²⁺ -linked and emits light by increasing the intracellular Ca ²⁺ ion concentration. The peptide of the present invention and the test compound of the present invention are measured by measuring the change in the luminescence intensity observed by the addition of the test compound as compared with the case where the peptide of the present invention and the test compound are added alone. Compounds that affect protein binding can be screened. The method is the same as described above except that the fluorescent substance is not incorporated.

(6) It has been known that the addition of a receptor agonist to cells expressing a receptor increases the intracellular inositol triphosphate concentration. By observing this reaction in the protein cell of the present invention caused by the peptide of the present invention, a compound that affects the binding of the peptide of the present invention to the protein of the present invention can be screened. After inoculating a 24-well plate and culturing the cells for 1 day in a medium in which myo- [2- ³ H] inositol (2.5 microCi / well) is added to the cells on the 1st day, the cells are thoroughly washed, and then the peptide of the present invention or After adding the peptide of the present invention and the test compound, 10% perchloric acid is added to stop the reaction.
Neutralize with 1.5 M KOH, 60 mM HEPES solution, pass through a column packed with 0.5 ml of AG1x8 resin (Bio-Rad), and pass 5 mM Na ₂ BO ₃ 60 mM
After washing with HCOONH _4, 1M HCOONH _4, the radioactivity eluted with 0.1 M HCOOH measured by a liquid scintillation counter. The peptide of the present invention and the protein of the present invention as test compounds, where the radioactivity in the medium by the reaction buffer to which the peptide of the present invention is not added is 0%, and the radioactivity in the medium when the peptide of the present invention is added is 100%. Calculate the effect on binding. A test compound having an inositol triphosphate-producing activity of, for example, 50% or less can be selected as a candidate substance capable of competitive inhibition. On the other hand, an agonist can be screened by observing an increase in inositol triphosphate production due to the addition of the test compound alone.

(7) A DNA containing a TRE (TPA response element) is inserted into a multicloning site upstream of a luciferase gene of a Picagene basic vector or a Picagene enhancer vector (Toyo Ink Mfg. Co., Ltd.), and this is inserted into a TRE- Reporter gene vector. In cells transfected with the TRE-reporter gene vector, intracellular Ca
Stimulation with increased ²⁺ ion concentration induces TRE-mediated luciferase gene expression and subsequent luciferase protein production. That is, by measuring the luciferase activity, a change in the amount of calcium ions in the cells into which the TRE-reporter gene vector has been introduced can be detected. A specific method for screening for a compound that alters the binding between the peptide of the present invention and the protein of the present invention using cells obtained by transfecting the TRE-reporter gene vector into the cells expressing the protein of the present invention is described below. The TRE-reporter gene-introduced protein-expressing cells of the present invention were placed in a 24-well plate at 5 × 10 ³ cells.
/ well and incubate for 48 hours. Cells are 0.05% BSA
After washing with Hanks buffer (pH 7.4) containing 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 were added thereto.
Let react for minutes. The cells are lysed with a cell lysing agent for Picker Gene (Toyo Ink Mfg. Co., Ltd.), and a luminescent substrate (Toyo Ink Mfg. Co., Ltd.) is added to the solution. Luminescence by luciferase is measured with a luminometer, liquid scintillation counter or top counter. The effect of the compound that alters the binding between the peptide of the present invention and the protein of the present invention can be measured by comparing the amount of luminescence by luciferase with the case where the peptide of the present invention is administered alone. At this time, the amount of luminescence increases due to an increase in intracellular Ca ²⁺ ion concentration by administration of the peptide of the present invention. A compound that suppresses this increase can be selected as a candidate substance having an antagonistic ability. On the other hand, agonists can also be screened by administering only the test compound and observing the same increase in luminescence as the peptide of the present invention. As a reporter gene, other than luciferase, for example, alkaline phosphatase, chloramphenicol acetyltransferase or β-galactosidase can also be used. The enzyme activities of the gene products of these reporter genes can be easily measured using a commercially available measurement kit as follows. Alkaline phosphatase activity is
For example, chloramphenicol acetyltransferase (chloramphe) by Wako Pure Chemical Lumi-Phos 530
nicol acetyltransferase) activity, for example, F
AST CAT chrolamphenicol Acetyltransferase Assay Ki
According to t, β-galactosidase activity can be measured by, for example, Aurora Gal-XE manufactured by Wako Pure Chemical Industries.

(8) Proliferation of cells expressing the protein of the present invention in response to the peptide of the present invention is observed by MAP kinase activation. This proliferation can be measured by MAP kinase activity, thymidine incorporation, and cell count (such as MTT). This can be used to screen for compounds that alter the binding of the peptide of the present invention to the protein of the present invention. The MAP kinase activity is determined by adding the peptide of the present invention or the peptide of the present invention and a test compound to cells, and then adding anti-MA to the cell lysate.
After obtaining a MAP kinase fraction by immunoprecipitation using a P kinase antibody, for example, MAP Kinase Assay K manufactured by Wako Pure Chemical Industries, Ltd.
It can be easily measured using it and γ- [ ³² P] -ATP. Thymidine uptake activity is seeded protein expressing cells of the present invention, after addition of the peptide or peptides of the present invention and a test compound of the present invention, [methyl- ³ H] - thymidine was added, it was then incorporated into the cells The radioactivity of labeled thymidine can be measured by lysing the cells and counting with a liquid scintillation counter. Proliferation of the protein-expressing cells of the present invention is performed by seeding the expressing cells, adding the peptide of the present invention or the peptide of the present invention and a test compound, and then adding MTT (3- (4,5-dimethyl-2-thiazolyl)-.
2,5-diphenyl-2H-tetrazolium bromide) is added, MTT formazan in which MTT is changed by being taken into the cell is dissolved, and the cell is lysed with isopropanol made acidic with hydrochloric acid. it can. A specific screening method using a labeled thymidine incorporation activity of a compound that alters the binding between the peptide of the present invention and the protein of the present invention is described below. Cells expressing the protein of the present invention were added to a 24-well plate at 5 / well.
000 seeds are cultured for one day. The cells are then starved in a serum-free medium for 2 days. After the peptide of the present invention or the peptide of the present invention and a test compound are added to the cells and cultured for 24 hours, [methyl- ³ H] -thymidine is added at 0.015 MBq per well and cultured for 6 hours. After the cells were washed with PBS, methanol was added for 1 hour.
Leave for 0 minutes. Then add 5% trichloroacetic acid and add 1
After standing for 5 minutes, the fixed cells are washed four times with distilled water.
The cells are lysed with a 0.3 N sodium hydroxide solution, and the radioactivity in the lysate is measured with a liquid scintillation counter. The effect of the compound that alters the binding between the peptide of the present invention and the protein of the present invention can be measured by comparing the increase in radioactivity due to thymidine incorporation with that of the case where the peptide of the present invention is administered alone. At this time, a compound that suppresses an increase in radioactivity due to administration of the peptide of the present invention can be selected as a candidate substance capable of competitive inhibition. On the other hand, an agonist can also be screened by administering only a test compound and observing an increase in radioactivity similar to that of the peptide of the present invention.

(9) When the peptide of the present invention is added to cells expressing the protein of the present invention, the K channel is activated,
Intracellular K ⁺ ions flow out of the cell. K ⁺ Rb ⁺ ions are ions and related elements, since through without K channel distinguished from the K ⁺ ions flow out of the cell, cells labeled Rb ^([86 Rb]) and allowed incorporated by adding Thereafter, the action of the peptide of the present invention can be measured by measuring the flow of [ ⁸⁶ Rb] flowing out upon stimulation of the peptide of the present invention.
A specific screening method for the compound that alters the binding between the peptide of the present invention and the protein of the present invention using [ ⁸⁶ Rb] efflux activity is described below. Protein expression cell of the present invention after 2 days plated into 24-well incubation for 2 hours in a medium containing ⁸⁶ RbCl of 1 mCi / ml. Wash the medium thoroughly and remove ⁸⁶
Remove RbCl completely. The peptide of the present invention or the peptide of the present invention and a test compound are added to the cells, the external solution is collected 30 minutes later, and the radioactivity is measured with a γ counter. The effect of the compound that alters the binding between the peptide of the present invention and the protein of the present invention can be measured by comparing the increase in radioactivity due to [ ⁸⁶ Rb] efflux with the case where the peptide of the present invention is administered alone. . At this time, a compound that suppresses an increase in radioactivity due to administration of the peptide of the present invention can be selected as a candidate substance capable of competitive inhibition. On the other hand, agonists can also be screened by administering only the test compound and observing the same increase in radioactivity as the peptide of the present invention.

(10) Extracellular pH (acid) which changes in response to the peptide of the present invention when the cell of the present invention expresses the protein
The activity of the peptide of the present invention can be measured by measuring the quantification rate using a Cytosensor device (Molecular Devices). A specific method for screening a compound that alters the binding between the peptide of the present invention and the protein of the present invention by measuring the change in extracellular pH using a Cytosensor device is described below. The protein-expressing cells of the present invention are cultured overnight in a capsule for a Cytosensor device, set in a chamber of the device, and RPMI 164 containing 0.1% BSA for about 2 hours until the extracellular pH is stabilized.
Permeate medium 0 (Molecular Devices). p
After H is stabilized, the pH change of the medium caused by perfusing the cells with the peptide of the present invention or the medium containing the peptide of the present invention and the test compound is measured. The effect of the compound that alters the binding of the peptide of the present invention to the peptide of the present invention can be measured by comparing the extracellular pH change of the protein-expressing cells of the present invention with that of the peptide of the present invention administered alone. it can. At this time,
A compound that suppresses extracellular pH change due to administration of the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability. On the other hand, an agonist can be screened by administering only a test compound and observing the same extracellular pH change as the peptide of the present invention.

(11) Yeast (Saccharomyces cerevisia)
e) The sex pheromone receptor Ste2 of the haploid α-mating type (MATα) is coupled with the G protein Gpa1, and responds to the sex pheromone α-mating factor
Activate AP kinase, and refer to Far1 (cell-cycle)
arrest) and the transcriptional activator Ste12 are activated. Ste12 induces the expression of various proteins including FUS1 involved in conjugation. On the other hand, the control factor Sst
2 functions suppressively in the above process. In this system,
Preparation of yeast into which a receptor gene has been introduced, activation of a signal transduction system in yeast cells by stimulation of a receptor agonist, and measurement of the reaction between the receptor agonist and the receptor using the resulting indicators such as proliferation. (Pausch, MH, Trends in Biotechnology, vol.
15, pp. 487-494 (1997)). Utilizing such a receptor gene-introduced yeast system, a compound that alters the binding of the peptide of the present invention and the protein of the present invention can be screened. The genes encoding Ste2 and Gpa1 of the MATα yeast are removed, and instead the gene of the protein of the present invention and the gene encoding the Gpa1-Gai2 fusion protein are introduced. The gene encoding Far is removed so that cell-cycle arrest does not occur, and the sensitivity of the response to the peptide of the present invention is improved by removing the gene encoding Sst. Further, a FUS1-HIS3 gene in which the histidine biosynthesis gene HIS3 is linked to FUS1 is introduced. The above-described genetic recombination operation is performed, for example, by Price et al. (Price, LA et al., Molecular and Cellular Biol.
ogy, vol. 15, pp. 6188-6195 (1995)), the somatostatin receptor type 2 (SST
R2) can be easily carried out by replacing the gene with the gene of the protein of the present invention. The transformed yeast thus constructed reacts with high sensitivity to the peptide of the present invention, which is a ligand of the protein of the present invention, and as a result, MAP kinase is activated to synthesize histidine biosynthetic enzyme. Viable in histidine-deficient medium. By utilizing this, the response of the protein-expressing yeast of the present invention by the peptide of the present invention can be observed using the growth of the yeast in a histidine-deficient medium as an index. The transformed yeast prepared as described above was cultured overnight in a liquid medium of a complete synthetic medium, and added to a dissolved agar medium from which histidine had been removed at a concentration of 2x10 ⁴ cells / ml, and 9x9 cm
Seeds in a 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 a test compound is placed on the agar surface, and cultured at 30 ° C. for 3 days. The effect of the compound that alters the binding between the peptide of the present invention and the protein of the present invention can be measured by comparing the growth of yeast around the filter paper with the case where the peptide of the present invention is administered alone. At this time, a compound that suppresses the growth of yeast by administration of the peptide of the present invention can be selected as a candidate substance having a competitive inhibition ability. On the other hand, an agonist can be screened by administering only a test compound and observing the growth of yeast similar to the peptide of the present invention. In addition, beforehand, the peptide of the present invention is added to the agar medium, and the test compound is impregnated on sterile filter paper and cultured. The effect of a compound that changes the binding between the peptide of the present invention and the protein of the present invention can also be examined.

(12) Gene RN of the protein of the present invention
A into Xenopus oocytes,
When stimulated by peptide, intracellular Ca²⁺High ion concentration
Ascending, calcium-activated chloride current occurs
You. This can be regarded as a change in membrane potential.
(K⁺The same applies when the ion concentration gradient changes). Departure
Protein transfection method of the present invention
Observing this response in Frog Xenopus oocytes
The connection between the peptide of the present invention and the protein of the present invention
Screening for compounds that may affect
Can be. Female Xenopus caught on ice and unable to move
The oocyte mass removed from the well was treated with MBS solution (88 mM N
aCl, 1mM KCl, 0.41mM CaCl_Two, 0.33mM Ca (NO_Three)_Two, 0.82m
MMgSO_Four, 2.4mM NaHCO_Three, 10 mM HEPES, pH 7.4)
1 until the egg mass is loosened with the collagenase (0.5mg / ml)
Treat at 150 rpm at 9 ° C for 1-6 hours. External solution is M
Wash three times by replacing with BS solution,
Poly (A)⁺SLT cRNA (50ng / 50nl)
Injection. The protein mRNA of the present invention
And in vitro transcription from plasmids
May be. This is cultured in MBS solution at 20 ° C. for 3 days.
You. This is the voltage clamp device flowing Ringer's solution.
Glass microelectrode for electric potential fixation, for electric potential measurement
A glass microelectrode is inserted into the cell, and the (-) pole is
Put. When the potential is stabilized, the peptide of the present invention or
Run Ringer's solution containing light peptide and test compound
Record the potential change. The peptide of the present invention and the tan of the present invention
The effects of compounds that alter protein binding are
Cell membrane potential of protein-introduced Xenopus oocytes
The change is compared to the case where the peptide of the present invention is administered alone.
Can be measured. At this time,
Inhibition of cell membrane potential change by administration of light peptide
Compound as a candidate substance with competitive inhibition ability
Can be. On the other hand, administration of the test compound alone
By observing a change in cell membrane potential similar to that of
Nist screening can also be performed. This system
To increase the amount of change in the reaction and make it easier to measure
Poly (A) of various G protein genes⁺ Introduce RNA
Can also be. Ca such as aequorin ²⁺In the presence of ions
Poly (A) of a protein gene that produces luminescence in⁺ RNA
Is not a membrane potential change
This reaction can also be measured by observing luminescence.

A kit for screening 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 comprises a protein of the present invention, a cell containing the protein of the present invention, or a protein containing the protein of the present invention. Containing the membrane fraction of the cells to be purified and the peptide of the present invention. Examples of the screening kit of the present invention include the following. 1. Screening reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
One containing 05% bovine serum albumin (manufactured by Sigma). Sterilized by filtration through a 0.45 μm filter, 4 ° C
Or may be prepared at the time of use. Standard of the protein of the present invention The CHO cells expressing the protein of the present invention were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well, at 37 ° C., 5% C
Cultured in O ₂ , 95% air for 2 days. Labeled ligand ^[3 H], ^[125 I], ^[14 C], the peptides of the present invention labeled with a ^[35 S]. The solution dissolved in an appropriate solvent or buffer is stored at 4 ° C. or −20 ° C., and diluted to 1 μM with a measuring buffer before use. Ligand standard solution The peptide of the present invention is dissolved to a concentration of 1 mM in PBS containing 0.1% bovine serum albumin (manufactured by Sigma).
Store at 20 ° C. 2. Assay Method The cells expressing the protein of the present invention cultured on a 12-well tissue culture plate are washed twice with 1 ml of the measurement buffer solution, and then 490 μl of the measurement buffer solution is added to each well. After adding 5 μl of a 10 ⁻³ to 10 ⁻¹⁰ M test compound solution, 5 μl of the labeled peptide of the present invention is added, and the mixture is reacted at room temperature for 1 hour. To determine the amount of non-specific binding, 5 μl of 10 ⁻³ M peptide of the present invention was used instead of the test compound.
I will add it. Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled peptide of the present invention bound to cells was 0.2
Dissolve in N NaOH-1% SDS and mix with 4 ml of liquid scintillator A (Wako Pure Chemical Industries). Liquid scintillation counter (Beckman)
Radioactivity was measured using Percent Maximum Binding
(PMB) is obtained by the following equation (Equation 1). [Equation 1] PMB = [(B−NSB) / (B ₀ −NSB)] × 10
0 PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding (Non-specific binding amount) B ₀ : Maximum binding amount

The compound or a salt thereof that can be 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. Specifically, 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 (a ZAQ agonist)), or a compound having no stimulating activity (a so-called protein of the present invention) Antagonists (ZAQ antagonists)
It is. Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, and the like, and these compounds may be novel compounds,
Known compounds may be used. The specific method of evaluating whether the protein of the present invention is an agonist or an antagonist may be in accordance with the following (i) or (ii). (I) After performing a binding assay shown in the above-mentioned screening method 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, It is determined whether or not 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 having no such activity or a salt thereof is an antagonist of the protein of the present invention. (Ii) (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) a case where 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 cells containing the protein of the present invention; When a compound to be converted and a test compound are brought into contact with cells containing the protein of the present invention, cell stimulating activities mediated by the protein of the present invention are measured and compared. The compound capable of decreasing the cell stimulating activity by 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. . Since 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, it is useful as a safe and low-toxic drug for suppressing the receptor activity.

As described above, since the peptide of the present invention has an activity of controlling intestinal contraction and the like, treatment and prevention of diseases such as digestive diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.). Among the compounds obtained using the above-described screening method or screening kit, agonists (ZAQ agonists) of the protein of the present invention can be used as digestive diseases (eg, enteritis, diarrhea, constipation) , A malabsorption syndrome, etc.). Further, the antagonist of the protein of the present invention (ZAQ antagonist) is used for digestive diseases (eg, enteritis,
It can be used as an agent for treating or preventing diseases such as diarrhea, constipation, malabsorption syndrome and the like. As a salt of a compound that can be obtained using the above-described screening method or screening kit, for example, a pharmaceutically acceptable salt or the like is used. Examples include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and aluminum salt and ammonium salt. Preferred examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine,
Examples thereof include salts with ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N'-dibenzylethylenediamine, and the like. Preferred examples of salts with inorganic acids 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. Preferred examples of the salt with a basic amino acid include, for example, salts with arginine, lysine, ortin, and the like, and preferred examples with the acidic amino acid include
For example, salts with aspartic acid, glutamic acid and the like can be mentioned. 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, a sugar-coated or enteric-coated tablet as required, a capsule, a elixir, orally as a microcapsule, or a sterile solution with water or another pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of injections such as suspensions. For example, it can be prepared 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. it can. The amount of the active ingredient in these preparations is such that a proper dosage in the specified range can be obtained. Examples of additives that can be incorporated into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry are used. When the preparation unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice such as dissolving or suspending the active substance in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. . Examples of aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents. For example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 ^™ , HCO
-50). The oily liquid includes sesame oil, soybean oil and the like, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent.
In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage Agents (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule.

The preparations thus obtained are safe and have low toxicity, and are, for example, mammals (eg, human, mouse, rat, guinea pig, rabbit, sheep, pig, cow, cat, dog, monkey, chimpanzee, 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, and the like. For administration, it is convenient to administer the ZAQ antagonist by intravenous injection in an amount of 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 amount can be administered in terms of 60 kg.

(3) Quantification of the Peptide of the Present Invention or a Salt Thereof Since the antibody of the present invention can specifically recognize the peptide of the present invention, the quantification of the peptide of the present invention in a test solution, particularly sandwich It can be used for quantification by immunoassay and the like. That is, the present invention provides (i) a method of competitively reacting an antibody of the present invention with a test solution and a labeled peptide of the present invention, and the ratio of the labeled peptide of the present invention bound to the antibody. And (ii) a test solution and an antibody of the present invention insolubilized on a carrier and another antibody of the present invention labeled with the test solution. And measuring the activity of the labeling agent on the insolubilized carrier after simultaneous or continuous reaction of the peptide of the present invention in a test solution.

In addition to the quantification of the peptide of the present invention using a monoclonal antibody against the peptide of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like can also be performed. . For these purposes, the antibody molecule itself may be used, or F (ab ') ₂ , Fab', or Fab fraction of the antibody molecule may be used. The method for quantifying the peptide of the present invention using the antibody of the present invention is not particularly limited. Any measurement method may be used as long as the amount of the complex 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, nephelometry, competition method, immunometric method and sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use the sandwich method described later. As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ³ H],
[ ¹⁴ C] and the like are used. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. Examples of the fluorescent substance include fluorescamine,
Fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For insolubilization of antigen or antibody,
Physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing a peptide or an enzyme may be used. As a carrier, agarose, dextran, insoluble polysaccharides such as cellulose, polystyrene,
Examples thereof include synthetic resins such as polyacrylamide and silicon, and glass. In the sandwich method, the 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 peptide of the present invention in the test solution can be determined. Primary reaction and 2
The next reaction may be performed in the reverse order, simultaneously, or at a different time. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not always one antibody.
It does not need to be a type, and a mixture of two or more types of antibodies may be used for the purpose of improving measurement sensitivity and the like. In the method for measuring the peptide of the present invention by the sandwich method of the present invention, as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different binding site to the peptide of the present invention is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the peptide of the present invention, the antibody used in the primary reaction is preferably C
For example, an antibody that recognizes an N-terminal other than the end 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, a nephelometry and the like. In the competition method, an antigen in a test solution and a labeled antigen are allowed to react competitively with an antibody, and then the unreacted labeled antigen is reacted.
(F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody or the like to the antibody, or an immobilized antibody is used as the first antibody, or
A solid-phase method using a soluble first antibody and a solid-phased antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. In nephelometry, the amount of insoluble precipitates generated as a result of 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 nephelometry utilizing laser scattering is preferably used.

In applying these individual immunoassays to the quantification method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the peptide of the present invention 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 and books. For example, Hiroe Irie, "Radio Immunoassay" (Kodansha, published in 1974), Hiroshi Irie, "Continuing Radio Immunoassay" (Kodansha, published in 1974),
Ed. Eiji Ishikawa et al. “Enzyme immunoassay” (Medical Publishing, Showa 53
Published in 1980), edited by Eiji Ishikawa et al. “Enzyme immunoassay” (second edition)
(Medical Shoin, published in 1982), Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical Shoin, published in 1987),
“Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Tec
hniques (Part A)), ibid.Vol. 73 (ImmunochemicalTechn.
iques (Part B)), Ibid.Vol. 74 (Immunochemical Techn)
iques (Part C)), ibid.Vol. 84 (Immunochemical Techni
ques (Part D: Selected Immunoassays)), ibid.Vol. 92
(Immunochemical Techniques (Part E: Monoclonal Antib
odies and General Immunoassay Methods)), ibid.Vol.
121 (Immunochemical Techniques (Part I: Hybridoma Te
chnology and Monoclonal Antibodies)) (both published by Academic Press). As described above, the peptide of the present invention can be quantified with high sensitivity by using the antibody of the present invention. Furthermore, by quantifying the concentration of the peptide of the present invention using the antibody of the present invention, if (1) an increase in the concentration of the peptide of the present invention is detected, for example, gastrointestinal diseases (eg, enteritis, diarrhea) , Constipation, malabsorption syndrome, etc.) or are likely to be affected in the future. Further, (2) when a decrease in the concentration of the peptide of the present invention is detected, for example, gastrointestinal diseases (eg, enteritis,
Diarrhea, constipation, malabsorption syndrome, etc.)
Alternatively, it can be diagnosed that the possibility of illness is high in the future. Further, the antibody of the present invention can be used for detecting the peptide of the present invention present in a subject such as a body fluid or a tissue. Further, for preparation of an antibody column used for purifying the peptide of the present invention, detection of the peptide of the present invention in each fraction at the time of purification, analysis of the behavior of the peptide of the present invention in test cells, etc. Can be used.

(4) Gene Diagnostic Agent The DNA of the present invention can be used, for example, as a probe to produce a human or warm-blooded animal (eg, rat, mouse, guinea pig, rabbit, bird, sheep, pig, cow,
DNA or mRNA abnormalities (genetic abnormalities) encoding the peptide of the present invention in horses, cats, dogs, monkeys, etc.) can be detected.
Or mRNA damage, mutation or decreased expression,
It is useful as a diagnostic agent for a gene such as an 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 known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989)).
Years), Proceedings of the National Academy of Sciences o
f the USA), Vol. 86, pp. 2766-2770 (19
1989)), a DNA microarray or the like. For example, when a decrease in expression is detected by Northern hybridization or a DNA microarray, or when a mutation in DNA is detected by the PCR-SSCP method or a DNA microarray, for example, gastrointestinal diseases (eg, enteritis, diarrhea, constipation, absorption) Diagnosis, etc.). (5) Medicine containing antisense DNA The antisense DNA capable of binding to the DNA of the present invention complementarily and suppressing the expression of the DNA is a function of the peptide of the present invention or the DNA of the present invention in vivo. Can be used as a therapeutic / prophylactic agent for diseases caused by excessive expression of the peptide of the present invention (for example, digestive diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.)). be able to. The above-mentioned antisense DNA can be used as the above-mentioned therapeutic / prophylactic agent in the same manner as the aforementioned therapeutic / prophylactic agent for various diseases containing the DNA of the present invention. For example, the antisense DNA
Can be administered alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector or the like, and then administered according to a conventional method. The antisense DNA
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. Furthermore, the antisense DNA 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.

(6) Pharmaceuticals Containing Antibody of the Present Invention Antibodies of the present invention, which have an activity of neutralizing the activity of the peptide of the present invention, are useful for, for example, diseases caused by overexpression of the peptide of the present invention (eg, digestion). It can be used as a medicament such as an agent for treating or preventing organ diseases (eg, enteritis, diarrhea, constipation, malabsorption syndrome, etc.). The therapeutic / prophylactic agent for the above-mentioned diseases containing the antibody of the present invention can be used directly as a liquid or as a pharmaceutical composition in an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, Dogs, monkeys, etc.) can be administered orally or parenterally. The dosage depends on the administration target, target disease, symptoms,
Although it differs depending on the administration route and the like, for example, for the purpose of treating digestive diseases, the antibody of the present invention is usually administered in a single dose of 0.0.
About 1-20 mg / kg body weight, preferably 0.1-10
mg / kg body weight, more preferably 0.1-5 mg
It is convenient to administer intravenously about 1 to 5 times a day, preferably about 1 to 5 times a day, preferably about 1 to 3 times a day. 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 according to the symptoms. The antibodies of the present invention can be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the above 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 and the like). Syrup, emulsion, suspension and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the field of pharmaceuticals. For example, carriers for tablets, excipients include lactose, starch,
Sucrose, magnesium stearate and the like are used.

Examples of the composition for parenteral administration include injections, suppositories and the like.
It includes dosage forms such as subcutaneous injections, intradermal injections, intramuscular injections, and infusions. 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. Examples of the aqueous liquid for injection include, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, and a suitable solubilizing agent, for example,
Alcohol (eg, ethanol), polyalcohol (eg,
Propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-
50 (polyoxyethylene (50mol) adduct of hydrogenat
ed castor oil)]. 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 in combination as a solubilizing agent. The prepared injection is usually filled in a suitable ampoule. A suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a usual suppository base. The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like.
It is preferable that the above antibody is contained in about 100 mg, and about 10 to 250 mg in other dosage forms. Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.

(7) Production of Non-Human Animal Containing the DNA of the Present Invention Using the DNA of the present invention, a transgenic non-human animal expressing the protein or the like of the present invention can be produced. Non-human animals include mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) (hereinafter abbreviated as animals), and in particular, mice, rabbits, etc. Is preferred. In introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells. For example, when a rabbit-derived DNA of the present invention is to be introduced, a gene construct in which the DNA of the present invention derived from an animal having high homology with the gene is bound to the downstream of various promoters capable of expressing the same in animal cells is used, for example, in a rabbit fertilized egg. By microinjecting the DNA into an animal, a DNA-transferred animal that highly produces the protein of the present invention can be produced. As this promoter, for example, a ubiquitous expression promoter such as a virus-derived promoter or metallothionein may be used, but an NGF gene promoter or an enolase gene promoter that is specifically expressed in the brain is preferably used.

The introduction of the 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 animal. The presence of the protein of the present invention in the germinal cells of the produced animal after DNA introduction
This means that all the offspring of the animal produced contain the protein or the like of the present invention in all of the germ cells and somatic cells. The progeny of this type of animal that has inherited the gene contains the protein of the present invention in all of its germinal and somatic cells. After confirming that the DNA-transferred animal of the present invention stably retains the gene by mating, the animal can be subcultured in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA,
By obtaining a homozygous animal having the transgene on both homologous chromosomes and mating the male and female animals, it is possible to breed and passage so that all progeny contain the DNA. The animal into which the DNA of the present invention has been introduced is useful as an animal for screening for an agonist or antagonist for the protein or the like of the present invention since the protein or the like of the present invention is highly expressed. The DNA-introduced animal of the present invention can also be used as a cell source for tissue culture. For example, analyzing the protein or the like of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-introduced mouse of the present invention or analyzing the tissue in which the protein of the present invention expressed by a gene is present. Can be. Culturing cells of a tissue containing the protein or the like of the present invention by standard tissue culture techniques, and using them to study the function of cells from a generally difficult-to-culture tissue such as, for example, from brain or peripheral tissue; Can be. In addition, by using the cells, for example, a drug that enhances the function of various tissues can be selected. In addition, if there is a high expression cell line, the protein of the present invention can be isolated and purified therefrom.

(8) Use of the peptide of the present invention, etc. The peptide of the present invention and SEQ ID NO: 36, SEQ ID NO: 4
0, a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, a partial peptide thereof, or a salt thereof, Peptides of the invention and SEQ ID NO: 36, SEQ ID NO: 4
0, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, of a compound having an amino acid sequence identical to or substantially identical to the amino acid sequence represented by SEQ ID NO: 49 or a salt thereof, The screening method can be performed in the same manner as the screening method of the present invention described in the above (2). The peptide of the present invention and a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, or a protein thereof Identical to the amino acid sequence represented by SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, which is characterized by containing a partial peptide or a salt thereof. Alternatively, a screening kit for a compound or a salt thereof that changes the binding property to a protein or a salt thereof having substantially the same amino acid sequence may be carried out in the same manner as the screening kit of the present invention described in (2) above. Can be. Peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 34 or a salt thereof, and SEQ ID NO: 1, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO:
47, a protein or its partial peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48 or SEQ ID NO: 49, or a salt thereof, wherein SEQ ID NO: 34
SEQ ID NO: 1, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO:
47, a method of screening for a compound or a salt thereof that alters the binding property to a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 48 or SEQ ID NO: 49,
It can be carried out in the same manner as the screening method of the present invention described in the above (2). Peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 34 or a salt thereof, and SEQ ID NO: 1, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: No .: 48 or SEQ ID NO: 49, characterized by containing a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 49, a partial peptide thereof, or a salt thereof. SEQ ID NO: 1, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49 with a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence or a salt thereof. Alters the binding to a protein or a salt thereof that has the same or substantially the same amino acid sequence as the represented amino acid sequence. Kit for screening a compound or its salt that is the (2) can be carried out similarly to the screening kit of the invention described. The compound or its salt obtainable by using the above-described screening method or screening kit is the same as the compound or its salt obtainable by using the screening method of the present invention or the screening kit of the present invention described in the above (2). Can be used.

In the present specification and drawings, when bases, amino acids and the like are represented by abbreviations, IUPAC-IUB
Abbreviations from the Commission on Biochemical Nomenclature or common abbreviations in the field,
An example is given below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine Y: thymine or cytosine N: thymine, cytosine, adenine or guanine R: adenine or guanine M: cytosine or adenine W: thymine or adenine Adenine S: cytosine or guanine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine triphosphate dCTP: deoxycytidine triphosphate ATP: adenosine triphosphate Gly Or G: glycine Ala or A: alanine Val or V: valine Leu or L: leucine Ile or I: isoleucine Ser or S: serine T r or T: threonine Cys or C: cysteine Met or M: methionine Glu or E: glutamic acid Asp or D: aspartic acid Lys or K: lysine Arg or R: arginine His or H: histidine Phe or F: phenylalanine Tyr Tyrosine Trp or W: tryptophan Pro or P: proline Asn or N: asparagine Gln or Q: glutamine pGlu: pyroglutamic acid Xaa: unidentified amino acid residue

Further, substituents, protecting groups, reagents and the like frequently used in the present specification are represented by the following symbols. Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group Ac: acetyl group TC: thiazolidine-4 (R) -carboxamide group Bom: benzyloxymethyl Bzl: benzyl Z: benzyloxycarbonyl Br-Z: 2- bromobenzyloxycarbonyl Cl-Z: 2-chloro benzyloxycarbonyl Cl ₂ Bzl: 2,6-dichlorobenzyl Boc: t-butyloxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy - 4-oxo-1,2,3-benzotriazine PAM: phenylacetamidomethyl Tos: p-toluenesulfonyl Fmoc: N-9-fluorenylmethoxycarbonyl DNP: dinitrophenyl Bum: tert-butoxymethyl T rt: trityl Bom: benzyloxymethyl Z: benzyloxycarbonyl MeBzl: 4-methylbenzyl DCC: N, N'-dicyclohexylcarbodiimide HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide NMP: N-methyl Pyrrolidone HONB: N-hydroxy-5-norbornene-2,3-dicarboxyimide NMP: N-methylpyrrolidone TFA: Trifluoroacetic acid CHAPS: 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate PMSF : Phenylmethylsulfonyl fluoride GDP: guanosine-5'-diphosphate Fura-2AM: 1- [6-amino-2- (5-carboxy-2-oxazolyl) -5-benzofuranyloxy] -2- ( 2-amino-5-methylfur Nonoxy) -ethane-N, N, N ', N'-tetraacetic acid pentaacetoxymethyl ester Fluo-3AM: 1- [2-amino-5- (2,7-dichloro-6-hydroxy-3-oxy- 9-xanthenyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ', N'-tetraacetoxymethylester HEPES: 2- [4- (2-hydroxyethyl ) -1-Piperazinyl] ethanesulfonic acid EDTA: ethylenediaminetetraacetic acid SDS: sodium dodecyl sulfate BSA: bovine serum albumin HBSS: Hanks balanced salt solution EIA: enzyme immunoassay

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the amino acid sequence of the human brain-derived protein of the present invention. [SEQ ID NO: 2] This shows the base sequence of the DNA encoding the human brain-derived protein of the present invention containing the amino acid sequence represented by SEQ ID NO: 1 (ZAQC). [SEQ ID NO: 3] This shows the base sequence of the DNA encoding the human brain-derived protein of the present invention containing the amino acid sequence represented by SEQ ID NO: 1 (ZAQT). [SEQ ID NO: 4] This shows the base sequence of primer 1 used in EXAMPLE 1, which will be described later. [SEQ ID NO: 5] This shows the base sequence of primer 2 used in EXAMPLE 1, which will be described later. [SEQ ID NO: 6] This shows the base sequence of primer 3 used in EXAMPLE 2, which will be described later. [SEQ ID NO: 7] This shows the base sequence of primer 4 used in EXAMPLE 2, which will be described later. [SEQ ID NO: 8] ZAQp used in Example 2 described later
2 shows the nucleotide sequence of the probe. [SEQ ID NO: 9] This shows the base sequence of primer ZAQC Sal used in EXAMPLE 2, which will be described later. [SEQ ID NO: 10] This shows the base sequence of primer ZAQC Spe used in EXAMPLE 2, which will be described later. [SEQ ID NO: 11] This shows the N-terminal amino acid sequence of the ZAQ activating peptide purified in Example 3 (3-8) described later. [SEQ ID NO: 12] This shows the base sequence of primer ZF1 used in EXAMPLE 4, which will be described later. [SEQ ID NO: 13] This shows the base sequence of primer ZF2 used in EXAMPLE 4, which will be described later. [SEQ ID NO: 14] This shows the base sequence of primer ZF3 used in EXAMPLE 4, which will be described later. [SEQ ID NO: 15] Human Z obtained in Example 4 described later
2 shows the base sequence at the 3 ′ end of DNA encoding the AQ ligand peptide. [SEQ ID NO: 16] This shows the base sequence of primer ZAQL-CF used in Example 4, which will be described later. [SEQ ID NO: 17] This shows the base sequence of primer ZAQL-XR1 used in EXAMPLE 4, which will be described later. [SEQ ID NO: 18] This shows the base sequence of the DNA fragment obtained in Example 4 described later. [SEQ ID NO: 19] This shows the base sequence of the DNA fragment obtained in Example 4 described later. [SEQ ID NO: 20] This shows the amino acid sequence of mature human ZAQ ligand peptide. [SEQ ID NO: 21] This shows the amino acid sequence of human mature ZAQ ligand mature peptide. [SEQ ID NO: 22] This shows the amino acid sequence of human ZAQ ligand precursor peptide. [SEQ ID NO: 23] This shows the amino acid sequence of human ZAQ ligand precursor peptide. [SEQ ID NO: 24] This shows the base sequence of DNA containing DNA encoding human ZAQ ligand precursor peptide represented by SEQ ID NO: 28. [SEQ ID NO: 25] This shows the base sequence of DNA containing DNA encoding human ZAQ ligand precursor peptide represented by SEQ ID NO: 29. [SEQ ID NO: 26] This shows the base sequence of DNA encoding the human type ZAQ ligand mature peptide represented by SEQ ID NO: 20. [SEQ ID NO: 27] This shows the base sequence of DNA encoding the human ZAQ ligand mature peptide represented by SEQ ID NO: 21. [SEQ ID NO: 28] This shows the base sequence of DNA encoding the human ZAQ ligand precursor peptide represented by SEQ ID NO: 22. [SEQ ID NO: 29] This shows the base sequence of DNA encoding the human ZAQ ligand precursor peptide represented by SEQ ID NO: 23. [SEQ ID NO: 30] This shows the base sequence of the DNA fragment used in Example 5 (5-1) described later. [SEQ ID NO: 31] This shows the N-terminal amino acid sequence of human ZAQ ligand peptide analyzed in Example 6 (6-2) described later. [SEQ ID NO: 32] This shows the base sequence of a primer used in EXAMPLE 7, which will be described later. [SEQ ID NO: 33] This shows the base sequence of a primer used in EXAMPLE 7, which will be described later. [SEQ ID NO: 34] This shows the amino acid sequence of snake venom MIT1 purified in Example 8 described later. [SEQ ID NO: 35] This shows the base sequence of cDNA encoding the human I5E receptor protein. [SEQ ID NO: 36] This shows the amino acid sequence of human I5E receptor protein. [SEQ ID NO: 37] This shows the base sequence of a primer used in EXAMPLE 9, which will be described later. [SEQ ID NO: 38] This shows the base sequence of a primer used in EXAMPLE 9, which will be described later. [SEQ ID NO: 39] This shows the base sequence of cDNA encoding a novel G protein-coupled receptor protein (rZAQ1). [SEQ ID NO: 40] This shows the amino acid sequence of novel G protein-coupled receptor protein (rZAQ1). [SEQ ID NO: 41] This shows the base sequence of a probe used in EXAMPLE 10, which will be described later. [SEQ ID NO: 42] This shows the base sequence of a probe used in EXAMPLE 10, which will be described later. [SEQ ID NO: 43] This shows the base sequence of a primer used in EXAMPLE 10, which will be described later. [SEQ ID NO: 44] This shows the base sequence of a primer used in EXAMPLE 10, which will be described later. [SEQ ID NO: 45] This shows the base sequence of a primer used in EXAMPLE 10, which will be described later. [SEQ ID NO: 46] This shows the base sequence of cDNA encoding a novel G protein-coupled receptor protein (rZAQ2). [SEQ ID NO: 47] This shows the amino acid sequence of a novel G protein-coupled receptor protein (rZAQ2). [SEQ ID NO: 48] This shows the amino acid sequence of mouse-derived G protein-coupled receptor protein (GPR73). [SEQ ID NO: 49] This shows the amino acid sequence of mouse-derived G protein-coupled receptor protein (mI5E). [SEQ ID NO: 50] This shows the base sequence of cDNA encoding mouse-derived G protein-coupled receptor protein (GPR73). [SEQ ID NO: 51] This shows the base sequence of cDNA encoding mouse-derived G protein-coupled receptor protein (mI5E). [SEQ ID NO: 52] DNA fragment # used in Reference Example 1
1 shows the base sequence of Example 1. [SEQ ID NO: 53] DNA fragment # used in Reference Example 1
2 shows the nucleotide sequence of 2. [SEQ ID NO: 54] DNA fragment # used in Reference Example 1
2 shows the base sequence of No. 3. [SEQ ID NO: 55] DNA fragment # used in Reference Example 1
4 shows the base sequence of Example 4. [SEQ ID NO: 56] DNA fragment # used in Reference Example 1
5 shows the base sequence of Example 5. [SEQ ID NO: 57] DNA fragment # used in Reference Example 1
6 shows the base sequence of No. 6. [SEQ ID NO: 58] This shows the base sequence of synthetic DNA encoding the human ZAQ ligand represented by SEQ ID NO: 21.

The transformant Escherichia coli DH5α / pCR obtained in Example 1 described later.
2.1-ZAQC has been in effect since August 23, 1999, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan. Biotechnology Industrial Technology Research Institute (NIB
H)) under the deposit number FERM BP-6855 from August 4, 1999, 2-17-85 Jusanhoncho, Yodogawa-ku, Osaka-shi, Japan Japan Fermentation Research Institute (IFO)
Deposit No. IFO 16301.
The transformant Escherichia coli DH5α / pCR2.1-ZA obtained in Example 1 described later.
QT has been registered with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (formerly Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology (NIBH)) from August 23, 1999 under the deposit number FERM BP-6856. 1999, 8
It has been deposited with the Fermentation Research Institute (IFO) under the deposit number IFO16302 from April 4. The transformant Escheric coli obtained in Example 4 described below (Escheric
hia coli) TOP10 / pHMITA has been deposited with the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary Center (formerly National Institute of Advanced Industrial Science and Technology (NIBH)) from July 13, 2000. BP-7219 and deposited on May 26, 2000 at the Institute of Fermentation (IFO) under the deposit number IFO 16440. The transformant Escherichia coli TOP10 / pHMITG obtained in Example 4 described below has been used since July 13, 2000 by the National Institute of Advanced Industrial Science and Technology (AIST) Patent Organism Depositary Center (formerly Ministry of International Trade and Industry). Deposit No. FER with National Institute of Bioscience and Biotechnology (NIBH)
MBP-7220, deposited on May 26, 2000 at the Institute of Fermentation (IFO) under the deposit number IFO 1
No. 6441. The transformant Escherichia coli (Escherichia col) obtained in Example 9 described later is used.
i) DH5α / pCR2.1-rZAQ1 was
Deposit No. FERM from the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center (formerly National Institute of Advanced Industrial Science and Technology (NIBH))
BP-7275, deposited on August 1, 2000 with the Institute of Fermentation (IFO) under the deposit number IFO16459
Has been deposited as The transformant Escherichia coli DH obtained in Example 10 described later
10B / pCMV-rZAQ2 was obtained on August 21, 2000.
Deposited from the National Institute of Advanced Industrial Science and Technology (AIST) at the Patent Organism Depositary Center (formerly the National Institute of Advanced Industrial Science and Technology (NIBH), Ministry of International Trade and Industry)
No. 76 has been deposited with the Fermentation Research Institute (IFO) from August 1, 2000 as a deposit number IFO16460. Escherichia coli MM294 (DE3) / p obtained in Reference Example 1 described below.
TCh1ZAQ has been registered with the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary from April 27, 2001 under the deposit number FERMBP-7571, and from January 16, 2001 under the deposit number IFO 16527. Deposited with the Fermentation Research Institute (IFO).

[0096]

The present invention will be described in more detail with reference to the following Examples and Reference Examples, which do not limit the scope of the present invention. Genetic manipulation using Escherichia coli is based on molecular cloning.
g) was followed.

Example 1 Cloning of cDNA encoding G protein-coupled receptor protein ZAQ and determination of nucleotide sequence Using human pituitary gland cDNA (CLONTECH) as a template, two primers, primer 1 (5'-GTCGACATGGAGACCACCACCA)
TGGGGTTCATGG-3 ′; SEQ ID NO: 4) and primer 2
(5′-ACTAGTTTATTTTAGTCTGATGCAGTCCACCTCTTC-3 ′; SEQ ID NO: 5) was used to carry out a PCR reaction. The composition of the reaction solution in this reaction uses 1/10 amount of the above cDNA as a template, and uses Advantage2 Polymerase Mix (CLONTEC
H company) 1/50 volume, primer 1 and primer 2 each
0.2 μM, dNTPs 200 μM, and the attached buffer were added to the enzyme to make a liquid volume of 25 μl. The PCR reaction was performed at 94 ° C
After 2 minutes, three cycles of 94 ° C for 20 seconds, 72 ° C for 100 seconds, three cycles of 94 ° C for 20 seconds, 68 ° C for 100 seconds, 94 ° C for 20 seconds, 64 ° C for 20 seconds , 68 ℃ ・
A 100 second cycle is repeated 38 times, and finally at 68 ° C.
A 7-minute extension reaction was performed. The reaction product after the PCR reaction was subcloned into a plasmid vector pCR2.1 (Invitrogen) according to the prescription of a TA cloning kit (Invitrogen). This was introduced into Escherichia coli DH5α, and cD
After selecting clones having NA in LB agar medium containing ampicillin, the sequences of the individual clones were analyzed. As a result, two types of cDNA sequences ZAQC encoding a novel G protein-coupled receptor protein (SEQ ID NO: 2) And ZAQT (SEQ ID NO: 3). Since the proteins containing the amino acid sequence derived from this cDNA all had the same sequence (SEQ ID NO: 1), they were named ZAQ, and the transformant containing the DNA represented by SEQ ID NO: 2 was used as a transformant for Escherichia coli. ) DH5α / pCR
A transformant containing 2.1-ZAQC and the DNA represented by SEQ ID NO: 3 was transformed into Escherichia coli DH5α / pCR2.1-
It was named ZAQT.

Example 2 Z by Taqman PCR
Analysis of AQ expression distribution Primers and probes used for Taqman PCR were obtained from Primer Express ver. 1.0 (PE Biosystems Japan) and search for primer 3 (5'-TCATGTTGCT
CCACTGGAAGG-3 '(SEQ ID NO: 6)), primer 4 (5'-C
CAATTGTCTTGAGGTCCAGG-3 '(SEQ ID NO: 7)), ZAQprobe
(5'-TTCTTACAATGGCGGTAAGTCCAGTGCAG-3 '(SEQ ID NO:
8)) was selected. As a reporter dye for the probe,
FAM (6-carboxyfluorescein) was added. As standard DNA, pAK-ZAQ obtained in Example 3 described later was used.
Using C as a template, primer ZAQC Sal (5'-GTCGACATGGAGACCA
CCATGGGGTTCATGG-3 '(SEQ ID NO: 9)) and ZAQC Spe
The PCR fragment amplified using (5′-ACTAGTTTATTTTAGTCTGATGCAGTCCACCTCTTC-3 ′ (SEQ ID NO: 10)) was converted to CHROMAS
PIN200 (CLONTECH Laboratories, Inc. (CA, USA))
Purified using, 10 ⁰ -10 ⁶ copies - was used to adjust the / [mu] l. As a cDNA source for each organization, Human Multiple T
issue cDNA Panel I and Panel II (CLONTECH Laborat
ories, Inc. )It was used. Primers, probes,
Add Taqman Universal PCR Master Mix (PE Biosystems Japan) to the template in the specified amount described in the attached
PCR reaction and analysis were performed using I PRISM 7700 Sequence Detection System (PE Biosystems Japan). The results are shown in FIG. ZAQ expression was mainly observed in the testis, then in the lungs, brain and the like.

[Table 1]

Example 3 Isolation of ZAQ Activating Peptide (3-1) Preparation of Milk Extract A commercially available pasteurized milk was used to prepare an extract as follows. Using a high-speed centrifuge (CR26H, R10A rotor: Hitachi, Ltd.), 2 liters of milk at 10,000 rpm, 15 rpm
For 4 minutes at 4 ° C., and the resulting supernatant was filtered through gauze.
The lipid pieces were removed. Acetic acid was added to the supernatant to a final concentration of 1 M, and the mixture was stirred at 4 ° C. for 30 minutes.
After centrifugation at 000 rpm for 15 minutes, the supernatant was filtered with gauze to remove insolubles. A double volume of acetone was added to the supernatant while stirring, followed by stirring at 4 ° C. for 3 hours. Next, a high-speed centrifuge (CR26
10,000 rp using H, R10A rotor: Hitachi, Ltd.
After centrifugation at m for 15 minutes, the resulting supernatant was filtered with gauze to remove insolubles. The obtained supernatant was put on a rotary evaporator to remove acetone, and finally 1350 ml
Concentrated. The obtained concentrate is added to 675 ml for 3 times.
The mixture was mixed with 38 ml of diethyl ether, mixed vigorously in a separating funnel, and an aqueous phase was obtained after two-phase separation. The same operation was repeated once more for the obtained aqueous phase to obtain a clear aqueous phase. The obtained aqueous phase was concentrated to 800 ml using a rotary evaporator to obtain a final extract.

(3-2) Crude Fractionation of Milk Extract by C18 Reversed-Phase Chromatography A column packed with silica gel having octadecyl groups fixed thereon.
After swelling 10 g of Sep-Pak C18 (Waters) with methanol, the mixture was equilibrated with 1 M acetic acid. In this column, (3-1)
(2 liters of milk) prepared as described above. Subsequently, 100 ml of 1 M acetic acid was passed through the column to wash the gel. Next, 200 ml of 60% acetonitrile / 0.1% trifluoroacetic acid was passed through the column to elute the crude peptide component of interest. The obtained eluate was concentrated using an evaporator and then freeze-dried (12EL;
(VirTis).

(3-3) Crude Fractionation of Milk Extract by Sulfopropyl Ion Exchange Chromatography A polypropylene column was swollen in 100 mM hydrochloric acid.
SP Sephadex C-25 (Amersham Pharmacia Biotech)
Was filled to a volume of 2 ml, washed with distilled water and 2 M ammonium formate (pH 4.0), and then equilibrated with solution I (2 M ammonium formate: acetonitrile: water = 1: 25: 74). The freeze-dried product obtained in the above (3-2) is
It was dissolved in 0 ml and loaded on 2 ml of SP Sephadex C-25. After washing with 10 ml of solution I, solution II (2 M ammonium formate: acetonitrile: water = 1: 2.5: 6.5), solution III (2
M ammonium formate: acetonitrile: water = 1: 1: 2),
IV solution (2M ammonium formate: acetonitrile: water =
1: 0.5: 0.5) Elution was performed sequentially at 10 ml each. Each of the obtained solutions I to IV was lyophilized (12EL; VirTis
Lyophilized.

(3-4) TSKgel O of milk extract
Fractionation by DS80Ts reversed-phase high-performance liquid chromatography A TSKgel ODS-80Ts reverse-phase high-performance liquid chromatography column (Tosoh Corporation, 4.6 mm x 25 cm) was used at 40 ° C at a flow rate of 1 ml / min for solution A (0.1% Trifluoroacetic acid / distilled water) volume 91.7% / B solution (0.1% trifluoroacetic acid /
A 60% acetonitrile) volume of 8.3% was run to equilibrate.
The freeze-dried products of Solution I to Solution IV obtained in the above (3-3) were each dissolved in 4 ml of 1 M acetic acid and subjected to a chromatography operation. That is, after 4 ml of the lyophilized product solution was applied to the column, the solution A volume was increased to 67% / solution B volume 33% over 1 minute at a flow rate of 1 ml / min, and then the solution A volume 67% over 40 minutes. The solution B concentration was increased in a linear gradient from 33%% / solution B volume to 0% solution A / 100% solution B volume. The eluate was fractionated with 1 ml fraction numbers, and 2 μl of each fraction was collected.
was mixed with 150 μl of 0.2% Bovine Serum Albumin (BSA) / distilled water and freeze-dried. This dried product is described in (3-
An assay sample for measuring the intracellular Ca ²⁺ ion concentration increasing activity described in 5) was used.

(3-5) Intracellular Ca using FLIPR
Measurement of ²⁺ ion concentration increasing activity A ZAQ stable expression cell line was prepared as follows. That is, one clone of DH5α / pCR2.1-ZAQC obtained in Example 1 was cultured with shaking in an LB medium containing ampicillin.
The plasmid pCR2.1-ZAQC was obtained. This is the restriction enzyme Sal
Treatment with I and SpeI cut out the insert coding for ZAQC. Similarly, pAKKO-1.11H (Biochemica et Bi) treated with restriction enzymes SalI and SpeI.
ophysicaActa 1219 (1994) 251-259) and the insert part using the Ligation Express Kit (CLONTECH Laboratori
es, Inc. (CA, USA)).
H10B was introduced by electroporation.
The structure of the plasmid contained in the obtained clone was confirmed by restriction enzyme treatment and sequence analysis, and a correctly constructed plasmid was used as a plasmid for expressing CHO cells, pAK-ZAQC. This plasmid pAK-ZAQC was transferred to CHO / dhfr ^- cells (American
an Type Culture Collection) to CellPhect Transfecti
It was obtained by transduction using an on kit (Amersham Pharmacia Biotech). First, Buffer A (CeF) was added to 4 μg of plasmid DNA dissolved in 120 μl of distilled water.
Add 120 μl of llPhect Transfection Kit)
After stirring and standing for 10 minutes, Buffer B (CellPhect Transf
240 μl) was added thereto, and the mixture was stirred vigorously to form a DNA-calcium phosphate complex containing the DNA. 5 x 10 ⁵ CHO / dhfr ^- cells 60 mm
Seed in a Petri dish, 10% fetal bovine serum (BIO WHITTAKE
R) in a Ham's F-12 medium (Nissui Pharmaceutical Co., Ltd.) for 1 day at 37 ° C. in 5% carbon dioxide.
480 μl of the suspension of the A-calcium phosphate complex was dropped on the cells in a petri dish. This was cultured at 37 ° C. in 5% carbon dioxide for 6 hours, and then the serum-free Ham's
The cells are washed twice with F-12 medium, and a buffer containing 15% glycerol (140 mM NaCl, 25 mM
HEPES, 1.4 mM Na ₂ HPO ₄ , pH 7.1) was added and treated for 2 minutes. This was again washed twice with a serum-free Ham's F-12 medium, and then Ham's containing 10% fetal bovine serum.
s The cells were cultured overnight in an F-12 medium at 37 ° C and 5% carbon dioxide. The cells were dispersed by trypsin treatment and collected from a Petri dish, and 2 × 10 ⁴ cells were inoculated in a 6-well plate, and dialyzed 10% fetal bovine serum (JRH BIOSCIENCES).
), 1 mM MEM non-essential amino acid solution (Dainippon Pharmaceutical Co., Ltd.), 100 units / ml Penicillin, 100 μg / ml Streptom
Dulbecco's modified Eagle medium (DME containing ycin)
M) Culture was started in a medium (Nissui Pharmaceutical Co., Ltd.) at 37 ° C. in 5% carbon dioxide gas. The transformed CHO cells into which the plasmid was introduced grow in the medium, but the non-transfected cells gradually died. Therefore, the medium was replaced every two days after the start of the culture to remove dead cells. After about 8 to 10 days from the start of culture, about 21 colonies of the transformed CHO cells were grown.
I chose one. RNA was recovered from each of the selected cells using a commercially available RNA isolation kit, and subsequently, a known RT-
A ZAQ-expressing CHO cell B-1 clone (hereinafter abbreviated as ZAQC-B1 cell) that highly expresses ZAQ was selected by PCR. As a control, clone 24 of CHO cells expressing ETA (endothelin A receptor) (hereinafter referred to as E
Abbreviated as TA24 cells. Journal of Pharmacology an
d Experimental Therapeutics, 279, 675-685, 199
6 years). For the assay sample obtained in the above (3-4), ZAQC-B1 cells and ETA
The intracellular Ca ²⁺ ion concentration increasing activity of 24 cells was measured using FLIPR (Molecular Devices). DM containing 10% dialyzed fetal bovine serum (hereinafter referred to as dFBS) was added to both ZAQC-B1 cells and ETA24 cells.
Those that were subcultured in EM were used. ZAQC-B1
Cells and ETA24 cells were suspended in a medium (10% d FBS-DMEM) at a concentration of 15 × 10 ⁴ cells / ml.
96-well plate (Black plate clear bottom, Coster)
Using a pipettor, inoculate 200 μl into each well (3.0
(× 10 ⁴ cells / 200 μl / well) The cells were cultured overnight in a 5% CO ₂ incubator at 37 ° C. before use (hereinafter referred to as a cell plate). H / HBSS (Nissui 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 μl and 200 μl of fetal bovine serum (FBS) were mixed. Fluo 3-AM (Dojindo Research Laboratories) 2 vials (50 μg)
To 40 μl of dimethyl sulfoxide, 20% Pluronic a
dissolved in 40 μl of cid (Molecular Probes), added to the above-mentioned H / HBSS-Probenecid-FBS, mixed, and then added to each well of the cell plate from which the culture solution had been removed using an 8-pipette.
Dispense 00 μl each and add 3 in a 5% CO ₂ incubator.
Incubated for 1 hour at 7 ° C (dye loading).
For the assay sample obtained in the above (3-4), 2.5 mM Probenecid, 0.1% CHA
150 μl of H / HBSS containing PS was added and diluted, and transferred to a 96-well plate for FLIPR (V-Bottom plate, Coster)
(Hereinafter referred to as a sample plate). After dye loading on the cell plate, plate washers (Molecular) with H / HBSS plus 2.5 mM Probenecid wash buffer.
The cell plate was washed four times with the aid of Devices, Inc., leaving 100 μl of wash buffer after washing. The cell plate and the sample plate were set on the FLIPR and an assay was performed.
μl of sample is transferred to the cell plate). As a result, the fraction (No. 5) obtained by separating the above (3-3) IV solution from the above (3-4) reverse phase high performance liquid chromatography was obtained.
3 showed an activity of increasing intracellular Ca ²⁺ ion concentration specific to ZAQC-B1 cells.

(3-6) TSKgel Super-P
Purification by henyl reversed-phase high-performance liquid chromatography TSKgel Super-Phenyl reversed-phase high-performance liquid chromatography
Column (Tosoh Corporation, 0.46 cm x 10 cm)
At 0 ° C, solution A (0.1%
91.7% capacity of acetic acid / distilled water / B liquid (0.1% trifluoro)
(Acetic acid / 60% acetonitrile) Equilibrate by flowing 8.3% volume
did. In the fraction No.53 obtained in the above (3-4)
Then, a chromatography operation was performed. That is,
After applying 1 ml of the solution of No. 53 to the column,
At a flow rate of 1 ml / min, solution A volume 75% /
Solution B volume is raised to 25%, then A over 75 minutes
Solution B concentration is linear up to solution volume 67% / solution B volume 33%
Raised with a gradient. Pipette the eluate in 500 μl aliquots.
A fraction was attached with a fraction number. Preparative fraction
25 μl each and mix with 150 μl of 0.2% BSA and freeze-dry
(12EL; VirTis). This dried product
H / HBSS containing 2.5 mM Probenecid and 0.1% CHAPS
Add 0 μl to dissolve, and use 50 μl of this solution
According to the test method of (3-5), intracellular Ca ²⁺ On ion concentration
By measuring the elevation activity, ZAQC-B1 cells
Receptor activating action was measured. As a result,
Has a receptor activating effect on ZAQC-B1 cells
Components, that is, ZAQ activating components, are mainly
It was found that the protein was eluted in Section No. 103-105.

(3-7) μRPC C2 / C18 ST
4.6 Purification by Reversed Phase High Performance Liquid Chromatography μRPC C2 / C18 ST 4.6 / 100 Column for Reversed Phase High Performance Liquid Chromatography (Amersham Pharmacia Biotech, 0.46
cm x 10 cm) at 40 ° C at a flow rate of 1 ml / min.
Liquid (heptafluorobutyric acid / distilled water) volume 95% / B liquid (0.
1% heptafluorobutyric acid / 100% acetonitrile) Capacity 5
% To equilibrate. TSKgel obtained in (3-6) above
Super-Phenyl reversed-phase high-performance liquid chromatography
After being applied to an RPC C2 / C18 ST 4.6 / 100 reverse phase column, solution A (0.1% heptafluorobutyric acid / distilled water) volume 95% / solution B (0.1% heptafluorobutyric acid / 100 % Acetonitrile) from 5% volume to 65% solution volume A / 35% solution volume, which is then increased at a flow rate of 1 ml / min for 60 minutes over a 60 minute period.
The eluate was recovered by increasing the linear gradient to 0% / B solution volume to 50%. The eluate was detected as a single peak at 210 nm UV absorption. Eluate 500 μl
Each fraction was numbered and fractionated, and 10 μl each of the fractions was mixed with 150 μl of 0.2% BSA and freeze-dried with a freeze dryer (12EL; VirTis). H / HBS containing 2.5 mM Probenecid, 0.1% CHAPS
75 μl of S was added and dissolved, and 50 μl of this solution was used to measure the receptor activating effect on ZAQC-B1 cells by the test method described in (3-5) above. As a result, a component having a receptor activating effect on the intended ZAQC-B1 cells, ie, a ZAQ activating component,
o.82-84. This activity peak completely coincides with the ultraviolet absorption peak at 210 nm,
It was determined that it was purified to a single peptide.

(3-8) Structural Analysis of Purified ZAQ Activating Peptide The structure of the ZAQ activating component obtained in the above (3-7) was determined by the following method. The purified product of ZAQ active ingredient was freeze-dried, and the obtained freeze-dried product was dissolved in DMSO solvent.
(Dimethylsulfoxide). A part of this solution was used as a protein sequencer (PerkinElmer,
E Biosystems Procise 491cLC) was used for amino acid sequence analysis from the N-terminus. As a result, 14 of the 16 amino acid residues from the N-terminal amino acid residue could be identified (Ala Val IleThr Gly Ala Xaa Glu
Arg Asp Val Gln Xaa Arg Ala Gly (SEQ ID NO: 1
1: Xaa is an unidentified residue)).

Example 4 Cloning of cDNA of Human ZAQ Ligand Peptide N-terminal Amino Acid Sequence of ZAQ Activating Peptide Purified from Milk Obtained in Example 3 (SEQ ID NO: 11)
A Blast search of the database using as a query revealed that a human EST (X40467) containing a sequence equivalent to the base sequence of DNA encoding a peptide having the amino acid sequence represented by SEQ ID NO: 11 was found. Since this sequence did not have a full-length open reading frame, the sequence of the undetermined portion was determined by the RACE method, and a cDNA clone having the full-length open reading frame was subsequently obtained. E
Based on information of ST (X40467), primers ZF1 (SEQ ID NO: 12), ZF2 (SEQ ID NO: 13) and ZF3
(SEQ ID NO: 14), and the human testis Marathon-Ready
3'RACE described below using cDNA (CLONTECH) as a template
An experiment was performed. ZF1: 5'-GGTGCCACGCGAGTCTCAATCATGCTCC-3 '(SEQ ID NO: 12) ZF2: 5'-GGGGCCTGTGAGCGGGATGTCCAGTGTG-3' (SEQ ID NO: 13) ZF3: 5'-CTTCTTCAGGAAACGCAAGCACCACACC-3 '(SEQ ID NO: 14) PCR of 3' RACE Reaction solution is 50 x Advantage 2 Polymerase
Mix (CLONTECH) 1 μl, attached 10 x Advantage 2
PCR buffer (400 mM Tricine-KOH, 150 mM KOAc, 35 mM
Mg (OAc) ₂ , 37.5μg / ml BSA, 0.05% Tween-20, 0.05%
Nonidet-P40), 5 μl, dNTP mixture (2.5 mM each,
(Takara Shuzo) 4 μl, 10 μM primer ZF1 1 μl, 10 μ
M Primer AP1 (Primer AP1 is from Maratho of CLONTECH)
1 μl of the template cDNA attached to the n-Ready cDNA Kit)
(CLONTECH, human testis Marathon-Ready cDNA)
1 and 33 μl of distilled water. The reaction conditions were: initial denaturation at 94 ° C for 60 seconds, 5 cycles of 94 ° C for 30 seconds to 72 ° C for 4 minutes, and 5 cycles of 94 ° C for 30 seconds to 70 ° C for 4 minutes.
The cycle reaction was repeated 25 times at 94 ° C. for 30 seconds-68 ° C. for 44 minutes. Subsequently, nest is performed using the reaction solution of the PCR reaction as a template.
ed PCR was performed. Reaction solution is 50 x Advantage 2 Polymer
1 μl of ase Mix (CLONTECH), 10 x Advantag attached
e 2 PCR buffer (400 mM Tricine-KOH, 150 mM KOAc, 3
5 mM Mg (OAc) ₂ , 37.5 μg / ml BSA, 0.05% Tween-20, 0.0
5% Nonidet-P40), dNTP mixture (2.5 mM eac
h, Takara Shuzo) 4 μl, 10 μM Primer ZF2 1 μl, 10 μl
μM Primer AP2 (Primer AP2 is CLONTECH's Mara
Thon-Ready cDNA Kit) (1 μl), template DN
A (50-fold diluted solution of the PCR reaction solution) was mixed with 5 μl of distilled water and 33 μl of distilled water. Reaction conditions are 94 ° C / 60
After the initial denaturation for 2 seconds, 5 cycles of 94 ° C./30 seconds-72 ° C./4 minutes, 5 cycles of 94 ° C./30 seconds-70 ° C./4 minutes, 9 cycles
A cycle reaction at 4 ° C. for 30 seconds-68 ° C. for 44 minutes was performed 25 times. Subsequently, a second nested PCR was performed using the reaction solution of the PCR reaction as a template. Reaction solution is 50 x Advantage 2
1 μl of Polymerase Mix (CLONTECH), 10 x
Advantage 2 PCR buffer (400 mM Tricine-KOH, 150 mM
KOAc, 35 mM Mg (OAc) ₂ , 37.5 μg / ml BSA, 0.05% Twee
5 μl of n-20, 0.05% Nonidet-P40), dNTP mixture (2.
5 mM each, Takara Shuzo) and 4 μl of 10 μM primer ZF3
μl, 10 μM Primer AP2 (Primer AP2 is CLONTECH
The one attached to Marathon-Ready cDNA Kit was used. ), 5 μl of template DNA (50-fold diluted solution of the PCR reaction solution), and 33 μl of distilled water were prepared. The reaction conditions were as follows: after initial denaturation at 94 ° C for 60 seconds,
The cycle reaction was carried out 5 times at 4 ° C. for 4 minutes, 5 times at 94 ° C. for 30 seconds to 70 ° C. for 4 minutes, and 25 times at 94 ° C. for 30 seconds at 68 ° C. for 44 minutes. TOPO TA Cloning
Cloning was performed using Kit (Invitrogen) according to the method described in the attached manual. The nucleotide sequence of the cloned DNA was decoded using ABI377 DNA sequencer to obtain a 3′-end sequence (SEQ ID NO: 15). SEQ ID NO:
Base sequence represented by No. 15 and EST (X40467)
The primers ZAQL-CF (SEQ ID NO: 16) and ZAQL-XR1 (SEQ ID NO: 17) were prepared based on the above information. Human testis Ma
PCR was performed using rathon-Ready cDNA (CLONTECH) as a template and primers ZAQL-CF and ZAQL-XR1. ZAQL-CF: 5'-CCACCATGAGAGGTGCCACG-3 '(SEQ ID NO: 16) ZAQL-XR1: 5'-CTCGAGCTCAGGAAAAGGATGGTG-3' (SEQ ID NO: 17) The PCR reaction solution was PfuTurbo DNA polymerase (Stratagene
1 μl), 5 μl of the attached 10 x PCR buffer, 2.5 mM dN
4 μl of TP mixture, 10 μM primer ZAQL-CF and ZAQL
-XR1 2.5 μl each, template DNA 5 μl, and distilled water 30
μl was prepared by mixing. The reaction conditions were as follows: initial denaturation at 95 ° C for 1 minute, cycle reaction at 95 ° C for 1 minute-60 ° C for 1 minute-72 ° C for 1 minute
And a final extension reaction at 72 ° C. for 10 minutes. The obtained DNA fragment was cloned using a TOPO TA Cloning Kit (Invitrogen) according to the method described in the attached manual. As a result of decoding the base sequence of the cloned DNA fragment using ABI 377 DNA sequencer, 371 bp of SEQ ID NO:
18 and SEQ ID NO: 19. The plasmid having the DNA fragment containing the nucleotide sequence represented by SEQ ID NO: 18 was named pHMITA, and the plasmid having the DNA fragment containing the nucleotide sequence represented by SEQ ID NO: 19 was named pHMITG. Escherichia coli (Escher) by plasmids pHMITA and pHMITG
(Escherichia coli) TOP10 / pHMITA and Escherichia coli TOP10 / pHMITG, respectively. As a result of analyzing the base sequences of these DNA fragments, the DNA fragment represented by SEQ ID NO: 18
The human ZAQ ligand precursor peptide (A
Type, 105 amino acid residues), and the DNA fragment represented by SEQ ID NO: 19 is the human ZA represented by SEQ ID NO: 23.
It was found that the DNA contained a DNA (SEQ ID NO: 29) encoding a Q ligand precursor peptide (G type, 105 amino acid residues). The base sequences represented by SEQ ID NO: 28 and SEQ ID NO: 29 have a typical signal sequence, and the DNA containing the base sequence represented by SEQ ID NO: 28 is represented by SEQ ID NO: 20. Human type ZAQ ligand mature peptide (A type, 8
D consisting of 258 base pairs encoding 6 amino acid residues)
NA (SEQ ID NO: 26) and SEQ ID NO: 29
The DNA containing the base sequence represented by SEQ ID NO:
Human ZAQ ligand mature peptide represented by 21
(G type, 86 amino acid residues) and 258 base pairs of DNA (SEQ ID NO: 27).

Example 5 Production of Human ZAQ Ligand Peptide in Mammalian Cells (1) (5-1) Construction of Human ZAQ Ligand Precursor Peptide Mammalian Cell Expression Vector Ecologically derived from plasmid pHMITG obtained in Example 4
382 containing cDNA encoding human ZAQ ligand precursor peptide by RI, XhoI restriction enzyme digestion
A bp DNA fragment (SEQ ID NO: 30) was cut out. That is, the plasmid pHMITG was digested with EcoRI and XhoI, and the obtained DNA was electrophoresed on a 1.5% agarose gel to give a cyber green stain of about 3%.
A piece of gel containing the 82 bp band was cut with a razor. Gene Clean spin DNA extraction kit (BIO 101
DNA fragments were recovered using the method described in US Pat. The obtained DNA fragment is used for CMV-IE enhancer and chicken beta-actin pro
EcoRI and XhoI were used for the mammalian cell expression vector pCAN618 (FIG. 11) using moter as an expression promoter.
It was cloned into the restriction enzyme cleavage site according to a standard method. As a result of decoding the nucleotide sequence of the cloned DNA fragment by the method described above, it was confirmed that the DNA fragment had the nucleotide sequence represented by SEQ ID NO: 30. The mammalian cell expression vector containing the DNA encoding the human ZAQ ligand precursor peptide was named pCANZAQLg2.

(5-2) Introduction of Expression Vector into COS7 Cells COS7 cells were purchased from the ATCC, and DMEM medium (1
(To which 0% FBS was added). 1.5 × 1 COS7 cells in DMEM medium
Spread it on a 10cm Petri dish so that it becomes 0 ⁶ cells / dish, 37
° C., and cultured overnight in 5% CO ₂ incubator. Human ZAQ ligand precursor peptide expression plasmid (pCAN
ZAQLg2) 2 µg (dissolved in 2 µl of TE buffer) in buffer EC (Effectene transfection reagent, QIAGEN) 29
8 μl was added, and further 16 μl of Enhancer was added, mixed for 1 second, and left at room temperature for 3 minutes. Further Effectene Transfecti
60 μl of on Reagent was added, mixed for 10 seconds, and left at room temperature for 10 minutes. Remove the supernatant of the cells sown the previous day and remove
After washing once with 10 ml of medium, 9 ml of DMEM medium was added. After adding 1 ml of DMEM medium to the plasmid solution and mixing, the mixture was added dropwise to the cells. After the whole was mixed, the cells were cultured overnight at 37 ° C. in a 5% CO ₂ incubator. DMEM medium 10m
After washing twice with l, 10 ml of DMEM medium was added,
° C., and cultured overnight in 5% CO ₂ incubator. Two days later, the culture supernatant was collected.

(5-3) Partial purification of ZAQ-activating peptide from culture supernatant of COS7 cell expressing human ZAQ ligand precursor peptide (5-3-1) COS7 cell culture expressing human ZAQ ligand precursor peptide Preparation of supernatant extract The culture supernatant of the COS7 cell expressing the human ZAQ ligand precursor peptide was collected, and the following operation was performed to prepare an extract. First, 1.1 ml of acetic acid was added dropwise to the cell culture supernatant (about 18.5 ml) so that the final concentration was 1 M, and the mixture was stirred for 1 hour.
Further, twice the volume of acetone was added thereto, and the mixture was stirred at 4 ° C. for 30 minutes.
The mixture was centrifuged at 15,000 rpm for 30 minutes using Hitachi, Ltd. to obtain a supernatant. The obtained supernatant was subjected to an evaporator to remove acetone, and then freeze-dried (12EL; VirTis).
Lyophilized.

(5-3-2) Sephade of COS7 cell culture supernatant expressing human ZAQ ligand precursor peptide
x G50 gel filtration chromatography and SepPa
k column chromatography The lyophilized powder obtained in the above (5-3-1) was dissolved in 2 ml of 1 M acetic acid, and then equilibrated with 1 M acetic acid.
(3 cm in diameter, 35 ml, Pharmacia Biotech) After adsorbing to the column, 1 M acetic acid was passed through the column, and the eluate was fractionated in 5 ml fractions with a fraction No., and freeze-dried (12 ml).
EL; VirTis). The SepPak C18-5g column (10 ml) was swollen with methanol, and then equilibrated by flowing 0.1% trifluoroacetic acid / distilled water. Sephadex G50
Among the fractions obtained by gel filtration chromatography, freeze-dried fractions Nos. 1 to 16 were combined and dissolved in 3 ml of 0.1% trifluoroacetic acid / distilled water, and applied to a SepPak C18-5g column. After washing with 24 ml of distilled water, 0.1% trifluoroacetic acid / 60% acetonitrile
Elution was at 20 ml. The obtained eluate was applied to a servant.

(5-3-3) Purification by Super ODS Reversed-Phase High-Performance Liquid Chromatography A TSKgel Super ODS reversed-phase high-performance liquid chromatography column (Tosoh Corporation, 0.46 cm × 10 cm) was flowed at 40 ° C. Solution A (0.1% trifluoroacetic acid / distilled water) was flowed at 1 ml / min to equilibrate. Obtained in (5-3-2)
After applying the SepPak C18-5g column fraction to a servant, it is applied to Super ODS reversed-phase high-performance liquid chromatography, and the solution A (0.1% trifluoroacetic acid / distilled water) volume 100% / solution B at a flow rate of 1 ml / min for 60 minutes. (0.1% trifluoroacetic acid / 60% acetonitrile) Volume 0% to Solution A volume 0%
/ B liquid volume up to 100% with a linear gradient,
The eluate was collected. The eluate was collected in 1 ml fraction N
The sample was collected with an o., and the whole fraction was freeze-dried with a freeze dryer (12EL; VirTis). To the dried product, 150 μl of H / HBSS to which 2.5 mM Probenecid and 0.2% BSA were added was added and dissolved.
The receptor activating effect on ZAQC-B1 cells was measured by the test method described in 3-4).

(5-3-4) Measurement of Intracellular Ca ²⁺ Ion Concentration Activity Using FLIPR The sample obtained in the above (5-3-3) was obtained in Example 3 (3-5). ZAQ-expressing cells (ZAQC-
The activity of increasing intracellular Ca ²⁺ ion concentration in B1) was measured using FLIPR. HOT7T1 as a control
75-expressing cells (hOT7T175-16; described in WO 00/24890) were used. For both ZAQC-B1 cells and hOT7T175-16 cells, those subcultured in DMEM supplemented with 10% dialyzed fetal bovine serum (hereinafter referred to as dFBS) were used. ZA
QC-B1 cells and hOT7T175-16 cells were each 15 × 10 ⁴ ce
lls / ml in a medium (10% dFBS-DMEM).
96-well plate for LIPR (Black plate clear bottom, C
(oster company) using a dispenser to seed 200 μl into each well.
(3.0 × 10 ⁴ cells / 200 μl / well) After culturing overnight at 37 ° C. in a 5% CO ₂ incubator, it was used (hereinafter referred to as a cell plate). H / HBSS (HANKS '9.8 g, sodium hydrogen carbonate 0.35 g, HEPES 4.77 g, pH with sodium hydroxide
After adjusting to 7.4, filter sterilization treatment) 21 ml, 250 mM Pr
Obenecid 210 μl and fetal bovine serum (FBS) 210 μl were mixed. Fluo3-AM 2 vial (50 μg) was added to 42 μl of dimethyl sulfoxide and 42 μl of 20% Pluronicacid
Dissolved in, the mixture was added to the H / HBSS-Probenecid-FBS, after mixing, dispensed to each well 100μl the cell plate except the culture solution using an 8 channel pipette, 37 in a 5% CO ₂ incubator Incubated for 1 hour at ℃
(Dye loading). For the assay sample obtained in the above (5-3-3), each fraction was subjected to H / HBSS.
150 μl of 2.5 mM Probenecid and 0.2% BSA was added and dissolved, and transferred to a 96-well plate for FLIPR (V-Bottom plate, Coster) (hereinafter referred to as a sample plate). After dye loading on the cell plate, H / HBSS
The cell plate was washed four times with a washing buffer containing 2.5 mM Probenecid using a plate washer (Molecular Devices), and after washing, 100 μl of washing buffer was left. FLIP the cell plate and sample plate
R and the assay was performed (by FLIPR,
0.05 ml of sample is transferred from the sample plate to the cell plate). ZAQC-B in fraction No.48-68
One cell-specific intracellular Ca ²⁺ ion concentration increasing activity was observed. From this, the target component having a receptor activating effect on ZAQC-B1 cells, namely, ZAQC-B1 cells
It was found that the Q-activating component was eluted in fractions No. 48-68.

Example 6 Production of Human ZAQ Ligand Peptide in Mammalian Cells (2) (6-1) Preparation of Culture Supernatant Human ZAQ was added to COS7 cells by the method described in Example 5.
Ligand precursor peptide expression plasmid (pCANZAQLg2)
Was introduced. That is, COS7 using DMEM medium
The cells were seeded on a 15 cm petri dish at 3.0 × 10 ⁶ cells / dish and cultured overnight at 37 ° C. in a 5% CO ₂ incubator. Buffer EC (Effectene transfection reage) was added to 4 μg of human ZAQ ligand precursor peptide expression plasmid (pCANZAQLg2) (dissolved in 4 μl of TE buffer).
(nt, QIAGEN) (600 μl), further added Enhancer (32 μl), mixed for 1 second, and allowed to stand at room temperature for 3 minutes. Further Effect
120 μl of eneTransfection Reagent was added, mixed for 10 seconds, and left at room temperature for 10 minutes. The supernatant of the cells sowed the day before was removed, washed once with 10 ml of DMEM medium, and 30 ml of DMEM medium was added. After adding 1 ml of DMEM medium to the plasmid solution and mixing, the mixture is added dropwise to the cells, and the whole is mixed.
The cells were cultured overnight in a 5% CO ₂ incubator at 7 ° C. D
After washing once with 10 ml of MEM medium,
and cultured overnight at 37 ° C. in a 5% CO ₂ incubator. One day later, the culture supernatant was collected and further added to DMEM.
After adding 20 ml of the medium and culturing overnight in a 5% CO ₂ incubator at 37 ° C., the culture supernatant was recovered.

(6-2) Purification of Human ZAQ Ligand Peptide from Culture Supernatant Culture supernatant of 80 15 cm dishes was collected by the method described in (6-1), and acetic acid was added thereto to a final concentration of 1M. Was added so that After stirring for 1 hour, twice the volume of acetone was added to precipitate the protein. The mixture was stirred at 4 ° C. for 30 minutes, and then centrifuged at 10,000 rpm for 30 minutes using a high-speed centrifuge (CR26H, RR10A type rotor: Hitachi, Ltd.) to obtain a supernatant. The obtained supernatant was subjected to an evaporator to remove acetone, and the supernatant was applied to a reversed phase column (Waters C18, 100 g) which had been equilibrated with 0.1% trifluoroacetic acid / distilled water in advance. 1000 ml of 0.1% trifluoroacetic acid / distilled water, then 0.1
After washing the column with 1000 ml of trifluoroacetic acid / 20% acetonitrile, the peptide was eluted with 1000 ml of 0.1% trifluoroacetic acid / 60% acetonitrile. After the obtained eluate was subjected to an evaporator, a freeze dryer (12EL; V)
(IrTis). TSKgel ODS80TM Reversed-Phase High Performance Liquid Chromatography Column (Tosoh Corporation, 2
1.5 mm x 30 cm) at 40 ° C at a flow rate of 4 ml / min
Then, the solution A (0.1% trifluoroacetic acid / distilled water) was flowed to equilibrate. After dissolving the obtained lyophilized powder in solution A, the solution was applied to the ODS80TM column, and the solution was dried at a flow rate of 4 ml / min.
0% solution A (0.1% trifluoroacetic acid / distilled water) volume 60% / solution B (0.1% trifluoroacetic acid / 60% acetonitrile) volume 40% to solution A volume 0% / solution B 100%
The peptide was eluted with a linear gradient up to. The eluate was fractionated by giving a fraction No. every 8 ml, and 50 μl was taken from the fractionated fraction and freeze-dried with a freeze dryer (12EL; VirTis). To this dried product, 200 μl of 2.5 mM Probenecid and 0.2% BSA added to H / HBSS was added and dissolved.
The receptor activating effect on ZAQC-B1 cells was measured by the test method of -3-4). As a result, a component having a receptor activating effect on the intended ZAQC-B1 cells, ie, a ZAQ activating component,
o. It was found to be eluted at 32. A TSKgel CM-2SW ion-exchange high-performance liquid chromatography column (Tosoh Corporation, 4.6 mm x 25 cm) was applied at 25 ° C at a flow rate of 1 m.
Solution A (10 mM ammonium formate / 10% acetonitrile) was flowed at 1 / min to equilibrate. No.3 above
2 was attached to the CM-2SW column at a flow rate of 1 ml / min.
Solution A (10 mM ammonium formate / 10% acetonitrile) volume 100% / solution B (1000 mM ammonium formate /
10% acetonitrile) Volume 0% to solution A volume 0% / B
With a linear gradient up to 100% liquid volume,
The peptide was eluted. The eluate was fractionated with a 1 ml fraction number, and 1.5 μl
And transfer it to H / HBSS with 2.5 mM Probenecid, 0.2% BSA
Dilute 200 µl, and use this solution to prepare (5-3-4)
The receptor activating effect on ZAQC-B1 cells was measured according to the test method described above. As a result, the desired ZAQC-
A component having a receptor activating effect on B1 cells, that is, a ZAQ activating component, is represented by fraction Nos. 56 and
57 was found to be eluted. TSKgel Super pheny
l A reverse-phase high performance liquid chromatography column (Tosoh Corporation, 4.6 mm x 10 cm) was flowed at 40 ° C at a flow rate of 1 ml.
Solution A (0.1% trifluoroacetic acid / distilled water) was allowed to flow at / min to equilibrate. Fractions No. 56 and 57 above
Is applied to the Super phenyl column, and the flow rate is 1 ml / mi.
Solution A (0.1% trifluoroacetic acid / distilled water) volume 70% / Solution B (0.1% trifluoroacetic acid / 60%
The peptide was eluted with a linear gradient from 30% acetonitrile) volume to 50% solution A / 50% solution B volume. The eluate was fractionated by 1 ml.
A 1.5 μl sample was collected from the fraction, and 2.5 μM Probenecid, 0.2% BSA 200 was added to H / HBSS.
After diluting μl, using this solution, the receptor activating effect on ZAQC-B1 cells was measured by the test method described in (5-3-4). As a result, the desired ZAQC-B1
It was found that the component having a receptor activating effect on cells, that is, the ZAQ activating component, was eluted in fractions Nos. 54, 55 and 56. This activity coincided with a single ultraviolet absorption peak, and it was determined that the active ingredient had been purified to a single level. The solvent in the purified sample of the ZAQ-activating component is removed by freeze-drying, and the obtained freeze-dried product is dissolved in solvent D
Dissolved in MSO (dimethyl sulfoxide). A portion (about 7.5 pmol) of this solution was transferred to a protein sequencer.
(PerkinElmer, PE Biosystems Procise 491cLC)
For N-terminal amino acid sequence analysis. as a result,
Of the tenth amino acid residue from the N-terminal amino acid residue, nine residues could be identified (Ala Val Ile Thr
Gly Ala Xaa Glu Arg Asp (SEQ ID NO: 31; Xaa is an unidentified residue)). The amino acid sequence obtained was consistent with the predicted N-terminal amino acid sequence of the mature human ZAQ ligand peptide. In addition, mass spectrometry of the purified sample of the ZAQ-activating component was performed using a Finnigan LCQ LC / MS device (Thermoquest, San Jose,
Using CA), electrospray ionization was performed, and it was confirmed that the molecular weight was 9657.6. This is 1
This is in good agreement with the theoretical value of 9657.3 of the human type ZAQ ligand mature peptide (SEQ ID NO: 21) of 86 residues in which all 0 cysteine residues form disulfide bonds.
It was confirmed that the purified Q-activating component preparation had a humanized ZAQ ligand mature peptide having the amino acid sequence represented by SEQ ID NO: 21.

(6-3) Measurement of ZAQ Activating Effect of Purified Human ZAQ Ligand Peptide It measured by the test method of (5-3-4). As a result, in ZAQ-expressing CHO cells (ZAQC-B1 cells), the mature human ZAQ ligand peptide caused an increase in intracellular calcium concentration in a concentration-dependent manner. The EC ₅₀ value was 96 pM, which revealed that the mature human ZAQ ligand peptide exhibited a very strong agonist activity.
The results are shown in FIG.

Example 7 Production of Human ZAQ Ligand Peptide in Mammalian Cells (3) (7-1) Human ZAQ ligand peptide stably expressed CH
Establishment of an O cell line Using the plasmid pHMITG described in Example 4 as a template, the following primers 5 ′ GTCGACCACCATGAGAGGTGCCACGC 3 ′ (SEQ ID NO: 32) 5 ′ ACTAGTCGCAGAACTGGTAGGTATGG 3 ′ (SEQ ID NO: 33) The PCR was amplified and cloned into pCR Blunt II vector (Invitrogen). The insert cDNA was excised from the obtained clone having the correct sequence using SalI and SpeI restriction enzymes, and inserted into a pAKKO1.11H expression vector.
Transfer the plasmid to CHO / dhFr ^- cell (AmericanType Cultur
e Collection), CellPhec described in (3-5) above
t Transfection kit (Amersham Pharmacia Biotech)
Was transduced according to the method using. Culture supernatants were collected from several clones of the transduced strain, and the ZAQ ligand activity for increasing the intracellular Ca ²⁺ ion concentration of ZAQC-B1 cells was measured by the test method described in (3-5). Thus, ZAQL-1 expressing C, which highly expresses ZAQ ligand
HO cell clone No. 4 was selected. (7-2) Preparation of serum-free culture supernatant of CHO cells expressing human ZAQ ligand (ZAQL-1) Dialyzed 10% fetal bovine serum (JRH BIOSCIENCES), 1
mM MEM non-essential amino acid solution, 100 units / ml Penicilli
n, Dulbecco's Modifie containing 100 μg / ml Streptomycin
d Eagle Medium (DMEM) Medium (Nissui Pharmaceutical Co., Ltd.)
The ZAQL-1-expressing CHO cell clone No. 4 cultured to 4 confluent Single Tray (Nunc) was trypsinized, dispersed, and then centrifuged and collected. Single T above
After suspending one ray of cells in 1.5 L of the above medium, use Cell Factori
es 10 (Nunc) and 4 Cell Factories 10
Was cultured in 5% carbon dioxide at 37 ° C. for 3 days. After removing the culture supernatant, 1 Cell Fac was added with 1 L of H / HBSS described above.
The tories 10 cells were washed. After removing H / HBSS, 2 L of serum-free medium (1 mM MEM)
Non-essential amino acid solution, 100 units / ml Penicillin, 100
Dulbecco's Modified Eagl containing μg / ml Streptomycin
e Medium medium) and further cultured for 2 days. The collected culture supernatant was centrifuged at 1,000 rpm for 10 minutes using a Hitachi high-speed centrifuge, and then filtered using gauze to obtain a clear supernatant. Acetic acid was added to this to a final concentration of 1 M, and the following operation was performed.

(7-3) Crude fractionation of serum-free culture supernatant of CHO cells expressing ZAQL-1 by octadodecyl reverse phase chromatography Prep packed with silica gel having octadodecyl groups fixed
C18 (Waters) was swollen with methanol and packed in a glass column (50 mm × 100 mm). Thereafter, the extract prepared in (6-2) was applied to a column equilibrated with 1 M acetic acid. The column was then washed with 800 ml of 1M acetic acid. Next, 1000 ml of 60% acetonitrile / 0.1% trifluoroacetic acid was passed through the column to elute the crude peptide component of interest. The obtained eluate was concentrated using an evaporator and then freeze-dried (12EL; Vir
(Tis). (7-4) Wakosil-II 5C18HG Pre
p Separation by reversed-phase high-performance liquid chromatography A Wakosil-II 5C18HG Prep reverse-phase high-performance liquid chromatography column (Wako Pure Chemical Industries, 20 mm × 250 mm) was used at 40 ° C. at a flow rate of 5 ml / min. Acetonitrile / distilled water) volume of 91.7% / solution B (0.1% trifluoroacetic acid / 60% acetonitrile) volume of 8.3% was flowed for equilibration. Chromatography was performed on the freeze-dried product obtained in the above (7-3). That is, the lyophilized product is 1M
After adding and dissolving 36 ml of acetic acid and centrifuging, 1/3 of the solution was applied to the column, and the volume of solution A was increased to 66.7% / volume of solution B over 3 minutes at a flow rate of 5 ml / ml for 1 minute. Solution A volume 16.7% Solution B volume 8 over 120 minutes
Solution B concentration was increased with a linear gradient to 3.3%. The eluate was fractionated with a 5 ml fraction number. 0.2% BSA 150 in 3 μl each from the preparative fraction
μl and freeze-dried with a freeze dryer (12EL; VirTis). This dried product is added to an assay buffer [H / HBSS
(Nissui 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), 2.5 mM Probenecid and 0.1% CHAPS Was added and dissolved, and 50 μl of this solution was used to measure the ZAQ-B1 receptor activating effect according to the test method described in (3-5) above. As a result, the desired ZA
It was found that the component having the Q-B1 receptor activating action was mainly eluted in fraction Nos. 73-75.

(7-5) Separation by TSKgel CM-2SW Ion Exchange High Performance Liquid Chromatography A TSKgel CM-2SW ion exchange high performance liquid chromatography column (Tosoh, 7.8 × 300 mm) was used at 25 ° C. at a flow rate of 2 ml.
Flow A solution (4 M ammonium formate: distilled water: acetonitrile = 1: 299: 100) 100% volume and Solution B (4 M ammonium formate: distilled water: acetonitrile = 1: 2: 1) volume 0% at / min. Equilibrated. Wakosil obtained in (7-4)
-II 5C18HG Prep Preparative fraction of reversed-phase high-performance liquid chromatography, fractionated fractions No.73-75 were lyophilized, dissolved in 4 ml of solution A, applied to a TSKgel CM-2SW ion exchange column, and then flowed at 1 ml / min. The eluate was recovered by raising the volume of solution A to 25% and the volume of solution B to 75% in a linear gradient over 120 minutes. 2 ml eluate
Each fraction was numbered and fractionated, and 10 μl of each fraction was mixed with 100 μl of 0.2% BSA and freeze-dried with a freeze dryer (12EL; VirTis). 100 μl of the above-mentioned assay buffer was added to the dried product to dissolve it.
According to the test method of 5), the ZAQ receptor activating action was measured. As a result, it was found that the component having the desired ZAQ receptor activating action was eluted in fraction No. 95-100. (7-6) Purification by TSKgel ODS-80Ts reversed-phase high-performance liquid chromatography A TSKgel ODS-80Ts reverse-phase high-performance liquid chromatography column (Tosoh, 4.6 mm x 100 mm) was used at 40 ° C at a flow rate of 1 ml / min. Solution (0.1% trifluoroacetic acid / distilled water) Capacity 91.7% / B solution (0.1% trifluoroacetic acid / 60%
(Acetonitrile) 8.3% of the volume was flowed to equilibrate. The chromatography operation was performed on the freeze-dried product of the fraction No. 95-100 obtained in the above (7-5). That is,
The lyophilized product was dissolved by adding 4 ml of 1 M acetic acid and attached to the column, and the solution was increased to 75% solution A / 25% solution B over 1 minute at a flow rate of 1 ml / min.
The eluate was collected by increasing the concentration of solution B in a linear gradient to 25% solution A volume and 75% solution B volume over a period of minutes. The eluate was fractionated with a 1 ml fraction number.
The ZAQ ligand activity was measured according to the method described in (3-5), and it was confirmed that the activity was eluted in a fraction that coincided with a single ultraviolet absorption peak. From this, it was determined that the ZAQ ligand was single purified.

(7-7) Structural Analysis of Purified ZAQ Ligand Peptide The structure of the ZAQ ligand peptide obtained in the above (7-6) was determined by the following method. It was freeze-dried with a freeze dryer (12EL; VirTis) in a purified sample of ZAQ-activated main component. The obtained lyophilized product was dissolved in the solvent DMSO (dimethyl sulfoxide). A portion of this solution was transferred to a protein sequencer (PerkinElmer, PE Biosys
tems Procise 491cLC) for amino acid sequence analysis from the N-terminus. As a result, an N-terminal amino acid sequence corresponding to the expected mature human ZAQ ligand peptide (SEQ ID NO: 21) was obtained. In addition, mass spectrometry was performed by electron spray ionization using a Finnigan LCQ LC / MS apparatus, and the molecular weight was calculated to be 9658.0. This measurement is based on the mature human ZAQ ligand peptide (SEQ ID NO: 2).
This was in good agreement with the theoretical value of 9657.3 in 1), and it was confirmed that the desired peptide containing the amino acid sequence represented by SEQ ID NO: 21 could be obtained.

Example 8 Snake Venom MIT1 and Human Z
Reactivity of AQ ligand with ZAQ and I5E (8-1) Isolation of snake venom MIT1 (8-1-1) Wakosil-II 5C18HGP
Separation by rep reversed-phase high-performance liquid chromatography Wakosil-II 5C18HG Prep Reverse-phase high-performance liquid chromatography column (Wako Pure Chemical Industries, 20 mm x 250 mm)
Solution A (0.1% trifluoroacetic acid / distilled water) volume 91.7% / solution B (0.1% trifluoroacetic acid / 60%) at a flow rate of 5 ml / min
(Acetonitrile) 8.3% of the volume was flowed to equilibrate. Black M
4 ml of 1 MAcOH was added to and dissolved in 50 mg of a freeze-dried amba venom (Sigma), and the supernatant obtained by centrifugation at 15,000 rpm for 10 minutes was subjected to chromatography. After the sample was applied to the column, the volume of solution A was increased to 91.7% / volume of solution 8.3% over 1 minute at a flow rate of 5 ml / min, and then the volume of solution A was 33.4% and the volume of solution B was 66.6% over 120 minutes. Until,
Solution B concentration was increased with a linear gradient. Eluate
After 20 minutes, fraction numbers were assigned by 10 ml each and fractionated. 1 μl of each fraction was collected, diluted 10,000-fold with the above-mentioned assay buffer, and the activation of the ZAQ-B1 receptor was measured according to the test method described in (3-5).
As a result, it was found that the target component having the ZAQ-B1 receptor activating action was mainly eluted in fraction Nos. 21-23. (8-1-2) Separation by TSKgel CM-2SW Ion Exchange High Performance Liquid Chromatography A column for TSKgel CM-2SW ion exchange high performance liquid chromatography (Tosoh, 4.6 × 250 mm) at 25 ° C. and a flow rate of 1 m
Solution A (4 M ammonium formate: distilled water: acetonitrile = 1: 299: 100) 100% volume and Solution B (4 M ammonium formate: distilled water: acetonitrile = 1: 2: 1) volume 0% at l / min The sink was equilibrated. Wakosil obtained in (7-1)
-II 5C18HG Prep Reversed-phase high-performance liquid chromatography, fractionated fractions, lyophilized fraction No. 21-23 were dissolved in 4 ml of solution A, applied to a TSKgelCM-2SW ion exchange column, and then flowed at 1 ml / min. The eluate was recovered by raising the volume of solution A to 0% / solution B to 100% in a linear gradient over 90 minutes. The eluate was fractionated with a 1 ml fraction number. 1 μl of each fraction was collected, diluted 10,000-fold with the above-mentioned assay buffer, and the activation of the ZAQ-B1 receptor was measured according to the test method described in (3-5) above. As a result, it was found that the target component having the ZAQ-B1 receptor activating action was mainly eluted in fraction Nos. 50-51.

(8-1-3) Vydac238TP34
10 Separation by reversed-phase high-performance liquid chromatography A Vydac238TP3410 reverse-phase high-performance liquid chromatography column (Vydac, 4.6 mm x 100 mm) was applied at 40 ° C at a flow rate of 1 ml /
Solution A (0.1% trifluoroacetic acid / distilled water) capacity 91.7 min
% / B solution (0.1% trifluoroacetic acid / 60% acetonitrile) 8.3% by volume was flowed to equilibrate. After directly applying fraction No. 50-51 of the TSKgel CM-2SW ion-exchange high performance liquid chromatography preparative fraction obtained in (8-1-2) to the column, the flow rate was 1 ml / min for 1 minute. Solution A to 75% / solution B to 25%, then to solution A 41.7% solution B to 58.3% solution B over 75 minutes.
The liquid concentration was increased with a linear gradient. Eluate to 0.
Each 5 ml was fractionated with a fraction number. 1 μl was collected from each fraction, diluted 10,000-fold with the above-mentioned assay buffer, and then subjected to the test method described in (3-5) above.
AQ-B1 receptor activating action was measured. As a result, it was found that the target component having the ZAQ-B1 receptor activating action was mainly eluted in fractions No. 108-115. (8-1-4) Structural analysis of purified snake venom MIT1 The structure of snake venom MIT1 obtained in (8-1-3) was determined by the following method. Purified MIT1
Was freeze-dried with a freeze dryer (12EL; VirTis). The obtained lyophilized peptide was dissolved in the solvent DMSO (dimethylsulfoxide). A part of this solution was used as a protein sequencer (PerkinElmer, PE Biosystems).
Procise 491cLC) for amino acid sequence analysis from the N-terminus. As a result, an N-terminal amino acid sequence corresponding to the predicted snake venom MIT1 (SEQ ID NO: 34) was obtained. In addition, using a Finnigan LCQ LC / MS instrument, mass spectrometry was performed by electron spray ionization, and the molecular weight was 850.
It was calculated to be 6.8. This measured value was in good agreement with the theoretical value of 8506.4 for snake venom MIT1, confirming that the desired peptide containing the amino acid sequence represented by SEQ ID NO: 34 could be obtained.

(8-2) Establishment of a stable I5E expression cell line The human I5E receptor cDNA (SEQ ID NO: 35) was cloned by a known PCR method and incorporated into a pAKKO1.11H expression vector. According to the method described in (3-5), CHO / dhFr ^- cells (American Type Cul
Culture Collection). About 20 colonies of the transformed CHO cells that grew 10 to 14 days after the start of the culture were selected. The selected cells were seeded at 3 × 10 ⁴ cells / well in a 96-well plate, and the MI was determined according to the test method described in (3-5).
The reactivity to T1 was examined. The 4th clone of CHO cell expressing I5E with good reactivity (abbreviated as I5E-4 cell) was selected. The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 35 is shown in SEQ ID NO: 36. (8-3) Human ZAQ ligand peptide and MIT
1. Measurement of ZAQ Receptor and I5E Receptor Activating Actions Regarding human ZAQ ligand peptide purified by the method described in Example 7 and snake venom MIT1 purified by the method described in (8-1) above, Their ZAQ receptor activating action and I5E receptor activating action were measured according to the method described in (3-5). The results are shown in FIG. 12 and FIG. As a result, human ZAQ ligand peptide and snake venom MIT1 caused an increase in intracellular calcium concentration in a concentration-dependent manner. ZAQ of snake venom MIT1
The receptor activating effect was 10 times stronger than that of the human ZAQ ligand peptide. In addition, the snake venom MIT1 had a human-type I5E receptor activating action about 100 times stronger than that of the human-type ZAQ ligand peptide.

Example 9 Cloning of cDNA Encoding a Novel G Protein-Coupled Receptor Protein (rZAQ1) Derived from Rat Brain cDNA and Determination of Nucleotide Sequence Using a rat whole brain cDNA library (CLONTECH) as a template, two types of primers were used. (SEQ ID NO: 37 and SEQ ID NO: 38) were used to carry out a PCR reaction. The composition of the reaction solution in this reaction was determined using the above cDNA as a 1/10 template, and the Advantage-2 cDNA Polymerase Mix (CLON
TECH) 1/50 volume, each primer 0.2μM, dNTPs 200μM,
Then, the attached buffer was added to the enzyme to make a liquid volume of 25 μl. The PCR reaction was carried out at 94 ° C for 20 seconds after
3 cycles of 2 ° C for 1 minute 30 seconds, 94 ° C for 20 seconds, 68 ° C
3 cycles of 1 minute and 30 seconds, 94 ° C for 20 seconds, 62 ° C for 20 seconds, 6
A cycle of 8 ° C. for 1 minute was repeated 36 times, and finally an extension reaction was performed at 68 ° C. for 7 minutes. The reaction product after the PCR reaction is TOPO
-Subcloned into a plasmid vector pCR2.1-TOPO (Invitrogen) according to the prescription of a TA cloning kit (Invitrogen). This is introduced into E. coli DH5α,
Clones with cDNA were selected on LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, cDNA (SEQ ID NO: 39) encoding a novel G protein-coupled receptor protein was obtained. The cDNA
Amino acid sequence (SEQ ID NO:
40) has the amino acid sequence represented by SEQ ID NO: 1 and 8
3.7% homology was observed. The novel G protein-coupled receptor protein containing this amino acid sequence was named rZAQ1. In addition, a transformant (Escherichia coli) containing a DNA containing the nucleotide sequence represented by SEQ ID NO: 39 was prepared from Escherichia coli DH5α /
It was named pCR2.1-rZAQ1.

Example 10 Cloning of cDNA encoding novel G protein-coupled receptor protein (rZAQ2) derived from rat brain cDNA and determination of nucleotide sequence A clone encoding rZAQ2 was obtained by the genetrapper method. That is, the probes (SEQ ID NO: 41 and SEQ ID NO: 42) were biotinylated, and then hybridized with a single-stranded rat whole brain cDNA library (GIBCO-BRL) to obtain the obtained single-stranded gene. The primers (SEQ ID NO: 43 and SEQ ID NO: 44) were used to repair double-stranded DNA. This gene was electroporated into Escherichia coli DH10B to obtain a transformant using ampicillin resistance as an index. Furthermore, colony PCR using a probe (SEQ ID NO: 41) and a primer (SEQ ID NO: 45)
Thus, a clone encoding the target nucleotide sequence was selected. ORF (op) predicted from the nucleotide sequence of this clone
The amino acid sequence (SEQ ID NO: 47) derived from the nucleotide sequence (SEQ ID NO: 46) of the en reading frame) is rZAQ
10.6% homology was found. A novel G protein-coupled receptor protein containing this amino acid sequence is called rZAQ
Named 2. The transformant (Escherichia coli) obtained by the genetrapper method was used to transform Escherichia coli (Escherichia coli).
a. coli) DH10B / pCMV-rZAQ2.

Example 11 Shrinkage test using guinea pig ileum extirpated specimen Guinea pig (std: Hartley, 7-8 weeks old, male, body weight 450 g)
The carotid artery was cut with scissors to kill the blood loss, and then the ileum was removed by laparotomy. The ileum was infused with Tyrode solution injected with 95% O ₂ -5% CO ₂ gas (composition: 138 mM NaCl, 2.7 mM KCl,
1.8 mM CaCl ₂ , 0.5 mM MgCl ₂ , 1.1 mM NaH ₂ PO ₄ , 11.9 m
M NaHCO ₃ , 5.6 mM glucose) in a glass dish containing surgical scissors and tweezers to remove fat debris and connective tissue attached to the ileum. Using. This sample was suspended in an organ bath (20 ml) filled with the above Tyrode solution warmed to 37 ° C., and was stabilized under a load of 0.5 g for 30 minutes or more. The contraction response of the ileum specimen was measured by NEC Sanei's AMPLIFIE
It was measured using R CASE 7747. After administration of 1 μM of acetylcholine and measurement of the maximal contractile response, a human ZAQ ligand peptide (ZAQL-
1) or the snake venom MIT obtained in the above (8-1-3)
1 was cumulatively administered and the contractile response was measured. For dilution of ZAQL-1 and MIT1, physiological saline containing 0.05% bovine serum albumin was used. The results are shown in FIG. ZA
The contractile response elicited by QL-1 and MIT1 is
The contractile response elicited by 1 μM of acetylcholine was
It was expressed as a percentage as 0%. As a result, ZAQL-
1 and MIT1 induced a strong contractile response in a dose-dependent manner. ZAQL-1 and MI calculated from the dose response curve
The EC ₅₀ values of T1 were 1.79 nM and 0.40 nM, respectively.

Example 12 CH expressing ZAQ and I5E
Binding assay using O cell membrane fraction (12-1) Preparation of ¹²⁵ I-MIT1 [ ¹²⁵ I] -Bolton-Hunter Reagent (monoiodinated, 37 MB
q, Benzene contained in NEX120 (NEN) was distilled off with nitrogen gas, then 20 μl of DMSO was added, and the solid was dissolved by pipetting. Here, a borate buffer (composition: 100 m
80 μl of a solution containing 4 nmol of MIT1 diluted with MH ₃ BO ₃ (pH 8.5) was added, mixed immediately, and incubated at room temperature for 2 hours. Then, 10% acetonitrile-0.1
% Trifluoroacetic acid (200 μl) was added to prepare a sample for HPLC separation. This was equilibrated by flowing 100% solution A (10% acetonitrile / 0.1% trifluoroacetic acid) / 0% solution B (0.1% trifluoroacetic acid / 40% acetonitrile) at room temperature at a flow rate of 1 ml / min. TSKgel Super-O
After being attached to a column for DS reversed-phase high-performance liquid chromatography (Tosoh Corporation, 0.46 cm x 10 cm), the flow rate was 1 ml.
/ Ml in 1 minute to raise the volume of solution A to 60% / volume of solution B to 40%, then increase the concentration of solution B in a linear gradient to 40% of solution A / volume of solution B to 60% in 60 minutes. I let it. The MIT1 into which [ ¹²⁵ I] -Bolton-Hunter Reagent to be eluted was introduced was manually separated, and the buffer (composition: 20 m
M Tris, 1 mM EDTA, 0.2% BSA, 0.1% CHAPS, 0.03%
After diluting with NaN ₃ , pH 7.4), the solution was stored at -80 ° C.

(12-2) Preparation of Cell Membrane Fraction The cell membrane fraction was prepared according to Journal of Pharmacology and Experime.
ntal Therapeutics, 279, 675-685, 1996. 10% dialyzed fetal calf serum (JRH
BIOSCIENCES), 1 mM MEM non-essential amino acid solution, 100
units / ml Penicillin, and 100 μg / ml Streptomycin
Contains Dulbecco's modified Eagle medium (DMEM)
Receptor-expressing CHO cells, ie, ZAQC-B1 cells or I5E-4 cells, were cultured in Singletray (Nunc) in a medium (Nissui Pharmaceutical Co., Ltd.) at 37 ° C. and 5% carbon dioxide.
When the cell density reaches 80-90%, discard the culture supernatant.
Add 30 ml of PBS (Takara Shuzo) containing 1 g / l EDTA to the tray,
After washing the cells, the supernatant was discarded, 30 ml of the above PBS-EDTA was added, and the mixture was allowed to stand at room temperature. The tray was shaken, the cells were detached from the tray, and the cell suspension was collected. Further, 10 ml of the above PBS-EDTA was again added to the tray for washing, the cells remaining in the tray were peeled off, and mixed with the cell suspension cells obtained first. The mixed cell suspension is transferred to a high-speed centrifuge (TOMY RL60).
Using 1), the mixture was centrifuged at 1,000 rpm for 5 minutes. Discard the supernatant,
The precipitated cells were stored at -80C. This precipitate (Single
Crushing buffer (composition: 10 mM NaHC) in one tray
O ₃ , 5 mM EDTA, 0.5 mM phenylmethylsulfonylfluoride
(PMSF), 10 μg / ml pepstatin-A, 20 μg / ml leupepti
n, 10 μg / ml E-64) was added, the precipitate was suspended by pipetting, and then crushed using a Polytron (blade type PTA7) under the conditions of a scale setting of 4, 20 seconds and three times. Next, the mixture was centrifuged at 2,500 rpm for 10 minutes using a No. 26 rotor with a Hitachi high-speed cooling centrifuge CR26H. The obtained supernatant was further centrifuged at 35,000 rpm for 1 hour using an SRP70AT rotor with an ultracentrifuge (Hitachi Koki SCP70H). A binding assay buffer (composition: 20 mM Tri
s, 1 mM EDTA, 0.5 mM PMSF, 10 μg / ml pepstatin-A, 4
0 μg / ml leupeptin, 10 μg / ml E-64, 0.03% NaN ₃ , pH
7.4) Add 5 ml, suspend and add Cell Strainer (FALCON 2
350) to obtain a ZAQC-B1 cell membrane fraction (ie, ZAQ membrane fraction) or an I5E-4 cell membrane fraction (ie, I5E membrane fraction). The protein concentration of each membrane fraction obtained was determined by Coomassie Protein Assay Reagen.
After measuring using t (PIERCE), dispense 100 μl each,
Stored at 0 ° C.

(12-3) Binding Assay The binding assay buffer described in (12-2) containing 0.1% BSA was used to obtain a ZAQ membrane fraction of 10 μg / ml.
Then, the I5E membrane fraction was diluted to 20 μg / ml,
Each μl was dispensed into a tube (FALCON 2053). Add 2 μl of test compound and 10 nM ¹²⁵ IM to each of the diluted membrane fractions.
2 μl of IT1 was added and incubated at 25 ° C. for 1 hour. As a test compound, a human ZAQ ligand peptide (ZAQL-
1) or the snake venom MIT obtained in the above (8-1-3)
1 (unlabeled MIT1) was used. Next, a filter buffer (composition; 20 mM Tris, EDTA, 0.1% BSA, 0.05
% CHAPS, 0.03% NaN ₃ , pH 7.4) and add buffer (composition: 20 mM Tris, 0.3% polyethyleneimin)
e, pH 7.4) glass fiber filter paper GF / F (Whatm
An) was immediately filtered, and 1.5 ml of a filtration buffer was again added to the reaction tube, followed by filtration in the same manner. The radioactivity of the glass fiber filter was measured using a gamma counter (COBRA, Packard), and the amount of ¹²⁵ I-MIT1 bound was measured. ¹²⁵ IM when unlabeled MIT1 (final concentration 1 μM) is used as a test compound
The amount of IT1 binding was determined as non-specific binding (NSB), and the amount of ¹²⁵ I-MIT1 binding when no test compound was added was defined as total binding (TB), and the maximum specific binding (TB-NSB) was calculated. . Also, the binding amount obtained when the test compound was added (B),
The specific binding amount (B-NSB) was calculated, and the percentage of the maximum specific binding amount ｛% SPB = (B-NSB) / (TB-NSB) × 100｝
Indicates the amount of binding in the presence of the test compound. ZAQ
The result using the membrane fraction is shown in FIG. 15 and the result using the I5E membrane fraction is shown in FIG. The IC ₅₀ values of unlabelled MIT1 calculated in this binding assay system, 38 pM (ZA
Q membrane fraction) and 32 pM (I5E membrane fraction). ZAQL-1
The IC ₅₀ values were 35 nM (ZAQ membrane fraction) and 93 nM (I5E membrane fraction).

Reference Example 1 Production of human ZAQ ligand (SEQ ID NO: 21) in Escherichia coli (Reference example 1-1) Construction of human ZAQ ligand expression plasmid in Escherichia coli (a) Six types of DNA fragments # 1 shown below Using # 6, the structural DNA of the ZAQ ligand was prepared. # 1: 5'-TATGGCGGTGATTACCGGTGCGTGCGAACGTGATGTGCAGTGCGGTGCGGGTACCTGCTGCGCGATTAG CCTGTGGCTGCGTGGTCTG-3 '(SEQ ID NO: 52), # 2: 5'-CGTATGTGCACCCCGCTGGGTCGTGAAGGTGAAGAATGCCATCCGGGTAGCCATAAAGTGCCGTTCTTC CGTAAACGTAAACATCATACCTG-3' (SEQ ID NO: 53), # 3: 5'-CCCGTGCCTGCCGAACCTGCTGTGCAGCCGTTTCCCGGATGGTCGTTATCGTTGCAGCATGGATCTGAA AAACATTAACTTTTAGG-3 '(SEQ ID NO: : 54), # 4: 5'-CACATACGCAGACCACGCAGCCACAGGCTAATCGCGCAGCAGGTACCCGCACCGCACTGCACATCACGT TCGCACGCACCGGTAATCACCGCCA-3 '(SEQ ID NO: 55), # 5: 5'-AGGCACGGGCAGGTATGATGTTTACGTTTACGGAAGAACGGCACTTTATGGCTACCCGGATGGCATTCT TCACCTTCACGACCCAGCGGGGTG-3' (SEQ ID NO: 56), # 6: 5'-GATCCCTAAAAGTTAATGTTTTTCAGATCCATGCTGCAACGATAACGACCATCCGGGAAACGGCTGCAC AGCAGGTTCGGC-3 '(SEQ ID NO: 57).

(B) Phosphorylation of DNA oligomer Four types of D except for the above # 1 and # 6 to be 5 'side
Each of the NA oligomers (# 2 to # 5) was added to 25 μl of a phosphorylation reaction solution [10 μg of DNA oligomer, 50 mM Tris
-HCl, pH 7.6, 10 mM MgCl ₂ , 1 mM spermidine, 10 mM dithiothreitol (hereinafter abbreviated as DTT), 0.1 mg / ml bovine serum albumin (hereinafter abbreviated as BSA), 1 mM ATP, 10 units T4 polynucleotide kinase ( (Takara Shuzo)] at 37 ° C. for 1 hour to phosphorylate the 5 ′ end of each oligomer. After the phenol treatment, double the amount of ethanol was added, and
After cooling to ° C., the DNA was precipitated by centrifugation. (c) Ligation of DNA fragment The DNA fragment obtained in (b) above and # 1 and # 6 were combined to make 120 μl. 9
After holding at 0 ° C. for 10 minutes, the mixture was gradually cooled to room temperature and annealed, and then a ligation reaction was performed using TaKaRa DNA Ligation Kit ver.2 (Takara Shuzo). To 30 μl of the annealing solution, add 30 μl of the II solution attached to the kit,
After mixing well, add 60 μl of solution I included in the kit,
The reaction was carried out at 37 ° C. for 1 hour to perform ligation. Thereafter, the mixture was subjected to phenol treatment, the aqueous layer was collected, twice the amount of ethanol was added, the mixture was cooled to -70 ° C, and centrifuged to remove D.
NA was precipitated. The DNA fragment thus obtained was subjected to phosphorylation with T4 polynucleotide kinase (Takara Shuzo) and then subjected to the following step (d).

(D) Construction of ZAQ ligand expression vector As an expression vector, pTCII (JP-A-2000-178297) was used.
No. 3) with NdeI and BamHI (Takara Shuzo)
After digestion at 7 ° C. for 2 hours, a 4.3 kb DNA fragment was subjected to QIAquick Gel Extraction by 1% agarose gel electrophoresis.
Extract using Kit (Qiagen), 25 μl TE
Dissolved in buffer. NdeI and Bam of this pTCII
The HI fragment and the structural gene (SEQ ID NO: 58) of the ZAQ ligand prepared as described above were combined with the TaKaRa DNA ligation kit ve.
A ligation reaction was performed using r.2 (Takara Shuzo).
Escherichia coli JM109 competent cells (Toyobo) were transformed with 10 µl of this reaction solution, seeded on an LB agar medium containing 10 µg / ml tetracycline, cultured overnight at 37 ° C, and the resulting tetracycline-resistant colonies were selected. This transformant was cultured overnight in LB medium, and
Plasmid pTC using Miniprep Kit (Qiagen)
h1ZAQ was prepared. The base sequence of this ZAQ ligand DNA was applied to Applied Biosystems model 377D.
It was confirmed using an NA sequencer. Plasmid pTC
h1ZAQ was transformed into Escherichia coli MM294.
(DE3), and a ZAQ ligand-expressing strain Escher
ichia coli MM294 (DE3) / pTCh1ZAQ was obtained.

Reference Example 1-2 Production of ZAQ Ligand The above Escherichia coli MM294 (DE3) / pTCh1ZAQ
1 L of LB medium containing 0 mg / L tetracycline
(1% peptone, 0.5% yeast extract, 0.5% sodium chloride) and cultured in a 2 L flask with shaking at 37 ° C. for 8 hours. The obtained culture solution was diluted with 19 L of a main fermentation medium (1.68% sodium monohydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.1% ammonium chloride, 0.05%).
% Sodium chloride, 0.05% magnesium sulfate, 0.1%
02% antifoam, 0.00025% ferrous sulfate, 0.00
Transplanted into a 50 L fermenter charged with 05% thiamine hydrochloride, 1.5% glucose, 1.5% high case amino)
At 30 ° C., aeration and stirring were started. When the turbidity of the culture broth reached 500 klet units, isopropyl-β-D-
Thiogalactopyranoside was added so that the final concentration became 12 mg / L, and culturing was further performed for 4 hours. After completion of the culture, the culture solution was centrifuged to obtain about 200 g of wet cells and stored at -80 ° C. This transformed E. coli MM29
4 (DE3) / pTCh1ZAQ has accession number IFO
No. 16527 has been deposited with the Fermentation Research Institute (IFO). (Reference Example 1-3) Activation of ZAQ ligand 200 m was added to 200 g of the cells obtained in Reference Example (1-2).
M Tris / HCl, 7M guanidine hydrochloride (pH 8.
0) After adding 400 ml to lyse the cells, centrifugation (10000 rpm, 1 hour) was performed. Add 0% to the supernatant.
4 M arginine, 50 mM Tris / HCl, 0.2 mM
GSSG, 10 liters of 1 mM GSH (pH 8.0) was added, and activation was performed at 4 ° C. overnight.

(Reference Example 1-4) Purification of ZAQ Ligand The regenerated solution, which had been activated in Reference Example (1-3), was adjusted to pH 6.0.
0 and adjusted to 50 mM phosphate buffer (pH 6.0) with an SP-Sepharose column (11.3 cm × 1).
5 cm), then 600 mM NaCl / 50
Elution was performed with mM phosphate buffer (pH 6.0), and the fractions containing the ZAQ ligand were pooled. 50 fractions of this fraction
CM-equilibrated with mM phosphate buffer (pH 6.0)
After passing through 5PW (21.5mm x 150mmL), adsorbing and washing, elution was carried out with a step gradient (60 minutes) of 0-100% B (B = 50mM phosphate buffer + 1M NaCl, pH 6.05). A ZAQ ligand fraction (elution time about 40 minutes) was obtained. This fraction was further purified with C4P-50 (21.5 mm ID × 300 mm) equilibrated with 0.1% trifluoroacetic acid.
L, Showa Denko), and after adsorption and washing, 25-5
Elution was performed with a step gradient (60 minutes) of 0% B (B: 80% acetonitrile / 0.1% trifluoroacetic acid), and ZAQ
After pooling the ligand fractions (elution time about 40 minutes), lyophilization was performed to obtain about 80 mg of a lyophilized ZAQ ligand powder.

(Reference Example 1-5) Characterization of ZAQ ligand (a) Analysis using SDS polyacrylamide gel electrophoresis The ZAQ ligand obtained in Reference Example (1-4) was 100
Sample buffer [La
emmli, Nature, 227, 680 (1979)], heated at 95 ° C. for 1 minute, and electrophoresed on Multigel 15/25 (Daiichi Pure Chemicals). Coomassie gel after electrophoresis
As a result of staining with brilliant blue (Coomassie brilliant blue), a single protein band was observed at the same position as the recombinant ZAQ ligand sample derived from COS7 cells obtained in Example 5. From this, the reference example (1
The recombinant ZAQ ligand sample derived from Escherichia coli obtained in -4) was found to be extremely high in purity and identical in molecular weight to the recombinant ZAQ ligand prepared from COS7 cells.

(B) Analysis of Amino Acid Composition The amino acid composition was analyzed using an amino acid analyzer (Hitachi L-8500A).
Amino Acid Analyzer). as a result,
It was consistent with the amino acid composition deduced from the DNA base sequence of the ZAQ ligand (peptide consisting of the amino acid sequence represented by SEQ ID NO: 21) (Table 2).

[Table 2] Acid hydrolysis (6N HCl-1% phenol, 110
C, 24 and 48 hours hydrolysis average) 1) extrapolated to 0 hours 2) undetected

(C) Analysis of N-terminal amino acid sequence The N-terminal amino acid sequence was analyzed using a gas-phase protein sequencer (P
E-Applied Biosystems model 492). As a result, the DN of the resulting ZAQ ligand
It was consistent with the N-terminal amino acid sequence of ZAQ ligand deduced from the nucleotide sequence of A (Table 3).

[Table 3] 1) The analysis was performed using phenylthiohydantoin 150 pmol. ND Indicates undetected.

(D) C-terminal amino acid analysis The C-terminal amino acid was analyzed using an amino acid analyzer (Hitachi L-8500A).
Amino Acid Analyzer). The obtained ZAQ ligand coincided with the C-terminal amino acid deduced from the nucleotide sequence of DNA (Table 4).

[Table 4] (E) Mass Spectrometry Mass spectrometry was performed using an LCQ ion trap mass spectrometer (manufactured by ThermoQuest) equipped with a nanoESI ion source.
As a result, a molecular weight of 9657.55 ± 0.89 was obtained.
21 and 10 residues of C contained in SEQ ID NO: 21
The ys was in good agreement with the theoretical molecular weight (9657.3) of the ZAQ ligand in which 5 pairs of disulfide bonds were formed.

(Reference Example 1-6) Measurement of ZAQ ligand activity (measurement of intracellular Ca ion concentration increasing activity using FLIPR) Using the method of Example 5, a ZAQ ligand standard was prepared,
Activity measurement (measurement of intracellular Ca ion concentration increasing activity using FLIPR) was performed. As a result, it had an activity equivalent to that of the COS7 cell-derived recombinant sample (purified ZAQ ligand) obtained in Example 5.

[0140]

The peptide of the present invention, a DNA encoding the peptide of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention) and an antibody against the peptide of the present invention (hereinafter abbreviated as the antibody of the present invention) Are) therapeutic or prophylactic agents for various diseases associated with the peptide of the present invention, screening for compounds or salts thereof that alter the binding between the peptide of the present invention and the protein of the present invention, peptides of the present invention or salts thereof Quantification, genetic diagnostics, pharmaceuticals containing antisense DNA, pharmaceuticals containing the antibody of the present invention, production of non-human animals containing the DNA of the present invention, and comparison with structurally similar ligands / receptors Useful for implementing drug design based on.

[0141]

[Sequence List] [SEQUENCE LISTING] <110> Takeda Chemical Industries, Ltd. <120> Novel Phisiological Active Peptide and Its Use <130> P2001-175 <150> JP 2000-217442 <151> 2000-07-18 <150> JP 2001-26779 <151> 2001-02-02 <160> 58 <210> 1 <211> 393 <212> PRT <213> Human <400> 1 Met Glu Thr Thr Met Gly Phe Met Asp Asp Asn Ala Thr Asn Thr Ser 5 10 15 Thr Ser Phe Leu Ser Val Leu Asn Pro His Gly Ala His Ala Thr Ser 20 25 30 Phe Pro Phe Asn Phe Ser Tyr Ser Asp Tyr Asp Met Pro Leu Asp Glu 35 40 45 Asp Glu Asp Val Thr Asn Ser Arg Thr Phe Phe Ala Ala Lys Ile Val 50 55 60 Ile Gly Met Ala Leu Val Gly Ile Met Leu Val Cys Gly Ile Gly Asn 65 70 75 80 Phe Ile Phe Ile Ala Ala Leu Val Arg Tyr Lys Lys Leu Arg Asn Leu 85 90 95 Thr Asn Leu Leu Ile Ala Asn Leu Ala Ile Ser Asp Phe Leu Val Ala 100 105 110 Ile Val Cys Cys Pro Phe Glu Met Asp Tyr Tyr Val Val Arg Gln Leu 115 120 125 Ser Trp Glu His Gly His Val Leu Cys Thr Ser Val Asn Tyr Leu Arg 130 135 140 Thr Val Ser Leu Tyr Val Ser Thr Asn Ala Leu Leu Ala Ile Ala Ile 145 150 155 160 Asp Arg Tyr Leu Ala Ile Val His Pro Leu Arg Pro Arg Met Lys Cys 165 170 175 Gln Thr Ala Thr Gly Leu Ile Ala Leu Val Trp Thr Val Ser Ile Leu 180 185 190 Ile Ala Ile Pro Ser Ala Tyr Phe Thr Thr Glu Thr Val Leu Val Ile 195 200 205 Val Lys Ser Gln Glu Lys Ile Phe Cys Gly Gln Ile Trp Pro Val Asp 210 215 220 Gln Gln Leu Tyr Tyr Lys Ser Tyr Phe Leu Phe Ile Phe Gly Ile Glu 225 230 235 240 Phe Val Gly Pro Val Val Thr Met Thr Leu Cys Tyr Ala Arg Ile Ser 245 250 255 Arg Glu Leu Trp Phe Lys Ala Val Pro Gly Phe Gln Thr Glu Gln Ile 260 265 270 Arg Lys Arg Leu Arg Cys Arg Arg Lys Thr Val Leu Val Leu Met Cys 275 280 285 Ile Leu Thr Ala Tyr Val Leu Cys Trp Ala Pro Phe Tyr Gly Phe Thr 290 295 300 Ile Val Arg Asp Phe Phe Pro Thr Val Phe Val Lys Glu Lys His Tyr 305 310 315 320 Leu Thr Ala Phe Tyr Ile Val Glu Cys Ile Ala Met Ser Asn Ser Met 325 330 335 Ile Asn Thr Leu Cys Phe Val Thr Val Lys Asn Asp Thr Val Lys Tyr 340 345 350 Phe Lys Lys Ile Met Leu Leu His Trp Lys Ala Ser Tyr Asn Gly Gly 355 360 365 Lys Ser Ser Ala Asp Leu Asp Leu Lys Thr Ile Gly Met Pro Ala Thr 370 375 380 Glu Glu Val Asp Cys Ile Arg Leu Lys 385 390 <210> 2 <211> 1179 <212> DNA <213> Human <400> 2 atggagacca ccatggggtt catggatgac aatgccacca acacttccac cagcttcctt 60 tctgtgctca accctcatgg agcccatgcc acttccttcc cattcaactt cagctacagc 120 gactatgata tgcctttgga tgaagatgag gatgtgacca attccaggac gttctttgct 180 gccaagattg tcattgggat ggccctggtg ggcatcatgc tggtctgcgg cattggaaac 240 ttcatcttta tcgctgccct ggtccgctac aagaaactgc gcaacctcac caacctgctc 300 atcgccaacc tggccatctc tgacttcctg gtggccattg tctgctgccc ctttgagatg 360 gactactatg tggtgcgcca gctctcctgg gagcacggcc acgtcctgtg cacctctgtc 420 aactacctgc gcactgtctc tctctatgtc tccaccaatg ccctgctggc catcgccatt 480 gacaggtatc tggctattgt ccatccgctg agaccacgga tgaagtgcca aacagccact 540 ggcctgattg ccttggtgtg gacggtgtcc atcctgatcg ccatcccttc cgcctacttc 600 accaccgaga cggtcctcgt cattgtcaag agccaggaaa agatcttctg cggccagatc 660 tggcctgtgg accagcagct ctactacaag tcctacttcc tctttatctt tggcatagaa 720 ttcgtgggcc ccgtggtcac catgaccctg tgctatgcca ggatctcccg ggagctctgg 780 ttcaaggcgg tccctggatt ccagacagag cagatccgca agaggctgcg ctgccgcagg 840 aagacggtcc tg gtgctcat gtgcatcctc accgcctacg tgctatgctg ggcgcccttc 900 tacggcttca ccatcgtgcg cgacttcttc cccaccgtgt tcgtgaagga gaagcactac 960 ctcactgcct tctacatcgt cgagtgcatc gccatgagca acagcatgat caacactctg 1020 tgcttcgtga ccgtcaagaa cgacaccgtc aagtacttca aaaagatcat gttgctccac 1080 tggaaggctt cttacaatgg cggtaagtcc agtgcagacc tggacctcaa gacaattggg 1140 atgcctgcca ccgaagaggt ggactgcatc agactaaaa 1179 <210> 3 <211> 1179 <212> DNA <213> Human <400> 3 atggagacca ccatggggtt catggatgac aatgccacca acacttccac cagcttcctt 60 tctgtgctca accctcatgg agcccatgcc acttccttcc cattcaactt cagctacagc 120 gactatgata tgcctttgga tgaagatgag gatgtgacca attccaggac gttctttgct 180 gccaagattg tcattgggat ggccctggtg ggcatcatgc tggtctgcgg cattggaaac 240 ttcatcttta tcgctgccct ggtccgctac aagaaactgc gcaacctcac caacctgctc 300 atcgccaacc tggccatctc tgacttcctg gtggccattg tctgctgccc ctttgagatg 360 gactactatg tggtgcgcca gctctcctgg gagcacggcc acgtcctgtg cacctctgtc 420 aactacctgc gcactgtctc tctctatgtc tccaccaatg ccctgctggc catcgccatt 480 gacaggtatc tggctattgt ccatccgctg agaccacgga tgaagtgcca aacagccact 540 ggcctgattg ccttggtgtg gacggtgtcc atcctgatcg ccatcccttc cgcctacttc 600 accaccgaga cggtcctcgt cattgtcaag agccaggaaa agatcttctg cggccagatc 660 tggcctgtgg accagcagct ctactacaag tcctacttcc tctttatctt tggcatagaa 720 ttcgtgggcc ccgtggtcac catgaccctg tgctatgcca ggatctcccg ggagctctgg 780 ttcaaggcgg tccctggatt ccagacagag cagatccgca agaggctgcg ctgccgcagg 840 aagacggtcc tg gtgctcat gtgcatcctc accgcctacg tgctatgctg ggcgcccttc 900 tacggcttca ccatcgtgcg cgacttcttc cccaccgtgt ttgtgaagga gaagcactac 960 ctcactgcct tctacatcgt cgagtgcatc gccatgagca acagcatgat caacactctg 1020 tgcttcgtga ccgtcaagaa cgacaccgtc aagtacttca aaaagatcat gttgctccac 1080 tggaaggctt cttacaatgg cggtaagtcc agtgcagacc tggacctcaa gacaattggg 1140 atgcctgcca ccgaagaggt ggactgcatc agactaaaa 1179 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> <400> 4 gtcgacatgg agaccaccat ggggttcatg g 31 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> <400> 5 actagtttat tttagtctga tgcagtccac ctcttc 36 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 6 tcatgttgct ccactggaag g 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> <400> 7 ccaattgtct tgaggtccag g 21 <210> 8 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> <400> 8 ttcttacaat ggcggtaagt ccagtgcag 39 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> <400> 9 gtcgacatgg agaccaccat ggggttcatg g 31 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> <400> 10 actagtttat tttagtctga tgcagtccac ctcttc 36 <210> 11 <211> 16 <212> PRT <213> Bovine <400> 11 Ala Val Ile Thr Gly Ala Xaa Glu Arg Asp Val Gln Xaa Arg Ala Gly 5 10 15 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> <400> 12 ggtgccacgc gagtctcaat catgctcc 28 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> <400> 13 ggggcctgtg agcgggatgt ccagtgtg 28 <210> 14 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> <400> 14 cttcttcagg aaacgcaagc accacacc 28 <210> 15 <211> 409 <212> DNA <213> Human <400> 15 cttcttcagg aaacgcaagc accacacctg tccttgcttg cccaacctgc tgtgctccag 60 gttcccggac ggcaggtacc gctgctccat ggacttgaag aacatcaatt tttaggcgct 120 tgcctggtct caggataccc accatccttt tcctgagcac agcctggatt tttatttctg 180 ccatgaaacc cagctcccat gactctccca gtccctacac tgactaccct gatctctctt 240 gtctagtacg cacatatgca cacaggcaga catacctccc atcatgacat ggtccccagg 300 ctggcctgag gatgtcacag cttgaggctg tggtgtgaaa ggtggccagc ctggttctct 360 tccctgctca ggctgccaga gaggtggtaa atggcagaaa ggacattcc 409 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> <400> 16 ccaccatgag aggtgccacg 20 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> <400> 17 ctcgagctca ggaaaaggat ggtg 24 <210> 18 <211> 371 <212> DNA <213> Human <400> 18 ccaccatgag aggtgccacg cgagtctcaa tcatgctcct cctagtaact gtgtctgact 60 gtgctgtgat cacaggggcc tgtgagcggg atgtccagtg tggggcaggc acctgctgtg 120 ccatcagcct gtggcttcga gggctgcgga tgtgcacccc gctggggcgg gaaggcgagg 180 agtgccaccc cggcagccac aagatcccct tcttcaggaa acgcaagcac cacacctgtc 240 cttgcttgcc caacctgctg tgctccaggt tcccggacgg caggtaccgc tgctccatgg 300 acttgaagaa catcaatttt taggcgcttg cctggtctca ggatacccac catccttttc 360 ctgagctcga g 371 <210> 19 <211> 371 <212> DNA <213> Human <400> 19 ccaccatgag aggtgccacg cgagtctcaa tcatgctcct cctagtaact gtgtctgact 60 gtgctgtgat cacaggggcc tgtgagcggg atgtccagtg tggggcaggc acctgctgtg 120 ccatcagcct gtggcttcga gggctgcgga tgtgcacccc gctggggcgg gaaggcgagg 180 agtgccaccc cggcagccac aaggtcccct tcttcaggaa acgcaagcac cacacctgtc 240 cttgcttgcc caacctgctg tgctccaggt tcccggacgg caggtaccgc tgctccatgg 300 acttgaagaa catcaatttt taggcgcttg cctggtctca ggatacccac catccttttc 360 ctgagctcga g 371 <210> 20 <211> 86 <212> PRT <213> Human <400> 20 Ala Val Ile Thr Gly Ala Cys Glu Arg Asp Val Gln Cys Gly Ala Gly 5 10 15 Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg Met Cys Thr 20 25 30 Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser His Lys Ile 35 40 45 Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro Cys Leu Pro Asn 50 55 60 Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg Cys Ser Met Asp 65 70 75 80 Leu Lys Asn Ile Asn Phe 85 <210> 21 <211> 86 <212> PRT <213> Human <400> 21 Ala Val Ile Thr Gly Ala Cys Glu Arg Asp Val Gln Cys Gly Ala Gly 5 10 15 Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg Met Cys Thr 20 25 30 Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser His Lys Val 35 40 45 Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro Cys Leu Pro Asn 50 55 60 Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg Cys Ser Met Asp 65 70 75 80 Leu Lys Asn Ile Asn Phe 85 <210> 22 <211> 105 <212> PRT <213> Human <400> 22 Met Arg Gly Ala Thr Arg Val Ser Ile Met Leu Leu Leu Val Thr Val 5 10 15 Ser Asp Cys Ala Val Ile Thr Gly Ala cys Glu Arg Asp Val Gln Cys 20 25 30 Gly Ala Gly Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg 35 40 45 Met Cys Thr Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser 50 55 60 His Lys Ile Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro Cys 65 70 75 80 Leu Pro Asn Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg Cys 85 90 95 Ser Met Asp Leu Lys Asn Ile Asn Phe 100 105 <210> 23 <211> 105 <212> PRT <213> Human <400> 23 Met Arg Gly Ala Thr Arg Val Ser Ile Met Leu Leu Leu Val Thr Val 5 10 15 Ser Asp Cys Ala Val Ile Thr Gly Ala cys Glu Arg Asp Val Gln Cys 20 25 30 Gly Ala Gly Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg 35 40 45 Met Cys Thr Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser 50 55 60 His Lys Val Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro Cys 65 70 75 80 Leu Pro Asn Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg Cys 85 90 95 Ser Met Asp Leu Lys Asn Ile Asn Phe 100 105 <210> 24 <211> 678 <212> DNA <213> Human <400> 24 aaggctgagc gggaggaagc gagaggcatc taagcaggca gtgttttgcc ttcaccccaa 60 gtgaccatga gaggtgccac gcgagtctca atcatgctcc tcctagtaac tgtgtctgac 120 tgtgctgtga tcacaggggc ctgtgagcgg gatgtccagt gtggggcagg cacctgctgt 180 gccatcagcc tgtggcttcg agggctgcgg atgtgcaccc cgctggggcg ggaaggcgag 240 gagtgccacc ccggcagcca caagatcccc ttcttcagga aacgcaagca ccacacctgt 300 ccttgcttgc ccaacctgct gtgctccagg ttcccggacg gcaggtaccg ctgctccatg 360 gacttgaaga acatcaattt ttaggcgctt gcctggtctc aggataccca ccatcctttt 420 cctgagcaca gcctggattt ttatttctgc catgaaaccc agctcccatg actctcccag 480 tccctacact gactaccctg atctctcttg tctagtacgc acatatgcac acaggcagac 540 atacctccca tcatgacatg gtccccaggc tggcctgagg atgtcacagc ttgaggctgt 600 ggtgtgaaag gtggccagcc tggttctctt ccctgctcag gctgccagag aggtggtaaa 660 tggcagaaag gacattcc 678 <210> 25 <211> 678 <212> DNA <213> Human <400> 25 aaggctgagc gggaggaagc gagaggcatc taagcaggca gtgttttgcc ttcaccccaa 60 gtgaccatga gaggtgccac gcgagtctca atcatgctcc tcctagtaac tgtgtctgac 120 tgtgctgtga tcacaggggc ctgtgagcgg gatgtccagt gtggggcagg cacctgctgt 180 gccatcagcc tgtggcttcg agggctgcgg atgtgcaccc cgctggggcg ggaaggcgag 240 gagtgccacc ccggcagcca caaggtcccc ttcttcagga aacgcaagca ccacacctgt 300 ccttgcttgc ccaacctgct gtgctccagg ttcccggacg gcaggtaccg ctgctccatg 360 gacttgaaga acatcaattt ttaggcgctt gcctggtctc aggataccca ccatcctttt 420 cctgagcaca gcctggattt ttatttctgc catgaaaccc agctcccatg actctcccag 480 tccctacact gactaccctg atctctcttg tctagtacgc acatatgcac acaggcagac 540 atacctccca tcatgacatg gtccccaggc tggcctgagg atgtcacagc ttgaggctgt 600 ggtgtgaaag gtggccagcc tggttctctt ccctgctcag gctgccagag aggtggtaaa 660 tggcagaaag gacattcc 678 <210> 26 <211> 258 <212> DNA <213> Human <400> 26 gctgtgatca caggggcctg tgagcgggat gtccagtgtg gggcaggcac ctgctgtgcc 60 atcagcctgt ggcttcgagg gctgcggatg tgcaccccgc tggggcggga aggcgaggag 120 tgccaccccg gcagccacaa gatccccttc ttcaggaaac gcaagcacca cacctgtcct 180 tgcttgccca acctgctgtg ctccaggttc ccggacggca ggtaccgctg ctccatggac 240 ttgaagaaca tcaatttt 258 <210> 27 <211> 258 <212> DNA <213> Human <400> 27 gctgtgatca caggggcctg tgagcgggat gtccagtgtg gggcaggcac ctgctgtgcc 60 atcagcctgt ggcttcgagg gctgcggatg tgcaccccgc tggggcggga aggcgaggag 120 tgccaccccg gcagccacaa ggtccccttc ttcaggaaac gcaagcacca cacctgtcct 180 tgcttgccca acctgctgtg ctccaggttc ccggacggca ggtaccgctg ctccatggac 240 ttgaagaaca tcaatttt 258 <210> 28 <211> 315 <212> DNA <213> Human <400> 28 atgagaggtg ccacgcgagt ctcaatcatg ctcctcctag taactgtgtc tgactgtgct 60 gtgatcacag gggcctgtga gcgggatgtc cagtgtgggg caggcacctg ctgtgccatc 120 agcctgtggc ttcgagggct gcggatgtgc accccgctgg ggcgggaagg cgaggagtgc 180 caccccggca gccacaagat ccccttcttc aggaaacgca agcaccacac ctgtccttgc 240 ttgcccaacc tgctgtgctc caggttcccg gacggcaggt accgctgctc catggacttg 300 aagaacatca atttt 315 <210> 29 <211> 315 <212> DNA <213> Human <400> 29 atgagaggtg ccacgcgagt ctcaatcatg ctcctcctag taactgtgtc tgactgtgct 60 gtgatcacag gggcctgtga gcgggatgtc cagtgtgggg caggcacctg ctgtgccatc 120 agcctgtggc ttcgagggct gcggatgtgc accccgctgg ggcgggaagg cgaggagtgc 180 caccccggca gccacaaggt ccccttcttc aggaaacgca agcaccacac ctgtccttgc 240 ttgcccaacc tgctgtgctc caggttcccg gacggcaggt accgctgctc catggacttg 300 aagaacatca atttt 315 <210> 30 <211> 382 <212> DNA <213> Human <400> 30 gaattcgccc ttccaccatg agaggtgcca cgcgagtctc aatcatgctc ctcctagtaa 60 ctgtgtctga ctgtgctgtg atcacagggg cctgtgagcg ggatgtccag tgtggggcag 120 gcacctgctg tgccatcagc ctgtggcttc gagggctgcg gatgtgcacc ccgctggggc 180 gggaaggcga ggagtgccac cccggcagcc acaaggtccc cttcttcagg aaacgcaagc 240 accacacctg tccttgcttg cccaacctgc tgtgctccag gttcccggac ggcaggtacc 300 gctgctccat ggacttgaag aacatcaatt tttaggcgct tgcctggtct caggataccc 360 accatccttt cctgagctcg ag 382 <210> 31 <211> 10 <212> PRT <213> Human <400> 31 Ala Val Ile Thr Gly Ala Xaa Glu Arg Asp 5 10 <210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> <400> 32 gtcgaccacc atgagaggtg ccacgc 26 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> <400> 33 actagtcgca gaactggtag gtatgg 26 <210> 34 <211> 80 <212> PRT <213> Dendroaspis polylepis <400> 34 Ala Val Ile Thr Gly Ala Cys Glu Arg Asp Leu Gln Cys Gly Lys Gly 5 10 15 Thr Cys Cys Ala Val Ser Leu Trp Ile Lys Ser Val Arg Val Cys Thr 20 25 30 Pro Val Gly Thr Ser Gly Glu Asp Cys His Pro Ala Ser His Lys Ile 35 40 45 Pro Phe Ser Gly Gln Arg Met His His Thr Cys Pro Cys Ala Pro Asn 50 55 60 Leu Ala Cys Val Gln Thr Ser Pro Lys Lys Phe Lys Cys Leu Ser Lys 65 70 75 80 <210> 35 <211> 1152 <212> DNA <213> Human <400> 35 atggcagccc agaatggaaa caccagtttc acacccaact ttaatccacc ccaagaccat 60 gcctcctccc tctcctttaa cttcagttat ggtgattatg acctccctat ggatgaggat 120 gaggacatga ccaagacccg gaccttcttc gcagccaaga tcgtcattgg cattgcactg 180 gcaggcatca tgctggtctg cggcatcggt aactttgtct ttatcgctgc cctcacccgc 240 tataagaagt tgcgcaacct caccaatctg ctcattgcca acctggccat ctccgacttc 300 ctggtggcca tcatctgctg ccccttcgag atggactact acgtggtacg gcagctctcc 360 tgggagcatg gccacgtgct ctgtgcctcc gtcaactacc tgcgcaccgt ctccctctac 420 gtctccacca atgccttgct ggccattgcc attgacagat atctcgccat cgttcacccc 480 ttgaaaccac ggatgaatta tcaaacggcc tccttcctga tcgccttggt ctggatggtg 540 tccattctca ttgccatccc atcggcttac tttgcaacag aaaccgtcct ctttattgtc 600 aagagccagg agaagatctt ctgtggccag atctggcctg tggatcagca gctctactac 660 aagtcctact tcctcttcat ctttggtgtc gagttcgtgg gccctgtggt caccatgacc 720 ctgtgctatg ccaggatctc ccgggagctc tggttcaagg cagtccctgg gttccagacg 780 gagcagattc gcaagcggct gcgctgccgc aggaagacgg tcctggtgct catgtgcatt 840 ctcacggcct a tgtgctgtg ctgggcaccc ttctacggtt tcaccatcgt tcgtgacttc 900 ttccccactg tgttcgtgaa ggaaaagcac tacctcactg ccttctacgt ggtcgagtgc 960 atcgccatga gcaacagcat gatcaacacc gtgtgcttcg tgacggtcaa gaacaacacc 1020 atgaagtact tcaagaagat gatgctgctg cactggcgtc cctcccagcg ggggagcaag 1080 tccagtgctg accttgacct cagaaccaac ggggtgccca ccacagaaga agtggactgt 1140 atcaggctga ag 1152 <210> 36 <211> 384 <212> PRT <213> Human <400> 36 Met Ala Ala Gln Asn Gly Asn Thr Ser Phe Thr Pro Asn Phe Asn Pro 5 10 15 Pro Gln Asp His Ala Ser Ser Leu Ser Phe Asn Phe Ser Tyr Gly Asp 20 25 30 Tyr Asp Leu Pro Met Asp Glu Asp Glu Asp Met Thr Lys Thr Arg Thr 35 40 45 Phe Phe Ala Ala Lys Ile Val Ile Gly Ile Ala Leu Ala Gly Ile Met 50 55 60 Leu Val Cys Gly Ile Gly Asn Phe Val Phe Ile Ala Ala Leu Thr Arg 65 70 75 80 Tyr Lys Lys Leu Arg Asn Leu Thr Asn Leu Leu Ile Ala Asn Leu Ala 85 90 95 Ile Ser Asp Phe Leu Val Ala Ile Ile Cys Cys Pro Phe Glu Met Asp 100 105 110 Tyr Tyr Val Val Arg Gln Leu Ser Trp Glu His Gly His Val Leu Cys 115 120 125 Ala Ser Val Asn Tyr Leu Arg Thr Val Ser Leu Tyr Val Ser Thr Asn 130 135 140 Ala Leu Leu Ala Ile Ala Ile Asp Arg Tyr Leu Ala Ile Val His Pro 145 150 155 160 Leu Lys Pro Arg Met Asn Tyr Gln Thr Ala Ser Phe Leu Ile Ala Leu 165 170 175 Val Trp Met Val Ser Ile Leu Ile Ala Ile Pro Ser Ala Tyr Phe Ala 180 185 190 Thr Glu Thr Val Val Leu Phe Ile Val Lys Ser Gln Glu Lys Ile Phe Cys 195 200 205 Gly Gln Ile Tr p Pro Val Asp Gln Gln Leu Tyr Tyr Lys Ser Tyr Phe 210 215 220 Leu Phe Ile Phe Gly Val Glu Phe Val Gly Pro Val Val Thr Met Thr 225 230 235 240 Leu Cys Tyr Ala Arg Ile Ser Arg Glu Leu Trp Phe Lys Ala Val Pro 245 250 255 Gly Phe Gln Thr Glu Gln Ile Arg Lys Arg Leu Arg Cys Arg Arg Lys 260 265 270 Thr Val Leu Val Leu Met Cys Ile Leu Thr Ala Tyr Val Leu Cys Trp 275 280 280 285 Ala Pro Phe Tyr Gly Phe Thr Ile Val Arg Asp Phe Phe Pro Thr Val 290 295 300 Phe Val Lys Glu Lys His Tyr Leu Thr Ala Phe Tyr Val Val Glu Cys 305 310 315 320 Ile Ala Met Ser Asn Ser Met Ile Asn Thr Val Cys Phe Val Thr 325 330 335 Lys Asn Asn Thr Met Lys Tyr Phe Lys Lys Met Met Leu Leu His Trp 340 345 350 Arg Pro Ser Gln Arg Gly Ser Lys Ser Ser Ala Asp Leu Asp Leu Arg 355 360 365 Thr Asn Gly Val Pro Thr Thr Glu Glu Val Asp Cys Ile Arg Leu Lys 370 375 380 384 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> <400> 37 gtcgacatgg agaccactgt ggggaccctg 30 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> <400> 38 actagtttat ttcagtcgga tgcagtccac 30 <210> 39 <211> 1179 <212> DNA <213> Rat <400> 39 atggagacca ctgtggggac cctgggcgag aataccacaa acactttcac cgacttcttt 60 tctgcacgtg atggcagtgg agccgaaacc tcccccttgc cattcacttt cagctatggt 120 gactatgaca tgccctcgga tgaagaggag gatgtgacca actctcggac tttctttgct 180 gccaagattg tcattggcat ggctttggtg ggcatcatgc tggtgtgtgg catcggcaac 240 ttcatcttca tcactgcgct ggcccgctac aaaaagcttc gcaacctcac caacctgctt 300 atcgccaacc tggccatttc ggacttcctg gtagccatcg tgtgctgccc ctttgagatg 360 gactactatg tggtacgcca gctctcctgg gagcacggcc atgtcctgtg cgcctccgtc 420 aactacttgc gcaccgtctc cctctacgtg tccactaacg ccctactggc cattgccatt 480 gacaggtatc tggccattgt gcacccgctg agaccgcgga tgaagtgtca aacggctgca 540 ggcctgatct tcctggtgtg gtctgtgtcc atcctcatcg ccatcccagc cgcctacttc 600 accactgaga cggtgttggt catcgtggaa agccaggaga agatcttctg cggccagatc 660 tggccggtgg atcagcagtt ctactacagg tcctatttcc ttttggtctt cggcctcgag 720 ttcgtgggtc ctgtaatcgc catgaccctg tgctatgcca gggtgtcccg agagctctgg 780 ttcaaggcgg tgcccggctt ccagacagag cagatccgcc ggaggctgcg ctgtcgccga 840 cggacggtac t ggggctcgt gtgcgtcctt tccgcctatg tgctgtgctg ggctcccttc 900 tatggcttca ccatcgtgcg tgacttcttc ccctccgtgt ttgtgaaaga gaagcactac 960 ctcaccgcct tttatgtggt ggagtgcatc gccatgagca acagtatgat caatacgctg 1020 tgctttgtga ctgtcaggaa taacaccagt aagtacctca agaggatcct gcggctccag 1080 tggagggcct ctcctagcgg gagcaaggcc agcgctgacc tcgacctcag gaccacgggg 1140 attcctgcca cggaggaggt ggactgcatc cgactgaaa 1179 <210> 40 <211> 393 <212> PRT <213> Rat <400> 40 Met Glu Thr Thr Val Gly Thr Leu Gly Glu Asn Thr Thr Asn Thr Phe 5 10 15 Thr Asp Phe Phe Ser Ala Arg Asp Gly Ser Gly Ala Glu Thr Ser Pro 20 25 30 Leu Pro Phe Thr Phe Ser Tyr Gly Asp Tyr Asp Met Pro Ser Asp Glu 35 40 45 Glu Glu Asp Val Thr Asn Ser Arg Thr Phe Phe Ala Ala Lys Ile Val 50 55 60 Ile Gly Met Ala Leu Val Gly Ile Met Leu Val Cys Gly Ile Gly Asn 65 70 75 80 Phe Ile Phe Ile Thr Ala Leu Ala Arg Tyr Lys Lys Leu Arg Asn Leu 85 90 95 Thr Asn Leu Leu Ile Ala Asn Leu Ala Ile Ser Asp Phe Leu Val Ala 100 105 110 Ile Val Cys Cys Pro Phe Glu Met Asp Tyr Tyr Val Val Arg Gln Leu 115 120 125 Ser Trp Glu His Gly His Val Leu Cys Ala Ser Val Asn Tyr Leu Arg 130 135 140 Thr Val Ser Leu Tyr Val Ser Thr Asn Ala Leu Leu Ala Ile Ala Ile 145 150 155 160 Asp Arg Tyr Leu Ala Ile Val His Pro Leu Arg Pro Arg Met Lys Cys 165 170 175 Gln Thr Ala Ala Gly Leu Ile Phe Leu Val Trp Ser Val Ser Ile Leu 180 185 190 Ile Ala Ile Pro Ala Ala Tyr Phe Thr Thr Glu Thr Val Leu Val Ile 195 200 205 Val Glu Ser Gln Glu Lys Ile Phe Cys Gly Gln Ile Trp Pro Val Asp 210 215 220 Gln Gln Phe Tyr Tyr Arg Ser Tyr Phe Leu Leu Val Phe Gly Leu Glu 225 230 235 240 Phe Val Gly Pro Val Ile Ala Met Thr Leu Cys Tyr Ala Arg Val Ser 245 250 255 Arg Glu Leu Trp Phe Lys Ala Val Pro Gly Phe Gln Thr Glu Gln Ile 260 265 270 Arg Arg Arg Leu Arg Cys Arg Arg Arg Thr Val Leu Gly Leu Val Cys 275 280 285 Val Leu Ser Ala Tyr Val Leu Cys Trp Ala Pro Phe Tyr Gly Phe Thr 290 295 300 Ile Val Arg Asp Phe Phe Pro Ser Val Phe Val Lys Glu Lys His Tyr 305 310 315 320 Leu Thr Ala Phe Tyr Val Val Glu Cys Ile Ala Met Ser Asn Ser Met 325 330 335 Ile Asn Thr Leu Cys Phe Val Thr Val Arg Asn Asn Thr Ser Lys Tyr 340 345 350 Leu Lys Arg Ile Leu Arg Leu Gln Trp Arg Ala Ser Pro Ser Gly Ser 355 360 365 Lys Ala Ser Ala Asp Leu Asp Leu Arg Thr Thr Gly Ile Pro Ala Thr 370 375 380 Glu Glu Val Asp Cys Ile Arg Leu Lys 385 390 <210> 41 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> <400> 41 cctcaccaay ctgctyatyg ccaacctggc c 31 <210> 42 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> <400> 42 gtggtrcgsc agctctcctg ggagca 26 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> <400> 43 tcccgggagc tctggttcaa ggc 23 <210> 44 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> <400> 44 gagtgcatcg ccatgagcaa cagcatg 27 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> <400> 45 ggcttgaacc agagctcccg gga 23 <210> 46 <211> 1266 <212> DNA <213> Rat <400> 46 atggtatcag ttctgtccaa cagggacctc cacacactgg ccccagctga agtgctgaac 60 tccacgtggg cctatctccc tgacacatac cagcctacct gccacatcat caacatggga 120 gaccagaacg gaaacacaag ctttgcacca gacttgaacc caccccaaga ccacgtctcc 180 ttgctcccct taaactacag ttatggagat tatgacatcc ccctggatga cgatgaggat 240 gtgaccaaga cacagacctt ctttgcagcc aaaatcgtca ttggcgtagc cctggcaggc 300 atcatgctag tctgcggcgt tggcaacttt gtcttcattg ctgccctcgc ccgctacaag 360 aagctgcgca accttaccaa cctcctcatc gctaacctgg ccatctctga cttcctggtg 420 gcgatcgtct gctgcccctt tgagatggac tactacgtag tacgtcagct ttcctgggag 480 catggtcacg tgctttgtgc ctccgtcaac taccttcgta cagtctccct gtacgtctcc 540 accaatgctc tgctggccat cgctattgac agatatctcg ctattgtcca ccccttaaaa 600 cggatgaatt accagaccgc ctccttcctg atcgctttgg tctggatggt ctccatcctc 660 atcgccatcc catctgccta cttcaccaca gaaaccatcc ttgttatcgt caagaatcag 720 gaaaagctct tctgtggtca gatctggccc gtggaccagc agctctacta caaatcctac 780 ttcctcttcg tcttcgggct tgagttcgtg ggtcccgtgg tcactatgac cctgtgctat 840 gccaggatct c ccaggagct ctggttcaag gctgtacctg gtttccagac ggagcagatc 900 cgcaagcgac tgcgctgccg ccgaaagaca gtgctattgc tcatgggtat cctcacagcc 960 tacgtgctgt gctgggcgcc tttctatggc tttaccatag tgcgagactt cttccccacg 1020 ctggttgtga aggagaagca ctacctcacc gccttctatg tcgtcgagtg catcgccatg 1080 agcaacagca tgatcaatac tatatgcttc gtgacggtca agaacaacac catgaaatac 1140 ttcaagaaga tgctgctgct gcactggcgg ccctctcact acgggagtaa gtccagcgcg 1200 gacctcgacc tcaaaaccag tggggttcct gccaccgaag aggtggactg tatcaggcta 1260 aagtag 1266 <210> 47 <211> 421 <212> PRT <213> Rat <400> 47 Met Val Ser Val Leu Ser Asn Arg Asp Leu His Thr Leu Ala Pro Ala 5 10 15 Glu Val Leu Asn Ser Thr Trp Ala Tyr Leu Pro Asp Thr Tyr Gln Pro 20 25 30 Thr Cys His Ile Ile Asn Met Gly Asp Gln Asn Gly Asn Thr Ser Phe 35 40 45 Ala Pro Asp Leu Asn Pro Pro Gln Asp His Val Ser Leu Leu Pro Leu 50 55 60 Asn Tyr Ser Tyr Gly Asp Tyr Asp Ile Pro Leu Asp Asp Asp Glu Asp 65 70 75 80 Val Thr Lys Thr Gln Thr Phe Phe Ala Ala Lys Ile Val Ile Gly Val 85 90 95 Ala Leu Ala Gly Ile Met Leu Val Cys Gly Val Gly Asn Phe Val Phe 100 105 110 Ile Ala Ala Leu Ala Arg Tyr Lys Lys Leu Arg Asn Leu Thr Asn Leu 115 120 125 Leu Ile Ala Asn Leu Ala Ile Ser Asp Phe Leu Val Ala Ile Val Cys 130 135 140 Cys Pro Phe Glu Met Asp Tyr Tyr Val Val Arg Gln Leu Ser Trp Glu 145 150 155 160 His Gly His Val Leu Cys Ala Ser Val Asn Tyr Leu Arg Thr Val Ser 165 170 175 Leu Tyr Val Ser Thr Asn Ala Leu Leu Ala Ile Ala Ile Asp Arg Tyr 180 185 190 Leu Ala Ile Val His Pro Leu Lys Arg Met Asn Tyr Gln Thr Ala Ser 195 200 205 Phe Leu Ile Ala Leu Val Trp Met Val Ser Ile Leu Ile Ala Ile Pro 210 215 220 Ser Ala Tyr Phe Thr Thr Glu Thr Ile Leu Val Ile Val Lys Asn Gln 225 230 235 240 Glu Lys Leu Phe Cys Gly Gln Ile Trp Pro Val Asp Gln Gln Leu Tyr 245 250 255 Tyr Lys Ser Tyr Phe Leu Phe Val Phe Gly Leu Glu Phe Val Gly Pro 260 265 270 Val Val Thr Met Thr Leu Cys Tyr Ala Arg Ile Ser Gln Glu Leu Trp 275 280 285 Phe Lys Ala Val Pro Gly Phe Gln Thr Glu Gln Ile Arg Lys Arg Leu 290 295 300 300 Arg Cys Arg Arg Lys Thr Val Leu Leu Leu Met Gly Ile Leu Thr Ala 305 310 315 320 Tyr Val Leu Cys Trp Ala Pro Phe Tyr Gly Phe Thr Ile Val Arg Asp 325 330 335 Phe Phe Pro Thr Leu Val Val Lys Glu Lys His Tyr Leu Thr Ala Phe 340 345 350 Tyr Val Val Glu Cys Ile Ala Met Ser Asn Ser Met Ile Asn Thr Ile 355 360 365 Cys Phe Val Thr Val Lys Asn Asn Thr Met Lys Tyr Phe Lys Lys Met 370 375 380 Leu Leu Leu His Trp Arg Pro Ser His Tyr Gly Ser Lys Ser Ser Ala 385 390 395 400 Asp Leu Asp Leu Lys Thr Ser Gly Val Pro Ala Thr Glu Glu Val Asp 405 410 415 Cys Ile Arg Leu Lys 420 <210> 48 <211> 393 <212> PRT <213> Mouse <400> 48 Met Glu Thr Thr Val Gly Ala Leu Gly Glu Asn Thr Thr Asp Thr Phe 1 5 10 15 Thr Asp Phe Phe Ser Ala Leu Asp Gly His Glu Ala Gln Thr Gly Ser 20 25 30 Leu Pro Phe Thr Phe Ser Tyr Gly Asp Tyr Asp Met Pro Leu Asp Glu 35 40 45 Glu Glu Asp Val Thr Asn Ser Arg Thr Phe Phe Ala Ala Lys Ile Val 50 55 60 Ile Gly Met Ala Leu Val Gly Ile Met Leu Val Cys Gly Ile Gly Asn 65 70 75 80 Phe Ile Phe Ile Thr Ala Leu Ala Arg Tyr Lys Lys Leu Arg Asn Leu 85 90 95 Thr Asn Leu Leu Ile Ala Asn Leu Ala Ile Ser Asp Phe Leu Val Ala 100 105 110 Ile Val Cys Cys Pro Phe Glu Met Asp Tyr Tyr Val Val Arg Gln Leu 115 120 125 Ser Trp Glu His Gly His Val Leu Cys Ala Ser Val Asn Tyr Leu Arg 130 135 140 Thr Val Ser Leu Tyr Val Ser Thr Asn Ala Leu Leu Ala Ile Ala Ile 145 150 155 160 Asp Arg Tyr Leu Ala Ile Val His Pro Leu Arg Pro Arg Met Lys Cys 165 170 175 Gln Thr Ala Ala Gly Leu Ile Phe Leu Val Trp Ser Val Ser Ile Leu 180 185 190 Ile Ala Ile Pro Ala Ala Tyr Phe Thr Thr Glu Thr Val Leu Val Ile 195 200 205 Val Glu Arg G ln Glu Lys Ile Phe Cys Gly Gln Ile Trp Pro Val Asp 210 215 220 Gln Gln Phe Tyr Tyr Arg Ser Tyr Phe Leu Leu Val Phe Gly Leu Glu 225 230 235 240 Phe Val Gly Pro Val Val Ala Met Thr Leu Cys Tyr Ala Arg Val Ser 245 250 255 Arg Glu Leu Trp Phe Lys Ala Val Pro Gly Phe Gln Thr Glu Gln Ile 260 265 270 Arg Arg Thr Val Arg Cys Arg Arg Arg Thr Val Val Leu Gly Leu Val Cys 275 280 285 Val Leu Ser Ala Tyr Val Leu Cys Trp Ala Pro Phe Tyr Gly Phe Thr 290 295 300 Ile Val Arg Asp Phe Phe Pro Ser Val Phe Val Lys Glu Lys His Tyr 305 310 315 320 Leu Thr Ala Phe Tyr Val Val Glu Cys Ile Ala Met Ser Asn Ser Met 325 330 335 Ile Asn Thr Leu Cys Phe Val Thr Val Arg Asn Asn Thr Ser Lys Tyr 340 345 350 Leu Lys Arg Ile Leu Arg Leu Gln Trp Arg Ala Ser Pro Ser Gly Ser 355 360 365 Lys Ala Ser Ala Asp Leu Asp Leu Arg Thr Thr Gly Ile Pro Ala Thr 370 375 380 Glu Glu Val Asp Cys Ile Arg Leu Lys 385 390 393 <210> 49 <211> 381 <212> PRT <213> Mouse <400> 49 Met Gly Pro Gln Asn Arg Asn Thr Ser Phe Ala Pro Asp Leu Asn Pro 1 5 10 15 Pro Gln Asp His Val Ser Leu Asn Tyr Ser Tyr Gly Asp Tyr Asp Leu 20 25 30 Pro Leu Gly Glu Asp Glu Asp Val Thr Lys Thr Gln Thr Phe Phe Ala 35 40 45 Ala Lys Ile Val Ile Gly Val Ala Leu Ala Gly Ile Met Leu Val Cys 50 55 60 Gly Ile Gly Asn Phe Val Phe Ile Ala Ala Leu Ala Arg Tyr Lys Lys 65 70 75 80 Leu Arg Asn Leu Thr Asn Leu Leu Ile Ala Asn Leu Ala Ile Ser Asp 85 90 95 Phe Leu Val Ala Ile Val Cys Cys Pro Phe Glu Met Asp Tyr Tyr Val 100 105 110 Val Arg Gln Leu Ser Trp Ala His Gly His Val Leu Cys Ala Ser Val 115 120 125 Asn Tyr Leu Arg Thr Val Ser Leu Tyr Val Ser Thr Asn Ala Leu Leu 130 135 140 Ala Ile Ala Ile Asp Arg Tyr Leu Ala Ile Val His Pro Leu Lys Pro 145 150 155 160 Arg Met Asn Tyr Gln Thr Ala Ser Phe Leu Ile Ala Leu Val Trp Met 165 170 175 Val Ser Ile Leu Ile Ala Val Pro Ser Ala Tyr Phe Thr Thr Glu Thr 180 185 190 Ile Leu Val Ile Val Lys Asn Gln Glu Lys Ile Phe Cys Gly Gln Ile 195 200 205 Trp Ser Val A sp Gln Gln Leu Tyr Tyr Lys Ser Tyr Phe Leu Phe Val 210 215 220 Phe Gly Leu Glu Phe Val Gly Pro Val Val Thr Met Thr Leu Cys Tyr 225 230 235 240 Ala Arg Ile Ser Gln Glu Leu Trp Phe Lys Ala Val Pro Gly Phe Gln 245 250 255 Thr Glu Gln Ile Arg Lys Arg Leu Arg Cys Arg Arg Lys Thr Val Leu 260 265 270 Leu Leu Met Gly Ile Leu Thr Ala Tyr Val Leu Cys Trp Ala Pro Phe 275 280 285 Tyr Gly Phe Thr Ile Val Arg Asp Phe Phe Pro Thr Val Val Val Lys 290 295 300 Glu Lys His Tyr Leu Thr Ala Phe Tyr Val Val Glu Cys Ile Ala Met 305 310 315 320 Ser Asn Ser Met Ile Asn Thr Ile Cys Phe Val Thr Val Lys Asn Asn 325 330 335 Thr Met Lys Tyr Phe Lys Lys Met Leu Arg Leu His Trp Arg Pro Ser 340 345 350 His Tyr Gly Ser Lys Ser Ser Ala Asp Leu Asp Leu Lys Thr Ser Gly 355 360 365 Val Pro Ala Thr Glu Glu Val Asp Cys Ile Arg Leu Lys 370 375 380 <210> 50 <211> 1179 <212> DNA <213> Mouse <400> 50 atggagacca ctgtcggggc tctgggtgag aataccacag acaccttcac cgacttcttt 60 tctgcactcg atggccatga agcccaaacc ggctcgttac cattcacttt cagctacggt 120 gactatgaca tgcccctgga tgaagaggaa gatgtgacca attctcggac tttctttgct 180 gccaagattg tcattggcat ggctttggtg ggtatcatgc tagtgtgtgg catcggcaac 240 ttcatcttta tcactgccct ggcccgctac aaaaagctcc gcaacctcac caacctgctt 300 atcgccaacc tggccatttc agacttcctc gtggccatcg tgtgctgccc ctttgagatg 360 gactactatg tggtgcgcca gctctcctgg gagcatggtc atgtcctgtg cgcctctgtc 420 aactacttgc gtaccgtctc cctctacgtc tccactaacg ccctactggc cattgccatt 480 gacaggtatc tggccattgt gcacccgctg agaccgcgga tgaagtgtca aacagccgcc 540 ggcctgatct tcctggtgtg gtcagtatcc atcctcatcg ccattccagc tgcctacttc 600 accactgaga ccgtgctggt catcgtggag agacaggaga agatcttctg tggtcagatc 660 tggccggtgg atcagcagtt ctactacagg tcctatttcc ttttggtttt cggcctcgag 720 ttcgtgggcc ccgtagtcgc catgaccttg tgctatgcca gggtgtcccg ggagctctgg 780 ttcaaggcgg tgccaggctt ccagacagag cagatccgcc ggacggtgcg ctgccgccgc 840 aggacggtgc t ggggctcgt gtgcgtcctc tctgcctatg tgctgtgctg ggctcccttc 900 tatggcttca ctatcgtgcg tgacttcttc ccctccgtgt ttgtgaagga gaagcactac 960 ctcaccgcct tctatgtggt ggagtgcatc gccatgagca acagcatgat caatacgctc 1020 tgctttgtga ctgtcaggaa taacaccagt aagtacctca agaggatcct gcggcttcag 1080 tggagggcct ctcccagcgg gagcaaggcc agcgctgacc tcgacctcag gaccacggga 1140 atacctgcca ccgaggaggt ggactgcatc cgactgaaa 1179 <210> 51 <211> 1143 <212> DNA <213> Mouse <400> 51 atgggacccc agaacagaaa cactagcttt gcaccagact tgaatccacc ccaagaccat 60 gtctccttaa actacagtta tggtgattat gacctccccc tgggtgagga tgaggatgtg 120 accaagacac agaccttctt tgcagccaaa attgtcattg gcgtggcact ggcaggcatc 180 atgctggtct gcggcattgg caactttgtc ttcattgctg ccctcgcccg ctacaagaag 240 ctgcgcaacc ttaccaacct cctcattgct aacctggcca tctctgactt cctggtggcg 300 atcgtctgct gcccctttga gatggactat tatgtagtac ggcagctttc ctgggcgcat 360 ggtcacgtgc tttgtgcctc cgtcaactac cttcgtacgg tctccctgta cgtctccacc 420 aacgctctgc tggccatcgc tattgacaga tacctcgcta ttgtccaccc tttgaaacca 480 cggatgaatt atcagaccgc ttccttcctg atcgctttgg tctggatggt ctccatcctc 540 atcgctgtcc catctgccta cttcaccaca gaaaccatcc tcgttatcgt caagaatcaa 600 gaaaaaatct tctgtggtca gatctggtcg gtggaccagc agctctacta caaatcctac 660 ttcctcttcg tcttcgggct tgagttcgtg ggtcccgtgg tcactatgac cctgtgctat 720 gccaggatct cccaagagct ctggttcaag gctgtacctg gcttccagac ggagcaaatc 780 cgcaagcggc tgcgttgccg ccgcaagaca gtgctactgc tcatgggcat cctcacagcc 840 tacgtgctgt g ctgggcgcc gttctatggc tttaccatag tgcgagactt cttccccacg 900 gtagttgtga aggagaagca ctacctcacc gccttctacg tcgtggagtg cattgccatg 960 agcaacagca tgatcaatac tatatgcttc gtgacggtca agaacaacac catgaaatac 1020 ttcaagaaga tgctgcggct ccactggcgg ccctctcact acgggagtaa gtccagcgct 1080 gacctcgacc tcaaaaccag cggggtgcct gccactgaag aggtggattg tatcagacta 1140 aag 1143 <210> 52 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 52 tatggcggtg attaccggtg cgtgcgaacg tgatgtgcag tgcggtgcgg gtacctgctg 60 cgcgattagc ctgtggctgc gtggtctg 88 <210> 53 <211> 92 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 53 cgtatgtgca ccccgctggg tcgtgaaggt gaagaatgcc atccgggtag ccataaagtg 60 ccgttcttcc gtaaacgtaa acatcatacc tg 92 <210> 54 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 54 cccgtgcctg ccgaacctgc tgtgcagccg tttcccggat ggtcgttatc gttgcagcat 60 ggatctgaaa aacattaact tttagg 86 <210> 55 <211> 94 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 55 cacatacgca gaccacgcag ccacaggcta atcgcgcagc aggtacccgc accgcactgc 60 acatcacgtt cgcacgcacc ggtaatcacc gcca 94 <210> 56 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 56 aggcacgggc aggtatgatg tttacgttta cggaagaacg gcactttatg gctacccgga 60 tggcattctt caccttcacg acccagcggg gtg 93 <210> 57 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 57 gatccctaaa agttaatgtt tttcagatcc atgctgcaac gataacgacc atccgggaaa 60 cggctgcaca gcaggttcgg c 81 <210> 58 <211> 258 <212> DNA <213> Artificial Sequence <220> <223> <400> 58 gcggtgatta ccggtgcgtg cgaacgtgat gtgcagtgcg gtgcgggtac ctgctgcgcg 60 attagcctgt ggctgcgtgg tctgcgtatg tgcaccccgc tgggtcgtga aggtgaagaa 120 tgccatccgg gtagccataa agtgccgttc ttccgtaaac gtaaacatca tacctgcccg 180 tgcctgccga acctgctgtg cagccgtttc ccggatggtc gttatcgttg cagcatggat 240 ctgaaaaaca ttaacttt 258

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence (ZAQC) of the DNA encoding the human brain-derived protein of the present invention obtained in Example 1, and the amino acid sequence deduced therefrom (following FIG. 2).

FIG. 2 shows the nucleotide sequence (ZAQC) of the DNA encoding the human brain-derived protein of the present invention obtained in Example 1 and the amino acid sequence deduced therefrom (continued from FIG. 1 and continued from FIG. 3).

FIG. 3 shows the nucleotide sequence (ZAQC) of the DNA encoding the human brain-derived protein of the present invention obtained in Example 1, and the amino acid sequence deduced therefrom (continuation of FIG. 2).

FIG. 4 shows the nucleotide sequence (ZAQT) of the DNA encoding the human brain-derived protein of the present invention obtained in Example 1 and the amino acid sequence deduced therefrom (continued from FIG. 5).

FIG. 5 shows the nucleotide sequence (ZAQT) of the DNA encoding the human brain-derived protein of the present invention obtained in Example 1, and the amino acid sequence deduced therefrom (continued from FIG. 4 and continued from FIG. 6).

FIG. 6 shows the nucleotide sequence (ZAQT) of the DNA encoding the human brain-derived protein of the present invention obtained in Example 1, and the amino acid sequence deduced therefrom (continuation of FIG. 5).

FIG. 7 shows a hydrophobicity plot of the human brain-derived protein of the present invention.

FIG. 8 shows the results of analysis of the expression distribution of ZAQ performed in Example 2.

FIG. 9 shows the amino acid sequences of MIT1, human ZAQ ligand precursor peptide (A type) and human ZAQ ligand precursor peptide (G type). In the figure, "MI
“T1” represents the amino acid sequence of MIT1 as “Human (A
"type)" represents the amino acid sequence of human ZAQ ligand mature peptide (A type) as "Human (Gt
ype) "is a humanized ZAQ ligand mature peptide (G
The amino acid sequence of (type) is shown respectively.

FIG. 10: Purified ZAQ performed in Example 6 (6-3)
The measurement result of the ZAQ activation effect of a ligand peptide is shown.

FIG. 11 shows a plasmid pC used in Example 5 (5-1).
3 shows a restriction map of AN618.

FIG. 12 shows human ZAQ performed in Example 8 (8-3).
The measurement results of the ZAQ receptor activating effect of the ligand peptide and MIT1 are shown. In the figure, ○ indicates a human ZAQ ligand peptide, and ● indicates MIT1.

FIG. 13: Humanized ZAQ performed in Example 8 (8-3)
The measurement result of the I5E receptor activation effect of a ligand peptide and MIT1 is shown. In the figure, ○ indicates a human ZAQ ligand peptide, and ● indicates MIT1.

14 shows the measurement results of the contraction effect performed in Example 11. FIG. In the figure, closed squares indicate human ZAQ ligand peptide, and open circles indicate MIT1.

FIG. 15 shows the measurement results of a binding assay using a ZAQ membrane fraction performed in Example 12. In the figure, solid squares indicate the ¹²⁵ I-MIT1 specific binding amount when a human ZAQ ligand peptide at a variable concentration (shown on the horizontal axis) was added as a test compound, and open circles also indicate when MIT1 was added as a test compound. Shows the amount of ¹²⁵ I-MIT1 specific binding.

FIG. 16 shows a measurement result of a binding assay using an I5E membrane fraction performed in Example 12. In the figure, □ indicates the amount of ¹²⁵ I-MIT1 specific binding when a human ZAQ ligand peptide at a variable concentration (shown on the horizontal axis) was added as a test compound, and ○ indicates a variable concentration of MIT1 at the test compound (shown on the horizontal axis). Shows the ¹²⁵ I-MIT1 specific binding amount when added as.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/10 A61P 1/14 4C085 1/12 C07K 14/705 4C086 1/14 16/28 4C087 C07K 14 / 705 C12N 1/15 4H045 16/28 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 G01N 33/15 Z 5/10 33/50 Z C12P 21/02 A61K 31/711 G01N 33/15 35/76 33/50 39/395 D // A61K 31/711 48/00 35/76 C12N 15/00 ZNAA 39/395 5/00 A 48/00 A61K 37/02 (72) Inventor Yoshihiro Takatsu 1-6-2, Takezono, Tsukuba, Ibaraki Prefecture Tsukuba / Sakura Danchi 905-505 (72) Inventor Takuya Watanabe 6-14-9-B 904, Shintaka, Yodogawa-ku, Osaka-shi, Osaka (72) Inventor Neko Terao 985 Onozaki, Tsukuba, Ibaraki, Japan Ground RO YAL ZOA Nakayama 307 (72) Inventor Yasushi Shinya 1-7-9 Kasuga Heights, Tsukuba, Ibaraki Prefecture 703 Takeda Kasuga Heights 703 (72) Inventor Shuji Hinuma 1-7-7 Kasuga Takeda, Tsukuba City, Ibaraki Prefecture Kasuga Takeda Heights 1402 F-term (Reference) 2G045 AA40 BA11 BB50 DA12 DA13 DA14 DA36 FB02 4B024 AA01 AA11 BA63 CA04 DA02 DA06 EA04 GA11 HA11 HA15 4B064 AG20 CA02 CA10 CA19 CC24 DA01 DA13 4B065 AA26X AA90A AACA AA24A02 AA17 BA01 BA08 BA22 BA23 CA18 CA20 DB63 NA14 ZA661 ZA721 ZC012 ZC412 4C085 AA13 BB11 CC03 CC21 DD62 EE01 4C086 AA01 AA03 EA16 MA01 MA04 NA14 ZA66 ZA72 ZC01 ZC41 4C087 AA01 AA03 ZA13A14 ZA14A14 DA75 EA25 EA50 FA72 FA74

Claims (27)

[Claims] 1. A peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 20 or SEQ ID NO: 21, or a salt thereof. 2. The peptide according to claim 1, which is a peptide containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 21, or a salt thereof. 3. The peptide according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 20, or a salt thereof. 4. The peptide according to claim 1, which comprises the amino acid sequence represented by SEQ ID NO: 21, or a salt thereof. 5. The peptide according to claim 1, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 22 or SEQ ID NO: 23, or a salt thereof. 6. A polynucleotide comprising a polynucleotide encoding the peptide according to claim 1. 7. The polynucleotide according to claim 6, which is a DNA. 8. The DNA according to claim 7, which comprises the nucleotide sequence represented by SEQ ID NO: 26 or SEQ ID NO: 27. 9. The DNA according to claim 7, which comprises the nucleotide sequence represented by SEQ ID NO: 28 or SEQ ID NO: 29. [10] A recombinant vector containing the polynucleotide according to [6]. [11] A transformant transformed with the recombinant vector according to [10]. (12) culturing the transformant according to (11),
A method for producing a peptide or a salt thereof according to claim 1, wherein the peptide according to claim 1 is produced and accumulated. 13. An antibody against the peptide according to claim 1 or a salt thereof. (14) A peptide or a salt thereof according to (1), and a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof or a salt thereof. A compound or a salt thereof, which alters the binding property to a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 with the peptide of claim 1 or a salt thereof. Screening method. 15. A peptide or a salt thereof according to claim 1, and a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, a partial peptide thereof or a salt thereof. A compound or a salt thereof, which alters the binding of a peptide or a salt thereof to a protein or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, or a salt thereof. Screening method. (16) A peptide or a salt thereof according to (1), and a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof or a salt thereof. A compound or a compound thereof which changes the binding property to a protein or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a peptide or a salt thereof according to claim 1 or a salt thereof. Salt screening kit. 17. A protein or a salt thereof, and a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36. A compound or a compound thereof which alters the binding property to a protein or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 36, or a peptide thereof or the salt thereof according to claim 1. Salt screening kit. 18. The peptide according to claim 1, which can be obtained by using the screening method according to claim 14 or the screening kit according to claim 16, the same as the amino acid sequence represented by SEQ ID NO: 1 or A compound or a salt thereof that alters the binding property to a protein containing substantially the same amino acid sequence or a salt thereof. (19) a peptide or salt thereof according to (1), which can be obtained by using the screening method according to (15) or the screening kit according to (17); A compound or a salt thereof that alters the binding property to a protein containing substantially the same amino acid sequence or a salt thereof. [20] a pharmaceutical comprising the compound or the salt thereof according to [18] or [19]; 21. The medicament according to claim 20, which is an agent for preventing or treating digestive diseases. A pharmaceutical comprising the peptide of claim 1 or a salt thereof. 23. The medicament according to claim 22, which is a prophylactic / therapeutic agent for digestive diseases. 24. A peptide according to claim 1 or a salt thereof, which can be obtained from a mammal using the screening method according to claim 14 or the screening kit according to claim 16, and represented by SEQ ID NO: 1. A method for preventing or treating gastrointestinal diseases, which comprises administering an effective amount of a compound or a salt thereof that alters the binding to a protein or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence to be used. . 25. The peptide according to claim 1 or a salt thereof, which can be obtained from a mammal using the screening method according to claim 15 or the screening kit according to claim 17, and represented by SEQ ID NO: 36. A method for preventing or treating gastrointestinal diseases, which comprises administering an effective amount of a compound or a salt thereof that changes the binding property to a protein or a salt thereof, which contains the same or substantially the same amino acid sequence as the amino acid sequence. . 26. The peptide according to claim 1 or a salt thereof, which can be obtained by using the screening method according to claim 14 or the screening kit according to claim 16 for producing an agent for preventing or treating digestive diseases. And SEQ ID NO:
Use of a compound having an amino acid sequence identical or substantially identical to the amino acid sequence represented by 1, or a compound or a salt thereof, which alters the binding property to a protein or a salt thereof. (27) the peptide or the salt thereof according to (1), which can be obtained by using the screening method according to (15) or the screening kit according to (17) for producing a prophylactic / therapeutic agent for a gastrointestinal disease; And SEQ ID NO:
Use of a compound having an amino acid sequence identical or substantially identical to the amino acid sequence represented by No. 36 or a compound or a salt thereof, which alters the binding property to a protein or a salt thereof.